Department of Clinical ChemistryUniversity of Helsinki

and Wihuri Reasearch Institute

THE EFFECTS OF ETHANOL AND

ACETALDEHYDE ON APOPTOSIS AND EXPRESSION OF

MODULATORY GENES IN HEART

Heidi Jänkälä

Academic dissertation

To be publicly discussed, with the permission of the Medical Faculty of the University of Helsinki, in auditorium 2 at the

Meilahti Hospital, Haartmaninkatu 4, HelsinkiOn September 23, 2005, at 12 noon

Helsinki 2005

SUPERVISORS

Docent Tiina MäkiDepartment of Clinical ChemistryUniversity of Helsinki

Professor Matti HärkönenDepartment of Clinical ChemistryUniversity of Helsinki

REVIEWERS

Professor Onni NiemeläDepartment of Clinical ChemistryUniversity of Tampere

Docent Kari PulkkiDepartment of Clinical ChemistryUniversity of Turku

OPPONENT

Professor Mikko SalaspuroResearch Unit of Substance Abuse MedicineInstitute of MedicineUniversity of Helsinki

ISBN 952-91-9196-0 (nid.)ISNBN 952-10-2658-8 (PDF)Yliopistopaino, Helsinki

CONTENTS

ABBREVIATIONS .................................................................................... 6

LIST OF ORIGINAL PUBLICATIONS ....................................................... 7

INTRODUCTION ..................................................................................... 8

REVIEW OF THE LITERATURE .............................................................. 9

1. ETHANOL AND ACETALDEHYDE METABOLISM ................................... 9

1.1. Pathways of alcohol metabolism .................................................. 9 1.2. Gender differences in metabolism ................................................ 9

2. EFFECTS OF ETHANOL AND ACETALDEHYDE ON HEART MUSCLE .......10

2.1. Mechanisms of cardiac muscle adaptation to ethanol and acetaldehyde ................................................................................10 2.2. Alcoholic cardiac muscle disease ..................................................12 2.2.1. General ...................................................................... 12 2.2.2. Gender differences in alcoholic heart muscle disease ...12

3. APOPTOSIS IN THE HEART ..................................................................13

3.1. General mechanisms .................................................................... 13 3.2. Hypertrophy, cell death and cell survival .....................................15 3.3. Gender differences ......................................................................16

4. ETHANOL AND APOPTOSIS .................................................................17

4.1. Heart ........................................................................................... 17 4.2. Other tissues ................................................................................17

5. ACETALDEHYDE AND APOPTOSIS ......................................................18

5.1. Heart and other tissues ................................................................18

6. GENES ASSOCIATED WITH CARDIAC CELL ADAPTATION, APOPTOSIS

AND HYPERTROPHY ................................................................................18

6.1. Atrial and brain natriuretic peptides ...........................................18 6.1.1. General ......................................................................18 6.1.2. Natriuretic peptides and gender ................................20 6.1.3. Natriuretic peptides and ethanol ................................20 6.2. p53 .............. ..............................................................................21 6.2.1. General ......................................................................21 6.2.2. p53 and gender ..........................................................22

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6.2.3. p53 and ethanol .........................................................23 6.3. p21 .............................................................................................23 6.3.1. General ......................................................................23 6.3.2. p21 and gender .........................................................25 6.3.3. p21 and ethanol .........................................................25 6.4. Bcl-2 and bax .............................................................................25 6.4.1. General ......................................................................25 6.4.2. Bcl-2, bax and gender .................................................27 6.4.3. Bcl-2, bax and ethanol ................................................27 6.5. Tumor necrosis factor-alpha .........................................................27 6.5.1. General ......................................................................27 6.5.2. TNF-alpha and gender ...............................................29 6.5.3. TNF-alpha and ethanol ..............................................29

AIMS OF THE STUDY ........................................................................... 30

MATERIALS AND METHODS ...............................................................31

1. ANIMALS AND EXPERIMENTAL DESIGN ..............................................31

1.1. One-week immobilization and skeletal muscle: a methodological study (I) ..................................................................31 1.2. Short-term experiment in vivo (II,IV) ..........................................31 1.3. Acute experiment with isolated heart (III) ...................................32 1.4. Chronic experiment in vivo (V) ....................................................32 1.5. Ethical considerations ..................................................................33

2. BLOOD AND TISSUE SAMPLING ..........................................................33

2.1. Measurement of blood ethanol and acetaldehyde levels ................33 2.2. Measurement of blood N-terminal ANP levels ...........................33 2.3. Analysis of mRNA levels by quantitative RT-PCR method .........33 2.4. Quantifi cation of cytosolic DNA fragmentation by sandwich enzyme immunoassay .......................................................................... 35

3. STATISTICAL ANALYSIS .......................................................................35

RESULTS .............................................................................................36

1. VALIDATION OF QUANTITATIVE RT-PCR METHOD (I) ..........................36

2. THE EFFECT OF SHORT-TERM ETHANOL AND CALCIUM CARBIMIDE

TREATMENT ON APOPTOSIS AND EXPRESSION OF CARDIAC

MODULATORY GENES IN VIVO (II, IV) ..................................................... 36

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3. THE EFFECT OF ACUTE ETHANOL AND ACETALDEHYDE INFUSION ON

APOPTOSIS AND EXPRESSION OF CARDIAC MODULATORY GENES IN

LANGENDORFF PREPARATION (III) .......................................................... 41

4. THE EFFECT OF CHRONIC ETHANOL EXPOSURE ON APOPTOSIS

AND EXPRESSION OF CARDIAC MODULATORY GENES IN FEMALE AND

MALE RATS IN VIVO (V) ........................................................................... 43

DISCUSSION .......... .............................................................................. 46

1. ETHANOL, ACETALDEHYDE AND LEFT VENTRICULAR APOPTOSIS ....... 46

2. ETHANOL, ACETALDEHYDE AND LEFT VENTRICULAR p53

GENE EXPRESSION .................................................................................. 47

3. ETHANOL, ACETALDEHYDE AND LEFT VENTRICULAR p21

GENE EXPRESSION .................................................................................. 48

4. ETHANOL, ACETALDEHYDE AND LEFT VENTRICULAR ANP

GENE EXPRESSION .................................................................................. 49

5. ETHANOL, ACETALDEHYDE AND LEFT VENTRICULAR BNP

GENE EXPRESSION .................................................................................. 50

6. ETHANOL, ACETALDEHYDE AND LEFT VENTRICULAR BCL-2 AND BAX

GENE EXPRESSION AND BCL-2/BAX RATIO ............................................. 50

7. ETHANOL, ACETALDEHYDE AND LEFT VENTRICULAR TNF-ALPHA

GENE EXPRESSION .................................................................................. 51

8. LIMITATIONS OF THE STUDY ............................................................... 52

SUMMARY AND CONCLUSION ........................................................... 53

ACKNOWLEDGEMENTS ....................................................................... 54

REFERENCES .......... .............................................................................. 56

ORIGINAL PUBLICATIONS ................................................................... 79

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ABBREVIATIONS

AA rats alcohol preferring (Alko, Alcohol) rat lineAcH acetaldehydeADH alcohol dehydrogenaseAHMD alcoholic heart muscle diseaseAkt a serine/threonine protein kinase also known as protein kinase BALDH acetaldehyde dehydrogenaseANA rats alcohol avoiding (Alko, Non-Alcohol) rat lineAng II angiotensin IIANP atrial natriuretic peptideATP adenosine triphosphateBax gene for proto-oncogene bax, a proapoptotic member of the bcl-2 familyBcl-2 gene for B cell lymphoma-2 protein, an antiapoptotic member of the bcl-2 familyBNP brain natriuretic peptidebp base pairbw body weightC controlCC calcium carbimidecDNA complementary deoxyribonucleic acidCNS central nervous systemcRNA complementary ribonucleic acidDNA deoxyribonucleic acidE ethanolELISA enzyme linked immunosorbent assayFAEE fatty acid ethyl estersLV left ventriclemRNA messenger ribonucleic acidNAD+ nicotinamide adenine dinucleotideNADP+ nicotinamide adenine dinucleotide phosphateNADPH reduced nicotinamide adenine dinucleotide phosphateNF-kB nuclear factor-kBn.s. non-signifi cantNT-ANP N-terminal ANPp21 cyclin-dependent kinase inhibitor, also known as ras, waf1, cip1p53 gene for protein p53, a tumor suppressor genePCR polymerase chain reactionRIA radioimmunoassayRT reverse transcriptionRT-PCR reverse transcription of RNA followed by polymerase chain reactionS.E.M. standard error of the meanSHR spontaneously hypertensive rat lineTNF-alpha tumor necrosis factor-alphaWKY normotensive Wistar-Kyoto ratline

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LIST OF ORIGINAL PUBLICATIONS

This thesis is based on the following papers referred to in the text by their Ro-man numerals:

I Jänkälä H, Harjola V-P, Petersen NE, Härkönen M. Myosin heavy chain mRNA transform to faster isoforms in immobilized skeletal muscle: a quanti-tative PCR study. J Appl Physiol 1997;82:977-982.

II Jänkälä H, Eriksson CJP, Petersen NE, Härkönen M, Mäki T. Role of acetaldehyde in the induction of heart left ventricular atrial natriuretic peptide gene expression in rats. Alcohol Alcohol 2000;35:331-335.

III Jänkälä H, Eklund KK, Kokkonen JO, Kovanen PT, Lindstedt KA, Härkönen M, Mäki T. Ethanol infusion increases ANP and p21 gene expression in isolated perfused rat heart. Biochem Biophys Res Commun 2001;281:328-333.

IV Jänkälä H, Eriksson CJP, Eklund KK, Härkönen M, Mäki T. Combined calcium carbimide and ethanol treatment induces high blood acetaldehyde lev-els, myocardial apoptosis and altered expression of apoptosis-regulating genes in rat. Alcohol Alcohol 2002;37:222-228.

V Jänkälä H, Eriksson CJP, Eklund KK, Sarviharju M, Härkönen M, Mäki T. Effect of chronic ethanol ingestion and gender on heart left ventricular p53 gene expression. Alcohol Clin Exp Res 2005;29:1368-1373.

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INTRODUCTION

Although benefi cial effects of mild to moderate ethanol consumption have been reported in relation to cardiovascular diseases (for references see Im-hof & Koenig 2003), alcohol abuse has been shown to be a major cause of non-ischemic cardiomyopathy in Western society. Outcome in those affected is poor, and mortality can reach 40-60% within three to fi ve years (Dancy & Maxwell 1986; Schoppet & Maisch 2001; Piano 2002). However, the biochem-ical and molecular mechanisms involved in the pathological effects of ethanol and its highly reactive metabolite, acetaldehyde, remain largely unknown.

During the last decade, roles of apoptosis and apoptosis regulating genes have been described in relation to many types of ethanol induced organ damage, for example in the liver (Goldin et al. 1993). Apoptosis is a stepwise, tightly regu-lated mechanism through which damaged or unwanted cells are eliminated without evoking an infl ammatory response (Bishopric et al. 2001). However, excessive apoptosis has been associated with many cardiac diseases, including cardiomyopathy. Adult cardiomyocytes are terminally differentiated and, once destroyed, are sparsely replaced. Their loss can therefore contribute to func-tional decline of the myocardium and lead to heart disease (Gill et al. 2002).

The aim of the work described here was to study the effects of ethanol and acetaldehyde on apoptosis and some genes associated with cardiac cell adapta-tion, apoptosis and hypertrophy in the rat heart, in vivo and in vitro.

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REVIEW OF LITERATURE

1. ETHANOL AND ACETALDEHYDE METABOLISM

1.1. Pathways of alcohol metabolism

In healthy individuals ethanol is mostly oxidized by alcohol dehydrogenase (ADH). ADH is a NAD+(NADP+)-dependent enzyme expressed as numerous isoenzymes, and is most abundant in liver tissue (Jörnvall & Höög 1995). Al-most all organs are capable of oxidizing small amounts of ethanol. Trace amounts of ADH have also been found in the heart (for references see Regan 1984).

The microsomal ethanol oxidizing system is an ethanol-inducible, NADPH-dependent pathway for ethanol oxidation. Expression of genes for these mono-oxygenases has been demonstrated in the heart, predominantly in the right side (Thum & Borlak 2000).

A third pathway of ethanol oxidation in mammals involves catalase, a hy-drogen-peroxide-dependent enzyme that acts as an intracellular antioxidant (Bai & Cederbaum 2001). Peroxisomes containing catalase have been found in the myocardium in mice, and in the hearts of several other mammalian spe-cies, including primates. Increased cardiac catalase activity has been observed in rats that have ingested ethanol over long periods, and this has been inter-preted as a protective compensatory mechanism. In rats, chronic inhibition of catalase during six weeks of ethanol consumption results in substantial histo-logic abnormality in the myocardium (for references see Regan 1984).

A nonoxidative pathway of ethanol metabolism has been identifi ed in the heart. This metabolic pathway results in potentially toxic products known as fatty acid ethyl esters (FAEE). These are thought to accumulate in cell mi-tochondria, facilitate uncoupling of oxidative phosphorylation and result in ineffi ciency of energy production, leading to cell damage. FAEEs are rapidly synthesized by FAEE synthase (Beckemeier & Bora 1998). Ethanol exposure results in signifi cant increases in levels of cardiac FAEE when ethanol reacts with nonesterifi ed fatty acids (Calabrese et al. 2001).

Oxidation of acetaldehyde to acetate is catalysed by acetaldehyde dehydro-genase (ALDH), a NAD+(NADP+)-dependent enzyme. There are four to fi ve classes of ALDH, including both constitutive and inducible isoenzymes. A low Km (1 µmol/l) ALDH activity has been found in rat heart mitochondria but an acetaldehyde concentration of 50 µmol/l or higher is necessary to reduce tis-sue NAD+ in the perfused heart (Peuhkurinen et al. 1983).

1.2. Gender differences in metabolism

Women develop, for example, alcohol-related cirrhosis and alcohol-related hepatitis at a younger age and after a lower cumulative alcohol intake than

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men. Differences between males and females in connection with ethanol-re-lated pharmacokinetics could explain observed gender differences concerning blood concentrations and toxicity of ethanol (Thomasson 1995). The rate of gastric emptying of alcohol is lower in women than in men, and fi rst-pass me-tabolism is less extensive in women than in men because of lower ADH ac-tivity in the female stomach. In consequence, the bioavailability of ethanol is higher in women than in men. The smaller volume of distribution for ethanol in the female body results in higher blood alcohol levels than in men. Blood alcohol levels are therefore higher and more sustained in women than in men after a given dose of ethanol per kg body weight. However, the rate of etha-nol oxidation in the liver is higher in women than in men. The maximal rate of elimination is 10% higher in women than in men (Baraona et al. 2001). Women therefore exhibit higher ethanol-induced plasma acetaldehyde levels than men, which may result in tissues being exposed to higher levels of acetal-dehyde (Thomasson 1995). It has also been suggested that estrogen-related el-evations of acetaldehyde levels may help explain gender differences relating to the adverse effects of ethanol (Eriksson et al. 1996).

2. EFFECTS OF ETHANOL AND ACETALDEHYDE ON HEART MUSCLE

2.1. Mechanisms of cardiac muscle adaptation to ethanol and acetaldehyde

Mitochondrial membrane damage may be one of the most important effects of alcohol on the heart. Ethanol changes mitochondrial membrane permeabil-ity and function, depresses mitochondrial respiration (for references see Regan 1984), inhibits mitochondrial protein synthesis (Dancy & Maxwell 1986) and induces oxidative stress (Reinke et al. 1987).

Both ethanol and acetaldehyde affect cardiac protein synthesis. In one study, short-term exposure to acetaldehyde but not to ethanol reduced cardiac protein synthesis (Schreiber et al. 1972). In other studies reductions in car-diac protein synthesis in response to short-term administration of ethanol have been observed (Siddiq et al. 1993; Preedy et al. 1994). More prolonged inges-tion of ethanol has been shown to either inhibit (Tiernan et al. 1985; Vary et al. 2001) or increase protein synthesis (Preedy et al. 1994). Animals that have inhaled acetaldehyde for 21 days exhibit no diminution in the rate of synthesis of cardiac proteins (Auton & Ward 1986).

Ethanol increases biomembrane fl uidity and selectively alters ionic con-ductance (Dancy & Maxwell 1986). For example, ethanol administration re-versibly inhibits both uptake and binding of calcium by isolated cardiac sar-coplasmic reticulum in vitro, and by mitochondria in vivo, and acetaldehyde, but not ethanol, inhibits calcium-activated myofi brillar ATPase (Bing 1978).

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Evidence that acetaldehyde plays a role in alcohol-induced cardiomyopa-thy stems almost entirely from experiments in animals. Consideration has been given to whether there may be an immunological basis for progression of alcoholic cardiomyopathy. Acetaldehyde is extremely reactive. It binds cova-lently to structural and contractile proteins. It forms protein adducts, which may cause DNA and cell malfunction through effects on transcription and/or by stimulating immune responses (Eriksson 2001; Niemelä 2001). Protein ad-ducts are generated both in heart and skeletal muscle in response to either acute (Niemelä et al. 2002; Niemelä et al. 2003) or chronic (Worrall et al. 2000; Worrall et al. 2001) ethanol exposure. Thirty-three per cent of patients with alcoholic heart muscle disease (AHMD) exhibit antibodies against car-diac protein-acetaldehyde adducts (Harcombe et al. 1995). Interesting work has been undertaken using transgenic mice with cardiac-specifi c over-expres-sion of ADH. After ethanol intake cardiac acetaldehyde levels are four times greater than in control mice, elevated ANP gene expression, cardiac lipid and protein damage, hypertrophy and contractile damage, all indicative of AHMD (Liang et al. 1999; Hintz et al. 2003). Evidence relating to human cardiac in-jury comes from observations of simultaneous consumption of alcohol and use of cyanamide or disulfi ram (acetaldehyde dehydrogenase inhibitors) (for review see Eriksson 2001). Administration of acetaldehyde is particularly problematic because of its extreme chemical and biological reactivity, and very high toxic-ity (Preedy & Richardson 1994).

In rats increases in heart weight are evident after only 48 hours of exposure to ethanol. After 96 hours of exposure increases of more than 20% over control values are observed (Adams & Hirst 1986). Chronic administration of alcohol for 12 weeks to eight months results in decreased heart weight (Capasso et al. 1991; Vary et al. 2001). In some studies heart weight to body weight ratios do not change (Capasso et al. 1991), as in others ratios decrease (Brown et al. 1996). In middle-aged men heart weight increases with increasing daily alco-hol use (Kajander et al. 2001). Acetaldehyde exposure for 21 days signifi cantly increases heart weight as a percentage of body weight (Auton & Ward 1986).

Recent data indicate that both acute (Krenz et al. 2002) and chronic (three to 12-weeks) (Miyamae et al. 1997) administration of ethanol can have a pro-tective effect on the heart that resembles preconditioning. Results of studies performed both in vivo and in vitro show that hearts that are exposed to etha-nol before ischemia recover better functionally and exhibit less myocyte dam-age after ischemia than hearts that are not exposed to ethanol. However, if ethanol remains present during ischemia it can nullify its own protective effect and the protection given by ischemic preconditioning. The anti-protective and protective effects occur at similar threshold levels, of about 5 mmol/l of etha-nol (Krenz et al. 2002). It has been suggested that Akt, a serine/threonine pro-tein kinase, may be a downstream mediator of the benefi cial effects of ethanol on myocardial function (Zhou et al. 2002).

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2.2. Alcoholic cardiac muscle disease

2.2.1. General

Alcohol abuse has been found to be a major cause of non-ischemic cardiomy-opathy in Western society. Alcoholic heart muscle disease (AHMD) accounts for about 3.8% of all cases of cardiomyopathy (Piano 2002). There are no specifi c immunohistochemical, immunological or other criteria for diagnosis of AHMD. Diagnosis is often simply presumptive, usually by exclusion. Like other dilat-ed cardiomyopathies, AHMD is characterized by increased left ventricular (LV) mass, dilatation of the ventricles, wall thinning and ventricular dysfunction. The diagnosis is established if the sole causative agent is consumption of more than 80 to 90 g of ethanol a day for more than fi ve to ten years (Urbano-Marquez et al. 1989; Preedy & Richardson 1994; Piano 2002). Some individuals are peculiarly resistant to AHMD. Little is known about the factors determining the severity of AHMD in individual alcoholics (Kajander et al. 2001).

It has been suggested that the mainstay of treatment should be permanent abstinence from alcohol (Piano 2002). However, Nicolas et al. (2002) report that both abstinence and controlled drinking are similarly effective in patients with AHMD in promoting improvement in cardiac function. They provide preliminary evidence that there is a threshold in relation to alcohol consump-tion, probably about 60 g per day (four standard drinks), below which the myocardium suffers no damage. No studies have been carried out in which pharmacotherapy has been specifi cally investigated in patients with AHMD. Guidelines recommend the use of agents such as angiotensin-converting en-zyme inhibitors and beta-adrenergic blockers that inhibit the LV remodelling process, and treat the patient’s symptoms (Piano 2002). The prognosis in pa-tients with AHMD is poor. Heart disease is an important cause of death in chronic alcoholics. Mortality is 40 to 60% within three to fi ve years of diag-nosis in a non-abstinent group (Dancy & Maxwell 1986; Schoppet & Maisch 2001; Piano 2002).

2.2.2. Gender differences in alcoholic heart muscle disease

In the United States, in both sexes, long-term heavy consumption of alcohol is the leading cause of nonischemic dilated cardiomyopathy. Women account for approximately 14% of cases of AHMD. Death rates from AHMD are greater in men than in women (Piano 2002). However, AHMD is equally common in chronically alcoholic women and men, even though the mean amount of ethanol ingested by female alcoholics has been found to be only 60% of that in male alcoholics (Fernandez-Sola & Nicolas-Arfelis 2002). Cardiac functional class and left ventricular mass index are lower in alcoholic women than al-coholic men (Fernandez-Sola et al. 1997). In a study by Kupari & Koskinen (1992) no statistically signifi cant differences were found in relation to LV anat-omy or function between alcoholic men and women, except for differences re-lated to body size.

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3. APOPTOSIS IN THE HEART

3.1. General mechanisms

Apoptosis was originally defi ned as a form of cell death with distinct phases of ultrastructural morphological features, such as nuclear chromatin condensation and cellular shrinkage (Majno & Joris 1995). However, apoptosis is probably best defi ned functionally, as a stepwise, tightly regulated mechanism for elimi-nating damaged or superfl uous cells without an infl ammatory response (Bisho-pric et al. 2001). Apoptosis occurs normally during development and tissue turnover, and in connection with functioning of the immune system. There are various points at which it can be regulated (Gill et al. 2002). At the cellular level, there is normally a balance between apoptotic and anti-apoptotic signals. Cell death occurs in response to a shift in this balance (Hunter & Chien 1999).

Two independent pathways lead to induction of apoptosis, with limited cross-talk between them. The type-I or ‘extrinsic’ apoptotic pathway is mediated by external factors that bind to members of the death receptor superfamily, of which tumor necrosis factor (TNF) receptor type 1 is an example. Specifi c bind-ing by TNF-alpha induces receptor aggregation, leading to cleavage and activa-tion of caspase 8 from procaspase 8, which in turn activates caspase 3. The second major pathway for apoptosis is the ‘intrinsic’, or type-II, mitochondria-depend-ent pathway. Mitochondria contain a number of highly lethal substances that can initiate apoptosis when released into the cytosol. Under conditions that remain partly mysterious, cytochrome c is released from mitochondria and forms a com-plex with procaspase 9 and its cofactor apoptotic protease-activating factor 1. In the presence of suffi cient ATP, caspase 9 then undergoes a conformational change that produces the active ‘apoptosome’. The apoptosome activates caspase 3 and execution of the apoptotic program follows (Bishopric et al. 2001). Some regula-tory mechanisms relating to apoptosis are shown in Figure 1.

Adult cardiomyocytes are terminally differentiated cells. Their loss can contribute to functional decline of the myocardium, leading to heart disease. Examination of human heart tissue has revealed apoptosis in conditions such as cardiomyopathy (Gill et al. 2002) and end-stage heart failure (Nadal-Gi-nard et al. 2003). Apoptosis is stimulated in myocytes by circumstances that include ischemia/reperfusion, hypoxia, presence of reactive oxygen species, calcium overload, mechanical factors (excessive stretching) and elevated lev-els of humoral factors, including norepinephrine and angiotensin II (Ang II) (Dispersyn & Borgers 2001). The extent of apoptosis has been found to vary widely. It depends on the model used and the area at risk examined (Kang & Izumo 2000). For example, in fatal myocarditis about 2% of cardiomyocytes are apoptotic (Kytö et al. 2004). In most studies of apoptotic cell death in end-stage heart failure variable low levels of apoptosis (0.1 to 0.5% of myocytes) have been recorded, and levels in non-failing myocardium have been negligi-ble. Conclusive evidence that cardiac myocyte apoptosis alone can cause cardiac

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Figure 1. Schematic representation of the apoptotic cascade in heart muscle. Two independent pathways lead to induction of apoptosis. Type-I or ‘extrinsic’ apoptotic pathway is mediated by external factors that bind to members of the death receptor superfamily, of which tumor necrosis factor (TNF) receptor type 1 is an example. Specifi c binding by TNF-alpha leads to activation of caspase 8 from procaspase 8, which in turn activates caspase 3. The second major path-way for apoptosis is the mitochondria-dependent pathway. Mitochondria contain a number of highly lethal substances that can initiate apoptosis when released into the cytosol. Anti-apoptotic proteins, including bcl-2, can inhibit apoptosis through mitochondria. Bax, on the other hand, is an important mediator of mi-tochondrial apoptotic process. When cytochrome c is released from mitochondria it forms a complex with procaspase 9 and its cofactor apoptotic protease-activat-ing factor 1 producing an active ‘apoptosome’. The apoptosome activates caspase 3 and execution of the apoptotic program follows. p53 regulates both transcrip-tion and function of bcl-2, bax and p21 genes. It is possible that Akt, a protein kinase, is involved in this regulatory process.

15

failure has been provided by studies involving genetically manipulated mouse models (Nadal-Ginard et al. 2003; Engel et al. 2004). Modest levels of myocyte apoptosis (affecting 23 myocytes per 105 nuclei as opposed to 1.5 myocytes per 105 nuclei in controls) are suffi cient to cause lethal dilated cardiomyopathy (Wencker et al. 2003). Apoptosis has also attracted considerable attention as a potentially reversible cause of cardiac functional deterioration.

3.2. Hypertrophy, cell death and cell survival

Cardiac remodelling is now recognized to be an important aspect of cardiovas-cular disease progression. Cardiac remodelling may be defi ned as genome ex-pression, molecular, cellular and interstitial changes that manifest themselves clinically as changes in the size, shape and function of the heart following cardiac insult. Cellular changes include, for example, myocyte hypertrophy, necrosis, apoptosis and fi brosis. The initial remodelling phase may, to some extent, be regarded as benefi cial. There are no data indicating when transition from possibly adaptive to maladaptive remodelling occurs (Cohn et al. 2000). Progressive remodelling, irrespective of the criteria used in its measurement, may always be considered deleterious and is associated with poor prognosis (Gaudron et al. 1993).

Left ventricular hypertrophy represents both an adaptive response to in-creased cardiac workload and a precursor of heart failure (Condorelli et al. 1999). Although initially compensatory, myocardial hypertrophy is an im-portant risk factor of subsequent cardiac morbidity and mortality (Hunter & Chien 1999). Adaptive hypertrophic response is characterized by increases in masses and volumes of individual myocytes, resulting in increases in heart weight without increases in numbers of cardiomyocytes (Morgan et al. 1987). Recent observations in humans and animals suggest that large myocytes lose ability to increase in size, and that telomeric shortening becomes apparent in hypertrophic old myocytes (Anversa et al. 2002). Once a critical level of short-ening occurs, sensors for DNA damage are triggered and cell-cycle arrest or apoptosis is induced (for references see Igney & Krammer 2002). In the human heart, telomere shortening with age may be a critical biological determinant of heart failure in the elderly (Leri et al. 2003). Evidence of myocyte apoptosis in association with left ventricular hypertrophy has been reported in relation to both experimental and clinical cardiac hypertrophy (Hunter & Chien 1999).

Cell death can occur in a destructive, uncontrolled manner via necrosis or controlled manner via apoptosis. Apoptosis occurs concomitantly with necrosis for example, in the failing heart (Guerra et al. 1999). It is often suggested that the two types of cell death can be triggered by the same factors but the degree of the insult determines whether the jeopardized cell will die by necrosis or apoptosis (Dispersyn & Borgers 2001). ‘Strong’ cell death stimuli usually lead to necrosis due to irreversible mitochondrial damage and energetic catastrophe. But ‘mild’ death stimuli may damage a subpopulation of mitochondria only;

16

resulting in apoptosis if the level of ATP produced by the spared mitochondria is high enough to activate the apoptosome (Martinou & Green 2001). Many stimuli, for example work overload and Ang II can result in both hypertrophy and apoptosis (Condorelli et al. 1999; Anversa et al. 2000; Zhang et al. 2000a). Sublethal injury on the other hand is likely to trigger a number of protec-tive mechanisms, which can be collectively termed ‘programmed cell survival’ (Dispersyn & Borgers 2001). Preconditioning and hibernation are typical ex-amples of such an adaptive response (Kloner et al. 1998). Findings therefore suggest a situation in which similar signal transduction pathways are involved in cardiomyocyte preconditioning, hypertrophy and apoptosis. They may rep-resent responses to various degrees of cardiac insult and different phases of a single process (Condorelli et al. 1999; Sabri et al. 2002). Cardiac response to a particular insult will also depend on the state of the organ before insult. For example, cyclic mechanical stretching results in apoptosis in myocytes from young rats but necrosis in myocytes from old rats (Husse et al. 2003). It is in-teresting that cardiac resistance to insult is increased through modifi cation of its gene expression (Kloner et al. 1998; Hochhauser et al. 2003).

3.3. Gender differences

Gender differences may infl uence susceptibility to apoptosis in the human heart. Percentages of apoptotic cardiomyocytes have been found to be two (Guerra et al. 1999) to three times (Mallat et al. 2001) higher in men than in women. Heart failure results in 13-fold and 27-fold increases in necrosis and 35-fold and 85-fold increases in apoptosis in women and men, respectively (Guerra et al. 1999). Ageing does not lead to myocyte loss and myocyte reac-tive hypertrophy in women. In contrast, men lose nearly 1% of their myocytes per year: between 17 and 89 years of age the male heart loses 45 million myo-cytes per year from the left ventricle. Myocyte loss is accompanied by progres-sive increases in average myocyte volume. This reactive hypertrophic response compensates, in part at least, for the destruction in muscle mass (Olivetti et al. 1995). In 50 patients with ischemic cardiomyopathy, the heart weight index is signifi cantly greater in men than in women. No similar difference is seen in patients with idiopathic cardiomyopathy. The differences between ischemic and idiopathic cardiomyopathy suggest that gender infl uences myocardial re-sponse to injury (Crabbe et al. 2003).

Telomerase replaces telomeric repeat DNA lost during the cell cycle, re-storing telomere length. Telomerase activity is detectable in both young and aging ventricular myocytes. However, aging decreases telomerase activity by 31% in male myocytes. In female myocytes activity of this enzyme increases by 72%. This different adaptation in the two sexes may be relevant to the myocyte loss that occurs in men as they age (Leri et al. 2000a). There is also a similar difference between the sexes in relation to myocardial activation of Akt, the serine/threonine protein kinase that regulates a wide range of physiological

17

responses, including metabolism, gene transcription and apoptosis (Camper-Kirby et al. 2001). Female hypertrophic hearts also express higher levels of, for example, alpha and beta-myosin heavy-chain mRNA and collagen type-I transcripts than male hypertrophic hearts (Rosenkranz-Weiss et al. 1994). In contrast, Zhao et al. (2002) have reported that cardiac fi broblasts from male rats express higher levels of the transcription factors c-jun and NF-kB than fi broblasts from female rats. In general, it has been reported that estrogens inhibit cardiac hypertrophy. However, this inhibition may not always be ben-efi cial, because a hypertrophic response to increased cardiac load is primarily adaptive (Vuolteenaho & Ruskoaho 2003). Estrogen replacement therapy after myocardial infarction in mice reduces infarct size but increases ventricular re-modelling and mortality (van Eickels et al. 2003). Both in vivo and in vitro apoptosis is decreased and Akt activated (Patten et al. 2004).

4. ETHANOL AND APOPTOSIS

4.1. Heart

Ethanol decreases the viability of primary cultured cardiomyocytes and in-creases apoptosis, dose-dependently (Chen et al. 2000). In vivo, prenatal ex-posure to ethanol increases myocardial apoptosis in rats (Ren et al. 2002). In contrast, when three weeks of administration of ethanol or water to rats was followed by an acute Langendorff experiment, infarct sizes were less and levels of apoptosis lower after ischemia-reperfusion in the hearts from the rats that had received ethanol than in the hearts from the controls. It would therefore appear that ethanol protects the heart from cellular injury under some circum-stances, through adaptation to oxidative stress (Sato et al. 2002).

4.2. Other tissues

No evidence of cell death has been found in skeletal muscle exposed acutely or chronically to ethanol (Paice et al. 2003). Exposure to ethanol induces apopto-sis in many other organs and various types of cell. For example, eight weeks of ethanol intake results in apoptosis in female and male rat livers. The effect is more evident in female than in male rats (Colantoni et al. 2003). In the devel-oping rat forebrain, ethanol triggers widespread apoptotic neurodegeneration. The apoptotic response induced by ethanol cannot be predicted from the dose but rather depends on how rapidly a dose is administered, and on how long blood ethanol levels remain above a toxic threshold, in a range between 39 and 43 mmol/l. Maintenance of blood ethanol concentrations at or above 43 mmol/l for four consecutive hours is the minimum condition for triggering of neu-rodegeneration (Ikonomidou et al. 2000). In neuroblastoma cells, ethanol con-centrations of 34 mmol/l or greater specifi cally increase DNA fragmentation but necrosis occurs only at concentrations of 103 mmol/l or more (McAlhany

18

et al. 2000). Treatment of endothelial cells with concentrations of ethanol of < 20 mmol/l activates a pathway promoting cell survival. Treatment with higher concentrations activates an apoptotic pathway (Liu et al. 2002). Chronic ad-ministration of alcohol to rats suppresses apoptosis in the pancreas (Fortunato & Gates 2000).

5. ACETALDEHYDE AND APOPTOSIS

5.1. Heart and other tissues

There are no reports of effects of acetaldehyde on cardiac apoptosis. With a rat as-trocyte cell culture exposure to acetaldehyde for four days signifi cantly increases DNA fragmentation (Holownia et al. 1999). With hamster ovary cell cultures apoptosis is increased dose- and time-dependently by exposure to acetaldehyde (Zimmerman et al. 1995). Interestingly, facilitation of acetaldehyde metabolism by aldehyde dehydrogenase-2 transgene over-expression prevents acetaldehyde-induced apoptosis in human endothelial cells (Li et al. 2004).

6. GENES ASSOCIATED WITH CARDIAC CELL ADAPTATION, APOPTOSIS AND HYPERTROPHY

6.1. Atrial and brain natriuretic peptides

6.1.1. General

Atrial and brain natriuretic peptides (ANP and BNP) are primarily produced by the heart. Both represent fi nal products of a precursor peptide. For exam-ple, after processing of a pre-signal peptide, subsequent cleavage of proANP releases a mature ANP together with a prosegment called N-terminal ANP (NT-ANP). NT-ANP has a longer half-life than ANP, and measurement of NT-ANP gives a better indication of average ANP level over time than meas-urement of ANP. Normally, the main site of secretion of BNP is the cardiac ventricle, that of ANP the atria (Ruskoaho 2003).

Evidence suggests that both ventricular structure and neurohumoral fac-tors are important in stimulating secretion of ANP and BNP. As would be expected, regulation of the biosynthesis and secretion of natriuretic pep-tides involves complex mechanisms that control vasomotor tone, systemic blood pressure and electrolyte homeostasis. Myocardial stretching leads to release of ANP and BNP. Neurohormones such as endothelin-1, Ang II, cat-echolamines, thyroid hormone and prostaglandins also stimulate natriuretic peptide gene expression and release (Yoshimura et al. 2001; Ruskoaho et al. 2003). However, there are differences in expression of the genes. For exam-ple, in a volume overloaded hypertrophy model ANP mRNA levels had al-ready increased on day 1 and left ventricular mass on day 7. BNP mRNA

19

levels remain unchanged throughout the study (Su et al. 1999). Under qui-escent culture conditions p21 stimulates ANP expression but in cells cul-tured under growth-activated conditions p21 suppresses ANP promoter ac-tivity (Kovacic-Milivojevic et al. 1997).On the other hand, TNF-alpha is a strong stimulant of BNP secretion (Yoshimura et al. 2001).

The natriuretic peptide system has emerged as one of the most important hormonal systems involved in the control of cardiovascular function. The phys-iological effects of ANP and BNP include vasodilatation, natriuresis, and inhi-bition of the renin-angiotensin-aldosterone and sympathetic nervous systems. ANP and BNP are also involved in neurotransmission, endocrine functions, and regulation of cell proliferation and hypertrophy (Filippatos et al. 2001; Ruskoaho 2003).

Induction of the natriuretic peptide genes is a feature of cardiac hypertro-phy in all mammalian species studied (Filippatos et al. 2001). ANP mRNA is found to correlate signifi cantly with the left-ventricular weight/body weight ratio, indicating that increased left ventricular ANP gene expression is related to cardiac hypertrophy. Similar results are found in relation to BNP (de Bold et al. 2001). However, hypertrophy and secretion of BNP do not always oc-cur simultaneously. BNP is not necessarily secreted by cells in primary car-diac hypertrophy but there is marked secretion of BNP with secondary car-diac hypertrophy, for example that seen in aortic stenosis and hypertension (Yoshimura et al. 2001). Low ventricular ANP gene expression can be linked genetically to high cardiac mass, independently of blood pressure, and is con-sistent with ANP playing a protective role against left ventricular hypertrophy (Masciotra et al. 1999). Results of experiments with transgenic mice in which the ANP gene has been deleted support this fi nding (John et al. 1995). In BNP knockout mice, ventricular BNP is undetectable but ventricular ANP concen-trations are 37 times higher than in controls. Severe cardiac fi brosis occurs in the animals but no signs of hypertrophy are found, suggesting that BNP may serve primarily as an antifi brotic factor and that enhanced expression of ANP protects the animals from cardiac hypertrophy (Tamura et al. 2000). ANP has also antiproliferative effects in various other cells, including, for example, rat cardiac fi broblasts (Cao & Gardner 1995), and vascular smooth muscle cells (Itoh et al. 1990). ANP and BNP inhibit the cardiac hypertrophy and fi brosis induced by aldosterone via both the endocrine and autocrine-paracrine systems (Yoshimura et al. 2001). ANP also attenuates the growth-promoting effects of noradrenaline on myocardial cells (Calderone et al. 1998).

In both mice and rat cardiac myocytes, ANP induces apoptosis dose- and time-dependently, and cell-type-specifi cally (Wu et al. 1997; Kang et al. 2000). In copper-defi cient mice, metallothionein, a protein that protects against oxi-dative injury, inhibits the transition from heart hypertrophy to failure by sup-pressing apoptosis through inhibition of both cardiac ANP production and its apoptotic effect (Kang et al. 2000). Transgenic mice that over-express ANP

20

exhibit a 27% reduction in total heart weight and it was clear that the net effect of ANP on the myocardium in vivo is limitation of myocyte growth (Klinger et al. 1993). ANP and BNP dose dependently induce apoptosis of, for example, rat endothelial cells, with equal potencies, and treatment of endothe-lial cells with ANP causes marked accumulation of p53 (Suenobu et al. 1999). However, the complexity of the system is highlighted by the fact that ANP also induces regeneration in human endothelial cells (Kook et al. 2003).

6.1.2. Natriuretic peptides and gender

Treatment with 17-beta-estradiol leads to increased production of ANP in rat heart ventricles in a pressure-overload hypertrophy model. The fi ndings sug-gest that the antihypertrophic effect of estradiol may be mediated by increased expression of ANP (van Eickels et al. 2001). However, no gender differences relating to ANP or BNP levels have been reported.

6.1.3. Natriuretic peptides and ethanol

In experiments in rats acutely administered ethanol, levels of left ventricular ANP mRNA and protein either remain constant (Wigle et al. 1995) or an increase in ANP tissue content is observed two hours after ethanol administra-tion (Guillaume et al. 1994). Six weeks of ethanol ingestion by rats results in both ventricular ANP mRNA and protein levels being higher than in control rats (Wigle et al. 1993a) but administration of ethanol for two or four months has no effect (Kim et al. 2003). Administration of ethanol for eight months signifi cantly reduces ANP mRNA and protein levels in the ventricles of SHR but not of WKY rats (Guillaume et al. 1996a). In transgenic mice hearts in which there is over-expression of alcohol dehydrogenase and cardiac acetalde-hyde levels are therefore four times higher than normal, ventricular ANP gene expression is several times higher than normal (Liang et al. 1999).

In atrial tissue, in acute in vivo experiments involving treatment with eth-anol, levels of ANP mRNA and protein remain constant (Wigle et al. 1993b) or ANP levels decrease (Guillaume et al. 1994). Moderate consumption of ethanol by rats for eight months increases atrial ANP levels, without altering ANP mRNA (Guillaume et al. 1996a). Consumption of alcohol for six weeks increases both atrial ANP mRNA and protein levels (Wigle et al. 1993a).

In plasma, the physiological response to acute administration of alcohol appears to depend on the volume of co-administered liquids. Plasma ANP levels correlate positively with atrial pressure in normal subjects (Sehested et al. 1998). The main mechanism involved in increased ANP release from atria after acute ethanol ingestion seems to be atrial stretching (Colantonio et al. 1991). Several reports have been published on the effects of acute ethanol ad-ministration on plasma ANP levels. Results have been confl icting (Colantonio et al. 1991; Hynynen et al. 1992; Leppäluoto et al. 1992; Ekman et al. 1994).

21

No alterations in plasma levels of ANP are evident after six weeks of ethanol treatment (Wigle et al. 1993a). After four or eight months of ethanol expo-sure, plasma ANP levels are signifi cantly lower than in controls (Guillaume et al. 1996a; Kim et al. 2003).

In cardiac ventricular tissue, acute ingestion of ethanol reduces BNP mRNA levels to 38% of control levels (Wigle et al. 1995). In contrast, admin-istration of ethanol for six weeks and two and four months results in no chang-es in BNP gene expression (Wigle et al. 1993c; Kim et al. 2003). Consump-tion of ethanol for eight months increased both ventricular BNP concentration and ventricular BNP mRNA in SHR (spontaneously hypertensive rat line) but not in normotensive WKY rats (Guillaume et al. 1996b).

In the atrial tissue of rats to which ethanol has been administered acute-ly, BNP mRNA levels are 43% of those in animals that have received water (Wigle et al. 1995). After chronic administration of ethanol to rats, elevated atrial BNP concentrations are observed without alterations in BNP mRNA levels (Guillaume et al. 1996b).

Circulating plasma BNP levels were high following six weeks of adminis-tration of ethanol to rats (Wigle et al. 1993c). Four or eight months of mod-erate ethanol consumption by rats resulted in circulating BNP levels below normal (Guillaume et al. 1996b; Kim et al. 2003).

6.2. p53

6.2.1. General

The protein p53 is a major element in a complex network that senses cellular stress, including DNA damage, hypoxia and hyperproliferation. Induction of apoptosis is the most conserved function of p53 but it also controls such cell functions as gene transcription, DNA synthesis, DNA repair, cell cycle arrest and aging. The activity of p53 is determined by stress type and individual cell/tissue characteristics. A stimulus that can result in elevation of p53 levels may not do so in all instances. In addition, when p53 levels are elevated, death does not always ensue. The mechanism by which the alternative fates of apop-tosis or cell-cycle arrest occur remains, however, to be elucidated (Hofseth et al. 2004; Slee et al. 2004).

Normal diploid cells have limited potential for replication. The number of times a cell can divide is determined by the lengths of the telomeres at the chromosome ends, which are shortened during each cell cycle by telomerase. Once a critical level of shortening has occurred, sensors for DNA damage are triggered and cell-cycle arrest or apoptosis is induced. p53 seems to be impor-tant in relation to this response (for references see Igney & Krammer 2002). Interestingly, it has been discovered that chronic constitutive expression of p53 accelerates aging in mice (Tyner et al. 2002). In the human heart, telomere shortening with increasing age could be a critical biological determinant of

22

heart failure in the elderly. Importantly, telomere loss leads to cardiac dilata-tion and heart failure associated with up-regulation of p53 (Leri et al. 2003).

The pro-apoptotic potential of p53 has been demonstrated in numerous systems, including the response of cardiac myocytes to stress. For example, in-duction of p53 down-regulates bcl-2 and up-regulates bax in myocytes, thus decreasing the bcl-2:bax ratio and increasing the susceptibility of cells to un-dergo apoptosis (Leri et al. 1998a; Leri et al. 1998b). Inhibition of functioning of p53 prevents activation of the renin-angiotensin system and stretch-medi-ated apoptosis in ventricular myocytes (Leri et al. 2000b). However, in some studies p53 levels do not increase with mechanical stretching, pressure over-load or cardiac hypertrophy (Kim et al. 1994; Fortuno et al. 1999). Prolonged hypoxia relating to neonatal cardiomyocytes results in substantial induction of expression of p53 and apoptosis (Long et al. 1997). Ischemic precondition-ing, in an ischemia-reperfusion model involving isolated working rat hearts (Maulik et al. 2000), and hypothermia (Ning et al. 2002) prevent p53 activa-tion and inhibit apoptosis. In contrast, myocyte apoptosis occurs as readily in the hearts of mice nullizygous for p53 as in wild-type littermates subjected to myocardial infarction or ischemia-reperfusion (Bialik et al. 1997; Webster et al. 1999). Results of numerous other studies also support both p53-depend-ent and -independent apoptotic pathways in the heart. In severe congestive heart failure expression of p53 and apoptosis are signifi cantly increased in hu-man cardiomyocytes (Song et al. 1999). In animal studies, for example pacing-induced heart failure in dogs enhances apoptosis, hypertrophy and expression of p53 and p53-dependent genes in ventricular myocytes (Leri et al. 1998b). These results suggest that p53 may play an important pathophysiological role in relation to heart failure.

6.2.2. p53 and gender

It has been reported that estradiol induces functional inactivation of p53 in breast cancer cells (Molinari et al. 2000). On the other hand, p53 infl uences the effects of estrogens, as it binds to multiple domains of the human estro-gen receptor and down-regulates expression of some estrogen-responsive genes such as bcl-2 (Liu et al. 1999). Cardiac fi broblasts are cell targets for estrogens. However, in both sexes, p53 protein levels are similar in cardiac fi broblasts (Zhao et al. 2002) and p53 mRNA levels are similar in skeletal hind limb muscle (Nakahara et al. 2003). In contrast, gender infl uences expression of p53 in MRL/lpr mice (autoimmune mouse model of Sjögren’s syndrome). The amount of p53 mRNA is higher in lacrimal and submandibular tissues of fe-male mice than in lacrimal and submandibular tissues of male mice (Toda et al. 1998).

23

6.2.3. p53 and ethanol

No reports on the effects of ethanol on cardiac levels of p53 have been pub-lished. In rat skeletal hind limb muscle, p53 mRNA levels are not affected by in vivo exposure to ethanol, acutely (2.5 hours) or chronically (six to seven weeks), even with cyanamide (a dehydrogenase inhibitor) predosing (Naka-hara et al. 2003). In regenerating rat liver, p53 transcript levels are increased (Lumpkin et al. 1995) or unchanged (Zhang et al. 2000b) following acute eth-anol exposure. In normal mice, exposure to ethanol for three weeks induces a signifi cant increase in the number of apoptotic hepatocytes. Apoptosis is com-pletely abrogated in p53-defi cient mice suggesting that p53 is necessary for ethanol-induced apoptosis, at least in the liver (Pani et al. 2004). Exposure to ethanol induces apoptosis and up-regulates expression of p53 also, for example, in neuronal cells (de la Monte et al. 2000). However, in human rectal mucosal cells, chronic alcohol abuse led to cell hyperproliferation with no change in p53 immunostaining (Simanowski et al. 2001). Acute exposure of human epi-thelial cells to non-cytotoxic ethanol concentrations does not affect p53 pro-tein levels (Guo et al. 1997).

6.3. p21

6.3.1. General

It has been suggested that p21 is involved in numerous important cell process-es, including DNA repair, differentiation and apoptosis (Gorospe et al. 1997). It is a p53 target gene. Expression of p21 has been shown to be up-regulated by p53 in response to the presence of DNA-damaging agents (El-Deiry et al. 1993). When DNA damage is limited and reparable, p53 activates genes, in-cluding p21, which relate to cell-cycle arrest and DNA repair. When DNA damage is very severe, p53 activates genes inducing apoptosis (Nakamura 2004). Apoptosis that is p53-dependent occurs normally in cells lacking p21. On the other hand, many reported pathways for induction of p21 expression are totally independent of p53 (Cox 1997). In various systems in which expo-sure to stressful stimuli results in p53-independent cell death, p21 has been found to be involved in survival responses. Adenovirus-driven ectopic expres-sion of p21 results in substantial protection against p53-induced apoptosis, in-dicating that p21 rescues cells from a path to programmed cell death (Gorospe et al. 1997). However, cell-fusion experiments have shown that apoptosis pre-dominates over cell-cycle arrest, irrespective of p21 levels and cell-cycle status (for references see Cox 1997). Introduction of exogenous p53 overcomes p21-mediated cell-cycle arrest at G1 and induces apoptosis, although p21 expres-sion is unaffected (Kagawa et al. 1997).

For example, mechanical loading, addition of Ang II and hypoxia rapidly ac-tivate p21 in cultured neonatal rat cardiac myocytes (Sadoshima & Izumo 1993; Sadoshima & Izumo 1996; Seko et al. 1996). p21 is also involved in the signal

24

transduction of a variety of hormones and growth factors (Kovacic-Milivojevic et al. 1997). Anti-p21 antibody completely blocks DNA synthesis and cell prolif-eration in response to Ang II, endothelin-1 and some other hormones (Schieffer et al. 1997). The p21 protein has also been reported to inhibit DNA replication and block cell-cycle progression (Ogawa et al. 1997). The importance of p21 as a regulator of senescence has been clearly established (Serrano & Blasco 2001). p21 stimulates rat ANP gene transcriptional activity in a cultured neonatal rat ventriculocyte model (Thorburn et al. 1993) and the p21-sensitive element is located within the ANP gene transcription start site. Under quiescent culture conditions, p21 stimulates ANP promoter activity, but in cells cultured under growth-activated (e.g., endothelin-1) conditions, p21 suppresses promoter activ-ity (Kovacic-Milivojevic et al. 1997).

Cardiomyocytes are terminally differentiated cells. Over 95% of adult car-diomyocytes are known to exist in the G0/G1 phase of the cell cycle (for refer-ences see McGill & Brooks 1995). p21 regulates the cell cycle by inhibiting the cyclin-dependent kinases required for progression from the G1 to the S phase (El-Deiry et al. 1993). In response to DNA damage, cells arrest cell cycle progression in a co-ordinated way, and induce expression of gene products that facilitate DNA repair (Deng et al. 1995). p21 is therefore normally an im-portant component of the checkpoint for cell DNA damage (Cox 1997). Mice null for the p21 gene develop normally but are defi cient in G1 cell-cycle arrest in response to DNA damage, and thus, defective in relation to DNA repair. However, the apoptotic response in such mice is unaltered (Deng et al. 1995; Brown et al. 1997).

Activated p21 has been linked to cardiac hypertrophy by a number of inves-tigators (Kovacic-Milivojevic et al. 1997). A p21-dependent pathway mediates hypertrophy, at least in part, following stimulation of a classical G-protein-cou-pled receptor in neonatal rat cardiac myocytes (Thorburn et al. 1993). Phenyle-phrine, Ang II and other hypertrophic agonists as well as mechanical loading cause biochemical activation of p21, and the p21-mediated pathway is suffi cient to induce growth or differentiation of mammalian cells (Sadoshima & Izumo 1993; Thorburn et al. 1995). Transgenic mice that over-express cardiac p21 ex-hibit morphological, physiological, and genetic markers, including elevated ANP gene expression of myocardial hypertrophy (Hunter et al. 1995). On the other hand, p21 is able to inhibit proliferating cell nuclear-antigen-mediated hypertro-phy in adult cardiomyocytes (Engel et al 2003). mRNA and protein expression of p21 are down-regulated signifi cantly but transiently during development of pres-sure-overload-induced LV hypertrophy in the rat. It has therefore been suggested that adjustment to intracellular balances between levels of positive and negative regulators of the cell cycle in cardiomyocytes is required to allow compensatory growth to occur during development of LV hypertrophy (Li & Brooks 1997). In contrast, in dogs with heart failure, hypertrophy is accompanied by p21 protein

25

up-regulation, and both hypertrophy and p21 levels decline after successful treat-ment, (Sabbah et al. 2003). In human idiopathic dilated cardiomyopathic hearts that have progressed to heart failure, p21 expression is decreased (Steenbergen et al. 2003).

The precise role of p21 in relation to apoptosis in myocytes is still not clear. p21 mRNA levels are increased in failing and non-failing hearts of SHR, and an elevated number of apoptotic cells accompanies the p21 mRNA increase (Li et al. 1997). In cultured neonatal rat cardiac myocytes exposure to hypoxia results in apoptosis and is accompanied by increased p53 and p21 levels (Long et al. 1997) or decreased p21 protein accumulation (Adachi et al. 2001). In a study by Hauck et al. (2002), down-regulation of p21 is required for hypoxia-induced cardiomyocyte apoptosis, and ectopic p21 expression is able to inhibit apoptotic cell death.

6.3.2. p21 and gender

The p21 exon includes an estrogen-responsive element (Pethe & Shekhar 1999). However, no gender-dependent differences in p21 levels have been reported.

6.3.3. p21 and ethanol

There are no reports relating to effects of ethanol or acetaldehyde on p21 ex-pression in the heart. In the liver, ethanol promotes accumulation of p21, and this may be an adaptive response that helps livers that have been exposed to ethanol to survive stress situations (Koteish et al. 2002). In another study, eth-anol treatment resulted in no change in p21 mRNA expression (Zhang et al. 2000b). In human epithelial cells (Guo et al. 1997) and rat aortic smooth mus-cle cells (Sayeed et al. 2002) exposure to ethanol results in p21 expression and inhibited cell proliferation.

6.4. Bcl-2 and bax

6.4.1. General

The B cell lymphoma gene-2 (bcl-2) family has over 20 members, and can be divided into anti-apoptotic (e.g. bcl-2) and pro-apoptotic proteins (e.g. bax) (Ig-ney & Krammer 2002). The chief function of bcl-2 family proteins is to regulate release of cytochrome c, apoptosis-inducing factor and other proteins from mi-tochondria, with pro-apoptotic bcl-2 family proteins promoting, and anti-apop-totic bcl-2 family members suppressing protein release by affecting the perme-ability of mitochondrial membranes. The relative ratios of anti- and pro-apop-totic bcl-2 family proteins dictate the sensitivities of cells to various apoptotic stimuli, including hypoxia, presence of oxidants and Ca2+ overload (Reed 2002). Although bax and bcl-2 heterodimerize and inhibit each other, both proteins can act independently in regulating apoptosis (Regula et al. 2003).

26

Hearts from rats subject to chronic hypoxia exhibit an increased rate of apoptosis and a decrease in bcl-2 protein level. In contrast, exposure of isolated neonatal rat cardiac myocytes to hypoxia also results in signifi cantly increased apoptosis but bcl-2 levels remain unchanged (Jung et al. 2001; Riva et al. 2001). Ischemia-reperfusion results in cardiomyocyte apoptosis in concert with down-regulation of bcl-2 mRNA (Cheng et al. 1996). In some studies, precon-ditioning results in up-regulation of the bcl-2 gene, and bcl-2 expression cor-relates with reduction of cardiomyocyte apoptosis and infarct size (Maulik et al. 1999; Xie et al. 2001). In transgenic mice, over-expression of bcl-2 reduces infarct size and renders the heart more resistant to apoptosis (Chen et al. 2001). As in other cells, therefore, over-expression of bcl-2 protects cardiac myocytes from apoptosis (Kirshenbaum & de Moissac 1997). Antisense bcl-2 oligode-oxynucleotides completely abolish the cardioprotective effects of adaptation by eliminating the “antideath signal” of bcl-2 (Hattori et al. 2001).

In LV hypertrophy and LV dysfunction bcl-2 levels are lower and apoptosis increased compared to controls (Condorelli et al. 1999). In man, heart failure is characterized by an increase in myocyte apoptosis, despite enhanced expression of bcl-2 (Olivetti et al. 1997; Latif et al. 2000). Enhanced expression of bcl-2 in the failing heart suggests that compensatory mechanisms are activated in the overloaded myocardium in an attempt to maintain cell survival (Olivetti et al. 1997). In apoptosis resulting from the presence of reactive oxygen spe-cies bcl-2 levels in myocytes decrease (Cook et al. 1999; Suzuki et al. 2001) or remain unchanged (von Harsdorf et al. 1999).

The bax gene promoter contains four p53-binding sites that can be spe-cifi cally activated by p53 (Miyashita & Reed 1995). For example, myocardial stretching (Leri et al. 1998a) and chronic hypoxia (Jung et al. 2001) induce expression of bax and myocyte apoptosis. Ischemic preconditioning induces down-regulation of bax, and cardioprotection, as evidenced by reduced num-bers of apoptotic cardiomyocytes (Nakamura et al. 2000; Hattori et al. 2001). In homozygotic knockout mice lacking the bax gene the heart is more protect-ed against myocardial ischemia-reperfusion injury than in normal wild-type mice (Hochhauser et al. 2003).

Marked up-regulation of bax and cardiomyocyte apoptosis contribute to the transition from LV hypertrophy to LV dysfunction in response to chronic pres-sure in rats (Condorelli et al. 1999). In groups with dilated cardiomyopathy and ischemic heart disease there are signifi cant increases in levels of bax and apoptosis (Anversa et al. 1998; Latif et al. 2000). Pacing-induced heart failure in dogs results in signifi cant increases in apoptosis and expression of bax (Leri et al. 1998b). In a study of myocytes from the hearts of patients with conges-tive heart failure, the level of bax is no different from normal (Olivetti et al. 1997). Bax is also up-regulated, for example, during Ang II-induced cardiac myocyte apoptosis (Diep et al. 2002).

27

Apoptosis is increased and the bcl-2/bax ratio decreased in infarcted areas of human hearts (Baldi et al. 2002). In rats, a reduction in the bcl-2/bax pro-tein ratio and an increase in apoptosis occurs, for example, in LV hypertrophy and LV dysfunction (Condorelli et al. 1999) and after exposure to oxidative stress (Suzuki et al. 2001).

6.4.2. Bcl-2, bax and gender

Bcl-2 is an estrogen-responsive gene (Dubal et al. 1999). Basal protein levels of bcl-2 are higher in cardiac fi broblasts from female rats than in cardiac fi -broblasts from male rats (Zhao et al. 2002). However, in rat skeletal hind limb muscle bcl-2 levels are similar in both sexes (Nakahara et al. 2003). Toda et al. (1998) reported that the amount of bcl-2 mRNA is higher in the lacrimal glands of female autoimmune mice than in the lacrimal glands of male au-toimmune mice. No reports on the effects of gender on bax gene expression have been published, although testosterone has been shown to induce bax and decrease bcl-2 protein levels and promote apoptosis in human renal tubular cells (Verzola et al. 2004).

6.4.3. Bcl-2, bax and ethanol

In primary cardiomyocyte cultures, exposure to ethanol decreases cell viabil-ity in a dose-dependent manner, and the content of bax protein increases (Chen et a. 2000).

In rat skeletal hind limb muscle, bcl-2 mRNA levels are unaffected by acute (2.5 hours) or chronic (six to seven weeks) exposure to ethanol in vivo, even after cyanamide (an inhibitor of dehydrogenase) predosing (Nakahara et al. 2003). Hepatic expression of bcl-2 and bax proteins is markedly higher than normal in patients with alcoholic liver disease and in mice to which etha-nol has been administered chronically (Rashid et al. 1999; Hong et al. 2002). In rats, bax mediates acute ethanol-induced hepatocytes apoptosis Adachi et al. 2004). However, chronic administration of alcohol induces apoptosis in the livers but does not affect bax or bcl-2 mRNA levels. Incubation of HepG2 cell line cells with ethanol in the absence of bcl-2 results in apoptosis. On the contrary, exposure to ethanol causes no apoptosis in cells over-expressing bcl-2 (Wu & Cederbaum 1999). There are numerous studies on the roles played by bcl-2 and bax in regulating ethanol-induced apoptosis in, for example, the CNS (Moore et al. 1999; de la Monte et al. 2000; Mooney & Miller 2001).

6.5. Tumor necrosis factor-alpha

6.5.1. General

TNF-alpha is a major mediator of apoptosis, infl ammation and immunity. It signals via two cell-surface receptors, TNF-R1 and TNF-R2. Binding of TNF-alpha to TNF-R1 triggers a series of intracellular events that result in

28

activation of two major transcription factors, nuclear factor-kB (NF-kB) and c-jun. These transcription factors are responsible for the inducible expression of genes important in relation to various biological processes, including cell growth and death, development, oncogenesis, and immune, infl ammatory and stress responses (Chen & Goeddel 2002). For example, myocardial stretching is followed by TNF-alpha production in the heart (Kapadia et al. 1997).

TNF-alpha activates extracellular matrix remodelling, and appears to con-trol both tissue destruction and rebuilding (Feldman et al. 2000). TNF-alpha depresses myocardial contractile function. In addition to calcium dyshomeos-tasis, mechanisms by which TNF-alpha causes myocardial dysfunction include, for example, oxidative stress, myocyte apoptosis and induction of other cardiac depressants such as IL-6 (Meldrum 1998). Accumulating evidence indicates that myocardial TNF-alpha is an autocrine contributor to myocardial dys-function and cardiomyocyte hypertrophy and death in, for example, ischemia-reperfusion injury, sepsis, heart failure, viral myocarditis, acute myocardial in-farction and cardiac allograft rejection (Meldrum 1998; Feldman et al. 2000). Transgenic mice with cardiac-restricted TNF-alpha over-expression develop cardiac failure and die prematurely (Kubota et al. 1997; Bryant et al. 1998).

Over-expression of TNF-alpha activates both anti- and pro-apoptotic path-ways in the myocardium of transgenic mice. Most of the cells suffering apop-tosis appeared not to be myocytes, even in mice with overt congestive heart failure. It would seem that TNF-alpha alone is not suffi cient to induce apopto-sis in cardiac myocytes in vivo (Kubota et al. 2001). In contrast, in a primary culture of rat cardiac myocytes, a dose-dependent increase in caspase-3 activity, indicative of apoptosis, is observed when cells are incubated with increasing concentrations of TNF-alpha (Carlson et al. 2002). Chronic infusion of TNF-alpha in vivo results in rapid development of dilated cardiomyopathy associ-ated with increased myocyte apoptosis (MacLellan & Schneider 1997). Adeno-viral-mediated gene transfer of bcl-2 prevents apoptosis of neonatal ventricular myocytes induced by TNF-alpha (deMoissac et al. 1998). In patients with con-gestive heart failure, TNF-alpha levels do not correlate with degree of cell loss (Rayment et al. 1999). Interestingly, soluble TNF type-2 receptor plasma lev-els identify a heart failure-patient subgroup with high levels of cardiomyocyte apoptosis (Saraste et al. 2002).

Emerging evidence suggests that TNF-alpha predominantly triggers apop-tosis in cells either defi cient in or defective in relation to NF-kB activation. In ventricular myocytes TNF-alpha is likely to activate dual signalling cascades, with one pathway leading to apoptosis and the other pathway, mediated via NF-kB, dominating to suppress pro-death signals and apoptosis (Mustapha et al. 2000; Bergmann et al. 2001). TNF-alpha protects the heart, for example, against ischemia-induced apoptosis (Kurrelmeyer et al. 2000). Pretreatment of rats with TNF-alpha has been shown to protect the heart from ischemia reper-fusion injury (Eddy et al. 1992).

29

6.5.2. TNF-alpha and gender

In humans, there are increased percentage TNF-alpha producing monocytes found in men as compared with women (Bouman et al. 2004). In transgen-ic mice that over-express cardiac-specifi c TNF-alpha, left ventricular func-tion and the six-month survival rate are signifi cantly better in females than in males, though TNF-alpha levels are identical in females and males (Kadokami et al. 2000). In rat liver, basal TNF-alpha protein levels are found to be similar in both sexes (Colantoni et al. 2003). In contrast, in the lacrimal glands of fe-male autoimmune mice amounts of TNF-alpha mRNA are higher than in the lacrimal glands of male autoimmune mice (Rocha et al. 1998).

6.5.3. TNF-alpha and ethanol

There are no reports on the effects of ethanol or acetaldehyde on TNF-alpha gene expression in the heart. In the liver, TNF-alpha is required for nor-mal hepatic regeneration, and in relation to repair of ethanol-related damage (Diehl 1999). Exposure to ethanol or acetaldehyde for 24 to72 hours results in expression of TNF-alpha in HepG2 hepatocytes (Gutierrez-Ruiz et al. 1999). Exposure of rats to ethanol for two weeks iv vivo does not result in up-regulation of basal hepatic gene expression of TNF-alpha (Järveläinen et al. 1999; Fleming et al. 2001) but eight weeks of exposure of rats to ethanol increases TNF-alpha protein levels and apoptosis. These effects of ethanol are more evident in female than in male livers, probably contributing to the greater sensitivity of female livers to ethanol-induced injury (Colantoni et al. 2003). The effects of chronic exposure to acetaldehyde are the opposite. Four weeks of treatment with ethanol and aldehyde dehydrogenase inhibitor re-sults in sustained increases in blood and liver acetaldehyde levels and down-regulates TNF-alpha gene expression in rats (Lindros et al. 1999). There are numerous other reports on the effects of ethanol on TNF-alpha mRNA and protein levels. For example, in cultured epidermoid skin cells, low concen-trations (40 to 100 mmol/l) of ethanol induce apoptosis by enhancing the effects of TNF-alpha (Neuman et al. 2002). Ethanol-induced subchronic gastritis results in activation of TNF-alpha expression followed by apoptosis in the gastric mucosa (Liu & Cho 2000).

30

AIMS OF THE STUDY

It has been suggested that excessive ethanol consumption is responsible for a number of deleterious effects on heart muscle, including the serious condition known as dilated cardiomyopathy. Mechanisms underlying ethanol-induced cardiac muscle damage are, however, largely unknown.

The aims of the study were:

1. To investigate whether ethanol is capable of triggering cardiac adaptation processes and changing the expression of the genes which regulate apopto-sis and hypertrophy.

2. To develop and employ a quantitative RT-PCR method for determining mRNAs of specifi c proteins in muscle samples.

3. To assess possible effects of short-term exposure to ethanol and acetalde-hyde derived from ethanol on apoptosis and genes associated with cardiac adaptation in vivo.

4. To evaluate acute effects of exposure to ethanol and acetaldehyde on apop-tosis and genes associated with cardiac cell adaptation in the isolated per-fused rat heart.

5. To explore the effects of chronic ethanol consumption on cardiac apoptosis and expression of genes associated with cardiac adaptation in both genders in vivo.

31

MATERIALS AND METHODS

1. ANIMALS AND EXPERIMENTAL DESIGN

1.1. One-week immobilization and skeletal muscle: a methodological study (I)

Male Wistar rats (Laboratory Animal Center, University of Helsinki) were housed in light and temperature controlled rooms with a 12 h light: 12 h dark cycle. Animals had free access to chow and tap water. At the age of 11 weeks animals were randomly assigned to control (n=6) and immobilization (n=6) groups. Immobilization for one week was performed by bilateral hind limb casting under anaesthesia (Booth & Kelso 1973). The ankles were fi xed in maximal plantar fl exion and the plaster cast extended around the knee and hip joints, which were immobilized at approximated resting angles. Strips of plaster of paris connected both hind limb casts dorsally, cranial to the tail to prevent the legs from slipping out of the cast. At the end of the experiment, gastrocnemius, plantaris and soleus were rapidly excised and frozen in liquid nitrogen. Muscle samples were stored in –80°C until mRNA analysis.

1.2. Short-term experiment in vivo (II,IV)

96 2-month-old male Wistar rats (Laboratory Animal Center, University of Helsinki) were randomly assigned to 12 experimental groups. Animals were housed in a temperature-controlled room with a 12 h light: 12 h dark cycle and provided with chow and water ad libitum. The four main experimental groups were as follows: controls (C: half of the group saline 8.3 ml/kg i.p. once a day; half of the group no treatment); ethanol (E: 1g/kg body weight i.p. as a 12% w/v solution in saline once a day); calcium carbimide (CC: half of the group calcium carbimide 100 mg/kg of diet; half of the group calcium carbim-ide 100 mg/ kg of diet + saline i.p.) and ethanol + calcium carbimide (E+CC: ethanol 1g/kg body weight i.p. once a day + calcium carbimide 100 mg/kg of diet). Each of these four experimental groups was divided into three subgroups based on duration of treatment, i.e. 2,5, or 8 days. The calcium carbimide diet started in the morning on day 1, ethanol on day 2. This way, in the 2-day ex-periment rats received calcium carbimide for 2 days and ethanol for 1 day, in the 5-day experiment rats received calcium carbimide for 5 days and ethanol for 4 days, and in the 8-day experiment for 8 days and 7 days, respectively. Blood samples for ethanol and acetaldehyde measurements were taken from the tail vein 2 hours after alcohol injection 1 day before decapitation and for N-terminal ANP at the time of decapitation. After decapitation, hearts were removed within 60 seconds, weighed and the free walls of the left ventricles were carefully dissected and frozen in liquid nitrogen for later RNA isolation. All samples were stored in –80°C until mRNA and ELISA analysis.

32

1.3. Acute experiment with isolated heart (III)

Male Wistar rats weighing from 260 to 410 g (Laboratory Animal Center, University of Helsinki) were maintained on rat chow and water ad libitum in a room with a lighting sequence of 12 h light and 12 h dark. The rats were anti-coagulated with heparin (500 IU/kg body weight i.p.) and killed by decapita-tion 20 min later in CO2 anaesthesia. The thoracic cavity was quickly opened and the heart was cooled with ice-cold perfusion fl uid. The aorta was cannu-lated superior to the aortic valve and retrograde perfusion was started with perfusion fl uid [DMEM with 25 mM HEPES (Gibco), supplemented with 2.5 mM L-carnitine, 5 mM creatine, 5 mM taurine, 2 mM sodium pyryvate (Sigma), 20 IU/l Insulin Actrapid (Novo Nordisk), pH 7.4] equilibrated with 95% O2 / 5% CO2 at 37°C. The hearts were perfused with a peristaltic pump (Perista AC 2110) at a fl ow rate of 7 ml/min and stimulated at 300 beats/min by Grass stimulator (Grass S88 stimulator, 5V, 0.5 ms). After the equilibration period (30 min) the hearts were perfused for 150 min in the absence (n=8) or presence of 500 µM acetaldehyde (n=9), or 5% (vol/vol) ethanol (n=7). After the perfusion period, the hearts were removed within 60 seconds and the free walls of the left ventricles were carefully dissected and immediately frozen in liquid nitrogen. All tissue samples were stored at –80°C until mRNA and ELISA analysis.

1.4. Chronic experiment in vivo (V)

Alcohol avoiding ANA (ALKO Non Alcohol) rats (F65) of both sexes were used. The rats were housed in groups of four to fi ve animals under a 12h/12h light/dark cycle. The animals received standard food ad libitum in pellet form during the forced exposure and in powder form during the self-selection peri-ods. The ethanol-exposed rats (females n=6; males n=8) were given 12% (v/v) ethanol as the only available fl uid from the age of 3 months to 24 months of age, except for the self selection period (3 weeks) at the beginning and at the end of the exposure, when rats were allowed a free choice between 10% etha-nol and tap water, to measure their individual voluntary ethanol consumption. The control rats (females n=7; males n=5) had only water available. The con-sumption of ethanol was carefully monitored monthly for a period of one week during the whole ethanol exposure period. During the forced ethanol period the ethanol consumption of the ANA males increased from 3.83 ± 0.26 g/kg to 4.99 ± 0.30 g/kg per day and of the ANA females from 6.16 ± 0.35 to 7.14 ± 0.28. A week before killing the rats, the ethanol-exposed rats were with-drawn from ethanol and had only food and water available for the last week of the experiment. Prior to decapitation, the rats were anaesthetized with sodium pentobarbital (60-120 mg/kg) intraperitoneally. After decapitation hearts were removed within 60 seconds, weighed and free walls of left ventricles were im-mediately frozen in liquid nitrogen for later RNA isolation. All tissue samples

33

were stored at –80°C until mRNA and apoptosis ELISA analysis were per-formed. The animal model and the protocol for ethanol administration have been previously described in detail (Rintala et al. 1997).

1.5. Ethical considerations

The animal studies were conducted in accordance with the principles of the Declaration of Helsinki and approved by the National Public Health Institu-tion Animal Care and Use Committee (II, IV, V) or by the Committee for ani-mal Care and Use of the Wihuri Research Institute (III).

2. BLOOD AND TISSUE SAMPLING

2.1. Measurement of blood ethanol and acetaldehyde levels

Blood samples were collected into tubes containing 22.5 mg of sodium fl uo-ride and 22.5 mg of potassium oxalate as anticoagulants for a volume of 10 ml of blood. Chilled tail blood samples were hemolyzed, and their ethanol and acetaldehyde contents measured with headspace gas chromatography (Perkin-Elmer F40). The conditions for analysis were as reported earlier (Eriksson et al. 1977). In short, the ethanol samples were diluted 1:10 in ice-cold water and all samples were stored in vials. Sealed vials were heated to 65°C and the con-ditions for analysis were the following: column 15% polyethylene glycol on Celite 60/100; oven temperature 75°C; transfer line and detector temperature 170°C; carrier gas fl ow rate (N2) 20 ml/min.

2.2. Measurement of blood N-terminal ANP levels

Blood for assay was taken in tubes containing aprotinin (500 IU/ml) and EDTA (1mg/ml) and immediately placed on ice. After centrifugation at 1000g for 10 min at 4°C, aliquots of plasma were stored at -80°C and later subjected to radioimmunoassay (RIA) by a commercial kit (Biotop, Oulu, Finland). All the samples of an experiment were run within the same assay.

2.3. Analysis of mRNA levels by quantitative RT-PCR method

Total RNA was extracted from frozen muscle samples by using a modifi cation of the acid guanidium thiocyanate-phenol-chloroform extraction (RNAzol B, Tel-Test, Friendswood, TX, USA). Frozen muscles were weighed and homoge-nised with Polytron (Kinematika, Littau, Switzerland) and RNA isolation was performed according to the manufactures recommendations except that the actual RNA extraction phase was performed twice to eliminate all genomic DNA contamination. This way, amplifi cation of non-reverse transcribed RNA produced no detectable signal. The total RNA concentration was assessed spec-trophotometrically (Gene Quant, Pharmacia Biotech, Finland).

34

One µg of total sample RNA was reverse transcribed to cDNA with both oligo-dT (Pharmacia Biotech) and random hexamer primers using Superscript II enzyme (Life Technologies). To evaluate the effi ciency of the reverse tran-scription reaction, i.e. the amount of cDNA synthesized, a known amount of control RNA was added to each reaction tube as an internal standard.

Control RNA was produced by in vitro transcription by fi rst construct-ing synthetic DNA templates that contained two primer pairs complementary to those used to amplify cDNAs of interest. DNA templates also contained sequences for the bacteriophage T7 promoter at the 5’ end for transcription into RNA and a poly A tail at the 3’ end to facilitate RT by oligo-dT. Con-trol RNA product was separated from synthetic DNA templates after in vitro transcription using phenol/chloroform extraction and ethanol precipitation af-ter incubation with RNAse-free DNAse (Promega, Madison, Wisconsin, USA) (Feldman et al. 1991). Control RNAs were tested for DNA contamination by performing RT-PCR in the absence of reverse transcriptase enzyme.

The concentration of the control cDNA can adversely affect the amplifi ca-tion effi ciency of the sample DNA, thus the optimal amount of control RNA for each gene was defi ned by including varying amounts of control RNA for each RT-PCR reaction. This was to ensure that the range of concentrations for both the sample cDNA and the control DNA allows amplifi cation within the exponential range.

The cDNA was amplifi ed in a DNA Thermal Cycler (Perkin Elmer) as de-scribed in detail in study III. A trace amount of γ32P-labeled 3’ primer was added to provide about 1.5x106 cpm per reaction to label the DNA. In this way, each synthesized DNA strand was radiolabeled and PCR primers ampli-fi ed both sample and control cDNA, as they both included sequences comple-mentary to the primer.

In using quantitative PCR, it is mandatory that colinear amplifi cation is obtained with the control cDNA and the sample. To ensure this and that measurements were performed during the exponential phase of amplifi cation, 10 µl of each PCR reaction mixture was removed every fourth cycle during cy-cles 12 to 32. PCR product aliquots were electrophoresed on 4.25% (w/v) Nu-SieveGTG gel (FMC Bioproducts). The amplifi cation products of the control cDNA and the cDNA of the sample were different in size, and therefore could be separated electrophoretically. Gels were stained with ethidium bromide and amplifi cation products were visualized by UV light. Bands representing the gene and the control were excised from the gel separately and the radioactiv-ity of each band was determined by Cerencov counting. The values from the cellular and control bands were plotted on a semi-logarithmic scale against the number of amplifi cation cycles. Parallel curves indicate that despite a differ-ence in size, the two templates were amplifi ed with comparable amplifi cation effi ciencies. Comparing the levels of radioactivity, the mRNA level of interest could then be calculated from the known amount of control RNA by using values from the exponential cycles (Feldman et al. 1991).

35

2.4. Quantifi cation of cytosolic DNA fragmentation by sandwich enzyme immunoassay

For quantifi cation of DNA fragmentation, specifi c determination of cytosolic mononucleosomes and oligonucleosomes was performed using a commercial quantitative sandwich ELISA kit (Boehringer Mannheim). In this procedure 10 mg of frozen heart left ventricular free wall tissue was homogenized at room temperature using a glass homogenizer in 200 µl of the lysis buffer supplied with the kit. After incubation for 30 min at room temperature the homoge-nate was centrifuged at 200 g for 10 min. The activity of the supernatant was measured immediately; all samples of an experiment in one assay, for mono- and oligonucleosomes according to the manufacturer’s recommendations.

3. STATISTICAL ANALYSIS

All data are presented as mean ± standard error of mean (S.E.M.). In studies I-IV the data were analyzed by the non-parametric Mann-Whitney U test us-ing the StatView 4.5 statistical package (Abacus Concepts, Ca, USA). In study V comparisons between groups were done with two-way analysis of variance (ANOVA) and correlation analysis was done with Pearson’s correlation coef-fi cients using SPSS for Windows 12.0 (SPSS Inc., Chicago, IL, USA). p<0.05 was considered statistically signifi cant.

36

RESULTS

1. VALIDATION OF QUANTITATIVE RT-PCR METHOD (I)

Contamination either with genomic DNA or previously amplifi ed DNA can greatly affect the results because of the high degree of sensitivity of PCR based methods (Bristow et al. 1993). For these reasons, the total RNA of the samples and internal stardard cRNA were assessed for DNA remnants by performing PCR and RT reactions without reverse transcriptase. As expected, no amplifi -cation products were seen. During measurements, RT and PCR reactions in-cluded a negative control. When performing the RT and PCR and measuring the same sample 10 times the coeffi cient of variation was 13.9%.

2. THE EFFECT OF SHORT-TERM ETHANOL AND CALCIUM CARBIMIDE TREATMENT ON APOPTOSIS AND EXPRESSION OF CARDIAC MODULATORY GENES IN VIVO (II, IV)

In this study, 96 6-week old rats were divided into three groups (2-day, 5-day and 8-day experiment) and each of these groups further to subgroups (C: control; E: ethanol 1g/kg of body weight daily; CC: calcium carbimide 100 mg/kg of diet daily; E+CC: treated with both ethanol and calcium carbimide daily). High blood acetaldehyde levels were measured in all of the E+CC treat-ment groups (Figure 2), but only in one ethanol treated group (2-day) was acetaldehyde concentration (2.1±1.6 µmol/l) detectable, although low. The blood samples for ethanol and acetaldehyde were taken 2 hours after ethanol injection and thus represent peak blood concentrations.

Total body weight growth was partly inhibited during combined E+CC treatment in all three (2-day, 5-day and 8-day) E+CC treatment groups com-pared with respective control groups (p<0.001 in all). In 2- and 5-day experi-ments calcium carbimide treatment alone also inhibited normal body weight increase (p<0.001) (Figure 3). The heart weight was lower in the E+CC treat-ment group at 8 days compared with the respective control group (p<0.05) (Figure 3). Also calcium carbimide treatment alone had a negative effect on heart weight at 8 days (Figure 4), but the heart to body weight ratio did not change signifi cantly, neither did it change in any other experimental group (Figure 5).

37

Figure 2. Blood ethanol (EtoH) and acetal-dehyde (AcH) concentrations after ethanol (E: 1 g/kg of body weight) or combined etha-nol + calcium carbimide (CC: 100 mg/ kg of diet) treatment for 2, 5 or 8 days. Values are expressed as means.

Figure 3. Total body weight increase af-ter exposure to either ethanol (E: 1 g/kg of body weight), calcium carbimide (CC: 100 mg/ kg of diet) or combined E+CC treat-ment for 2, 5 or 8 days. Values are expressed as means. *** p<0.001 as compared with respective control group.

Figure 4. Heart weights of control rats, and of rats after exposure to either ethanol (E: 1 g/kg of body weight), calcium carbim-ide (CC: 100 mg/ kg of diet), or combined E+CC treatment for 2, 5 or 8 days. Values are expressed as means. * p<0.05, ** p<0.01 as compared with respective control group.

Figure 5. Heart weight to body weight ra-tio in control rats and after exposure to ei-ther ethanol (E: 1 g/kg of body weight), cal-cium carbimide (CC: 100 mg/ kg of diet), or combined E+CC treatment for 2, 5 or 8 days. Values are expressed as means.

38

Both ethanol and calcium carbimide treatments alone tended to increase the relative amount of apoptotic nucleosomes, but only combined E+CC treat-ment at 5 days increased the index signifi cantly by 23% (p<0.05) compared with control rats (Figure 6).

Figure 6. The amount of apoptotic nucleosomes in control rats (C), and af-ter ethanol (E: 1 g/kg of body weight), calcium carbimide (CC: 100 mg/ kg of diet), or combined E+CC treatment at 5 days. Data are shown as means ± SEM. * p<0.05 as compared with the control group.

Combined E+CC treatment increased left ventricular ANP mRNA levels by 60% (p<0.05) at 2 days, and by 41% (p<0.05) at 8 days compared with respective control groups (Figures 7 and 8). On the other hand, BNP mRNA levels were not statistically signifi cantly changed in any of the experimental groups (Figures 7 and 8).

Combined E+CC treatment increased left ventricular p53 mRNA levels by 15% at 8 days as compared with the controls (p<0.05), but this induction was not seen in other experimental groups (Figures 7 and 8).

39

Figure 7. (a) The effects of ethanol (E: 1g/kg of body weight), calcium car-bimide (CC: 100 mg/kg of diet), and combined E+CC treatment on heart left ventricular mRNA levels of measured genes at 2 days. C= controls. Data are shown as means ± SEM. * p<0.05, ** p<0.01 as compared with respective control values. (b) Relative differences in mRNA expression of measured genes after ethanol (E: 1g/kg of body weight), calcium carbimide (CC: 100 mg/kg of diet), or combined E+CC treatment at 2 days. Values are expressed as % dif-ference relative to respective mRNA level in control group.

40

Figure 8. (a) The effects of ethanol (E: 1g/kg of body weight), calcium car-bimide (CC: 100 mg/kg of diet), and combined E+CC treatment on heart left ventricular mRNA levels of measured genes at 8 days. C= controls. Data are shown as means ± SEM. * p<0.05, ** p<0.01 as compared with respective control values. (b) Relative differences in mRNA expression of measured genes after ethanol (E: 1g/kg of body weight), calcium carbimide (CC: 100 mg/kg of diet), or combined E+CC treatment at 8 days. Values are expressed as % dif-ference relative to respective mRNA level in control group.

41

No statistically signifi cant changes could be observed in p21 mRNA levels in either ethanol or calcium carbimide treated groups at 2 or 8 days, although calcium carbimide treatment lowered the level by –26% at 8 days. Combined E+CC treatment, however, increased the left ventricular p21 mRNA level by 25% at 2 days (p<0.05) and, interestingly, decreased the level by 24% at 8 days (p<0.05) compared with respective controls (Figures 7 and 8).

Bcl-2 mRNA levels remained quite constant at 2 days, but at 8 days, a 36% increase was seen in the E+CC treatment group (p<0.05). On the oth-er hand, bax mRNA levels were more than 30% higher in all experimental groups compared with respective control groups. However, only certain of these changes were statistically signifi cant, probably due to the relative small experimental groups. It is noteworthy, that the only group in which the bcl-2 to bax ratio was signifi cantly decreased (by 36%; p<0.05) was the E+CC treat-ment group at 2 days (Figures 7 and 8).

TNF-alpha mRNA levels tended to be lower in all experimental groups at 2 days. However, none of these changes were statistically signifi cant (Figures 7 and 8).

3. THE EFFECT OF ACUTE ETHANOL AND ACETALDEHYDE INFUSION ON APOPTOSIS AND EXPRESSION OF CARDIAC MODULATORY GENES IN LANGENDORFF PREPARATION (III)

In this study, neither ethanol (5% v/v) nor acetaldehyde (500 µmol/l) infusion for 150 min in Langendorff preparation induced any changes in the relative amounts of apoptotic nucleosomes compared with control group (Figure 9; III: Table 3).

Figure 9. The amount of apoptotic nucleosomes in Langendorff preparation after infusion with vehicle (C), ethanol (E: 5% v/v), or acetaldehyde (A: 500 µmol/l) for 150 min. Data are shown as means ± SEM. There were no statisti-cally signifi cant differences between experimental groups.

42

Figure 10. (a) The effects of ethanol and acetaldehyde infusion on heart left ventricular mRNA levels of measure genes. Isolated rat hearts were infused with either vehicle (controls), ethanol (5% v/v), or acetaldehyde (500 µmol/l) for 150 min. Data are shown as means ± SEM. * p<0.05 as compared with control group. (b) Relative differences in mRNA expression of measured genes after 150 min of ethanol (E: 5% v/v) or acetaldehyde (A: 500 µmol/l) infusion in Langendorff preparation. Values are expressed as % difference relative to re-spective mRNA level in control group.

43

Ethanol infusion increased cardiac left ventricular ANP mRNA levels by 38% as compared with the control group (p<0.05). On the other hand, acetal-dehyde infusion had practically no effect on ANP mRNA levels. With respect to BNP mRNA levels, there were no signifi cant changes in either experimen-tal group (Figure 10).

No signifi cant differences in p53 mRNA levels were observed between the three groups (Figure 10).

Ethanol treatment increased p21 mRNA levels signifi cantly by 41% as compared with controls (p<0.05), but this induction was not seen in acetalde-hyde treated hearts (Figure 10).

The levels of the bcl-2 and bax mRNAs remained quite constant throughout the experiment. Similarly, there was not a marked change in bcl-2 to bax ratio in neither of the experimental groups compared with controls (Figure 10).

In the ethanol treated hearts the amount of TNF mRNA was 27% lower and in the acetaldehyde treated hearts 42% lower than in the control hearts but neither of these differences were statistically signifi cant (Figure 10).

4. THE EFFECT OF CHRONIC ETHANOL EXPOSURE ON APOPTOSIS AND EXPRESSION OF CARDIAC MODULATORY GENES IN FEMALE AND MALE RATS IN VIVO (V)

This 2-year study consisted of male and female rats, which were divided into ethanol-treated and control groups. The morning blood ethanol and acetalde-hyde levels were measured 2 months before the experiment ended (V: Table 1). No differences were observed in blood ethanol or acetaldehyde levels between the male and female ethanol fed groups. Likewise the chronic low dose etha-nol exposure had no effect on the heart weight to body weight ratios. On the contrary, the total body weights were dependent on both gender and ethanol. Body weights of the female rats were signifi cantly lower than the weights of the male rats (p<0.001) and the weights of the ethanol treated rats (ethanol fed male and female rats combined) were lower than the weights of the control rats (males and females combined) (p=0.042) (V: Table 1)

No signifi cant changes could be observed in the relative amounts of apop-totic nucleosomes in ethanol treated groups in either gender as compared with respective controls (Figure 11).

44

No statistically signifi cant differences were seen in ANP and BNP mRNA levels between any of the groups studied (Figure 12).

p53 mRNA levels were dependent on ethanol exposure (p<0.05) and gender (p<0.001). The chronic low dose ethanol treatment increased p53 mRNA levels in both males and females compared to their controls not exposed to ethanol. Interestingly, the p53 mRNA levels in males were higher (ethanol and control groups combined) than the p53 mRNA levels in females (ethanol and control groups combined) (Figure 12; V: Figure 1).

In contrast to p53 levels the p21 mRNA levels were not dependent either on gender or ethanol (Figure 12).

The levels of bcl-2 and bax mRNAs and the bcl-2 to bax ratios were not signifi cantly affected by either ethanol or gender (Figure 12).

No statistically signifi cant differences were seen in TNF-alpha mRNA lev-els between any of the groups studied (Figure 12).

Figure 11. The amount of apoptotic nucleosomes in control rats (MC= male control group, FC= female control group) and after two years of ethanol ex-posure in vivo (ME= male ethanol treated group, FE= female ethanol treated group). Data are shown as means ± SEM. There were no statistically signifi cant differences between experimental groups.

45

Figure 12. (a) The effect of two years of ethanol exposure on heart left ven-tricular mRNA levels of measured genes in male and female rats in vivo. Data are shown as means ± SEM. (b) Relative differences in mRNA expression of measured genes after two years of ethanol exposure in vivo. Values are ex-pressed as % difference relative to mRNA level in respective gender control hearts. M=male, F=female.

b

46

DISCUSSION

1. ETHANOL, ACETALDEHYDE AND LEFT VENTRICULAR APOPTOSIS

Alcohol abuse is a major cause of non-ischemic cardiomyopathy in Western society (Piano 2002). The mechanism by which exposure to ethanol results in cardiac damage is unknown, but apoptosis, which clearly plays a role in etha-nol-related injury of many other organs, may be involved. In the 150-minute study using a Langendorff preparation, no evidence was found that apoptosis occurred after ethanol or acetaldehyde infusion. Also exposure to ethanol alone for fi ve days or two years did not alter the degree of DNA fragmentation in vivo. In a study by Chen et al. (2000) using cultured neonatal cardiomyocytes, ethanol was found to increase apoptosis, dose-dependently. Prenatal exposure to ethanol has been found to increase myocardial apoptosis in rats (Ren et al. 2002). In contrast, ethanol has a protective effect on heart muscle under some experimental circumstances (Miyamae et al. 1997; Krenz et al. 2002). In an experiment, in which rats that had been exposed for three weeks to ethanol before Langendorff preparation, infarct sizes were smaller and apoptosis less after ischemia-reperfusion of the ethanol-treated hearts than in controls that had not been exposed to ethanol (Sato et al. 2002). Treatment of hearts with low concentrations of ethanol (< 5 mmol/l) activates the cell-survival-promot-ing pathway. Contrariwise, treatment with higher concentrations activates the pro-apoptotic pathway (Krenz et al. 2002). In the Langendorff experiment in the present study the ethanol concentration exceeded 5 mmol/l. However, the infusion time was only 150 minutes, which could have been too short for ap-optosis to be triggered. In the developing rat forebrain, triggering of apop-tosis by ethanol was found to require exposure to a concentration of at least 43 mmol/l for four consecutive hours (Ikonomidou et al. 2000). In the study group in which rats received fi ve days of treatment with both ethanol and cal-cium carbimide, blood ethanol concentrations were greater than 5 mmol/l, with concomitantly high blood acetaldehyde concentrations. In this group, the extent of DNA fragmentation was signifi cantly increased, refl ecting increased apoptosis. Acetaldehyde has been shown to accelerate apoptotic cell death and to inhibit cell growth in various other cells (Zimmerman et al. 1995; Holow-nia et al. 1999). This fi ndings therefore support a role for ethanol and/or the acetaldehyde produced from ethanol in inducing apoptosis at high concentra-tions in the heart. However, it cannot be concluded from our results whether ethanol, acetaldehyde or the combination of the two is the actual trigger of apoptosis. Also the direct effect of calcium carbimide itself remains unclear.

47

2. ETHANOL, ACETALDEHYDE AND LEFT VENTRICULAR p53 GENE EXPRESSION

Exposure of an isolated heart to ethanol alone for 150 minutes and adminis-tration of ethanol for two to eight days had no effects on p53 mRNA levels. There are reports in the literature that support both p53-dependent and p53-independent mechanisms of ethanol-related apoptosis and growth suppression in various organs. In many instances, p53 has been shown to be regulated at the level of protein expression. In the present study p53 protein levels were not measured. The possibility that p53 plays a role in ethanol-related cardiac adaptation at earlier time points cannot therefore be excluded.

In this study, rats to which ethanol had been administered for two years ex-hibited signifi cantly increased p53 mRNA levels, but no evidence of increased apoptosis was found. It has been shown previously that when p53 levels are elevated, cellular death does not always ensue (Hofseth et al. 2004; Slee et al. 2004). Also, occurrence of cardiac apoptosis depends on the intensity of a par-ticular stimulus (Kang & Izumo 2000). In the two-year study, levels of ethanol and acetaldehyde derived from ethanol were probably too low to cause detect-able cell death. It is also possible that apoptosis played a more signifi cant role at earlier time points in the adaptation process. The reason for the observed increased heart p53 levels after ethanol exposure is not clear at present. Oxida-tive stress increases p53 levels (Ueada et al. 2002) and oxidative stress has been implicated also in ethanol induced cell injury (Koch et al. 2002). Furthermore, acute ethanol exposure has been shown to induce oxidative stress in myocar-dium (Kannan et al. 2004). It has also been suggested that p53 participates in sensing oxidative DNA damage (Achanta & Huang 2004). Normal diploid cells have limited potential for replication. The number of cell divisions pos-sible is determined by the lengths of telomeres at chromosome ends. These shorten during each cell cycle. Once a critical length is reached, sensors for DNA damage are triggered and cell-cycle arrest or apoptosis is induced. p53 seems to be important in relation to this response to telomere erosion (for ref-erences see Igney & Krammer 2002). Interestingly, in mice, constitutive ex-pression of p53 itself accelerates aging, and telomere loss (Tyner et al. 2002). The elevated levels of p53 mRNA seen in the two-year study could therefore have refl ected ethanol-induced stress reaction of the heart.

In the present study combined treatment of rats with ethanol and calcium carbimide for eight days resulted in higher p53 mRNA levels than in the con-trols, and apoptosis after fi ve days. This fi nding is in accordance with earlier fi ndings suggesting that the p53 gene may play an important role in cardi-omyocyte apoptosis (Leri et al. 1998b; Cesselli et al. 2001). Conversely, our data suggest that subacute exposure to ethanol or acetaldehyde derived from ethanol at particular concentrations may trigger the p53 gene in rat hearts, and that p53-dependent injury may be one mechanism in the development of AHMD.

48

In the two-year study left ventricular p53 mRNA levels were approximately 40% higher in male rats than in female rats irrespective of exposure to ethanol. Myocyte death in failing hearts has been shown to be gender dependent with higher propensity to myocyte death in male hearts. In human beings, it has been reported that myocyte loss is related to increasing age in men, but remains un-changed with age in women (Guerra et al. 1999). Aging has also been shown to decrease telomerase activity in male rat myocytes but to increase the activity in female myocytes (Leri et al. 2000). In the human heart telomere loss leads to car-diac dilatation and heart failure associated with up-regulation of p53 (Leri et al. 2003). Therefore the observed gender dependence of p53 expression may be one factor explaining the differences in myocyte viability between genders in failing heart and during aging. However, in other cell and tissue types, results concern-ing p53 levels relative to gender are confl icting (Toda et al. 1998; Zhao et al. 2002; Natahara et al. 2003).

3. ETHANOL, ACETALDEHYDE AND LEFT VENTRICULAR p21 GENE EXPRESSION

In the study using a Langendorff preparation, infusion of ethanol for 150 min-utes increased p21 mRNA gene expression. p21 is a transcriptional regula-tor of ANP (Kovacic-Milivojevic et al. 1997) and ANP induction was already evident at 150 minutes. In contrast, in vivo exposure of rats to ethanol for two days, eight days and two years had no statistically signifi cant effects on p21 mRNA levels. It is possible that the p21 regulatory pathway does not play an important role at the later time points. The regulatory network also func-tions differently in organ models and in vivo. However, p21 has been linked to cardiac hypertrophy by a number of investigators (Kovacic-Milivojevic et al. 1997) and results in the present study showing that infusion of a 0.5% ethanol solution into an isolated heart can markedly induce expression of p21 and ANP genes is one of the fi rst direct pieces of evidence which links ethanol itself to a cardiomyocyte reaction related to cardiac hypertrophy.

In the study involving combined treatment with ethanol and calcium car-bimide, p21 gene expression was 25% greater than in a control group after two days but 24% less after eight days. At fi ve days apoptosis was triggered. p21 regulates the cell cycle by inhibiting the cyclin-dependent kinases re-quired for progression from the G1 to the S phase (El-Deiry et al. 1993) and is therefore important in relation to repair of damage to cell DNA (Cox 1997). p21 has been implicated in survival responses in a variety of systems in which exposure to stressful stimuli results in p53-independent cell death (Gorospe et al. 1997). In a study by Hauck et al. (2002), down-regulation of p21 was required for hypoxia-induced cardiomyocyte apoptosis to occur. In the present study, higher than normal p21 levels did not protect myocytes from apoptosis.

49

This result suggests that the high p21 mRNA levels seen after two days in the group treated with both ethanol and calcium carbimide may refl ect an attempt to diminish the environmental stress, at myocyte level. However, the DNA damage or biochemical stress resulting from exposure to ethanol or acetalde-hyde derived from ethanol was too great to be overcome by p21 up-regulation, and that induction of apoptosis therefore ensued.

4. ETHANOL, ACETALDEHYDE AND LEFT VENTRICULAR ANP GENE EXPRESSION

It has been suggested that ANP gene expression is a sensitive indicator of car-diac hypertrophic response (Chien et al. 1991). In the acute study using an isolated rat heart model, infusion of ethanol for 150 minutes signifi cantly in-creased left ventricular ANP (and p21) gene expression. Other investigators have reported an increase in ANP tissue protein content 120 minutes after exposure to ethanol (Guillaume et al. 1994) or no change in mRNA and pro-tein levels (Wigle et al. 1995). In the present study ethanol alone was capable of inducing genes involved in the regulation of cardiac hypertrophy, suggest-ing that some aspects of alcoholic heart disease could be due to direct cardiac effects of ethanol rather than refl ect alterations in neurohumoral, hormonal or nutritional status. In contrast to the 150-minute experiment, no signifi cant change in ANP mRNA levels were detected after exposure of rats to ethanol for periods ranging from two days to two years. In other studies, six weeks of ingestion of ethanol has been found to increase ANP mRNA and protein lev-els above those found in control rats (Wigle et al. 1993a) whereas two- or four-month exposures have no effect (Kim et al. 2003).

In the study using an isolated rat heart model, infusion of 500 µM acetal-dehyde for 150 minutes had no effect on left ventricular ANP gene expression. Effects of acetaldehyde at a later stage cannot, however, be excluded. Treatment of rats simultaneously with both ethanol and calcium carbimide resulted in high blood acetaldehyde levels and ANP mRNA concentrations that were signifi cant-ly higher than normal in the left ventricle wall. Elevated levels of expression of the ANP gene were detectable after only the second day of the treatment and remained evident after eight days of treatment. ANP protects against cardiac hypertrophy (Masciotra et al. 1999; Tamura et al. 2000) and the net effect of ANP on the myocardium in vivo is limitation of myocyte growth (Klinger et al. 1993). ANP has been shown to induce apoptosis of cardiac myocytes, dose- and time-dependently (Wu et al. 1997; Kang et al. 2000). Five days of combined treatment with ethanol and calcium carbimide resulted in apoptosis. These fi nd-ings suggest that high blood ethanol and/or acetaldehyde concentrations activate the gene encoding for ANP in rat ventricular myocardium, and that ANP might regulate apoptosis in the rat ventricular myocardium. Acetaldehyde could there-fore play a role in relation to development of alcoholic heart disease. This notion

50

is supported by the results of a study by Liang et al. (1999). They found that acetaldehyde levels in mice hearts that over-expressed cardiac-specifi c alcohol dehydrogenase were four times greater after exposure to ethanol and that these hearts exhibited a high incidence of signs of alcoholic myocardial disease and sig-nifi cantly increased ANP mRNA levels.

5. ETHANOL, ACETALDEHYDE AND LEFT VENTRICULAR BNP GENE EXPRESSION

In the present study, there were no consistent changes in cardiac BNP mRNA levels in any of the groups studied for 150 min to 2 years. This suggests that there was no sustained myocardial stretching, which stimulates BNP secretion (Ruskoaho 2003). It has been reported that administration of a 5g/kg dose of ethanol (40% w/v) to rats reduced left ventricular BNP mRNA levels to 38% of control levels after 120 minutes (Wigle et al. 1995). In studies lasting for six weeks or two, four or eight months, ethanol administration resulted in no changes in ventricular BNP gene expression (Wigle et al. 1993c;Guillaume et al. 1996b). Our fi ndings suggest that BNP was not involved in ethanol-in-duced myocardial adaptation at the time points studied in the experiments.

6. ETHANOL, ACETALDEHYDE AND LEFT VENTRICULAR BCL-2 AND BAX GENE EXPRESSION AND BCL-2/BAX RATIO

Overexpression of bcl-2 protects cardiac myocytes against apoptosis (Kirshen-baum & de Moissac 1997). In the present study, ethanol treatment alone had no effect on bcl-2 at any of the time points studied. Results reported in the litera-ture on the effects of ethanol on bcl-2 levels and apoptosis in other tissues are confl icting. Bax, on the other hand, accelerates apoptosis in ventricular cells, and it can regulate apoptosis independently of bcl-2 (Regula et al. 2003). Eight days of treatment with ethanol alone increased bax mRNA levels. This fi nding fi nd-ing suggest that ethanol regulates cardiac bax gene expression in vivo. Chen et al. (2000) have reported that ethanol increases bax protein levels dose-dependently in primary cardiomyocytes and decreases cell viability similarly.

In the group treated with both ethanol and calcium carbimide bax mRNA levels were signifi cantly greater than in controls after two days. This was fol-lowed by an elevated number of apoptotic nucleosomes after fi ve days. After eight days, bax gene expression was still being induced, and bcl-2 gene ex-pression was also elevated. These fi ndings provide further evidence that the bax gene may be involved in ethanol- and/or acetaldehyde-induced apoptosis. The elevated expression of bcl-2 after eight days suggests that compensatory mechanisms become activated in an attempt to control apoptosis and maintain cell survival.

51

The ratio of bcl-2 to bax can be regarded as a determinant of survival or death of ventricular cells (Reed 2002). In the present study the bcl-2/bax ratio after two days was signifi cantly lower in the rats treated with both ethanol and calcium carbimide than in the control group. In the developing rat CNS, prenatal exposure to ethanol lowered the bcl-2/bax ratio in the cortex, a fi nd-ing in accordance with anatomical data relating to ethanol toxicity (Mooney & Miller 2001). It is possible that the lower than normal cardiac bcl-2/bax ratio after two days was an element in the mechanism leading to apoptosis af-ter fi ve days. After eight days of ethanol exposure anti-apoptotic mechanisms were becoming activated as bcl-2 levels increased, and the bcl-2/bax ratio was at levels seen in controls. Ethanol and calcium carbimide were each capable on their own of increasing bax mRNA levels but their effects on the bcl-2/bax ra-tio were not signifi cant. This fi nding supports the view that in some instances the bcl-2 to bax ratio is a better indicator of susceptibility to cardiac apoptosis than bcl-2 or bax mRNA levels alone.

There were no differences in bcl-2 and bax gene expression or the bcl-2/bax ratio between female and male left ventricles. In contrast, in cardiac fi broblasts basal protein levels of bcl-2 are higher in female rats than in male rats (Zhao et al. 2002). Toda et al. (1998) have reported that amounts of bcl-2 mRNA were higher in the lacrimal glands of female autoimmune mice than in the lacrimal glands of male autoimmune mice. No reports on the effects of gender on bax expression have been published. Results in this study suggest that there are no major gender differences as far as the aging rat heart is concerned in relation to bcl-2 or bax gene expression.

7. ETHANOL, ACETALDEHYDE AND LEFT VENTRICULAR TNF-ALPHA GENE EXPRESSION

TNF-alpha mRNA levels did not alter consistently in any of these experi-ments. TNF-alpha is a pro-infl ammatory cytokine that has been associated with the genesis of numerous cardiovascular diseases, including heart failure (Feldman et al. 2000). There is also evidence that TNF-alpha plays a pro-sur-vival role in the heart through regulation of adaptive responses to biomechani-cal stress (Gill et al. 2002). The results suggest that TNF-alpha was not in-volved in the mechanism of ethanol- or acetaldehyde-induced cardiac adapta-tion under the circumstances studied. In contrast, ethanol has been found to affect TNF-alpha mRNA levels and signalling in other organs. In the liver, for example, TNF-alpha is required both for normal regeneration and in relation to ethanol-related liver damage (Diehl 1999).

Although amounts of TNF mRNA have been found to be higher in the lacrimal glands of female autoimmune mice than in the lacrimal glands of

52

male autoimmune mice (Rocha et al. 1998), no statistically signifi cant dif-ferences in cardiac TNF-alpha mRNA levels between female and male rats were found.

8. LIMITATIONS OF THE STUDY

A major limitation of this study is that it is not possible to differentiate be-tween the effects of ethanol and acetaldehyde in the group that was treated with both ethanol and calcium carbimide. It has been shown that treatment with concentrations of ethanol of < 5 mmol/l results in activation of path-ways that promote cardiac cell survival, and that treatment with concentra-tions of > 5 mmol/l activates pro-apoptotic pathways (Krenz et al. 2002). A concentration of 5 mmol/l was reached in all of the animal groups treated with both ethanol and calcium carbimide. In these groups acetaldehyde concentra-tion was also high. It is therefore diffi cult to say whether the effects of ethanol, acetaldehyde or the two together caused the changes in apoptosis and gene expression detected. Also the direct effect of calcium carbimide itself remains unclear. Methods of studying the effects of acetaldehyde itself in vivo are dif-fi cult due to its high toxicity.

Particularly in the two-year study, group sizes were small, and the number of experimental animals was limited. Also, in the two-year study, it would have been advantageous to determine p53 protein concentrations, since mRNA levels do not always refl ect changes at protein levels. Such experiments will be undertaken in future studies.

53

SUMMARY AND CONCLUSION

Dilated heart failure is the leading cause of death in the elderly, particularly in elderly men. Alcohol abuse has been proven to be a major cause of non-ischemic cardiomyopathy in Western society. However, neither the mechanism nor any specifi c toxic agent that results in alcoholic heart muscle disease has been established. During the last decade, roles played by apoptosis and apop-tosis-regulating genes have been clearly described in relation to other ethanol-induced organ damage, for example liver damage.

The key fi ndings in the study are:

1. A highly sensitive quantitative RT-PCR method was developed allowing determination of different mRNAs in very small amounts of cardiac tissue.

2. Combined ethanol and calcium carbimide treatment of rats for two to eight days resulted in high blood acetaldehyde levels, myocardial apoptosis and al-tered expression of ANP, p53, p21, bcl-2 and bax genes. Whether the trigger-ing molecule is ethanol or acetaldehyde formed from ethanol, or whether the changes observed result from the combined effects of both agents cannot be decided from the results of this study.

3. Acute ethanol infusion induced p21 and ANP gene expression in the iso-lated perfused rat heart.

4. Chronic exposure to ethanol in vivo induced cardiac left ventricular p53 gene expression. Expression of the p53 gene was also gender-dependent. Males had higher p53 mRNA levels than females.

In conclusion, the results of this study indicate that ethanol and acetaldehyde may both be capable of regulating myocardial apoptosis and expression of genes associated with cardiac cell adaptation to stress. Although it must be borne in mind that mRNA levels do not always refl ect changes in protein lev-els, our results shed new light on the basic mechanisms underlying the actions of ethanol and acetaldehyde on heart muscle.

54

ACKNOWLEDGEMENTS

This study was carried out at the Department of Clinical Chemistry at the Helsinki University Central Hospital, at the Department of Mental Health and Alcohol Research at the National Public Health Institute, Helsinki and at the Wihuri Research Institute, Helsinki. I wish to thank Professor Herman Adlercreutz, Professor Matti Härkönen, Professor Ulf-Håkan Stenman, Profes-sor Jouko Lönqvist, and Professor Petri Kovanen for allowing me to use the excellent research facilities of these institutions.

I am truly indebt to Professor Arthur M. Feldman, who gave me an oppor-tunity to be a part of his laboratory in1993 and to learn RT-PCR methodology. The half a year I spent at The Johns Hopkins University School of Medicine laid bases to my thesis and gave me a hint of the complex mechanisms regulat-ing the adaptation processes of cardiac muscle.

I wish to express my greatest gratitude to my supervisors Docent Tiina Mäki and Professor Matti Härkönen for their invaluable guidance and patience through these years.

Professor Onni Niemelä and Docent Kari Pulkki are gratefully ac-knowledged for improving my thesis by providing expert review of the fi nal manuscript.

I am most grateful to Dr. Veli-Pekka Harjola, for sharing both the fun and the misery during the times of learning principles of laboratory practice. Professor Aarno Palotie, Docent Hannu Somer, Docent Hannu Näveri, and Dr. Mervi Löfberg are to thank for introducing me into the world of science.

I wish to express my gratitude to Docent Peter Eriksson, and Maija Sarvi-harju, PhD, from the National Public Health Institute, for their collaboration, excellent facilities and samples provided for these studies. I am greatly indebted to Docent Kari Eklund from the Department of Medicine, Helsinki University Central Hospital, for help and collaboration during this work. My sincere thanks to Professor Petri Kovanen, Docent Jorma Kokkonen, and Ken Lindstedt, PhD, from the Wihuri Research Institute for fruitful collaboration.

I am very thankful to Professor Seppo Sarna, from the Department of Public Health, University of Helsinki, for his irreplaceable help with statistical problems.

I owe special thanks to the staff in our laboratory, Päivi Tuominen, Helena Taskinen and Anja Kallio. Ms. Päivi Tuominen shared with me the tears and the glory of the demanding RT-PCR method and also taught me the fi rst steps in laboratory work. Ms. Jaana Tuomikangas at the Wihuri Research Institute showed me the impossible by performing aorta cannnulation in hearts as tiny as the tips of our fi ngers. Ms. Aila Koponen gave me excellent assistance in collcting references.

55

This study was supported by grants from the Finnish Foundation for Al-cohol Studies, the Maud Kuistila Foundation, The Fund of Aino and Einari Haaki of the Finnish Cultural Foundation, and The Finnish Medical Society Duodecim.

Finally, this work would not have been possible without the support of my family and friends.

Kauniainen, September 2005

56

REFERENCES

Achanta G, Huang P. Role of p53 in sensing oxidative DNA damage in response to reactive oxygen species-generating agents. Cancer Res 2004;64:6233-6239.

Adachi M, Higuchi H, Miura S, Azuma T, Inokuchi S, Saito H, Kato S, Ishii H. Bax interacts with the voltage-dependent anion channel and medi-ates ethanol-induced apoptosis in rat hepatocytes. Am J Physiol Gastroint Liver Physiol 2004;287:G695-G705.

Adachi S, Ito H, Tamamori-Adachi M, Ono Y, Nozato T, Abe S, Ikeda MA, Marumo F, Hiroe M. Cyclin A/cdk2 activation is involved in hypoxia-in-duced apoptosis in cardiomyocytes. Circ Res 2001;88:408-414.

Adams MA, Hirst M. The infl uence of adrenal medullectomy on the develop-ment of ethanol-induced cardiac hypertrophy. Can J Physiol Pharmacol 1986;64:592-596.

Anversa P, Leri A, Beltrami CA, Guerra S, Kajstura J. Myocyte death and growth in the failing heart. Lab Invest 1998;78:767-786.

Anversa P, Leri A, Li B, Liu Y, Di Somma S, Kajstura J. Ischemic cardiomy-opathy and the cellular renin-angiotensin system. J Heart Lung Transplant 2000;19:S1-S11.

Anversa P, Leri A, Kajstura J, Nadal-Ginard B. Myocyte growth and cardiac repair. J Mol Cell Cardiol 2002;34:91-105.

Auton WP, Ward LC. Acetaldehyde and cardiac protein synthesis in the rat in vivo. Int J Biochem 1986;18:289-292.

Bai J, Cederbaum AI. Mitochondrial catalase and oxidative injury. Biol Sig-nals Recept 2001;10:189-199.

Baldi A, Abbate A, Bussani R, Patti G, Melfi R, Angelini A, Doprina A, Rossiello R, Silvestri F, Baldi F, Di Sciascio G. Apoptosis and post-infarc-tion left vetricular remodeling. J Mol Cell Cardiol 2002;34:165-174.

Baraona E, Abittan CS, Dohmen K, Moretti M, Pozzato G, Chayes ZW, Schaefer C, Lieber CS. Gender differences in pharmacokinetics of alcohol. Alcohol Clin Exp Res 2001;25:502-507.

57

Beckemeier ME, Bora PS. Fatty acid ethyl esters: potentially toxic products of myocardial ethanol metabolism. J Mol Cell Cardiol 1998;30:2487-2494.

Bergmann MW, Loser P, Dietz R, von Harsdorf R. Effect of NF-kappa B in-hibition on TNF-alpha-induced apoptosis and downstream pathways in cardiomyocytes. J Mol Cell Cardiol 2001;33:1223-1232.

Bialik S, Geenen DL, Sasson IE, Cheng R, Horner JW, Evans SM, Lord EM, Koch CJ, Kitsis RN. Myocyte apoptosis during acute myocardial infarc-tion in the mouse localizes to hypoxic regions but occurs independently of p53. J Clin Invest 1997;100:1363-1372.

Bing RJ. Cardiac metabolism: its contributions to alcoholic heart disease and myocardial failure. Circulation 1978;58:965-970.

Bishopric NH, Andreka P, Slepak T, Webster KA. Molecular mechanisms of apoptosis in the cardiac myocyte. Curr Opin Pharmacol 2001;1:141-150.

de Bold AJ, Ma KK, Zhang Y, de Bold ML, Bensimon M, Khoshbaten A. The physiological and pathophysiological modulation of the endocrine function of the heart. Can J Physiol Pharmacol 2001;79:705-714.

Booth FW, Kelso JR. Production of rat muscle atrophy by cast fi xation. J Appl Physiol 1973;34:404-406.

Bouman A, Schipper M, Heineman MJ, Faas MM. Gender difference in the non-specifi c immune response in humans. Am J Reprod Immunol 2004;52:19-26.

Bristow MR, Minobe WA, Raynolds MV, Port JD, Rasmussen R, Ray PE, Feldman AM. Reduced beta-1 receptor messenger RNA abundance in the failing human heart. J Clin Invest 1993;92:2737-2745.

Brown JP, Wei W, Sedivy JM. Bypass of senescence after disruption of p21CIP1/WAF1 gene in normal diploid human fi broblasts. Science 1997;277:831-834.

Brown RA, Savage AO, Lloyd TC. Acute and chronic effects of ethanol on papillary muscles from spontaneously hypertensive rats. Eur J Pharmacol 1996;302:61-67.

58

Bryant D, Becker L, Richardson J, Shelton J, Franco F, Peshock R, Thompson M, Giroir B. Cardiac failure in transgenic mice with myocardial expression of tumor nerosis factor-alpha. Circulation 1998;97:1375-1381.

Calabrese V, Scapagnini G, Catalano C, Dinotta F, Bates TE, Calvani M, Stella AMG. Effects of acetyl-L-carnitine on the formation of fatty acid ethyl es-ters in brain and peripheral organs after short–term ethanol administration in rat. Neurochem Res 2001;26:167-174.

Calderone A, Thaik CM, Takahashi N, Chang DLF, Colucci WS. Nitric oxide, atrial natriuretic peptide, and cyclic GMP inhibit the growth-promoting effects of norepinephrine in cardiac myocytes and fi broblasts. J Clin Invest 1998;101:812-818.

Camper-Kirby D, Welch S, Walker A, Shiraishi I, Setchell KDR, Schaefer E, Kajstura J, Anversa P, Sussman MA. Myocardial Akt activation and gender increased nuclear activity in females versus males. Circ Res 2001;88:1020-1027.

Cao L, Gardner DG. Natriuretic peptides inhibit DNA synthesis in cardiac fi broblasts. Hypertension 1995;25:227-234.

Capasso JM, Li P, Guideri G, Anversa P. Left ventricular dysfunction induced by chronic alcohol ingestion in rats. Am J Physiol 1991;261:H212-H219.

Carlson DL, Lightfoot E Jr, Bryant DD, Haudek SB, Maass D, Horton J, Gi-roir BP. Burn plasma mediates cardiac myocyte apoptosis via endotoxin. Am J Physiol Heart Circ Physiol 2002;282:H1907-H1914.

Cesselli D, Jakoniuk I, Barlucchi L, Beltrami AP, Hintze TH, Nadal-Ginard B, Kajstura J, Leri A, Anversa P. Oxidative stress-mediated cardiac cell death is a major determinant of ventricular dysfunction and failure in dog dilated cardiomyopathy. Circ Res 2001;89:279-286.

Chen DB, Wang L, Wang PH. Insulin-like growth factor I retards apoptotic signaling induced by ethanol in cardiomyocytes. Life Sci 2000;67:1683-1693.

Chen G, Goeddel DV. TNF-R1 signaling: a beautiful pathway. Science 2002;296:1634-1635.

59

Chen Z, Chua CC, Ho YS, Hamdy RC, Chua BHL. Overexpression of bcl-2 attenuates apoptosis and protects against myocardial I/R injury in trans-genic mice. Am J Physiol Heart Circ Physiol 2001;280:H2313-H2320.

Cheng W, Kajstura J, Nitahara JA, Li B, Reiss K, Liu Y, Clark WA, Kra-jewski S, Reed JC, Olivetti G, Anversa P. Programmed myocyte cell death affects the viable myocardium after infarction in rats. Exp Cell Res 1996;226:316-327.

Chien KR, Knowlton KU, Zhu H, Chien S. Regulation of cardiac gene ex-pression during myocardial growth and hypertrophy: molecular studies of an adaptive physiologic response. FASEB J 1991;5:3037-3046.

Cohn JN, Ferrari R, Sharpe N. Cardiac remodeling - concepts and clinical implications: a consensus paper from an international forum on cardiac re-modeling. J Am Coll Cardiol 2000;35:569-582.

Colantoni A, Idilman R, De Maria N, La Paglia N, Belmonte J, Wezeman F, Emanuele N, Van Thiel DH, Kovacs EJ, Emanuele MA. Hepatic apoptosis and proliferation in male and female rats fed alcohol: role of cytokines. Al-cohol Clin Exp Res 2003;27:1184-1189.

Colantonio D, Casale R, Desiati P, De Michele G, Mammarella M, Pasqualetti P. A possible role of atrial natriuretic peptide in ethanol-induced acute diuresis. Life Sci 1991;48:635-642.

Condorelli G, Morisco C, Stassi G, Notte A, Farina F, Sgaramella G, de Rien-zo A, Roncarati R, Trimarco B, Lembo G. Increased cardiomyocyte apop-tosis and changes in proapoptotic and antiapoptotic genes bax and bcl-2 during left ventricular adaptations to chronic pressure overload in the rat. Circulation 1999;99:3071-3078.

Cook SA, Sugden PH, Clerk A. Regulation of bcl-2 family proteins during development and in response to oxidative stress in cardiac myocytes. As-sociation with changes in mitochondrial membrane potential. Circ Res 1999;85:940-949.

Cox LS. Multiple pathways control cell growth and transformation: overlap-ping and independent activities of p53 and p21Cip1/WAF1/Sdi1. J Pathol 1997;183:134-140.

Crabbe DL, Dipla K, Ambati S, Zafeiridis A, Gaughan JP, Houser SR, Mar-gulies KB. Gender differences in post-infarction hypertrophy in end-stage failing hearts. J Am Coll Cardiol 2003;41:300-306.

60

Dancy M, Maxwell JD. Alcohol and dilated cardiomyopathy. Alcohol Alcohol 1986;21:185-198.

Deng C, Zhang P, Harper JW, Elledge SJ, Leder P. Mice lacking p21CIP1/WAF1 undergo normal development, but are defective in G1 checkpoint control. Cell 1995;82:675-684.

Diehl AM. Effect of ethanol on tumor necrosis factor signaling during liver regeneration. Clin Biochem 1999;32:571-578.

Diep QN, El Mabrouk M, Yue P, Schiffrin EL. Effect of AT(1) receptor block-ade on cardiac apoptosis in angiotensin II-induced hypertension. Am J Physiol Heart Circ Physiol 2002;282:H1635-H1641.

Dispersyn GD, Borgers M. Apoptosis in the heart: about programmed cell death and survival. News Physiol Sci 2001;16:41-47.

Dubal DB, Shughrue PJ, Wilson ME, Merchenthaler I, Wise PM. Estradiol modulates bcl-2 in cerebral ischemia: a potential role for estrogen recep-tors. J Neurosci 1999;19:6385-6393.

Eddy LJ, Goeddel DV, Wong GHW. Tumor necrosis factor-alpha pretreat-ment is protective in a rat model of myocardial ischemia-reperfusion in-jury. Biochem Biophys Res Comm 1992;184:1056-1059.

van Eickels M, Grohe C, Cleutjens JPM, Janssen BJ, Wellens HJJ, Doev-endans PA. 17beta-estradiol attenuates the development of pressure-over-load hypertrophy. Circulation 2001;104:1419-1423.

van Eickels M, Patten RD, Aronovitz MJ, Alsheikh–Ali A, Gostyla K, Cel-estin F, Grohe C, Mendelsohn ME, Karas RH. 17-beta-estradiol increases cardiac remodeling and mortality in mice with myocardial infarction. J Am Coll Cardiol 2003;41:2084-2092.

Ekman AC, Vakkuri O, Vuolteenaho O, Leppäluoto J. Ethanol decreases noc-turnal plasma levels of atrial natriuretic peptide (ANP 99-126) but not the N-terminal fragment of pro-atrial natriuretic peptide (ANP 1-98) in man. Clin Sci 1994;86:285-290.

El-Deiry WS, Tokino T, Velculescu VE, Levy DB, Parsons R, Trent JM, Lin D, Mercer WE, Kinzler KW, Vogelstein B. WAF1, a potential mediator of p53 tumor suppression. Cell 1993;75:817-825.

61

Engel D, Peshock R, Armstong RC, Sivasubramanian N, Mann DL. Cardiac myocyte apoptosis provokes adverse cardiac remodeling in transgenic mice with targeted TNF overexpression. Am J Physiol Heart Circ Physiol 2004;287:H1303-H1311.

Engel FB, Hauck L, Boehm M, Nabel EG, Dietz R, von Harsdorf R. p21CIP1 controls proliferating cell nuclear antigen level in adult cardiomyocytes. Mol Cell Biol 2003;23:555-565.

Eriksson CJP, Sippel HW, Forsander OA. The determination of acetaldehyde in biological samples by head-space gas chromatography. Anal Biochem 1977;80:116-124.

Eriksson CJP, Fukunaga T, Sarkola T, Lindholm H, Ahola L. Estrogen-related acetaldehyde elevation in women during alcohol intoxication. Alcohol Clin Exp Res 1996;20:1192-1195.

Eriksson CJP. The role of acetaldehyde in the actions of alcohol (Update 2000). Alcohol Clin Exp Res 2001;25:15S-32S.

Feldman AM, Ray PE, Silan CM, Mercer JA, Minobe W, Bristow MR. Selec-tive gene expression in failing human heart. Quantifi cation of steady-state levels of messenger RNA in endomyocardial biopsies using the polymerase chain reaction. Circulation 1991;83:1866-1872.

Feldman AM, Combes A, Wagner D, Kadakomi T, Kubota T, Li YY, McTi-ernan C. The role of tumor necrosis factor in the pathophysiology of heart failure. J Am Coll Cardiol 2000;35:537-544.

Fernandez-Sola J, Estruch R, Nicolas JM, Pare JC, Sacanella E, Antunez E, Urbano-Marquez A. Comparison of alcoholic cardiomyopathy in women versus men. Am J Cardiol 1997;80:481-485.

Fernandez-Sola J, Nicolas-Arfelis JM. Gender differences in alcoholic cardio-myopathy. J Gend Specif Med 2002;5:41-47.

Filippatos GS, Gangopadhyay N, Lalude O, Parameswaran N, Said SI, Spiel-man W, Uhal BD. Regulation of apoptosis by vasoactive peptides. Am J Physiol Lung Cell Mol Physiol 2001;281:L749-L761.

Fleming S, Toratani S, Shea-Donohue T, Kashiwabara Y, Vogel SN, Metcalf ES. Pro- and anti-infl ammatory gene expression in the murine small in-testine and liver after chronic exposure to ethanol. Alcohol Clin Exp Res 2001;25:579-589.

62

Fortunato F, Gates LK Jr. Alcohol feeding and lipopolysaccharide injec-tion modulate apoptotic effectors in the rat pancreas in vivo. Pancreas 2000;21:174-180.

Fortuno MA, Zalba G, Ravassa S, D’Elom E, Beaumont FJ, Fortuno A, Diez J. p53-mediated upregulation of bax gene transcription is not involved in bax-alpha protein overexpression in the left ventricle of spontaneously hy-pertensive rats. Hypertension 1999;33:1348-1352.

Gaudron P, Eilles C, Kugler I, Ertl G. Progressive left ventricular dysfunction and remodeling after myocardial infarction. Potential mechanisms and early predictors. Circulation 1993;87:755-763.

Gill C, Mestril R, Samali A. Losing heart: the role of apoptosis in heart dis-ease-a novel therapeutic target. FASEB J 2002;16:135-146.

Goldin RD, Hunt NC, Clark J, Wickramasinghe SN. Apoptotic bodies in a murine model of alcoholic liver disease: reversibility of ethanol-induced changes. J Pathol 1993;171:73-76.

Gorospe M, Cirielli C, Wang X, Seth P, Capogrossi MC, Holbrook NJ. P21Waf1/Cip1 protects against p53-mediated apoptosis of human melanoma cells. Oncogene 1997;14:929-935.

Guerra S, Leri A, Wang X, Finato N, Di Loreto C, Beltrami CA, Kajstura J, Anversa P. Myocyte death in the failing human heart is gender dependent. Circ Res 1999;85:856-866.

Guillaume P, Gutkowska J, Gianoulakis C. Increased plasma atrial natriu-retic peptide after acute injection of alcohol in rats. J Pharmacol Exp Ther 1994;271:1656-1665.

Guillaume P, Jankowski M, Gianoulakis C, Gutkowska J. Effect of chronic ethanol consumption on the atrial natriuretic system of spontaneously hy-pertensive rats. Alcohol Clin Exp Res 1996a;20:1653-1661.

Guillaume P, Jankowski M, Gutkowska J, Gianoulakis C. Effect of chronic moderate ethanol consumption on heart brain natriuretic peptide. Eur J Pharmacol 1996b;316:49-58.

Guo W, Baluda MA, Park NH. Ethanol upregulates the expression of p21 WAF1/CIP1 and prolongs G1 transition via a p53-independent pathway in human epithelial cells. Oncogene 1997;15:1143-1149.

63

Gutierrez-Ruiz MC, Quiroz SC, Souza V, Bucio L, Hernandez E, Olivares IP, Llorente L, Vargas-Vorackova F, Kerchenobich D. Cytokines, growth fac-tors, and oxidative stress in HepG2 cells treated with ethanol, acetalde-hyde and LPS. Toxicology 1999;134:197-207.

Harcombe AA, Ramsay L, Kenna JG, Koskinas J, Why HJ, Richardson PJ, Weissberg PL, Alexander GJ. Circulating antibodies to cardiac pro-tein-acetaldehyde adducts in alcoholic heart muscle disease. Clin Sci 1995;88:263-268.

von Harsdorf R, Li PF, Dietz R. Signaling pathways in reactive oxygen spe-cies-induced cardiomyocyte apoptosis. Circulation 1999;99:2934-2941.

Hattori R, Hernandez TE, Zhu L, Maulik N, Otani H, Kaneda Y, Das DK, An essential role of the antioxidant gene bcl-2 in myocardial adaptation to ischemia: an insight with antisense bcl-2 therapy. Antioxidant Redox Sig-nal 2001;3:403-413.

Hauck L, Hansmann G, Dietz R, von Harsdorf R. Inhibition of hypoxia-in-duced apoptosis by modulation of retinoblastoma protein-depedent signal-ing in cardiomyocytes. Circ Res 2002;91:782-789.

Hintz KK, Relling DP, Saari JT, Borgerding AJ, Duan J, Ren BH, Kato K, Epstein PN, Ren J. Cardiac overexpression of alcohol dehydroge-nase exacerbates cardiac contractile dysfunction, lipid peroxidation, and protein damage after chronic ethanol ingestion. Alcohol Clin Exp Res 2003;27:1090-1098.

Hochhauser E, Kivity S, Offen D, Maulik N, Otani H, Barhum Y, Pannet H, Shneyvays V, Shainberg A, Goldshtaub V, Tobar A, Vidne BA. Bax abla-tion protects against myocardial ischemia-reperfusion injury in transgenic mice. Am J Physiol Heart Circ Physiol 2003;284:H2351-H2359.

Hofseth LJ, Hussain SP, Harris CC. P53: 25 years after its discovery. Trends Pharmacol Sci 2004;25:177-181.

Holownia A, Ledig M, Braszko JJ, Menez JF. Acetaldehyde cytotoxicity in cultured rat astrocytes. Brain Res 1999;833:202-208.

Hong F, Kim WH, Tian Z, Jaruga B, Ishac E, Shen X, Gao B. Elevated in-terleukin-6 during ethanol consumption acts as a potential endogenous protective cytokine against ethanol-induced apoptosis in the liver: involve-ment of induction of bcl-2 and bcl-xl proteins. Oncogene 2002;21:32-43.

64

Hunter JJ, Tanaka N, Rockman HA, Ross J Jr, Chien KR. Ventricular expression of a MLC-2v-ras fusion gene induces cardiac hypertrophy and selective diasolic dysfunction in transgenic mice. J Biol Chem 1995;270:23173-23178.

Hunter JJ, Chien KR. Signaling pathways for cardiac hypertrophy and failure. N Engl J Med 1999;341:1276-1283.

Husse B, Sopart A, Isenberg G. Cyclical mechanical stretch-induced apoptosis in myocytes from young rats but necrosis in myocytes from old rats. Am J Physiol Heart Circ Physiol 2003;285:H1521-H1527.

Hynynen M, Kupari M, Salmenperä M, Koskinen P, Tikkanen I, Fyhrquist F. Plasma atrial natriuretic factor during ethanol ingestion in volume-loaded subjects. Alcohol Alcohol 1992;27:277-285.

Igney FH, Krammer PH. Death and anti-death: tumour resistance to apopto-sis. Nat Rev Cancer;2002;2:277-288.

Ikonomidou C, Bittigau P, Ishimaru MJ, Wozniak DF, Koch C, Genz K, Price MT, Stefovska V, Hörster F, Tenkova T, Dikranian K, Olney JW. Ethanol-induced apoptotic neurodegeneration and fetal alcohol syndrome. Science 2000;287:1056-1060.

Imhof A, Koenig W. Alcohol infl ammation and coronary heart disease. Addict Biol 2003;8:271-277.

Itoh H, Pratt RE, Dzau VJ. Atrial natriuretic polypeptide inhibits hypertro-phy of vascular smooth muscle cells. J Clin Invest 1990;86:1690-1697.

John SWM, Krege JH, Oliver PM, Hagaman JR, Hodgin JB, Pang SC, Flynn TG, Smithies O. Genetic decreases in atrial natriuretic peptide and salt-sensitive hypertension. Science 1995;267:679-681.

Jung F, Weiland U, Johns RA, Ihling C, Dimmeler S. Chronic hypoxia in-duces apoptosis in cardiac myocytes: a possible role for bcl-2-like proteins. Biochem Biophys Res Comm 2001;286:419-425.

Järveläinen HA, Fang C, Ingelman-Sundberg M, Lindros KO. Efffect of chronic coadministration of endotoxin and ethanol on rat liver pathol-ogy and proinfl ammatory and anti-infl ammatory cytokines. Hepatology 1999;29:1503-1510.

65

Jörnvall H, Höög JO. Nomenclature of alcohol dehydrogenases. Alcohol Alco-hol 1995;30:153-161.

Kadokami T, McTiernan CF, Kubota T, Frye CS, Feldman AM. Sex-related survival differences in murine cardiomyopathy are associated with differ-ences in TNF-receptor expression. J Clin Invest 2000;106:589-597.

Kagawa S, Fujiwara T, Hizuta A, Yasuda T, Zhang WW, Roth JA, Tanaka N. p53 expression overcomes p21WAF1/CIP1-mediated G1 arrest and induc-es apoptosis in human cancer cells. Oncogene 1997;15:1903-1909.

Kajander OA, Kupari M, Perola M, Pajarinen J, Savolainen V, Penttilä A, Karhunen PJ. Testing genetic susceptibility loci for alcoholic heart muscle disease. Alcohol Clin Exp Res 2001;25:1409-1413.

Kang PM, Izumo S. Apoptosis and heart failure. A critical review of the lit-erature. Circ Res 2000;86:1107-1113.

Kang YJ, Zhou ZX, Wu H, Wang GW, Saari JT, Klein JB. Metallothionein inhibits myocardial apoptosis in copper-defi cient mice: role of atrial natri-uretic peptide. Lab Invest 2000;80:745-757.

Kannan M, Wang L, Kang YJ. Myocardial oxidative stress and toxicity in-duced by acute ethanol exposure in mice. Exp Biol Med (Maywood) 2004;229:553-559.

Kapadia SR, Oral H, Lee J, Nakano M, Taffet GE, Mann DL. Hemodynamic regulation of tumor necrosis factor-alpha gene and protein expression in adult feline myocardium. Circ Res 1997;81:187-195.

Kim KK, Soonpaa MH, Daud AI, Koh GY, Kim JS, Field LJ. Tumor suppres-sor gene expression during normal and pathologic myocardial growth. J Biol Chem 1994;269:22607-22613.

Kim SD, Bieniarz T, Esser KA, Piano MR. Cardiac structure and function af-ter short-term ethanol consumption in rats. Alcohol 2003;29:21-29.

Kirshenbaum LA, de Moissac D. The bcl-2 gene product prevents programmed cell death of ventricular myocytes. Circulation 1997;96:1580-1585.

66

Klinger JR, Petit RD, Curtin LA, Warburton RR, Wrenn DS, Steinhelper ME, Field LJ, Hill NS. Cardiopulmonary responses to chronic hypoxia in transgenic mice that overexpress ANP. J Appl Physiol 1993;75:198-205.

Kloner RA, Bolli R, Marban E, Reinlib L, Braunwald E. Medical and cellular implications of stunning, hibernation, and preconditioning: an NHLBI workshop. Circulation 1998;97:1848-1867.

Koch OR, Pani G, Borrello S, Colavitti R, Cravero A, Farre S, Galeotti T. Ox-idative stress and antioxidant defences in ethanol-induced cell injury. Mol Aspects Med 2004;25:191-198.

Kook H, Itoh H, Choi BS, Sawada N, Doi K, Hwang TJ, Kim KK, Arai H, Baik YH, Nakao K. Physiological concentration of atrial natriuretic pep-tide induces endothelial regeneration in vitro. Am J Physiol Heart Circ Physiol 2003;284:H1388-H1397.

Koteish A, Yang S, Lin H, Huang J, Diehl AM. Ethanol induces redox-sensi-tive cell-cycle inhibitors and inhibits liver regeneration after partial hepa-tectomy. Alcohol Clin Exp Res 2002;26:1710-1718.

Kovacic-Milivojevic B, Zlock DW, Gardner DG. Ras inhibits jun-activated human atrial natriuretic peptide gene transcription in cultured ventricular myocytes. Circ Res 1997;80:580-588.

Krenz M, Cohen MV, Downey JM. The protective and anti-protective ef-fects of ethanol in a myocardial infarct model. Ann NY Acad Sci 2002;957:103-114.

Kubota T, McTiernan CF, Frye CS, Slawson SE, Lemster BH, Koretsky AP, Demetris AJ, Feldman AM. Dilated cardiomyopathy in transgenic mice with cardiac-specifi c overexpression of tumor necrosis factor-alpha. Circ Res 1997;81:627-635.

Kubota T, Miyagishima M, Frye CS, Alber SM, Bounoutas GS, Kadokami T, Watkins SC, McTiernan CF, Feldman AM. Overexpression of tumor necro-sis factor-alpha activates both anti- and pro-apoptotic pathways in the myocardium. J Moll Cell Cardiol 2001;33:1331-1344.

Kupari M, Koskinen P. Comparison of the cardiotoxicity of ethanol in women versus men. Am J Cardiol 1992;70:645-649.

67

Kurrelmeyer KM, Michael LH, Baumgarten G, Taffet GE, Peschon JJ, Sivas-ubramanian N, Entman ML, Mann DL. Endogenous tumor necrosis factor protects the adult cardiac myocyte against ischemic-induced apoptosis in a murine model of acute myocardial infarction. Proc Natl Acad Sci USA 2000;97:5456-5461.

Kytö V, Saraste A, Saukko P, Henn V, Pulkki K, Vuorinen T, Voipio-Pulkki LM. Apoptotic cardiomyocyte death in fatal myocarditis. Am J Cardiol 2004;94:746-750.

Latif N, Khan MA, Birks E, O’Farrell A, Westbrook J, Dunn MJ, Yacoub MH. Upregulation of the bcl-2 family of proteins in end stage heart fail-ure. J Am Coll Cardiol 2000;35:1769-1777.

Leppäluoto J, Vuolteenaho O, Arjamaa O, Ruskoaho H. Plasma immunoreac-tive atrial natriuretic peptide and vasopressin after ethanol intake in man. Acta Physiol Scand 1992;144:121-127.

Leri A, Claudio PP, Li Q, Wang X, Reiss K, Wang S, Malhotra A, Kajstura J, Anversa P. Stretch-mediated release of angiotensin II induces myocyte apoptosis by activating p53 that enhances the local renin-angiotensin sys-tem and decreases the bcl-2-to-bax protein ratio in the cell. J Clin Invest 1998a;101:1326-1342.

Leri A, Liu Y, Malhotra A, Li Q, Stiegler P, Claudio PP, Giordano A, Kajstura J, Hintze TH, Anversa P. Pacing-induced heart failure in dogs enhances the expression of p53 and p53-dependent genes in ventricular myocytes. Circulation 1998b;97:194-203.

Leri A, Malhotra A, Liew CC, Kajstura J, Anversa P. Telomerase activity in rat myocytes is age and gender dependent. J Mol Cell Cardiol 2000a;32:385-390.

Leri A, Fiordaliso F, Setoguchi M, Limana F, Bishopric NH, Kajstura J, Web-ster K, Anversa P. Inhibition of p53 function prevents renin-angiotensin system activation and stretch-mediated myocyte apoptosis. Am J Pathol 2000b;157:843-857.

Leri A, Franco S, Zacheo A, Barlucchi L, Chimenti S, Limana F, Nadal-Ginard B, Kajstura J, Anversa P, Blasco MA. Ablation of telomerase and telom-erase loss leads to cardiac dilatation and heart failure associated with p53 upregulation. EMBO J 2003;22:131-139.

68

Li JM, Brooks G. Downregulation of cyclin-dependent kinase inhibitors p21 and p27 in pressure-overload hypertrophy. Am J Physiol 1997;273:H1358-H1367.

Li SY, Gomelsky M, Duan J, Zhang Z, Gomelsky L, Zhang X, Epstein PN, Ren J. Overexpression of aldehyde dehydrogenase-2 (ALDH2) transgene prevents acetaldehyde-induced cell injury in human umbilical vein en-dothelial cells. J Biol Chem 2004;279:11244-11252.

Li Z, Bing OHL, Long X, Robinson KG, Lakatta EG. Increased cardiomyo-cyte apoptosis during the transition to heart failure in the spontaneosly hypertensive rat. Am J Physiol 1997;272:H2313-H2319.

Liang Q, Carlson EC, Borgerding AJ, Epstein PN. A transgenic model of acetaldehyde overproduction accelerates alcohol cardiomyopathy. J Phar-macol Exp Ther 1999;291:766-772.

Lindros KO, Jokelainen K, Nanji AA. Acetaldehyde prevents nuclear fac-tor-kappa B activation and hepatic infl ammation in ethanol-fed rats. Lab Invest 1999;79:799-806.

Liu ESL, Cho CH. Relationship between ethanol-induced gastritis and gastric ulcer formation in rats. Digestion 2000;62:232-239.

Liu G, Schwartz JA, Brooks SC. p53 down-regulates ER-responsive genes by interfering with the binding of ER to ERE. Biochem Biophys Res Comm 1999;264:359-364.

Liu J, Tian Z, Gao B, Kunos G. Dose-dependent activation of antiapoptotic and proapoptotic pathways by ethanol treatment in human vascular en-dothelial cells. J Biol Chem 2002;277:20927-20933.

Long X, Boluyt MO, de Lourdes Hipolito M, Lundberg MS, Zheng JS, O’Neill L, Cirielli C, Lakatta EG, Crow MT. P53 and the hypoxia-in-duced apoptosis of cultured neonatal rat cardiac myocytes. J Clin Invest 1997;99:2635-2643.

Lumpkin CK Jr, Moore TL, Tarpley MD, Taylor JM, Badger TM, McClung JK. Acute ethanol and selected growth suppressor transcripts in regenerat-ing rat liver. Alcohol 1995;12:357-362.

69

MacLellan WR, Schneider MD. Death by design. Programmed cell death in cardiovascular biology and disease. Circ Res 1997;81:137-144.

Majno G, Joris I. Apoptosis, oncosis, and necrosis. An overview of cell death. Am J Pathol 1995;146:3-15.

Mallat Z, Fornes P, Costagliola R, Esposito B, Belmin J, Lecomte D, Tedgui A. Age and gender effects on cardiomyocyte apoptosis in the normal hu-man heart. J Gerontol A Biol Sci Med Sci 2001;56A:M719-M723.

Martinou JC, Green DR. Breaking the mitochondrial barrier. Nat Rev Mol Cell Biol 2001;2:63-67.

Masciotra S, Picard S, Deschepper CF. Cosegregation analysis in genetic cross-es suggests a protective role for atrial natriuretic factor against ventricular hypertrophy. Circ Res 1999;84:1453-1458.

Maulik N, Engelman RM, Rousou JA, Flack III JE, Deaton D, Das DK. Ischemic preconditioning reduces apoptosis by upregulating anti-death gene bcl-2. Circulation 1999;100(Suppl II):II369-II375.

Maulik N, Sasaki H, Addya S, Das DK. Regulation of cardiomyocyte apopto-sis by redox-sensitive transcription factors. FEBS Lett 2000;485:7-12.

McAlhany RE Jr, West JR, Miranda RC. Glial-derived neutrotrophic factor (GDNF) prevents ethanol-induced apoptosis and JUN kinase phosphoryla-tion. Brain Res Dev Brain Res 2000;119:209-216.

McGill CJ, Brooks G. Cell cycle control mechanisms and their role in cardiac growth. Cardiovasc Res 1995;30:557-569.

Meldrum DR. Tumor necrosis factor in the heart. Am J Physiol 1998;274:R577-R595.

Miyamae M, Diamond I, Weiner MW, Camacho SA, Figueredo VM. Regular alcohol consumption mimics cardiac preconditioning by pro-tecting against ischemia-reperfusion injury. Proc Natl Acad Sci USA 1997;94:3235-3239.

Miyashita T, Reed JC. Tumor suppressor p53 is a direct transcriptional activa-tor of the human bax gene. Cell 1995;80:293-299.

70

de Moissac D, Mustapha S, Greenberg AH, Kirshenbaum LA. Bcl-2 activates the transcription factor NFkappaB through the degradation of the cyto-plasmic inhibitor IkappaBalpha. J Biol Chem 1998;273:23946-23951.

Molinari AM, Bontempo P, Schiavone EM, Tortora V, Verdicchio MA, Na-politano M, Nola E, Moncharmont B, Medici N, Nigro V, Armetta I, Ab-bondanza C, Puca GA. Estradiol induces functional inactivation of p53 by intracellular redistribution. Cancer Res 2000;60:2594-2597.

de la Monte SM, Ganju N, Banerjee K, Brown NV, Luong T, Wands JR. Par-tial rescue of ethanol-induced neuronal apoptosis by growth factor activa-tion of phosphoinositol-3-kinase. Alcohol Clin Exp Res 2000;24:716-726.

Mooney SM, Miller MW. Effects of prenatal exposure to ethanol on the ex-pression of bcl-2, bax and caspase 3 in the developing rat cerebral cortex and thalamus. Brain Res 2001;911:71-81.

Moore DB, Walker DW, Heaton MB. Neonatal ethanol exposure alters bcl-2 family mRNA levels in the rat cerebellar vermis. Alcohol Clin Exp Res 1999;23:1251-1261.

Morgan HE, Gordon EE, Kira Y, Chua HL, Russo LA, Peterson CJ, McDer-mott PJ, Watson PA. Biochemical mechanisms of cardiac hypertrophy. Ann Rev Physiol 1987;49:539-543.

Mustapha S, Kirshner A, De Moissac D, Kirshenbaum LA. A direct require-ment of nuclear factor-kappaB for suppression of apoptosis in ventricular myocytes. Am J Physiol Heart Circ Physiol 2000;279:H939-H945.

Nadal-Ginard B, Kajstura J, Anversa P, Leri A. A matter of life and death: cardiac myocyte apoptosis and regeneration. J Clin Invest 2003;111:1457-1459.

Nakahara T, Hashimoto K, Hirano M, Koll M, Martin CR, Preedy VR. Acute and chronic effects of alcohol exposure on skeletal muscle c-myc, p53, and bcl-2 mRNA expression. Am J Physiol Endocrinol Metab 2003;285:E1273-E1281.

Nakamura M, Wang NP, Zhao ZQ, Wilcox JN, Thourani V, Guyton RA, Vinten-Johansen J. Preconditioning decreases bax expression, PMN accumulation and apoptosis in reperfused rat heart. Cardiovasc Res 2000;45:661-670.

71

Nakamura Y. Isolation of p53-target genes and their functional analysis. Can-cer Sci 2004;95:7-11.

Neuman MG, Haber JA, Malkiewicz IM, Cameron RG, Katz GG, Shear NH. Ethanol signals for apoptosis in cultured skin cells. Alcohol 2002;26:179-190.

Nicolas JM, Fernandez-Sola J, Estruch R, Pare JC, Sacanella E, Urbano-Mar-quez A, Rubin E. The effect of controlled drinking in alcoholic cardiomy-opathy. Ann Intern Med 2002;136:192-200.

Niemelä O. Distribution of ethanol-induced protein adducts in vivo: relation-ship to tissue injury. Free Radic Biol Med 2001;31:1533-1538.

Niemelä O, Parkkila S, Koll M, Preedy VR. Generation of protein adducts with malondialdehyde and acetaldehyde in muscles with predominantly type I or type II fi bers in rats exposed to ethanol and the acetaldehyde de-hydrogenase inhibitor cyanamide. Am J Clin Nutr 2002;76:668-674.

Niemelä O, Parkkila S, Worrall S, Emery PW, Preedy CR. Generation of alde-hyde-derived protein modifi cations in ethanol-exposed heart. Alcohol Clin Exp Res 2003;27:1987-1992.

Ning XH, Chen SH, Xu CS, Li L, Yao LY, Qian K, Krueger JJ, Hyyti OM, Portman MA. Molecular biology of thermoregulation. Selected contri-bution: hypothermic protection of the ischemic heart via alterations in apoptotic pathways as assessed by gene array analysis. J Appl Physiol 2002;92:2200-2207.

Ogawa M, Maeda K, Chung YS, Sowa M. Loss of p21WAF1/CIP1 expres-sion correlates with disease progression in gastric carcinoma. Br J Cancer 1997;75:1617-1620.

Olivetti G, Giordano G, Corradi D, Melissari M, Lagrasta C, Gambert SR, Anversa P. Gender differences and aging: effects on the human heart. J Am Coll Cardiol 1995;26:1068-1079.

Olivetti G, Abbi R, Quaini F, Kajstura J, Cheng W, Nitahara JA, Quaini E, Di Loreto C, Beltrami CA, Krajewski S, Reed JC, Anversa P. Apoptosis in the failing human heart. N Engl J Med 1997;336:1131-1141.

72

Paice AG, Hesketh JE, Towner P, Hirako M, Peters TJ, Preedy VR. No change in apoptosis in skeletal muscle exposed acutely or chronically to alcohol. Addict Biol 2003;8:97-105.

Pani G, Fusco S, Colavitti R, Borrello S, Maggiano N, Cravero AA, Farre SM, Galeotti T, Koch OR. Abrogation of hepatocyte apoptosis and early appearance of liver dysplasia in ethanol-fed p53-defi cient mice. Biochem Biophys Res Comm 2004;325:97-100.

Patten RD, Pourati I, Aronovitz MJ, Baur J, Celestin F, Chen X, Michael A, Haq S, Nuedling S, Grohe C, Force T, Mendelsohn ME, Karas RH. 17beta-estradiol reduces cardiomyocyte apoptosis in vivo and in vitro via activation of phospho-inositide-3 kinase/Akt signaling. Circ Res 2004;95:692-699.

Pethe V, Shekhar PVM. Estrogen inducibility of c-ha-ras transcription in breast cancer cells. J Biol Chem 1999;274:30969-30978.

Peuhkurinen KJ, Kiviluoma KT, Hiltunen JK, Takala TES, Hassinen IE. Ef-fects of ethanol metabolites on intermediary metabolism in heart muscle. Pharmacol Biochem Behav 1983;18 (Suppl 1):279-283.

Piano MR. Alcoholic cardiomyopathy: incidence, clinical characteristics, and pathophysiology. Chest 2002;121:1638-1650.

Preedy VR, Siddiq T,Why HJF, Richardson PJ. Ethanol toxicity and cardiac protein synthesis in vivo. Am Heart J 1994;127:1432-1439.

Preedy VR, Richardson PJ. Ethanol induced cardiovascular disease. Br Med Bull 1994;50:152-163.

Rashid A, Wu TC, Huang CC, Chen CH, Lin HZ, Yang SQ, Lee FYJ, Diehl AM. Mitochondrial proteins that regulate apoptosis and necrosis are in-duced in mouse fatty liver. Hepatology 1999;29:1131-1138.

Rayment NB, Haven AJ, Madden B, Murday A, Trickey R, Shipley M, Davies MJ, Katz DR. Myocyte loss in chronic heart failure. J Pathol 1999;188:213-219.

Reed JC. Apoptosis-based therapies. Nat Rev Drug Discov 2002;1:111-121.

Regan TJ. Alcoholic cardiomyopathy. Prog Cardiovasc Dis 1984;27:141-152.

73

Regula KM, Ens K, Kirshenbaum LA. Mitochondria-assisted cell suicide: a license to kill. J Mol Cell Cardiol 2003;35:559-567.

Reinke LA, Lai EK, DuBose CM, McCay PB. Reactive free radical generation in vivo in heart and liver of ethanol-fed rats: correlation with radical for-mation in vitro. Proc Natl Acad Sci USA 1987;84:9223-9227.

Ren J, Wold LE, Natavio M, Ren BH, Hannigan JH, Brown RA. Infl uence of prenatal alcohol exposure on myocardial contractile function in adult rat hearts: role of intracellular calcium and apoptosis. Alcohol Alcohol 2002;37:30-37.

Rintala J, Jaatinen P, Lu W, Sarviharju M, Eriksson CJP, Laippala P, Kiianmaa K, Hervonen A. Effects of lifelong ethanol consumption on cerebellar layer volumes in AA and ANA rats. Alcohol Clin Exp Res 1997;21:311-317.

Riva C, Chevrier C, Pasqual N, Saks V, Rossi A. Bcl-2/bax protein expression in heart, slow-twitch and fast-twitch muscles in young rats growing under chronic hypoxia conditions. Mol Cell Biochem 2001;226:9-16.

Rocha EM, Wickham LA, Huang Z, Toda I, Gao J, deSilveira IA, Sullivan DA. Presence and testosterone infl uence on the levels of anti- and pro-infl ammatory cytokines in lacrimal tissues of a mouse model of Sjögren’s syndrome. Adv Exp Med Biol 1998;438:485-491.

Rosenkranz-Weiss P, Tomek RJ, Mathew J, Eghbali M. Gender-specifi c differ-ences in expression of mRNAs for functional and structural proteins in rat ventricular myocardium. J Mol Cell Cardiol 1994;26:261-270.

Ruskoaho H. Cardiac hormones as diagnostic tools in heart failure. Endocrine Rev 2003;24:341-356.

Sabbah HN, Sharov VG, Gupta RC, Mishra S, Rastogi S, Undrovinas AI, Chaudhry PA, Todor A, Mishima T, Tanhehco EJ, Suzuki G. Reversal of chronic molecular and cellular abnormalities due to heart failure by pas-sive mechanical ventricular containment. Circ Res 2003;93:1095-1101.

Sabri A, Wilson BA, Steinberg SF. Dual actions of the Galphaq agonist pas-teurella multocida toxin to promote cardiomyocyte hypertrophy and en-hance apoptosis susceptibility. Circ Res 2002;90:850-857.

74

Sadoshima JI, Izumo S. Mechanical stretch rapidly activates multiple signal transduction pathways in cardiac myocytes: potential involvement of an autocrine/paracrine mechanism. EMBO J 1993;12:1681-1692.

Sadoshima JI, Izumo S. The heterotrimeric Gq protein-coupled angiotensin II receptor activates p21ras via the tyrosine kinase-Shc-Grb2-Sos pathway in cardiac myocytes. EMBO J 1996;15:775-787.

Saraste A, Voipio-Pulkki LM, Heikkilä P, Laine P, Nieminen MS, Pulkki K. Soluble tumor necrosis factor receptor levels identify a subgroup of heart failure patients with increased cardiomyocyte apoptosis. Clin Chim Acta 2002;320:65-67.

Sato M, Maulik N, Das DK. Cardioprotection with alcohol. Role of both alco-hol and polyphenolic antioxidants. Ann NY Acad Sci 2002;957:122-135.

Sayeed S, Cullen JP, Coppage M, Sitzmann JV, Redmond EM. Ethanol dif-ferentially modulates the expresion and activity of cell cycle regulatory proteins in rat aortic smooth muscle cells. Eur J Pharmacol 2002;445:163-170.

Schieffer B, Drexler H, Ling BN, Marrero MB. G protein –coupled receptors control vascular smooth muscle cell proliferation via pp60c-src and p21ras. Am J Physiol 1997;272:C2019-C2030.

Schoppet M, Maisch B. Alcohol and the heart. Herz 2001;26:345-352.

Schreiber SS, Briden K, Oratz M, Rothschild MA. Ethanol, acetaldehyde, and myocardial protein synthesis. J Clin Invest 1972;51:2820-2826.

Sehested J, Heringlake M, Schmidt V. Neurohumoral cardiovascular re-sponses to alcohol and their modulation by peroral fl uid. Am J Cardiol 1998;81:761-765.

Seko Y, Tobe K, Takahashi N, Kaburagi Y, Kadowaki T, Yazaki Y. Hy-poxia and hypoxia/reoxygenation activate Src family tyrosine kinases and p21ras in cultured rat cardiac myocytes. Biochem Biophys Res Comm 1996;226:530-535.

Serrano M, Blasco MA. Putting the stress on senescence. Curr Opin Cell Biol 2001;13:748-753.

75

Siddiq T, Salisbury JR, Richardson PJ, Preedy VR. Synthesis of ventricular mitochondrial proteins in vivo: effect of acute ehanol toxicity. Alcohol Clin Exp Res 1993;17:894-899.

Simanowski UA, Homann N, Knuhl M, Arce L, Waldherr R, Conradt C, Bosch FX, Seitz HK. Increased rectal cell proliferation following alcohol abuse. Gut 2001;49:418-422.

Slee EA, O’Connor DJ, Lu X. To die or not to die: how does p53 decide. On-cogene 2004;23:2809-2818.

Song H, Conte JV Jr, Foster AH, McLaughlin JS, Wei C. Increased p53 pro-tein expression in human failing myocardium. J Heart Lung Transplant 1999;18:744-749.

Steenbergen C, Afshari CA, Petranka JG, Collins J, Martin K, Bennett L, Haugen A, Bushel P, Murphy E. Alterations in apoptotic signaling in hu-man idiopathic cardiomyopathic hearts in failure. Am J Physiol Heart Circ Physiol 2003;284:H268-H276.

Su X, Brower G, Janicki JS, Chen YF, Oparil S, Dell’Italia LJ. Differential expression of natriuretic peptides and their receptors in volume overload cardiac hypertrophy in the rat. J Mol Cell Cardiol 1999;31:1927-1936.

Suenobu N, Shichiri M, Iwashina M, Marumo F, Hirata Y. Natriuretic pep-tides and nitric oxide induce endothelial apoptosis via a cGMP-dependent mechanism. Arterioscler Thromb Vasc Biol 1999;19:140-146.

Suzuki K, Kostin S, Person V, Elsässer A, Schaper J. Time course of the apop-totic cascade and effects of caspase inhibitors in adult rat ventricular car-diomyocytes. J Mol Cell Cardiol 2001;33:983-994.

Tamura N, Ogawa Y, Chusho H, Nakamura K, Nakao K, Suda M, Kasahara M, Hashimoto R, Katsuura G, Mukoyama M, Itoh H, Saito Y, Tanaka I, Otani H, Katsuki M, Nakao K. Cardiac fi brosis in mice lacking brain natriuretic peptide. Proc Natl Acad Sci USA 2000;97:4239-4244.

Thomasson HR. Gender differences in alcohol metabolism. Physiological re-sponses to ethanol. Recent Dev Alcohol 1995;12:163-179.

76

Thorburn A, Thorburn J, Chen SY, Powers S, Shubeita HE, Feramisco JR, Chien KR. Hras-dependent pathways can activate morphological and genetic markers of cardiac muscle cell hypertrophy. J Biol Chem 1993;268:2244-2249.

Thorburn J, Carlson M, Mansour SJ, Chien KR, Ahn NG, Thorburn A. Inhi-bition of a signaling pathway in cardiac muscle cells by active mitogen-ac-tivated protein kinase kinase. Mol Biol Cell 1995;6:1479-1490.

Thum T, Borlak J. Gene expression in distinct regions of the heart. Lancet 2000;355:979-983.

Tiernan JM, Ward LC, Cooksley WG. Inhibition by ethanol of cardiac protein synthesis in the rat. Int J Biochem 1985;17:793-798.

Toda I, Wickham A, Sullivan DA. Gender and androgen treatment infl u-ence the expression of proto-oncogenes and apoptotic factors in lacrimal and salivary tissues of MRL/lpr mice. Clin Immunol Immunopathol 1998;86:59-71.

Tyner SD, Venkatachalam S, Choi J, Jones S, Ghebranious N, Igelmann H, Lu X, Soron G, Cooper B, Brayton C, Hee Park S, Thompson T, Karsenty G, Bradley A, Donehower LA. P53 mutant mice that display early ageing-as-sociated phenotypes. Nature 2002;415:45-53.

Ueada S, Masutani H, Nakamura H, Tanaka T, Ueno M, Yodoi J. Redox con-trol of cell death. Antioxid Redox Signal 2002;4:405-414.

Urbano-Marquez A, Estruch R, Navarro-Lopez F, Grau JM, Mont L, Rubin E. The effects of alcoholism on skeletal and cardiac muscle. N Engl J Med 1989;320:409-415.

Vary TC, Lynch CJ, Lang CH. Effects of chronic alcohol consumption on reg-ulation of myocardial protein synthesis. Am J Physiol Heart Circ Physiol 2001;281:H1242-H1251.

Verzola D, Gandolfo MT, Salvatore F, Villaggio B, Gianiorio F, Traverso P, De-ferrari G, Garibotto G. Testosterone promotes apoptotic damage in human renal tubular cells. Kidney Internat 2004;65:1252-1261.

Vuolteenaho O, Ruskoaho H. Gender matters: estrogen protects from cardiac hypertrophy. Trends Endocrinol Metab 2003;14:52-54.

77

Webster KA, Discher DJ, Kaiser S, Hernandez O, Sato B, Bishopric NH. Hy-poxia-activated apoptosis of cardiac myocytes requires reoxygenation or a pH shift and is independent of p53. J Clin Invest 1999;104:239-252.

Wencker D, Chandra M, Nguyen K, Miao W, Garantziotis S, Factor SM, Shirani J, Armstrong RC, Kitsis RN. A mechanistic role for cardiac myo-cyte apoptosis in heart failure. J Clin Invest 2003;111:1497-1504.

Wigle DA, Pang SC, Radakovic NN, Sarda IR, Watson JD, Roy RN, Flynn TG. Chronic ethanol ingestion modifi es the renin-aldosterone axis inde-pendent of alterations in the regulation of atrial natriuretic peptide. Alco-hol Clin Exp Res 1993a;17:841-846.

Wigle DA, Pang SC, Sarda IR, Watson JD, Radakovic NN, Roy R, Flynn TG. Acute ethanol ingestion modifi es the circulating plasma levels of atrial natriuretic peptide. Alcohol 1993b;10:275-280.

Wigle DA, Pang SC, Watson JD, Sarda IR, Radakovic NN, Flynn TG. Chronic ethanol treatment increases the circulating plasma levels of B-type natriuretic peptide (BNP-45) in the rat. Am J Hypertens 1993c;6:719-722.

Wigle DA, Watson JD, Pang SC, Sarda IR, Roy RN, Flynn TG. Gene expres-sion of A- and B-type natriuretic peptides in response to acute ethanol ingestion. Alcohol Clin Exp Res 1995;19:1317-1320.

Worrall S, Richardson PJ, Preedy VR. Experimental heart muscle damage in alcohol feeding is associated with increased amounts of reduced- and unre-duced-acetaldehyde, and malondialdehyde-acetaldehyde protein adducts. Addict Biol 2000;5:421-427.

Worrall S, Niemelä O, Parkkila S, Peters TJ, Preedy VR. Protein adducts in type I and type II fi bre predominant muscles of the ethanol-fed rat: pref-erential localisation in the sarcolemmal and subsarcolemmal region. Eur J Clin Invest 2001;31:723-730.

Wu CF, Bishopric NH, Pratt RE. Atrial natriuretic peptide induces apoptosis in neonatal rat cardiac myocytes. J Biol Chem 1997;272:14860-14866.

Wu D, Cederbaum AI. Ethanol-induced apoptosis to stable HepG2 cell lines expressing human cytochrome P-4502E1. Alcohol Clin Exp Res 1999;23:67-76.

78

Xie Z, Koyama T, Suzuki J, Fujii Y, Togashi H, Sawa H, Nagashima K. Coro-nary reperfusion following ischemia: different expression of bcl-2 and bax proteins, and cardiomyocyte apoptosis. Jpn Heart J 2001;42:759-770.

Yoshimura M, Yasue H, Ogawa H. Pathophysiological signifi cance and clini-cal application of ANP and BNP in patients with heart failure. Can J Physiol Pharmacol 2001;79:730-735.

Zhang D, Gaussin V, Taffet GE, Belaguli NS, Yamada M, Schwartz RJ, Michael LH, Overbeek PA, Schneider MD. TAK1 is activated in the myo-cardium after pressure overload and is suffi cient to provoke heart failure in transgenic mice. Nat Med 2000a;6:556-563.

Zhang MN, Gong Y, Minuk GY. The effects of acute ethanol exposure on inhibitors of hepatic regenerative activity in the rat. Mol Cel Biochem 2000b;207:109-114.

Zhao X, Eghbali-Webb M. Gender-related differences in basal and hypoxia-induced activation of signal transduction pathways controlling cell cycle progression and apoptosis, in cardiac fi broblasts. Endocrine 2002;18:137-145.

Zhou HZ, Karliner JS, Gray MO. Moderate alcohol consumption induces sustained cardiac protection by activating PKC-epsilon and Akt. Am J Physiol Heart Circ Physiol 2002;283:H165-H174.

Zimmerman BT, Crawford GD, Dahl R, Simon FR, Mapoles JE. Mechanisms of acetaldehyde-mediated growth inhibition: delayed cell cycle progression and induction of apoptosis. Alcohol Clin Exp Res 1995;19:434-440.