Research Article SERCA2 Haploinsufficiency in a Mouse ...downloads.hindawi.com/journals/bmri/2015/251598.pdf · Research Article SERCA2 Haploinsufficiency in a Mouse Model of Darier

Research ArticleSERCA2 Haploinsufficiency in a Mouse Model of DarierDisease Causes a Selective Predisposition to Heart Failure

Vikram Prasad1 John N Lorenz2 Valerie M Lasko2 Michelle L Nieman2 Wei Huang3

Yigang Wang3 David W Wieczorek1 and Gary E Shull1

1Department of Molecular Genetics Biochemistry and Microbiology University of Cincinnati College of MedicineCincinnati OH 45267 USA2Department of Cellular and Molecular Physiology University of Cincinnati College of Medicine Cincinnati OH 45267 USA3Department of Pathology and Laboratory Medicine University of Cincinnati College of Medicine Cincinnati OH 45267 USA

Correspondence should be addressed to Vikram Prasad prasadvikramgmailcom

Received 26 September 2014 Revised 18 December 2014 Accepted 23 December 2014

Academic Editor Monica Fedele

Copyright copy 2015 Vikram Prasad et alThis is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Null mutations in one copy of ATP2A2 the gene encoding sarcoendoplasmic reticulum Ca2+-ATPase isoform 2 (SERCA2)cause Darier disease in humans a skin condition involving keratinocytes Cardiac function appears to be unimpaired in Darierdisease patients with no evidence that SERCA2 haploinsufficiency itself causes heart disease However SERCA2 deficiency iswidely considered a contributing factor in heart failure We therefore analyzed Atp2a2 heterozygous mice to determine whetherSERCA2 haploinsufficiency can exacerbate specific heart disease conditions Despite reduced SERCA2a levels in heart Atp2a2heterozygous mice resembled humans in exhibiting normal cardiac physiology When subjected to hypothyroidism or crossedwith a transgenic model of reduced myofibrillar Ca2+-sensitivity SERCA2 deficiency caused no enhancement of the disease stateHowever when combined with a transgenic model of increased myofibrillar Ca2+-sensitivity SERCA2 haploinsufficiency causedrapid onset of hypertrophy decompensation and deathThese effects were associated with reduced expression of the antiapoptoticHax1 increased levels of the proapoptotic genes Chop and Casp12 and evidence of perturbations in energy metabolismThese datareveal myofibrillar Ca2+-sensitivity to be an important determinant of the cardiac effects of SERCA2 haploinsufficiency and raisethe possibility that Darier disease patients are more susceptible to heart failure under certain conditions

1 Introduction

In humans loss of one copy of theATP2A2 gene causesDarierdisease (DD) an acantholytic skin disease [1 2] ATP2A2encodes two alternatively spliced variants of sarcoendoplas-mic reticulum Ca2+-ATPase isoform 2 (SERCA2) These areSERCA2b the ubiquitous endoplasmic reticulum (ER) Ca2+pump and SERCA2a the cardiac and slow-twitch skeletalmuscle sarcoplasmic reticulum (SR) Ca2+ pump [3] In itscapacity as the primary cardiac SR Ca2+ pump SERCA2afacilitates muscle relaxation and replenishes SR Ca2+ storesneeded for contraction [3] Because reduced SERCA2 activityis often observed in heart disease [4] andDDmutations causereductions in SERCA2 expression and activity [5] one might

expect these mutations to lead to heart disease in humansHowever two studies of DD patients with a mean age ofsim47 years indicated that cardiac performance is normal andyielded no evidence of heart disease [6 7] these findings havebeen interpreted to suggest that a role for SERCA2 deficiencyin heart disease while relevant in rodent models may beless important in humans [7] However while these resultsprovide compelling evidence thatAtp2a2 heterozygosity doesnot impair cardiac function in middle-aged humans they donot rule out the possibility that SERCA2 haploinsufficiencycan increase susceptibility to disease progression and heartfailure

In earlier studies ablation of one copy of the Atp2a2 geneinmice of amixed 129Svj and Black Swiss background caused

Hindawi Publishing CorporationBioMed Research InternationalVolume 2015 Article ID 251598 21 pageshttpdxdoiorg1011552015251598

2 BioMed Research International

a reduction in cardiac SERCA2a protein expression to 65of wild-type (WT) levels with impaired contractility andrelaxation [8] However true heart disease was not observedin fact the major phenotype of the Atp2a2+minus mice was thedevelopment of squamous cell tumors in keratinized epithe-lial tissues [9 10] Later studies on these mice revealed anincreased susceptibility to pressure-overload cardiac hyper-trophy [11] and a reduction of rate-dependent inotropy inisolated mutant hearts relative to WT controls [12] In thecurrent study we used Atp2a2+minus mice on an inbred FVBNbackgroundwhile these heterozygous (HET)mice continuedto display effects of SERCA2 haploinsufficiency in keratinizedepithelia cardiac performance was apparently normal moreclosely reflecting findings in DD patients [6 7] The HETmodel was well suited to determine if SERCA2 deficiencywhile apparently benign under normal conditions couldexacerbate cardiac disease progression Specifically we inves-tigated the interaction of SERCA2 haploinsufficiency withhypothyroidism which is known to impair cardiac perfor-mance [13] and alterations in myofibrillar Ca2+-sensitivitywhich causes pathological hypertrophy and heart failure[14 15] For the latter double mutant mice were generatedby crossing HET mice with transgenic lines expressing theGlu54Lysmutant 120572-tropomyosin which reduces myofibrillarCa2+-sensitivity and leads to dilated cardiomyopathy [16 17]and the Glu180Gly mutant 120572-tropomyosin which increasesmyofibrillar Ca2+ sensitivity and causes hypertrophic car-diomyopathy [18 19] Our results reveal an unexpected selec-tivity in the effects of SERCA2 haploinsufficiency in heartwhich should be taken into consideration in themanagementof Darier disease patients

2 Materials and Methods

21 Animal Models The original Atp2a2+minus line was back-crossed onto the inbred FVBN background in excess of 15generations to generate the HET mutant line utilized in thisstudy This Atp2a2+minus mouse line has been made available toresearchers through the Jackson Labs repository All othermice used in this study including the WT the transgenicmouse models carrying the Glu154Lys mutant 120572-tropomyo-sin which has dilated cardiomyopathy (DCM) [16] and theGlu180Gly mutant 120572-tropomyosin which has hypertrophiccardiomyopathy (HCM) [18] were also on the inbred FVBNbackground HET mice were bred with the DCM andHCM lines to generate double (DCMHET and HCMHET)and single mutant offspring The hypothyroid model wasprepared by treating WT and HET mice with 6-n-propylthiouracil (PTU) exactly as previously described [20] Allprocedures conformed to guidelines published by the NIH(Guide for the Care and Use of Laboratory Animals publi-cation number 86-23 revised 1996) and were approved bythe Institutional Animal Care and Use Committee at theUniversity of Cincinnati

22 Evaluation of Cardiac Function Anesthesia of mice withketamine and inactin analysis of cardiovascular functionusing pressure transducers inserted into the left ventricle

and right femoral vein delivery of drugs via a cannula inthe right femoral vein and recording and analysis of datawere performed exactly as described previously [13] Analysisof cardiac function by M-mode echocardiography of miceanesthetized using isoflurane inhalation and analysis of datawere performed exactly as described previously [21]

23 Immunoblot Analyses Hearts were harvested from anes-thetized mice and processed for immunoblot analysis as pre-viously described [22] Phosphorylation of phospholamban(PLN) in response to 120573-adrenergic stimulation was assessedin ventricles from mice that were anesthetized and surgicallyinstrumented as described above and treated with dobu-tamine (16 ngg body weightmin) Estimation of proteinconcentration in total homogenates resolution of proteins bydiscontinuous reducing SDS-PAGE and immunoblot analy-ses were carried out as described [22] All primary and sec-ondary antibodies used have been previously described [22]

24 Real-Time Polymerase Chain Reaction Heartsventricleswere harvested from anesthetized mice and processed forreal-time PCR (RT-PCR) analysis as previously described[22] In addition to the primer pairs that have been pre-viously described [22 23] the following were used Hspa5(GRP78BiP) PrimerBank ID number 31981722a1 Hsp90b1(GRP94) PrimerBank ID number 6755863a1 Casp12 (Cas-pase 12) PrimerBank ID number 31981868a1Ddit3 (CHOP)PrimerBank ID number 31982415a1 Eif2ak3 (PERK) Primer-Bank ID number 6857781a1 Acox1 PrimerBank ID num-ber 26333821a1 Fabp3 PrimerBank ID number 6753810a1Orai1 PrimerBank ID number 93277106b1 Stim1 Primer-Bank ID number 31981983a2 Hax1 PrimerBank ID number6754160a1 Rcan2 PrimerBank ID number 46560586c1 andPpar120574 PrimerBank ID number 187960104c1 primers for Pln(phospholamban) were adapted from PrimerBank ID num-ber 213512815c1 (forward primer 51015840 AAGTGCAATACC-TCACTCG 31015840 reverse primer 51015840 GATCAGCAGCAGACA-TATC 31015840) mRNA levels forAtp2b1 (QT01072106) andAtp2b4(QT01076271) were determined using QuantiTect PrimerAssay Kits (Qiagen)

25 Statistics Results are presented as means plusmn standarderror (SE) Individual comparisons were performed using atwo-sided Studentrsquos 119905-test and a 119875 value of lt005 was con-sidered significant

3 Results

31 Cardiovascular Performance in WT and HET MiceCardiovascular performance of adult FVBN WT and HETmice was analyzed using a pressure transducer in the leftventricle under both basal conditions and upon 120573-adrenergicstimulation with dobutamine No significant differences wereobserved in basal heart rate mean arterial pressure left ven-tricular end-diastolic pressure or maximum rates of left ven-tricular pressure development or decay (Figure 1) Treatmentwith dobutamine led to similar changes in both genotypeswith no impairment of chronotropic inotropic or lusitropicresponses Rate of left ventricular pressure development was

BioMed Research International 3

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Figure 1 Cardiovascular function in Atp2a2 heterozygous mice Ventricular and arterial pressures were measured in anesthetized adultFVBN wild-type and Atp2a2+minus mice under baseline conditions and upon 120573-adrenergic stimulation using dobutamine (a) Heart rate(HR) (b) mean arterial pressure (MAP) (c) left ventricular end-diastolic pressure (LVEDP) (d) maximal rate of left ventricular pressuredevelopment (+dPdt Max) (e) rate of left ventricular pressure development at 40mmHg (dPdt40) and (f) maximal rate of decay of leftventricular developed pressure (minusdPdt Max) Values are means plusmn SE 119899 = at least 5 for each genotype


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Figure 2 Effects of Atp2a2 heterozygosity on expression of Ca2+ handling proteins in heart Adult WT and Atp2a2+minus (HET) hearts wereprocessed for analysis of mRNA and protein levels (a) Atp2a2 mRNA determined by RT-PCR (b) immunoblot analysis of SERCA2aryanodine receptor isoform 2 (RYR2) 1205722 subunit of L-type Ca2+ channel (LTCC1205722) andNa+Ca2+ exchanger isoform 1 (NCX1) Quantitationof SERCA2a (c) and LTCC1205722 (d) protein levels Immunoblot analyses of phospholamban (PLN) and PLN phosphorylated on Ser16 (PS16)and Thr17 (PT17) were performed using heart samples from anesthetized surgically instrumented mice under both baseline conditions (e)and after 120573-adrenergic stimulation with dobutamine at 16 ngg body weightmin (f g h) mRNA levels were normalized toGapdh and proteinlevels were normalized to sarcomeric actin (sactin) Values are means plusmn SE 119899 = at least 4 for each genotype lowast119875 lt 005 versus WT controls

also calculated at 40mmHg to assess possible effects of thesmall but nonsignificant difference in mean arterial pressure(Figure 1(b)) however the two genotypes had virtually iden-tical responses

32 Effects of Atp2a2 Heterozygosity on Proteins Implicated inCa2+-Handling RT-PCR analysis of total RNA fromWT andHET hearts revealed that mRNA levels of the Atp2a2 genewhich codes for SERCA2 were reduced to 48 plusmn 5 of WT

levels (Figure 2(a)) However immunoblot analysis of totalprotein homogenates revealed a much smaller reduction inSERCA2a protein levels (to 79plusmn3ofWT levels Figures 2(b)and 2(c)) in HET hearts This reduction was associated withan increase (to 137plusmn11ofWT levels Figures 2(b) and 2(d))in expression of the 1205722 subunit of the L-type Ca2+-channelwhich is the principal Ca2+-uptake mechanism on a beat-to-beat basis There were no changes in expression of either theryanodine receptor isoform 2 which mediates Ca2+-release


from the SR or the Na+Ca2+ exchanger isoform 1 (NCX1Slc8a1) which is the predominant Ca2+-efflux mechanism incardiac myocytes in HET hearts (Figure 2(b))

SERCA2 activity is negatively regulated by phospholam-ban (PLN) which binds to the Ca2+-pump in a phosphor-ylation-dependent manner [24] Reduced PLN expressionand increased PLN phosphorylation on residues Ser16 (PS16)and Thr17 (PT17) can both enhance SERCA2a-mediatedSR Ca2+-sequestration [24] However immunoblot analysisrevealed that PLN expression and baseline phosphorylation(Figure 2(e)) were unaltered in HET hearts The inotropicand lusitropic effects of 120573-adrenergic stimulation are medi-ated at least in part via increased PLN phosphorylationwhich facilitates more robust Ca2+-cycling [24] The normalincrease in the cardiovascular performance of stimulatedHET mice raised the possibility that 120573-adrenergic stimulatedPLN phosphorylation was augmented to compensate for thereduction in SERCA2a levels To test this hypothesis PS16and PT17 levels were determined in hearts fromWTandHETmice stimulated with dobutamine While PS16 levels werecomparable between stimulatedWTandHEThearts (Figures2(f) and 2(g)) PT17 levels were elevated in stimulated HEThearts (by 153 plusmn 13) when compared to similarly treatedWT hearts (Figures 2(f) and 2(h))

33 Atp2a2 Heterozygosity Does Not Exacerbate CardiacDysfunction Caused by Hypothyroidism Hypothyroidism isa well-recognized cardiovascular disease risk factor [25]Studies in rabbits and mice have shown that it is associ-ated with a reduction in cardiac SERCA2 expression [2026] raising the possibility that the impairment of cardiacfunction caused by hypothyroidism would be greater inAtp2a2 heterozygous individuals To test this hypothesishypothyroidism was induced in WT and HET mice andcardiac performance was assessed by in vivo catheterizationCardiovascular function was similarly diminished in bothWT and HET mice (Figures 3(a)ndash3(f)) under both baselineconditions and upon 120573-adrenergic stimulation

34 Atp2a2 Heterozygosity Has No Appreciable Effect on theCardiac Phenotype of TransgenicMice with Dilated Cardiomy-opathy Myofibrillar Ca2+-sensitivity is a major determinantof cardiac function in addition to altering force develop-ment changes in Ca2+-sensitivity can impact myofibrillarCa2+-buffering diastolic Ca2+-levels and SERCA2-mediatedcytosolic Ca2+-clearance [14 15 27ndash29] Transgenic expres-sion of Glu154Lys mutant 120572-tropomyosin in the DCMmouseheart lowers myofibrillar Ca2+ sensitivity impairs contrac-tility and leads to dilated cardiomyopathy [16] In order todetermine the effects of SERCA2 haploinsufficiency doublemutant DCMHET mice were analyzed DCMHET micewere viable and appeared normal The increase in heartweight body weight (HW BW) and heart weight tibiallength ratios determined in 8ndash10-week-old mice was similarin both single mutant DCM and double mutant DCMHETmice (Figure 4(a)) Echocardiographic analyses revealedthat cardiac function was not compromised in DCMHETmice when compared to DCM controls in fact fractional

shortening and ejection fraction trended slightly higher inDCMHET hearts (Figure 4(b)) RT-PCR analysis showedthat the increases in mRNA levels for Nppa (atrial natriureticpeptide) Myh7 (120573-myosin heavy chain) and Acta1 (skeletal120572-actin) as markers of pathological hypertrophy were notsignificantly different between DCM and DCMHET hearts(Figure 4(c)) Atp2a2 mRNA levels which were reduced to69 plusmn 4 of WT levels in DCM hearts were lower in DCMHET hearts (42 plusmn 4 of WT levels Figure 4(d)) Howeverthe reduction in SERCA2a protein was more modest withexpression at 86 plusmn 2 of DCM levels in DCMHET hearts(Figure 4(e))

35 Atp2a2 Heterozygosity Causes Rapid Progression ofHypertrophy and Decompensation in Transgenic HCM MiceIncreased myofibrillar Ca2+-sensitivity which impairs relax-ation is associated with hypertrophic cardiomyopathy andhas also been reported in end-stage heart failure [14 15]Cardiac expression of Glu180Gly mutant 120572-tropomyosin inthe HCM transgenic model increases myofibrillar Ca2+-sensitivity impairs relaxation leads to development of fibro-sis and hypertrophy and causes death at 5-6 months of age[18] To determine the effects of SERCA2 haploinsufficiencyon the HCM phenotype double mutant HCMHET micewere generated as described above SERCA2 haploinsuffi-ciency caused a rapid onset of heart failure and death 80of HCMHET mice were dead by 5 weeks of age and nonesurvived beyond 6-7 weeks (Figure 5(a)) When comparedto age-matched HCM controls HCMHET hearts displayedovert hypertrophy with pronounced left atrial remodelingas early as 4 weeks of age (Figure 5(b)) HW BW ratioswere higher in HCMHET mice (Figure 5(c)) as were ratiosof ventricular weight body weight (Figure 5(d)) indicatingthat the increase in HW BW was not simply a consequenceof atrial remodeling Consistent with these changes mRNAlevels for several markers of cardiac remodeling which havebeen shown to be elevated in 4-week-old HCM hearts [23]were further increased in HCMHET hearts these includedNppa (Figure 5(e)) Myh7 (Figure 5(f)) Acta1 (Figure 5(g))and Ctgf encoding connective tissue growth factor (Fig-ure 5(h))

36 Effect of Atp2a2 Heterozygosity on Regulators of Ca2+-Handling and Protein Phosphatases in HCMHET HeartsRT-PCR analysis of 4-week-old WT HCM and HCMHEThearts revealed that Atp2a2 mRNA levels which werereduced to 72plusmn5ofWT levels in HCMhearts were sharplyreduced in HCMHET hearts (to 18 plusmn 2 of WT levelsFigure 6(a)) mRNA levels for PLN which were maintainedatWT levels inHCMhearts were also reduced inHCMHEThearts (53 plusmn 3 of WT levels Figure 6(b)) However expres-sion of SERCA2a protein showed a smaller reduction of just23 plusmn 5 from HCM levels in HCMHET hearts (Figures6(c) and 6(d)) with no change seen in PLN protein levels(Figure 6(c))

In addition to its role in contractility Ca2+ is a reg-ulator of key signaling cascades in heart the Ca2+-pools


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Figure 3 Effects of hypothyroidism on cardiovascular performance ofAtp2a2 heterozygousmice Adult wild-type andAtp2a2+minus (HET)micewere rendered hypothyroid and cardiovascular performance was assessed under both baseline conditions and after 120573-adrenergic stimulation(a) Heart rate (HR) (b) mean arterial pressure (MAP) (c) systolic left ventricular pressure (systolic LVP) (d) maximal rate of left ventricularpressure development (+dPdt Max) (e) rate of left ventricular pressure development at 40mmHg (dPdt40) (f) maximal rate of decay ofleft ventricular developed pressure (minusdPdt Max) Values are means plusmn SE 119899 = at least 4 for each genotype


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Figure 4 Effects ofAtp2a2heterozygosity in a transgenicmodel of reducedmyofibrillar Ca2+ sensitivityWTmice transgenicmice expressingthe Glu154Lysmutant 120572-tropomyosin which causes dilated cardiomyopathy (DCM) and double mutant DCMAtp2a2+minus (DCMHET)micewere analyzedMorphometric analyses revealed similar heart weight bodyweight (HW BW) andheartweight tibial length (HW TL) ratiosin DCM and DCMHET mice (a) echocardiographic analysis shows fractional shortening and ejection fraction in DCM and DCMHETmice (b) RT-PCR analysis shows mRNA levels for (c) atrial natriuretic peptide (Nppa) 120573-myosin heavy chain (Myh7) and skeletal 120572-actin(Acta1) and for Atp2a2 (d) Immunoblot analysis of cardiac homogenates and quantitation show relative levels of SERCA2a (e) in DCM andDCMHEThearts mRNA levels were normalized toGapdh and protein levels were normalized to sarcomeric actin (sactin) Values aremeansplusmn SE 119899 = at least 4 for each genotype lowast119875 lt 005 versus WT controls 119875 = 006 versus WT controls +119875 = 005 versus DCM dagger119875 lt 005 versusDCM

implicated in such regulatory functions are thought to existin microdomains spatially distinct from bulk SR Ca2+-stores [30] which are probably regulated by sarcolemmalCa2+ pumps such as the plasma membrane Ca2+-ATPases(PMCA) Impairments in cytosolic bulk Ca2+-handling havethe potential to perturb the spatial isolation of these Ca2+-microdomains and can lead to a greater reliance on sar-colemmal Ca2+ pumps for Ca2+-clearance RT-PCR analysisrevealed that mRNA levels for PMCA4 were increased inHCM hearts (141 plusmn 4 ofWT levels) and remained similarlyelevated in HCMHET hearts (Figure 6(e)) In contrastmRNA levels for PMCA1 whichwere not significantly alteredin HCM hearts were reduced in HCMHET hearts (to 80 plusmn6 of WT levels Figure 6(f))

As described above increased myofibrillar Ca2+-buffer-ing in HCM hearts has the potential to antagonize SR Ca2+-sequestration with effects on SRER Ca2+-store levels How-ever as we have previously demonstrated [22] the amplitude

of stimulated Ca2+-transients is only modestly reduced inHCMmyocytes which raises the possibility that SRERCa2+-stores may be replenished by alternativemechanisms in thesehearts These can include mediators of store-operated Ca2+-entry (SOCE) indeed expression of mRNAs for both Orai1(to 155 plusmn 19 of WT levels) and Stim1 (to 178 plusmn 9 of WTlevels) which encode proteins with well-characterized rolesin stress-induced SOCE activity [31 32] was increased inHCMhearts Unexpectedly this increase was reversed toWTlevels in HCMHET hearts (Figures 6(g) and 6(h))

Increased expression or activity of the protein phos-phatases calcineurin protein phosphatase 1 (PP1) andprotein phosphatase 2A (PP2A) is strongly associated withpathological hypertrophy and heart failure [33ndash36] Proteinlevels of the catalytic subunits of calcineurin (CnA) PP1(PP1-C) and PP2A (PP2A-C) which reflect expression ofthe respective holoenzymes were assessed While CnA andPP2A-C expression were comparable between HCM and


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Figure 5 Effects of Atp2a2 heterozygosity in a transgenic model of increased myofibrillar Ca2+ sensitivity WT mice mice expressing theGlu180Gly mutant 120572-tropomyosin which causes hypertrophic cardiomyopathy (HCM) and double mutant HCMAtp2a2+minus (HCMHET)mice were analyzed Survival of HCM and HCMHETmice was assessed at 5 weeks of age (a) Gross morphometry at 4 weeks of age showed(b) overt remodeling (c) increased heart weight body weight ratios (HW BW) and (d) increased ventricular weight body weight ratios(VW BW) in HCMHET mice RT-PCR shows elevated mRNA levels in HCMHET hearts for (e) atrial natriuretic peptide (Nppa) (f) 120573-myosin heavy chain (Myh7) (g) skeletal 120572-actin (Acta1) and (h) connective tissue growth factor (Ctgf ) mRNA levels were normalized toGapdh expression Values shown are means plusmn SE 119899 = at least 4 for each genotype dagger119875 lt 005 versus HCM controls


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lowast

(l)

Figure 6 Effects of Atp2a2 heterozygosity on regulators of Ca2+-handling and phosphatases in HCM models Hearts from WT mice miceexpressing the Glu180Gly mutant 120572-tropomyosin (HCM) and HCMAtp2a2+minus double mutant (HCMHET) mice were processed for RT-PCR and immunoblots RT-PCR analysis of mRNA for (a) SERCA2 (Atp2a2) and (b) phospholamban (Pln) (c) immunoblot analysis ofSERCA2a and PLN and (d) quantitation of SERCA2a protein RT-PCR analyses of mRNA for (e) plasma membrane Ca2+-ATPase isoform 4(Atp2b4) (f) plasmamembrane Ca2+-ATPase isoform 1 (Atp2b1) (g) sarcolemmal calcium release-activated calciummodulator 1 (Orai1) and(h) stromal interaction molecule 1 (Stim1) and (i) immunoblot analyses of the catalytic subunits of calcineurin (CnA) protein phosphatase 1(PP1-C) and protein phosphatase 2A (PP2A-C) in HCM and HCMHET hearts (j) Quantitation of PP1-C protein levels RT-PCR analysesof mRNA for (k) regulator of calcineurin 1 (Rcan1) and (l) regulator of calcineurin 2 (Rcan2) mRNA levels were normalized to Gapdh andprotein levels were normalized to sarcomeric actin (sactin) Values are means plusmn SE 119899 = at least 4 for each genotype lowast119875 lt 005 versus WTcontrols dagger119875 lt 005 versus HCM 119875 = 008 versus WT controls

HCMHET hearts (Figure 6(i)) PP1-C levels were reducedin HCMHET hearts (Figures 6(i) and 6(j)) To addressthe possibility that calcineurin activity was augmentedin HCMHET hearts mRNA levels for regulator of calcin-eurin 1 (calcipressin-1 Rcan1) a marker of calcineurinactivity were determined by RT-PCR analysis While therewas no difference in Rcan1 levels between HCM and HCMHET hearts (Figure 6(k)) mRNA levels of Rcan2 whichcodes for calcipressin-2 were significantly reduced

in HCMHET hearts (58 plusmn 5 of WT levels Figure6(l))

37 Expression of ER Stress Markers and Regulators of Apopto-sis in HCMHET Hearts The reduction in SERCA2a proteinlevels coupled with the downregulation of SOCE-relatedgenes raised the possibility that ER stress was elevated inHCMHET hearts We initially assessed expression of keyER stress markers in HCM hearts RT-PCR analysis revealed


WT HCM

Hsp

90b1

(Grp94

) G

apdh

( le

vels)

100

50

100

50

Hsp

a4(B

iP)

Gap

dh(

leve

ls)

WT HCM

(a)

50

100

150

Eif2

ak3

(PER

K) G

apdh

( le

vels)

lowast

WT HCM

(b)

lowast

Ddi

t3(C

HO

P) G

apdh

( le

vels)

WT HCM

50

100

150

(c)

lowast

Casp

12 G

apdh

( le

vels)

WT HCM

50

150

100

(d)

50

150

100

lowast

Hax

1 G

apdh

( le

vels)

WT HCM

(e)

50

100

Hsp

a4(B

iP)

Gap

dh(

leve

ls)

HCM HCMHET HCM HCMHET

50

100

Hsp

90b1

(Grp94

) G

apdh

( le

vels)

(f)

HCM HCMHET

Eif2

ak3

(PER

K) G

apdh

( le

vels)

50

100

(g)

Figure 7 Continued


HCM HCMHET

50

150

100

dagger

Ddi

t3(C

HO

P) G

apdh

( le

vels)

(h)

HCM HCMHET

dagger

50

100

Casp

12 G

apdh

( le

vels)

(i)

HCM HCMHET

dagger

50

100

Hax

1 G

apdh

( le

vels)

(j)

Figure 7 Effect of Atp2a2 heterozygosity on markers of ER stress and apoptosis in HCM and HCMHET hearts Heart RNA fromWTmicemice expressing the Glu180Gly mutant 120572-tropomyosin (HCM) and HCMAtp2a2+minus double mutant (HCMHET) mice was analyzed by RT-PCR Panels (a)ndash(e) compare WT and HCM panels (f)ndash(j) compare HCM and HCMHET mRNA levels are shown for (a f) ER chaperonesBiPGRP78 (Hspa4) and GRP94 (Hsp90b1) (b g) PERK (Eif2ak3) (c h) CHOP (Ddit3) (d i) caspase 12 (Casp12) and (e j) HCLS1 associatedprotein X-1 (Hax1) in WT HCM and HCMHET hearts mRNA levels were normalized to Gapdh Values shown are means plusmn SE 119899 = at least4 for each genotype lowast119875 lt 005 versus WT controls dagger119875 lt 005 versus HCM

no increase in mRNA for the ER chaperones BiPGRP78 orGRP94 (Figure 7(a)) However mRNA for PERK a majorkinase involved in ER stress responses was increased inHCMhearts (to 147plusmn12ofWT levels Figure 7(b)) FurthermoremRNAs for the ER stress related proapoptotic proteinsCHOP(145 plusmn 12 of WT levels Figure 7(c)) and CASP12 (164 plusmn5 of WT levels Figure 7(d)) were also increased in HCMhearts These changes were associated with an increase (to148 plusmn 14 of WT levels Figure 7(e)) in mRNA for theantiapoptotic protein HCLS1 associated protein X-1 (HAX1)which is localized to mitochondria and the SR [37]

Therewas no increase inmRNA levels forGRP78GRP94or PERK in HCMHET hearts when compared to HCMcontrols (Figures 7(f) and 7(g)) However mRNA levels forCHOP (143 plusmn 16 of HCM levels Figure 7(h)) and CASP12(128 plusmn 9 of HCM levels Figure 7(i)) were further elevatedin HCMHET hearts The increase seen in HAX1 mRNA inHCM hearts was reversed in HCMHET hearts (to 64 plusmn 4of HCM levels Figure 7(j))

38 Effect of Atp2a2 Heterozygosity on Regulators of EnergyMetabolism in HCM Hearts Impaired relaxation resultingfrom an increase inmyofibrillar Ca2+-sensitivity can energet-ically stress the heart [38] The resultant dysregulation ofenergy metabolism has been suggested to contribute to thehypertrophic phenotype associated with sarcomeric muta-tions that increase myofibrillar Ca2+-sensitivity [39 40]mRNA levels of Ppar120574 encoding peroxisome proliferator-activated receptor gamma a regulator of lipid metabolism

that is implicated in the development of pathologicalhypertrophy [41] were normal in HCM hearts but reducedin HCMHET hearts (to 81 plusmn 6 of WT levels Figure 8(a))Hearts rely predominantly on lipids for their energy supply[42] and FABP3 is the cardiacmuscle-isoform of fatty acidbinding proteins which mediate the intracellular transportof long-chain fatty acids Expression of the Fabp3 gene wasdownregulated (to 76 plusmn 9 of WT levels) in HCM heartsand further reduced in HCMHET hearts (to 49 plusmn 5of WT levels Figure 8(b)) Mitochondrial uptake of long-chain fatty acids ismediated by carnitine palmitoyltransferase1b (CPT1b) the mRNA levels for which were reduced inHCM (to 88 plusmn 3 of WT levels) and further reduced inHCMHET (to 61 plusmn 2 of WT levels) hearts (Figure 8(c))CPT1b-mediated transfer of long-chain fatty acids can beinhibited by malonyl CoA which is generated from acetyl-CoA by acetyl-CoA carboxylase beta (encoded by Acacb)While Acacb mRNA levels trended lower in HCM heartsthey were downregulated in HCMHET hearts (to 58 plusmn 7of WT levels Figure 8(d)) In addition mRNA levels foracyl-CoA oxidase 1 (Acox1) which is the first enzyme ofthe 120573-oxidation pathway were also reduced (to 64 plusmn 5of WT levels) in HCMHET hearts (Figure 8(e)) Besideslipids hearts also utilize glucose as an energy source [42]Glucose uptake in heart is mediated by members of Slc2aglucose transporter family of which GLUT4 (Slc2a4) is thepredominant isoform in cardiac myocytes mRNA levels forGLUT4 were reduced (to 80 plusmn 2 of WT levels) in HCMhearts with a more pronounced reduction (to 47plusmn2 ofWTlevels) in HCMHET hearts (Figure 8(f))


50

100

Ppar120574

Gap

dh(

leve

ls)

daggerlowast

WT HCM HCMHET

(a)

Fabp

3 G

apdh

( le

vels)

WT HCM HCMHET

50

100

daggerlowast

lowast

(b)

WT HCM HCMHET

50

100

daggerlowast

lowast

Cpt1

b G

apdh

( le

vels)

(c)

WT HCM HCMHET

50

100

daggerlowast

Acac

b G

apdh

( le

vels)

(d)

WT HCM HCMHET

50

100

daggerlowast

Acox

1 G

apdh

( le

vels)

(e)

WT HCM HCMHET

50

100

daggerlowast

lowast

Slc2

a4(G

LUT4

) G

apdh

( le

vels)

(f)

Figure 8 Effect of Atp2a2 heterozygosity on regulators of energy metabolism in HCM and HCMHET hearts Heart RNA from WT micemice expressing the Glu180Gly mutant 120572-tropomyosin (HCM) and HCMAtp2a2+minus double mutant (HCMHET) mice was analyzed by RT-PCR mRNA levels are shown for (a) peroxisome proliferator-activated receptor gamma (Ppar120574) (b) fatty acid binding protein 3 (Fabp3) (c)the muscle-isoform of carnitine palmitoyltransferase 1 (Cpt1b) (d) acetyl CoA-carboxylase beta (Acacb) (e) acyl-CoA oxidase 1 (Acox1) and(f) the GLUT4 glucose transporter (Slc2a4) mRNA levels were normalized to Gapdh Values are means plusmn SE 119899 = at least 4 for each genotypelowast119875 lt 005 versus WT controls dagger119875 lt 005 versus HCM


Table 1 Effect of Atp2a2 heterozygosity on regulators of apoptosis and energy metabolism in DCM and DCMHET hearts

Gene Relative mRNA levels (normalized to Gapdh)WTpara DCM DCMHET

CHOP (Ddit3) 100 plusmn 10 103 plusmn 3 113 plusmn 10Caspase 12 (Casp12) 100 plusmn 7 122 plusmn 3lowast 120 plusmn 16HCLS1 associated protein X-1 (Hax1) 100 plusmn 5 91 plusmn 3 106 plusmn 12Fatty acid binding protein 3 (Fabp3) 100 plusmn 7 70 plusmn 4lowast 75 plusmn 7lowast

Carnitine palmitoyltransferase 1 (Cpt1b) 100 plusmn 2 77 plusmn 3lowast 70 plusmn 8lowast

Glucose transporter 4 (Slc2a4) 100 plusmn 12 88 plusmn 2 95 plusmn 8paraHeart RNA from wild-type (WT) mice transgenic mice expressing Glu154Lys mutant 120572-tropomyosin (DCM) and DCMAtp2a2+minus double mutant(DCMHET) mice was analyzed by RT-PCR Values are mean plusmn SE 119899 = at least 4 for each genotype lowast119875 lt 005 versus WT controls

39 Biochemical Effects of Atp2a2 Heterozygosity Seen inHCMHET Hearts Are Lacking in DCMHET Hearts Asdescribed above Atp2a2 heterozygosity did not exacerbatethe cardiac disease phenotype of DCM mice Given thedramatic effects of SERCA2 haploinsufficiency inHCMHEThearts at the RNAprotein levels we analyzed DCMHEThearts to identify possible underlying similarities SeveralmRNAs analyzed (Casp12 Fabp3 and Cpt1b) were alteredin DCM hearts relative to WT hearts However RT-PCRanalysis revealed that Atp2a2 heterozygosity did not impactmRNA levels for Ddit3 (CHOP) Casp12 Hax1 Fabp3 Cpt1bor Slc2a4 (GLUT4) in DCMHET hearts when comparedwith DCM hearts (Table 1) Immunoblot analyses of totalventricular homogenates of DCM and DCMHET miceshowed no change in protein levels of CnA PP1-C or PP2A-C(data not shown)

4 Discussion

While there is strong evidence that ATP2A2 heterozygositydoes not impair cardiovascular performance in humanseither at rest or during exercise [6 7] the opportunities toelucidate the molecular mechanisms that allow for cardiacfunction to be preserved in otherwise healthyDDpatients areobviously limited For example it remains unknown if lossof one ATP2A2 allele elicits the compensatory upregulationof the second functional allele or whether SERCA2a haploin-sufficiency in human heart leads to a greater and apparentlyeffective reliance on transsarcolemmal Ca2+-fluxThere havealso been no studies to determine if DDpatients aremore sus-ceptible to heart failure and decompensation in the contextof secondary pathological insults The Atp2a2 heterozygousmouse model on the inbred FVBN background offered aunique opportunity to address these questions as it resembleshumanDDpatients in displaying a skinkeratinized epithelialphenotype with no overt effects on cardiovascular function

Expression of SERCA2a protein levels at sim80 of WTlevels in HET hearts demonstrated a remarkable ability toupregulate expression of the single functional Atp2a2 alleleThis was unexpected given the evidence from transgeniclines that it is difficult to appreciably overexpress SERCA2protein in mouse hearts [43 44] Given that Atp2a2 mRNAlevels were reduced by sim50 in HET hearts it is reasonable

to assume that the increase in SERCA2a protein resultsprincipally from posttranscriptional adaptations these couldinclude augmented translation extended SERCA2 proteinhalf-life andor diminished SERCA2 protein degradationThe increase seen in LTCC1205722 protein levels is consistent withthe idea that even modest reductions in SERCA2a expressionmight lead to a greater reliance on Ca2+-handling acrossthe sarcolemma While such a shift would help preserveCa2+-homeostasis it is likely to place a greater energeticburden on the heart SERCA2 is unique in its stoichiometryof transporting 2 Ca2+ ionsATP hydrolyzed In comparisontranssarcolemmal flux of Ca2+ via LTCC-influx NCX1-efflux(with a stoichiometry of 3Na+ 1Ca2+) and Na+K+-ATPase-mediated Na+ removal effectively doubles the energy cost ofCa2+-clearance

Increased PLN phosphorylation can also serve to com-pensate for the reduction in SERCA2 levels [23] with evi-dence of a predominant role for phosphorylation at Ser16but not Thr17 in 120573-adrenergic stimulation of inotropy andlusitropy in mouse hearts [45] Therefore it is difficult toascribe any part of the normal 120573-adrenergic response seenin HET hearts to increased PLN phosphorylation given thatPS16 levels were unaltered and only PT17 levels were elevatedin stimulated HET hearts While phosphorylation at Thr17is mediated by Ca2+calmodulin-dependent kinase CaMKIIdephosphorylation is mediated by PP1 [46 47] Althoughimmunoblot analysis showed no evidence of a reductionin PP1 expression in HET hearts (data not shown) a clearreduction in PP1 expression was observed in HCMHEThearts relative to HCM controls probably providing somecompensation for the reduction in SERCA2 protein

Reduction in SERCA2 expressionactivity is closely asso-ciated with progression of heart disease and failure Whilerecent clinical trials provide strong evidence that enhancingSERCA2a expression can alleviate impairment of cardiacfunction [48ndash50] it remains unclear whether SERCA2adeficiency can itself precipitate decompensation and heartfailure This is highly relevant to DD patients particularlyin the context of aging or secondary pathological conditionsaffecting the heart Hypothyroidism which affects about46 of the US population [51] is one such condition knownto compromise cardiac function [13 24] and reduce SERCA2


expression [20 25] The finding that Atp2a2 heterozygositydoes not exacerbate the effects of hypothyroidism in micemay result from the switch in myosin heavy chain (MHC)isoforms from 120572-MHC to the slower 120573-MHC which occursin hypothyroidism and is known to be energetically favorable[52] Whether the effects of hypothyroidism are similarlyunaffected by SERCA2 haploinsufficiency in human DDpatients is an issue that remains to be clarified it should beevaluated with no preconceptions as the lack of an effect inthe rodent model does not discount possible consequences inhuman DD patients

A relative increase in 120573-MHC levels which commonlyoccurs in diseased conditions renders hearts more suscepti-ble to chronic myocardial stress [53] Although this outcomewas not tested in the context of hypothyroidism inHETmicewe investigated the effects ofAtp2a2 heterozygosity inmodelsof altered myofibrillar Ca2+-sensitivity which impose signifi-cant chronic contractile stress and are closely associated withthe pathogenesis of cardiomyopathies and heart failure [54]DCM mice with reduced myofibrillar Ca2+-sensitivity havebeen reported to develop hypertrophy at 2 months of agewith significant dilation and myocyte disarray occurring by5 months and mice starting to die at 4ndash6 months of age[16] These effects were shown to be associated with down-regulation of SERCA2a consistent with the possibility thatAtp2a2 heterozygosity would hasten disease pathogenesisThe absence of such an effect in DCMHET mice howeverrevealed that SERCA2 haploinsufficiency has limited effectsin the context of reduced myofibrillar Ca2+-sensitivity whichoccurs during pathological conditions such as inflammationand sepsis [55]

The rapid onset of hypertrophy and decompensationin HCMHET mice on the other hand strongly suggeststhat disease pathogenesis associated with increased myofib-rillar Ca2+-sensitivity may be exacerbated in DD patientsThe catastrophic effects of SERCA2 haploinsufficiency inHCMHET occurred despite SERCA2a protein levels beingreduced by just 23 compared to levels in HCM heartsThis indicates that relatively modest reductions in SERCA2aexpression can have profound effects in hearts with increasedmyofibrillar Ca2+-sensitivity This effect is likely due tothe fact that besides factors such as mechanical load andsarcolemmal Ca2+-flux dissociation of Ca2+ from myofibrilsis facilitated by SERCA2-mediated Ca2+-clearance [56] Theimportance of this function is expected to be amplified inthe context of elevated myofibrillar Ca2+-sensitivity makingeven small changes in SERCA2 expressionfunction highlyconsequential Recent reports that elevating SERCA2 activityeither by increased expression of SERCA2a or ablation ofphospholamban attenuates disease progression in HCMmice [57 58] are consistent with this hypothesis

Perturbations in cytosolic Ca2+-clearance are also asso-ciated with the generation of ventricular arrhythmias [56]which are strongly implicated in progression to heart failureand sudden death [59 60] Targeted SERCA2a gene therapyhas been shown in multiple models to reduce ventricu-lar arrhythmias in addition to improving cardiac function[4] The highly advanced disease condition observed in

HCMHET mice as young as 4 weeks of age precludedeffective functional analyses of these mice However weexamined left ventricular pressure measurements in HETmice and found no evidence of extrasystolic beats or otherarrhythmias under either basal conditions or upon maximal120573-adrenergic stimulation (data not shown)

The more rapid decompensation in HCMHET heartswas associated with a reversal of the increase in Orai1 andStim1 levels seen in HCM hearts The incidence of increasedSOCE via ORAI1 and STIM1 in pathological hypertrophyhas implicated these proteins in disease pathogenesis [31]However recent data from knockout models reveal a morenuanced role for these proteins in heart with effects on Ca2+-homeostasis and the development of compensatory hypertro-phy [61 62] Orai1 deficiency was found to exacerbate lossof cardiac function and hasten progression to dilation andwas associatedwith increased apoptosis [63] Indeed levels ofproapoptotic Chop and Casp12 were elevated in HCMHEThearts and notably expression of antiapoptotic Hax1 whichwas elevated in HCM hearts was reduced to WT levelsin HCMHET hearts There is increasing evidence that theantiapoptotic function of the HAX1 protein is linked to itsassociation with SR Ca2+-handling [37 64] where HAX1 hasbeen shown to localize to the SR in a PLN-dependentmanner[65] HAX1 overexpression which promotes cell survivaldownregulates SERCA2 protein levels whereas SERCA2overexpression has been shown to antagonize its protectiveeffects [66 67] Therefore the reversion of HAX1 expressionto WT levels may contribute to the preservation of SERCA2levels in HCMHET hearts with potentially maladaptiveeffects on myocyte cell survival Further detailed studies willbe necessary to fully elucidate possible interactions betweenHAX1 which has been localized to mitochondria as well[37] and SERCA2 during HCM pathogenesis Additionalperturbations in myocardial energy metabolism which arestrongly implicated in hypertrophy and heart failure [38ndash41] are also likely to contribute to the rapid decompensationobserved in HCMHET hearts Reductions in both CPT1band GLUT4 have been shown to promote pathologicalhypertrophy and heart failure [68ndash70] Conversely we haverecently demonstrated that protection against Tm180-relatedhypertrophy is associated with preservation of CPT1b andGLUT4 expression at WT levels [23]

In conclusion our data show that the loss of one copyof the Atp2a2 gene which causes reduced expression ofSERCA2a in heart appears to be benign under normalconditions and even in some disease states This finding isconsistent with the results of studies in human DD patientsin which the loss of one ATP2a2 allele caused no impairmentof cardiac performance [6 7] However our studies with themouse model of DD also reveal that loss of a single copy ofAtp2a2 can lead to much more rapid decompensation heartfailure and death in mice carrying an HCM mutation thatincreases myofibrillar Ca2+ sensitivity These results suggestthat DD patients may be less tolerant of the changes associ-ated with increased myofibrillar Ca2+-sensitivity in heart Inaddition to being relevant to pathological conditions such asend-stage heart failure [15] the implications of this finding


could extend to the use of therapeutic agents that enhancemyofibrillar Ca2+-sensitivity in DD patients For examplealthough long-term treatment with the Ca2+-sensitizer lev-osimendan improves cardiac function in a model withcardiomyocyte-specific ablation of SERCA2 it also signif-icantly increases fibrosis in SERCA2-deficient hearts [71]While extrapolation of results from rodent models to humandisease merits caution our results suggest that progressionof some types of heart disease is likely to be exacerbatedby DD mutations DD patients and their physicians shouldtherefore be aware of the possibility of adverse interactionsbetween SERCA2 haploinsufficiency and certain pathologicalconditions affecting cardiovascular health

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

This work was supported by National Institutes of HealthGrants HL061974 (GES) and HL107957 (YW) an AmericanHeart Association Beginning Grant-in-Aid 11BGIA77220005(VP) and a Near Horizons Pilot grant from the University ofCincinnati Heart Lung and Vascular Institute (VP and JNL)The authors thank Maureen Bender for excellent animalhusbandry

References

[1] A Sakuntabhai V Ruiz-Perez S Carter et al ldquoMutations inATP2A2 encoding a Ca2+ pump cause Darier diseaserdquo NatureGenetics vol 21 no 3 pp 271ndash277 1999

[2] A Hovnanian ldquoDarierrsquos disease from dyskeratosis to endo-plasmic reticulum calciumATPase deficiencyrdquo Biochemical andBiophysical Research Communications vol 322 no 4 pp 1237ndash1344 2004

[3] M Periasamy P Bhupathy andG J Babu ldquoRegulation of sarco-plasmic reticulum Ca2+ ATPase pump expression and its rele-vance to cardiac muscle physiology and pathologyrdquo Cardiovas-cular Research vol 77 no 2 pp 265ndash273 2008

[4] M B Sikkel C Hayward K T MacLeod S E Harding andA R Lyon ldquoSERCA2a gene therapy in heart failure an anti-arrhythmic positive inotroperdquo British Journal of Pharmacologyvol 171 no 1 pp 38ndash54 2014

[5] YMiyauchi T Daiho K Yamasaki et al ldquoComprehensive anal-ysis of expression and function of 51 sarco(endo)plasmic retic-ulum Ca2+-ATPase mutants associated with darier diseaserdquoTheJournal of Biological Chemistry vol 281 no 32 pp 22882ndash22895 2006

[6] S Tavadia R C Tait T A McDonagh and C S MunroldquoPlatelet and cardiac function in Darierrsquos diseaserdquo Clinical andExperimental Dermatology vol 26 no 8 pp 696ndash699 2001

[7] B MMayosi A Kardos C H Davies et al ldquoHeterozygous dis-ruption of SERCA2a is not associated with impairment of car-diac performance in humans implications for SERCA2a as atherapeutic target in heart failurerdquoHeart vol 92 no 1 pp 105ndash109 2006

[8] M Periasamy T D Reed L H Liu et al ldquoImpaired car-diac performance in heterozygous mice with a null mutationin the sarco(endo)plasmic reticulum Ca2+-ATPase isoform 2(SERCA2) generdquo Journal of Biological Chemistry vol 274 no4 pp 2556ndash2562 1999

[9] L H Liu G P Boivin V Prasad M Periasamy and G E ShullldquoSquamous cell tumors in mice heterozygous for a null alleleof Atp2a2 encoding the sarco(endo)plasmic reticulum Ca2+-ATPase isoform 2 Ca2+ pumprdquo Journal of Biological Chemistryvol 276 no 29 pp 26737ndash26740 2001

[10] V Prasad G P Boivin M L Miller et al ldquoHaploinsufficiencyof Atp2a2 encoding the sarco(endo)plasmic reticulum Ca2+-ATPase isoform 2 Ca2+ pump predisposes mice to squamouscell tumors via a novel mode of cancer susceptibilityrdquo CancerResearch vol 65 no 19 pp 8655ndash8661 2005

[11] J E J Schultz B J Glascock S AWitt et al ldquoAccelerated onsetof heart failure in mice during pressure overload with chroni-cally decreased SERCA2 calcium pump activityrdquoThe AmericanJournal of PhysiologymdashHeart and Circulatory Physiology vol286 no 3 pp H1146ndashH1153 2004

[12] SHuke LH Liu D BiniakiewiczW T Abraham andM Peri-asamy ldquoAltered force-frequency response in non-failing heartswith decreased SERCA pump-levelrdquo Cardiovascular Researchvol 59 no 3 pp 668ndash677 2003

[13] J N Lorenz and J Robbins ldquoMeasurement of intraventricularpressure and cardiac performance in the intact closed-chestanesthetized mouserdquo The American Journal of PhysiologymdashHeart and Circulatory Physiology vol 272 no 3 pp H1137ndashH1146 1997

[14] D Fatkin and R M Graham ldquoMolecular mechanisms of inher-ited cardiomyopathiesrdquo Physiological Reviews vol 82 no 4 pp945ndash980 2002

[15] J van der Velden Z Papp R Zaremba et al ldquoIncreased Ca2+-sensitivity of the contractile apparatus in end-stage humanheart failure results from altered phosphorylation of contractileproteinsrdquoCardiovascular Research vol 57 no 1 pp 37ndash47 2003

[16] S Rajan R P H Ahmed G Jagatheesan et al ldquoDilated cardio-myopathy mutant tropomyosin mice develop cardiac dysfunc-tion with significantly decreased fractional shortening andmyofilament calcium sensitivityrdquo Circulation Research vol 101no 2 pp 205ndash214 2007

[17] T M Olson N Y Kishimoto F G Whitby and V V MichelsldquoMutations that alter the surface charge of alpha-tropomyosinare associated with dilated cardiomyopathyrdquo Journal of Molecu-lar and Cellular Cardiology vol 33 no 4 pp 723ndash732 2001

[18] R Prabhakar G P Boivin I L Grupp et al ldquoA familial hyper-trophic cardiomyopathy 120572-tropomyosinmutation causes severecardiac hypertrophy and death in micerdquo Journal of Molecularand Cellular Cardiology vol 33 no 10 pp 1815ndash1828 2001

[19] L Thierfelder H Watkins C MacRae et al ldquoAlpha-Tropo-myosin and cardiac troponin Tmutations cause familial hyper-trophic cardiomyopathy A disease of the sarcomererdquo Cell vol77 no 5 pp 701ndash712 1994

[20] T D Reed G J Babu Y Ji et al ldquoThe expression of SR calciumtransport ATpase and the Na+Ca2+ exchanger are antithet-ically regulated during mouse cardiac development and inhypohyperthyroidismrdquo Journal of Molecular and Cellular Car-diology vol 32 no 3 pp 453ndash464 2000

[21] A N Wansapura V M Lasko J B Lingrel and J N LorenzldquoMice expressing ouabain-sensitive 1205721-NaK-ATPase haveincreased susceptibility to pressure overload-induced cardiac


hypertrophyrdquo The American Journal of PhysiologymdashHeart andCirculatory Physiology vol 300 no 1 pp H347ndashH355 2011

[22] N J AlMoamen V Prasad I Bodi et al ldquoLoss of the AE3 anionexchanger in a hypertrophic cardiomyopathy model causesrapid decompensation and heart failurerdquo Journal of Molecularand Cellular Cardiology vol 50 no 1 pp 137ndash146 2011

[23] V Prasad J N Lorenz V M Lasko et al ldquoAblation of plasmamembrane Ca2+-ATPase isoform 4 prevents development ofhypertrophy in a model of hypertrophic cardiomyopathyrdquoJournal of Molecular and Cellular Cardiology vol 77 pp 53ndash632014

[24] DHMacLennan and EG Kranias ldquoPhospholamban a crucialregulator of cardiac contractilityrdquoNature ReviewsMolecular CellBiology vol 4 no 7 pp 566ndash577 2003

[25] I Klein and S Danzi ldquoThyroid disease and the heartrdquo Circula-tion vol 116 no 15 pp 1725ndash1735 2007

[26] M Arai K Otsu D H MacLennan N R Alpert and MPeriasamy ldquoEffect of thyroid hormone on the expression ofmRNA encoding sarcoplasmic reticulum proteinsrdquo CirculationResearch vol 69 no 2 pp 266ndash276 1991

[27] H Kogler and J C Ruegg ldquoCardiac contractility modulationof myofibrillar calcium sensitivity by 120573-adrenergic stimulationrdquoIsrael Journal of Medical Sciences vol 33 no 1 pp 1ndash7 1997

[28] K Brixius P Savvidou-Zaroti U Mehlhorn W Bloch E GKranias and R H G Schwinger ldquoIncreased Ca2+-sensitivityof myofibrillar tension in heart failure and its functionalimplicationrdquo Basic Research in Cardiology vol 97 supplement1 pp I111ndashI117 2002

[29] M Endoh ldquoCardiac Ca2+ signaling and Ca2+ sensitizersrdquoCircu-lation Journal vol 72 no 12 pp 1915ndash1925 2008

[30] S A Goonasekera and J D Molkentin ldquoUnraveling the secretsof a double life contractile versus signaling Ca2+ in a cardiacmyocyterdquo Journal of Molecular and Cellular Cardiology vol 52no 2 pp 317ndash322 2012

[31] F R Giachini V V Lima J L Hannan F S Carneiro R CWebb and R C Tostes ldquoSTIM1Orai1-mediated store-operatedCa2+ entry the tip of the icebergrdquo Brazilian Journal of Medicaland Biological Research vol 44 no 11 pp 1080ndash1087 2011

[32] J Soboloff B S Rothberg M Madesh and D L Gill ldquoSTIMproteins dynamic calcium signal transducersrdquo Nature ReviewsMolecular Cell Biology vol 13 no 9 pp 549ndash565 2012

[33] J D Molkentin J R Lu C L Antos et al ldquoA calcineurin-dependent transcriptional pathway for cardiac hypertrophyrdquoCell vol 93 no 2 pp 215ndash228 1998

[34] J Neumann T Eschenhagen L R Jones et al ldquoIncreasedexpression of cardiac phosphatases in patients with end-stageheart failurerdquo Journal of Molecular and Cellular Cardiology vol29 no 1 pp 265ndash272 1997

[35] M Yamada Y IkedaM Yano et al ldquoInhibition of protein phos-phatase 1 by inhibitor-2 gene delivery ameliorates heart failureprogression in genetic cardiomyopathyrdquo The FASEB Journalvol 20 no 8 pp 1197ndash1199 2006

[36] U Gergs P Boknik I Buchwalow et al ldquoOverexpression ofthe catalytic subunit of protein phosphatase 2A impairs cardiacfunctionrdquo Journal of Biological Chemistry vol 279 no 39 pp40827ndash40834 2004

[37] S V Yap E Vafiadaki J Strong and A Kontrogianni-Kon-stantopoulos ldquoHAX-1 a multifaceted antiapoptotic proteinlocalizing in the mitochondria and the sarcoplasmic reticulumof striated muscle cellsrdquo Journal of Molecular and CellularCardiology vol 48 no 6 pp 1266ndash1279 2010

[38] A Kataoka C Hemmer and P B Chase ldquoComputational simu-lation of hypertrophic cardiomyopathy mutations in TroponinI influence of increased myofilament calcium sensitivity onisometric force ATPase and [Ca2+]119894rdquo Journal of Biomechanicsvol 40 no 9 pp 2044ndash2052 2007

[39] J G Crilley E A Boehm E Blair et al ldquoHypertrophic car-diomyopathy due to sarcomeric genemutations is characterizedby impaired energy metabolism irrespective of the degree ofhypertrophyrdquo Journal of the American College of Cardiology vol41 no 10 pp 1776ndash1782 2003

[40] C Ferrantini A Belus N Piroddi B Scellini C Tesi andC Poggesi ldquoMechanical and energetic consequences of HCM-causing mutationsrdquo Journal of Cardiovascular TranslationalResearch vol 2 no 4 pp 441ndash451 2009

[41] B N Finck ldquoThe PPAR regulatory system in cardiac physiologyand diseaserdquo Cardiovascular Research vol 73 no 2 pp 269ndash277 2007

[42] G D Lopaschuk J R Ussher C D L Folmes J S Jaswal andW C Stanley ldquoMyocardial fatty acid metabolism in health anddiseaserdquo Physiological Reviews vol 90 no 1 pp 207ndash258 2010

[43] H He F J Giordano R Hilal-Dandan et al ldquoOverexpressionof the rat sarcoplasmic reticulumCa2+ ATPase gene in the heartof transgenic mice accelerates calcium transients and cardiacrelaxationrdquo The Journal of Clinical Investigation vol 100 no 2pp 380ndash389 1997

[44] D L Baker K Hashimoto I L Grupp et al ldquoTargeted overex-pression of the sarcoplasmic reticulum Ca2+-ATPase increasescardiac contractility in transgenic mouse heartsrdquo CirculationResearch vol 83 no 12 pp 1205ndash1214 1998

[45] G Chu J W Lester K B YoungW Luo J Zhai and E G Kra-nias ldquoA single site (Ser16) phosphorylation in phospholambanis sufficient in mediating its maximal cardiac responses to 120573-agonistsrdquo Journal of Biological Chemistry vol 275 no 49 pp38938ndash38943 2000

[46] C Mundina-Weilenmann L Vittone M Ortale G C de Cin-golani and A Mattiazzi ldquoImmunodetection of phosphoryla-tion sites gives new insights into the mechanisms underlyingphospholamban phosphorylation in the intact heartrdquoThe Jour-nal of Biological Chemistry vol 271 no 52 pp 33561ndash335671996

[47] A Mattiazzi C Mundina-Weilenmann C Guoxiang L Vit-tone and E Kranias ldquoRole of phospholamban phosphorylationonThr17 in cardiac physiological and pathological conditionsrdquoCardiovascular Research vol 68 no 3 pp 366ndash375 2005

[48] A Papolos and W H Frishman ldquoSarcoendoplasmic reticulumcalcium transport ATPase 2a a potential gene therapy target inheart failurerdquo Cardiology in Review vol 21 no 3 pp 151ndash1542013

[49] K Zsebo A Yaroshinsky J J Rudy et al ldquoLong-term effects ofAAV1SERCA2a gene transfer in patients with severe heart fail-ure analysis of recurrent cardiovascular events and mortalityrdquoCirculation Research vol 114 no 1 pp 101ndash108 2014

[50] B Greenberg A Yaroshinsky K M Zsebo et al ldquoDesign ofa phase 2b trial of intracoronary administration of AAV1SERCA2a in patients with advanced heart failure the CUPID2 trial (calcium up-regulation by percutaneous administrationof gene therapy in cardiac disease phase 2b)rdquo Journal of theAmerican College of Cardiology Heart Failure vol 2 no 1 pp84ndash92 2014

[51] S H Golden K A Robinson I Saldanha B Anton andP W Ladenson ldquoPrevalence and incidence of endocrine andmetabolic disorders in the united states a comprehensive


reviewrdquo Journal of Clinical Endocrinology ampMetabolism vol 94no 6 pp 1853ndash1878 2009

[52] C Holubarsch R P Goulette R Z Litten B J Martin LA Mulieri and N R Alpert ldquoThe economy of isometricforce development myosin isoenzyme pattern and myofibrillarATPase activity in normal and hypothyroid rat myocardiumrdquoCirculation Research vol 56 no 1 pp 78ndash86 1985

[53] M Krenz and J Robbins ldquoImpact of beta-myosin heavy chainexpression on cardiac function during stressrdquo Journal of theAmerican College of Cardiology vol 44 no 12 pp 2390ndash23972004

[54] K A Palmiter and R J Solaro ldquoMolecular mechanisms regu-lating the myofilament response to Ca2+ implications of muta-tions causal for familial hypertrophic cardiomyopathyrdquo BasicResearch in Cardiology Supplement vol 92 supplement 1 pp63ndash74 1997

[55] L-L Wu C Tang andM-S Liu ldquoAltered phosphorylation andcalcium sensitivity of cardiac myofibrillar proteins during sep-sisrdquoTheAmerican Journal of PhysiologymdashRegulatory Integrativeand Comparative Physiology vol 281 no 2 pp R408ndashR4162001

[56] H E D J Ter Keurs and P A Boyden ldquoCalcium and arrhyth-mogenesisrdquo Physiological Reviews vol 87 no 2 pp 457ndash5062007

[57] J R Pena A C Szkudlarek CMWarren et al ldquoNeonatal genetransfer of Serca2a delays onset of hypertrophic remodeling andimproves function in familial hypertrophic cardiomyopathyrdquoJournal of Molecular and Cellular Cardiology vol 49 no 6 pp993ndash1002 2010

[58] RDGaffin J R PenaM S L Alves et al ldquoLong-term rescue ofa familial hypertrophic cardiomyopathy caused by a mutationin the thin filament protein tropomyosin via modulation ofa calcium cycling proteinrdquo Journal of Molecular and CellularCardiology vol 51 no 5 pp 812ndash820 2011

[59] J MMcLenachan E Henderson K I Morris and H J DargieldquoVentricular arrhythmias in patients with hypertensive leftventricular hypertrophyrdquoTheNew England Journal of Medicinevol 317 no 13 pp 787ndash792 1987

[60] A Curcio D Torella C Iaconetti et al ldquoMicroRNA-1 down-regulation increases connexin 43 displacement and inducesventricular tachyarrhythmias in rodent hypertrophic heartsrdquoPLoS ONE vol 8 no 7 Article ID e70158 2013

[61] H E Collins X Zhu-Mauldin R B Marchase and J CChatham ldquoSTIM1Orai1-mediated SOCE current perspectivesand potential roles in cardiac function and pathologyrdquo TheAmerican Journal of PhysiologymdashHeart and Circulatory Physi-ology vol 305 no 4 pp H446ndashH458 2013

[62] H E Collins L He L Zou et al ldquoStromal interactionmolecule1 is essential for normal cardiac homeostasis through modula-tion of ER and mitochondrial functionrdquo The American Journalof PhysiologymdashHeart and Circulatory Physiology vol 306 no 8pp H1231ndashH1239 2014

[63] J S Horton C L Buckley E M Alvarez A SchorlemmerandA J Stokes ldquoThe calcium release-activated calcium channelOrai1 represents a crucial component in hypertrophic com-pensation and the development of dilated cardiomyopathyrdquoChannels vol 8 no 1 pp 35ndash48 2014

[64] E Vafiadaki D A Arvanitis S N Pagakis et al ldquoThe anti-apoptotic protein HAX-1 interacts with SERCA2 and regulatesIts protein levels to promote cell survivalrdquoMolecular Biology ofthe Cell vol 20 no 1 pp 306ndash318 2009

[65] E Vafiadaki D Sanoudou D A Arvanitis D H Catino E GKranias and A Kontrogianni-Konstantopoulos ldquoPhospholam-ban interacts with HAX-1 a mitochondrial protein with anti-apoptotic functionrdquo Journal of Molecular Biology vol 367 no 1pp 65ndash79 2007

[66] E Vafiadaki D A Arvanitis S N Pagakis et al ldquoThe anti-apoptotic protein HAX-1 interacts with SERCA2 and regulatesits protein levels to promote cell survivalrdquoMolecular Biology ofthe Cell vol 20 no 1 pp 306ndash318 2009

[67] W Zhao J RWaggoner Z-G Zhang et al ldquoThe anti-apoptoticprotein HAX-1 is a regulator of cardiac functionrdquo Proceedings ofthe National Academy of Sciences of the United States of Americavol 106 no 49 pp 20776ndash20781 2009

[68] L He T Kim Q Long et al ldquoCarnitine palmitoyltransferase-1b deficiency aggravates pressure overload-induced cardiachypertrophy caused by lipotoxicityrdquo Circulation vol 126 no 14pp 1705ndash1716 2012

[69] K R Haynie B Vandanmagsar S E Wicks J Zhang andR L Mynatt ldquoInhibition of carnitine palymitoyltransferase1binduces cardiac hypertrophy and mortality in micerdquo DiabetesObesity amp Metabolism vol 16 no 8 pp 757ndash760 2014

[70] A A Domenighetti V R Danes C L Curl J M Favaloro JProietto and L M D Delbridge ldquoTargeted GLUT-4 deficiencyin the heart induces cardiomyocyte hypertrophy and impairedcontractility linked with Ca2+ and proton flux dysregulationrdquoJournal of Molecular and Cellular Cardiology vol 48 no 4 pp663ndash672 2010

[71] V Hillestad F Kramer S Golz A Knorr K B Andersson andG Christensen ldquoLong-term levosimendan treatment improvessystolic function and myocardial relaxation in mice withcardiomyocyte-specific disruption of the Serca2 generdquo Journalof Applied Physiology vol 115 no 10 pp 1572ndash1580 2013

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


MEDIATORSINFLAMMATION

of


Behavioural Neurology

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine

OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


a reduction in cardiac SERCA2a protein expression to 65of wild-type (WT) levels with impaired contractility andrelaxation [8] However true heart disease was not observedin fact the major phenotype of the Atp2a2+minus mice was thedevelopment of squamous cell tumors in keratinized epithe-lial tissues [9 10] Later studies on these mice revealed anincreased susceptibility to pressure-overload cardiac hyper-trophy [11] and a reduction of rate-dependent inotropy inisolated mutant hearts relative to WT controls [12] In thecurrent study we used Atp2a2+minus mice on an inbred FVBNbackgroundwhile these heterozygous (HET)mice continuedto display effects of SERCA2 haploinsufficiency in keratinizedepithelia cardiac performance was apparently normal moreclosely reflecting findings in DD patients [6 7] The HETmodel was well suited to determine if SERCA2 deficiencywhile apparently benign under normal conditions couldexacerbate cardiac disease progression Specifically we inves-tigated the interaction of SERCA2 haploinsufficiency withhypothyroidism which is known to impair cardiac perfor-mance [13] and alterations in myofibrillar Ca2+-sensitivitywhich causes pathological hypertrophy and heart failure[14 15] For the latter double mutant mice were generatedby crossing HET mice with transgenic lines expressing theGlu54Lysmutant 120572-tropomyosin which reduces myofibrillarCa2+-sensitivity and leads to dilated cardiomyopathy [16 17]and the Glu180Gly mutant 120572-tropomyosin which increasesmyofibrillar Ca2+ sensitivity and causes hypertrophic car-diomyopathy [18 19] Our results reveal an unexpected selec-tivity in the effects of SERCA2 haploinsufficiency in heartwhich should be taken into consideration in themanagementof Darier disease patients

2 Materials and Methods

21 Animal Models The original Atp2a2+minus line was back-crossed onto the inbred FVBN background in excess of 15generations to generate the HET mutant line utilized in thisstudy This Atp2a2+minus mouse line has been made available toresearchers through the Jackson Labs repository All othermice used in this study including the WT the transgenicmouse models carrying the Glu154Lys mutant 120572-tropomyo-sin which has dilated cardiomyopathy (DCM) [16] and theGlu180Gly mutant 120572-tropomyosin which has hypertrophiccardiomyopathy (HCM) [18] were also on the inbred FVBNbackground HET mice were bred with the DCM andHCM lines to generate double (DCMHET and HCMHET)and single mutant offspring The hypothyroid model wasprepared by treating WT and HET mice with 6-n-propylthiouracil (PTU) exactly as previously described [20] Allprocedures conformed to guidelines published by the NIH(Guide for the Care and Use of Laboratory Animals publi-cation number 86-23 revised 1996) and were approved bythe Institutional Animal Care and Use Committee at theUniversity of Cincinnati

22 Evaluation of Cardiac Function Anesthesia of mice withketamine and inactin analysis of cardiovascular functionusing pressure transducers inserted into the left ventricle

and right femoral vein delivery of drugs via a cannula inthe right femoral vein and recording and analysis of datawere performed exactly as described previously [13] Analysisof cardiac function by M-mode echocardiography of miceanesthetized using isoflurane inhalation and analysis of datawere performed exactly as described previously [21]

23 Immunoblot Analyses Hearts were harvested from anes-thetized mice and processed for immunoblot analysis as pre-viously described [22] Phosphorylation of phospholamban(PLN) in response to 120573-adrenergic stimulation was assessedin ventricles from mice that were anesthetized and surgicallyinstrumented as described above and treated with dobu-tamine (16 ngg body weightmin) Estimation of proteinconcentration in total homogenates resolution of proteins bydiscontinuous reducing SDS-PAGE and immunoblot analy-ses were carried out as described [22] All primary and sec-ondary antibodies used have been previously described [22]

24 Real-Time Polymerase Chain Reaction Heartsventricleswere harvested from anesthetized mice and processed forreal-time PCR (RT-PCR) analysis as previously described[22] In addition to the primer pairs that have been pre-viously described [22 23] the following were used Hspa5(GRP78BiP) PrimerBank ID number 31981722a1 Hsp90b1(GRP94) PrimerBank ID number 6755863a1 Casp12 (Cas-pase 12) PrimerBank ID number 31981868a1Ddit3 (CHOP)PrimerBank ID number 31982415a1 Eif2ak3 (PERK) Primer-Bank ID number 6857781a1 Acox1 PrimerBank ID num-ber 26333821a1 Fabp3 PrimerBank ID number 6753810a1Orai1 PrimerBank ID number 93277106b1 Stim1 Primer-Bank ID number 31981983a2 Hax1 PrimerBank ID number6754160a1 Rcan2 PrimerBank ID number 46560586c1 andPpar120574 PrimerBank ID number 187960104c1 primers for Pln(phospholamban) were adapted from PrimerBank ID num-ber 213512815c1 (forward primer 51015840 AAGTGCAATACC-TCACTCG 31015840 reverse primer 51015840 GATCAGCAGCAGACA-TATC 31015840) mRNA levels forAtp2b1 (QT01072106) andAtp2b4(QT01076271) were determined using QuantiTect PrimerAssay Kits (Qiagen)

25 Statistics Results are presented as means plusmn standarderror (SE) Individual comparisons were performed using atwo-sided Studentrsquos 119905-test and a 119875 value of lt005 was con-sidered significant

3 Results

31 Cardiovascular Performance in WT and HET MiceCardiovascular performance of adult FVBN WT and HETmice was analyzed using a pressure transducer in the leftventricle under both basal conditions and upon 120573-adrenergicstimulation with dobutamine No significant differences wereobserved in basal heart rate mean arterial pressure left ven-tricular end-diastolic pressure or maximum rates of left ven-tricular pressure development or decay (Figure 1) Treatmentwith dobutamine led to similar changes in both genotypeswith no impairment of chronotropic inotropic or lusitropicresponses Rate of left ventricular pressure development was


400

500

600H

R (b

pm)


(a)

50

70

90


2

MA

P (m

mH

g)

(b)

1

3

5


LVED

P (m

mH

g)

(c)

9k

15k

21k


+dPdt

max

(mm

Hg

s)

(d)

HETWild-type

9k

13k

17k


dPdt40

(mm

Hg

s)

(e)

HETWild-type


minusdPdt

max

(mm

Hg

s)

minus11k

minus9k

minus7k

(f)



50

WT HET

lowast

100

Atp2

a2 G

apdh

( le

vels)

(a)

SERCA2a

sactin

RYR2

LTCC1205722

NCX1

WT

HET

WT

HET

WT

HET

(b)

50

WT HET

lowast

100

SERC

A2

a s

actin

( le

vels)

(c)

WT HET

50

100

lowast s

actin

( le

vels)

LTCC

1205722

(d)

WT

HET

WT

HET

WT

HET

PS16

PT17

PLN

(e)

Figure 2 Continued


WT

HET

WT

HET

WT

HET

sactin

PS16

PT17

(f)

50

100

PS16

sac

tin (

leve

ls)

WT HET

(g)

50

100

PT17

sac

tin (

leve

ls)

150

WT HET

lowast

(h)















250

280

310

340H

R (b

pm)

0 1 2 4 8 16 32

Dobutamine (nggmin)

(a)

40

55

70

MA

P (m

mH

g)

0 1 2 4 8 16 32

Dobutamine (nggmin)

(b)

65

85

Systo

lic L

VP

(mm

Hg)

0 1 2 4 8 16 32

Dobutamine (nggmin)

(c)

9k

5k

13k

+dPdt

max

(mm

Hg

s)

0 1 2 4 8 16 32

Dobutamine (nggmin)

(d)

0 1 2 4 8 16 32

Dobutamine (nggmin)

HETWild-type

11k

8k

5k

dPdt40

(mm

Hg

s)

(e)

0 1 2 4 8 16 32

Dobutamine (nggmin)

minus8k

minus6k

minus4k

minusdPdt

max

(mm

Hg

s)

HETWild-type

(f)



2

4

6

DCM DCMHET

HW

BW

ratio

(mg

g)4

8

DCM DCMHET

HW

TL

ratio

(mg

mm

)

(a)

10

20

DCM DCMHET

Frac

tiona

l sho

rten

ing

()

30

60

DCM DCMHET

Ejec

tion

frac

tion

()

(b)

500

1000

WT DCM DCMHET

lowast

lowast

Nppa

Gap

dh(

leve

ls)

WT DCM DCMHET

Acta

1 G

apdh

( le

vels)

50

100

500

1000

WT DCM DCMHET

Myh

7 G

apdh

( le

vels)

lowast

(c)

Figure 4 Continued


WT DCM DCMHET

50

100

lowast

lowast

Atp2

a2 G

apdh

( le

vels)

dagger

(d)

DCM

DCM

DCM

DCM

HET

DCM

HET

DCM

HET

SERCA2a

sactin

50

100

DCM DCMHETSE

RCA2

a s

actin

( le

vels)

+

(e)







50

100

Surv

ival

( le

vels)

HCM HCMHET

(1416)

(315)

(a)

HCM HCMHET

(b)

4

8

HCM HCMHET

HW

BW

ratio

(mg

g)

dagger

(c)

4

2

HCM HCMHET

dagger

VW

BW

ratio

(mg

g)

(d)

100

200

HCM HCMHET

dagger

Nppa

Gap

dh(

leve

ls)

(e)

100

200

300

HCM HCMHET

dagger

Myh

7 G

apdh

( le

vels)

(f)

50

150

100

HCM HCMHET

dagger

Acta

1 G

apdh

( le

vels)

(g)

100

200

HCM HCMHET

dagger

Ctgf

Gap

dh(

leve

ls)

(h)



dagger

lowast

lowast

50

100

WT HCM HCMHET

Atp2

a2 G

apdh

( le

vels)

(a)

WT HCM HCMHET

daggerlowast

50

100

Gap

dh(

leve

ls)Pl

n

(b)

HCM

HCM

HET

HCM

HCM

HET

HCM

HCM

HET

SERCA2a

PLN

sactin

(c)

dagger

50

100

HCM HCMHET

SERC

A2

a s

actin

( le

vels)

(d)

Atp2

b4(P

mca

4) G

apdh

( le

vels)

lowast

50

100

WT HCM HCMHET

lowast

(e)

Figure 6 Continued


Atp2

b1(P

mca

1) G

apdh

( le

vels)

dagger

WT HCM HCMHET

50

100

(f)

WT HCM HCMHET

Ora

i1 G

apdh

( le

vels)

dagger

50

100

150

lowast

(g)

WT HCM HCMHET

Stim

1 G

apdh

( le

vels)

50

100

150

dagger

lowast

(h)

HCM

HCM

HET

HCM

HCM

HET

HCM

HCM

HET

sactin

PP1-C

PP2A-C

CnA

(i)

Figure 6 Continued


dagger50

100

PP1

-C s

actin

( le

vels)

HCM HCMHET

(j)

lowast

lowast

100

Rcan

1 G

apdh

( le

vels)

300

200

WT HCM HCMHET

(k)

Rcan

2 G

apdh

( le

vels)

dagger

50

100

WT HCM HCMHET

lowast

(l)






WT HCM

Hsp

90b1

(Grp94

) G

apdh

( le

vels)

100

50

100

50

Hsp

a4(B

iP)

Gap

dh(

leve

ls)

WT HCM

(a)

50

100

150

Eif2

ak3

(PER

K) G

apdh

( le

vels)

lowast

WT HCM

(b)

lowast

Ddi

t3(C

HO

P) G

apdh

( le

vels)

WT HCM

50

100

150

(c)

lowast

Casp

12 G

apdh

( le

vels)

WT HCM

50

150

100

(d)

50

150

100

lowast

Hax

1 G

apdh

( le

vels)

WT HCM

(e)

50

100

Hsp

a4(B

iP)

Gap

dh(

leve

ls)


50

100

Hsp

90b1

(Grp94

) G

apdh

( le

vels)

(f)

HCM HCMHET

Eif2

ak3

(PER

K) G

apdh

( le

vels)

50

100

(g)

Figure 7 Continued


HCM HCMHET

50

150

100

dagger

Ddi

t3(C

HO

P) G

apdh

( le

vels)

(h)

HCM HCMHET

dagger

50

100

Casp

12 G

apdh

( le

vels)

(i)

HCM HCMHET

dagger

50

100

Hax

1 G

apdh

( le

vels)

(j)







50

100

Ppar120574

Gap

dh(

leve

ls)

daggerlowast

WT HCM HCMHET

(a)

Fabp

3 G

apdh

( le

vels)

WT HCM HCMHET

50

100

daggerlowast

lowast

(b)

WT HCM HCMHET

50

100

daggerlowast

lowast

Cpt1

b G

apdh

( le

vels)

(c)

WT HCM HCMHET

50

100

daggerlowast

Acac

b G

apdh

( le

vels)

(d)

WT HCM HCMHET

50

100

daggerlowast

Acox

1 G

apdh

( le

vels)

(e)

WT HCM HCMHET

50

100

daggerlowast

lowast

Slc2

a4(G

LUT4

) G

apdh

( le

vels)

(f)









4 Discussion


















Acknowledgments


References

















































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















400

500

600H

R (b

pm)


(a)

50

70

90


2

MA

P (m

mH

g)

(b)

1

3

5


LVED

P (m

mH

g)

(c)

9k

15k

21k


+dPdt

max

(mm

Hg

s)

(d)

HETWild-type

9k

13k

17k


dPdt40

(mm

Hg

s)

(e)

HETWild-type


minusdPdt

max

(mm

Hg

s)

minus11k

minus9k

minus7k

(f)



50

WT HET

lowast

100

Atp2

a2 G

apdh

( le

vels)

(a)

SERCA2a

sactin

RYR2

LTCC1205722

NCX1

WT

HET

WT

HET

WT

HET

(b)

50

WT HET

lowast

100

SERC

A2

a s

actin

( le

vels)

(c)

WT HET

50

100

lowast s

actin

( le

vels)

LTCC

1205722

(d)

WT

HET

WT

HET

WT

HET

PS16

PT17

PLN

(e)

Figure 2 Continued


WT

HET

WT

HET

WT

HET

sactin

PS16

PT17

(f)

50

100

PS16

sac

tin (

leve

ls)

WT HET

(g)

50

100

PT17

sac

tin (

leve

ls)

150

WT HET

lowast

(h)















250

280

310

340H

R (b

pm)

0 1 2 4 8 16 32

Dobutamine (nggmin)

(a)

40

55

70

MA

P (m

mH

g)

0 1 2 4 8 16 32

Dobutamine (nggmin)

(b)

65

85

Systo

lic L

VP

(mm

Hg)

0 1 2 4 8 16 32

Dobutamine (nggmin)

(c)

9k

5k

13k

+dPdt

max

(mm

Hg

s)

0 1 2 4 8 16 32

Dobutamine (nggmin)

(d)

0 1 2 4 8 16 32

Dobutamine (nggmin)

HETWild-type

11k

8k

5k

dPdt40

(mm

Hg

s)

(e)

0 1 2 4 8 16 32

Dobutamine (nggmin)

minus8k

minus6k

minus4k

minusdPdt

max

(mm

Hg

s)

HETWild-type

(f)



2

4

6

DCM DCMHET

HW

BW

ratio

(mg

g)4

8

DCM DCMHET

HW

TL

ratio

(mg

mm

)

(a)

10

20

DCM DCMHET

Frac

tiona

l sho

rten

ing

()

30

60

DCM DCMHET

Ejec

tion

frac

tion

()

(b)

500

1000

WT DCM DCMHET

lowast

lowast

Nppa

Gap

dh(

leve

ls)

WT DCM DCMHET

Acta

1 G

apdh

( le

vels)

50

100

500

1000

WT DCM DCMHET

Myh

7 G

apdh

( le

vels)

lowast

(c)

Figure 4 Continued


WT DCM DCMHET

50

100

lowast

lowast

Atp2

a2 G

apdh

( le

vels)

dagger

(d)

DCM

DCM

DCM

DCM

HET

DCM

HET

DCM

HET

SERCA2a

sactin

50

100

DCM DCMHETSE

RCA2

a s

actin

( le

vels)

+

(e)







50

100

Surv

ival

( le

vels)

HCM HCMHET

(1416)

(315)

(a)

HCM HCMHET

(b)

4

8

HCM HCMHET

HW

BW

ratio

(mg

g)

dagger

(c)

4

2

HCM HCMHET

dagger

VW

BW

ratio

(mg

g)

(d)

100

200

HCM HCMHET

dagger

Nppa

Gap

dh(

leve

ls)

(e)

100

200

300

HCM HCMHET

dagger

Myh

7 G

apdh

( le

vels)

(f)

50

150

100

HCM HCMHET

dagger

Acta

1 G

apdh

( le

vels)

(g)

100

200

HCM HCMHET

dagger

Ctgf

Gap

dh(

leve

ls)

(h)



dagger

lowast

lowast

50

100

WT HCM HCMHET

Atp2

a2 G

apdh

( le

vels)

(a)

WT HCM HCMHET

daggerlowast

50

100

Gap

dh(

leve

ls)Pl

n

(b)

HCM

HCM

HET

HCM

HCM

HET

HCM

HCM

HET

SERCA2a

PLN

sactin

(c)

dagger

50

100

HCM HCMHET

SERC

A2

a s

actin

( le

vels)

(d)

Atp2

b4(P

mca

4) G

apdh

( le

vels)

lowast

50

100

WT HCM HCMHET

lowast

(e)

Figure 6 Continued


Atp2

b1(P

mca

1) G

apdh

( le

vels)

dagger

WT HCM HCMHET

50

100

(f)

WT HCM HCMHET

Ora

i1 G

apdh

( le

vels)

dagger

50

100

150

lowast

(g)

WT HCM HCMHET

Stim

1 G

apdh

( le

vels)

50

100

150

dagger

lowast

(h)

HCM

HCM

HET

HCM

HCM

HET

HCM

HCM

HET

sactin

PP1-C

PP2A-C

CnA

(i)

Figure 6 Continued


dagger50

100

PP1

-C s

actin

( le

vels)

HCM HCMHET

(j)

lowast

lowast

100

Rcan

1 G

apdh

( le

vels)

300

200

WT HCM HCMHET

(k)

Rcan

2 G

apdh

( le

vels)

dagger

50

100

WT HCM HCMHET

lowast

(l)






WT HCM

Hsp

90b1

(Grp94

) G

apdh

( le

vels)

100

50

100

50

Hsp

a4(B

iP)

Gap

dh(

leve

ls)

WT HCM

(a)

50

100

150

Eif2

ak3

(PER

K) G

apdh

( le

vels)

lowast

WT HCM

(b)

lowast

Ddi

t3(C

HO

P) G

apdh

( le

vels)

WT HCM

50

100

150

(c)

lowast

Casp

12 G

apdh

( le

vels)

WT HCM

50

150

100

(d)

50

150

100

lowast

Hax

1 G

apdh

( le

vels)

WT HCM

(e)

50

100

Hsp

a4(B

iP)

Gap

dh(

leve

ls)


50

100

Hsp

90b1

(Grp94

) G

apdh

( le

vels)

(f)

HCM HCMHET

Eif2

ak3

(PER

K) G

apdh

( le

vels)

50

100

(g)

Figure 7 Continued


HCM HCMHET

50

150

100

dagger

Ddi

t3(C

HO

P) G

apdh

( le

vels)

(h)

HCM HCMHET

dagger

50

100

Casp

12 G

apdh

( le

vels)

(i)

HCM HCMHET

dagger

50

100

Hax

1 G

apdh

( le

vels)

(j)







50

100

Ppar120574

Gap

dh(

leve

ls)

daggerlowast

WT HCM HCMHET

(a)

Fabp

3 G

apdh

( le

vels)

WT HCM HCMHET

50

100

daggerlowast

lowast

(b)

WT HCM HCMHET

50

100

daggerlowast

lowast

Cpt1

b G

apdh

( le

vels)

(c)

WT HCM HCMHET

50

100

daggerlowast

Acac

b G

apdh

( le

vels)

(d)

WT HCM HCMHET

50

100

daggerlowast

Acox

1 G

apdh

( le

vels)

(e)

WT HCM HCMHET

50

100

daggerlowast

lowast

Slc2

a4(G

LUT4

) G

apdh

( le

vels)

(f)









4 Discussion


















Acknowledgments


References

















































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















50

WT HET

lowast

100

Atp2

a2 G

apdh

( le

vels)

(a)

SERCA2a

sactin

RYR2

LTCC1205722

NCX1

WT

HET

WT

HET

WT

HET

(b)

50

WT HET

lowast

100

SERC

A2

a s

actin

( le

vels)

(c)

WT HET

50

100

lowast s

actin

( le

vels)

LTCC

1205722

(d)

WT

HET

WT

HET

WT

HET

PS16

PT17

PLN

(e)

Figure 2 Continued


WT

HET

WT

HET

WT

HET

sactin

PS16

PT17

(f)

50

100

PS16

sac

tin (

leve

ls)

WT HET

(g)

50

100

PT17

sac

tin (

leve

ls)

150

WT HET

lowast

(h)















250

280

310

340H

R (b

pm)

0 1 2 4 8 16 32

Dobutamine (nggmin)

(a)

40

55

70

MA

P (m

mH

g)

0 1 2 4 8 16 32

Dobutamine (nggmin)

(b)

65

85

Systo

lic L

VP

(mm

Hg)

0 1 2 4 8 16 32

Dobutamine (nggmin)

(c)

9k

5k

13k

+dPdt

max

(mm

Hg

s)

0 1 2 4 8 16 32

Dobutamine (nggmin)

(d)

0 1 2 4 8 16 32

Dobutamine (nggmin)

HETWild-type

11k

8k

5k

dPdt40

(mm

Hg

s)

(e)

0 1 2 4 8 16 32

Dobutamine (nggmin)

minus8k

minus6k

minus4k

minusdPdt

max

(mm

Hg

s)

HETWild-type

(f)



2

4

6

DCM DCMHET

HW

BW

ratio

(mg

g)4

8

DCM DCMHET

HW

TL

ratio

(mg

mm

)

(a)

10

20

DCM DCMHET

Frac

tiona

l sho

rten

ing

()

30

60

DCM DCMHET

Ejec

tion

frac

tion

()

(b)

500

1000

WT DCM DCMHET

lowast

lowast

Nppa

Gap

dh(

leve

ls)

WT DCM DCMHET

Acta

1 G

apdh

( le

vels)

50

100

500

1000

WT DCM DCMHET

Myh

7 G

apdh

( le

vels)

lowast

(c)

Figure 4 Continued


WT DCM DCMHET

50

100

lowast

lowast

Atp2

a2 G

apdh

( le

vels)

dagger

(d)

DCM

DCM

DCM

DCM

HET

DCM

HET

DCM

HET

SERCA2a

sactin

50

100

DCM DCMHETSE

RCA2

a s

actin

( le

vels)

+

(e)







50

100

Surv

ival

( le

vels)

HCM HCMHET

(1416)

(315)

(a)

HCM HCMHET

(b)

4

8

HCM HCMHET

HW

BW

ratio

(mg

g)

dagger

(c)

4

2

HCM HCMHET

dagger

VW

BW

ratio

(mg

g)

(d)

100

200

HCM HCMHET

dagger

Nppa

Gap

dh(

leve

ls)

(e)

100

200

300

HCM HCMHET

dagger

Myh

7 G

apdh

( le

vels)

(f)

50

150

100

HCM HCMHET

dagger

Acta

1 G

apdh

( le

vels)

(g)

100

200

HCM HCMHET

dagger

Ctgf

Gap

dh(

leve

ls)

(h)



dagger

lowast

lowast

50

100

WT HCM HCMHET

Atp2

a2 G

apdh

( le

vels)

(a)

WT HCM HCMHET

daggerlowast

50

100

Gap

dh(

leve

ls)Pl

n

(b)

HCM

HCM

HET

HCM

HCM

HET

HCM

HCM

HET

SERCA2a

PLN

sactin

(c)

dagger

50

100

HCM HCMHET

SERC

A2

a s

actin

( le

vels)

(d)

Atp2

b4(P

mca

4) G

apdh

( le

vels)

lowast

50

100

WT HCM HCMHET

lowast

(e)

Figure 6 Continued


Atp2

b1(P

mca

1) G

apdh

( le

vels)

dagger

WT HCM HCMHET

50

100

(f)

WT HCM HCMHET

Ora

i1 G

apdh

( le

vels)

dagger

50

100

150

lowast

(g)

WT HCM HCMHET

Stim

1 G

apdh

( le

vels)

50

100

150

dagger

lowast

(h)

HCM

HCM

HET

HCM

HCM

HET

HCM

HCM

HET

sactin

PP1-C

PP2A-C

CnA

(i)

Figure 6 Continued


dagger50

100

PP1

-C s

actin

( le

vels)

HCM HCMHET

(j)

lowast

lowast

100

Rcan

1 G

apdh

( le

vels)

300

200

WT HCM HCMHET

(k)

Rcan

2 G

apdh

( le

vels)

dagger

50

100

WT HCM HCMHET

lowast

(l)






WT HCM

Hsp

90b1

(Grp94

) G

apdh

( le

vels)

100

50

100

50

Hsp

a4(B

iP)

Gap

dh(

leve

ls)

WT HCM

(a)

50

100

150

Eif2

ak3

(PER

K) G

apdh

( le

vels)

lowast

WT HCM

(b)

lowast

Ddi

t3(C

HO

P) G

apdh

( le

vels)

WT HCM

50

100

150

(c)

lowast

Casp

12 G

apdh

( le

vels)

WT HCM

50

150

100

(d)

50

150

100

lowast

Hax

1 G

apdh

( le

vels)

WT HCM

(e)

50

100

Hsp

a4(B

iP)

Gap

dh(

leve

ls)


50

100

Hsp

90b1

(Grp94

) G

apdh

( le

vels)

(f)

HCM HCMHET

Eif2

ak3

(PER

K) G

apdh

( le

vels)

50

100

(g)

Figure 7 Continued


HCM HCMHET

50

150

100

dagger

Ddi

t3(C

HO

P) G

apdh

( le

vels)

(h)

HCM HCMHET

dagger

50

100

Casp

12 G

apdh

( le

vels)

(i)

HCM HCMHET

dagger

50

100

Hax

1 G

apdh

( le

vels)

(j)







50

100

Ppar120574

Gap

dh(

leve

ls)

daggerlowast

WT HCM HCMHET

(a)

Fabp

3 G

apdh

( le

vels)

WT HCM HCMHET

50

100

daggerlowast

lowast

(b)

WT HCM HCMHET

50

100

daggerlowast

lowast

Cpt1

b G

apdh

( le

vels)

(c)

WT HCM HCMHET

50

100

daggerlowast

Acac

b G

apdh

( le

vels)

(d)

WT HCM HCMHET

50

100

daggerlowast

Acox

1 G

apdh

( le

vels)

(e)

WT HCM HCMHET

50

100

daggerlowast

lowast

Slc2

a4(G

LUT4

) G

apdh

( le

vels)

(f)









4 Discussion


















Acknowledgments


References

















































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















WT

HET

WT

HET

WT

HET

sactin

PS16

PT17

(f)

50

100

PS16

sac

tin (

leve

ls)

WT HET

(g)

50

100

PT17

sac

tin (

leve

ls)

150

WT HET

lowast

(h)















250

280

310

340H

R (b

pm)

0 1 2 4 8 16 32

Dobutamine (nggmin)

(a)

40

55

70

MA

P (m

mH

g)

0 1 2 4 8 16 32

Dobutamine (nggmin)

(b)

65

85

Systo

lic L

VP

(mm

Hg)

0 1 2 4 8 16 32

Dobutamine (nggmin)

(c)

9k

5k

13k

+dPdt

max

(mm

Hg

s)

0 1 2 4 8 16 32

Dobutamine (nggmin)

(d)

0 1 2 4 8 16 32

Dobutamine (nggmin)

HETWild-type

11k

8k

5k

dPdt40

(mm

Hg

s)

(e)

0 1 2 4 8 16 32

Dobutamine (nggmin)

minus8k

minus6k

minus4k

minusdPdt

max

(mm

Hg

s)

HETWild-type

(f)



2

4

6

DCM DCMHET

HW

BW

ratio

(mg

g)4

8

DCM DCMHET

HW

TL

ratio

(mg

mm

)

(a)

10

20

DCM DCMHET

Frac

tiona

l sho

rten

ing

()

30

60

DCM DCMHET

Ejec

tion

frac

tion

()

(b)

500

1000

WT DCM DCMHET

lowast

lowast

Nppa

Gap

dh(

leve

ls)

WT DCM DCMHET

Acta

1 G

apdh

( le

vels)

50

100

500

1000

WT DCM DCMHET

Myh

7 G

apdh

( le

vels)

lowast

(c)

Figure 4 Continued


WT DCM DCMHET

50

100

lowast

lowast

Atp2

a2 G

apdh

( le

vels)

dagger

(d)

DCM

DCM

DCM

DCM

HET

DCM

HET

DCM

HET

SERCA2a

sactin

50

100

DCM DCMHETSE

RCA2

a s

actin

( le

vels)

+

(e)







50

100

Surv

ival

( le

vels)

HCM HCMHET

(1416)

(315)

(a)

HCM HCMHET

(b)

4

8

HCM HCMHET

HW

BW

ratio

(mg

g)

dagger

(c)

4

2

HCM HCMHET

dagger

VW

BW

ratio

(mg

g)

(d)

100

200

HCM HCMHET

dagger

Nppa

Gap

dh(

leve

ls)

(e)

100

200

300

HCM HCMHET

dagger

Myh

7 G

apdh

( le

vels)

(f)

50

150

100

HCM HCMHET

dagger

Acta

1 G

apdh

( le

vels)

(g)

100

200

HCM HCMHET

dagger

Ctgf

Gap

dh(

leve

ls)

(h)



dagger

lowast

lowast

50

100

WT HCM HCMHET

Atp2

a2 G

apdh

( le

vels)

(a)

WT HCM HCMHET

daggerlowast

50

100

Gap

dh(

leve

ls)Pl

n

(b)

HCM

HCM

HET

HCM

HCM

HET

HCM

HCM

HET

SERCA2a

PLN

sactin

(c)

dagger

50

100

HCM HCMHET

SERC

A2

a s

actin

( le

vels)

(d)

Atp2

b4(P

mca

4) G

apdh

( le

vels)

lowast

50

100

WT HCM HCMHET

lowast

(e)

Figure 6 Continued


Atp2

b1(P

mca

1) G

apdh

( le

vels)

dagger

WT HCM HCMHET

50

100

(f)

WT HCM HCMHET

Ora

i1 G

apdh

( le

vels)

dagger

50

100

150

lowast

(g)

WT HCM HCMHET

Stim

1 G

apdh

( le

vels)

50

100

150

dagger

lowast

(h)

HCM

HCM

HET

HCM

HCM

HET

HCM

HCM

HET

sactin

PP1-C

PP2A-C

CnA

(i)

Figure 6 Continued


dagger50

100

PP1

-C s

actin

( le

vels)

HCM HCMHET

(j)

lowast

lowast

100

Rcan

1 G

apdh

( le

vels)

300

200

WT HCM HCMHET

(k)

Rcan

2 G

apdh

( le

vels)

dagger

50

100

WT HCM HCMHET

lowast

(l)






WT HCM

Hsp

90b1

(Grp94

) G

apdh

( le

vels)

100

50

100

50

Hsp

a4(B

iP)

Gap

dh(

leve

ls)

WT HCM

(a)

50

100

150

Eif2

ak3

(PER

K) G

apdh

( le

vels)

lowast

WT HCM

(b)

lowast

Ddi

t3(C

HO

P) G

apdh

( le

vels)

WT HCM

50

100

150

(c)

lowast

Casp

12 G

apdh

( le

vels)

WT HCM

50

150

100

(d)

50

150

100

lowast

Hax

1 G

apdh

( le

vels)

WT HCM

(e)

50

100

Hsp

a4(B

iP)

Gap

dh(

leve

ls)


50

100

Hsp

90b1

(Grp94

) G

apdh

( le

vels)

(f)

HCM HCMHET

Eif2

ak3

(PER

K) G

apdh

( le

vels)

50

100

(g)

Figure 7 Continued


HCM HCMHET

50

150

100

dagger

Ddi

t3(C

HO

P) G

apdh

( le

vels)

(h)

HCM HCMHET

dagger

50

100

Casp

12 G

apdh

( le

vels)

(i)

HCM HCMHET

dagger

50

100

Hax

1 G

apdh

( le

vels)

(j)







50

100

Ppar120574

Gap

dh(

leve

ls)

daggerlowast

WT HCM HCMHET

(a)

Fabp

3 G

apdh

( le

vels)

WT HCM HCMHET

50

100

daggerlowast

lowast

(b)

WT HCM HCMHET

50

100

daggerlowast

lowast

Cpt1

b G

apdh

( le

vels)

(c)

WT HCM HCMHET

50

100

daggerlowast

Acac

b G

apdh

( le

vels)

(d)

WT HCM HCMHET

50

100

daggerlowast

Acox

1 G

apdh

( le

vels)

(e)

WT HCM HCMHET

50

100

daggerlowast

lowast

Slc2

a4(G

LUT4

) G

apdh

( le

vels)

(f)









4 Discussion


















Acknowledgments


References

















































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


























250

280

310

340H

R (b

pm)

0 1 2 4 8 16 32

Dobutamine (nggmin)

(a)

40

55

70

MA

P (m

mH

g)

0 1 2 4 8 16 32

Dobutamine (nggmin)

(b)

65

85

Systo

lic L

VP

(mm

Hg)

0 1 2 4 8 16 32

Dobutamine (nggmin)

(c)

9k

5k

13k

+dPdt

max

(mm

Hg

s)

0 1 2 4 8 16 32

Dobutamine (nggmin)

(d)

0 1 2 4 8 16 32

Dobutamine (nggmin)

HETWild-type

11k

8k

5k

dPdt40

(mm

Hg

s)

(e)

0 1 2 4 8 16 32

Dobutamine (nggmin)

minus8k

minus6k

minus4k

minusdPdt

max

(mm

Hg

s)

HETWild-type

(f)



2

4

6

DCM DCMHET

HW

BW

ratio

(mg

g)4

8

DCM DCMHET

HW

TL

ratio

(mg

mm

)

(a)

10

20

DCM DCMHET

Frac

tiona

l sho

rten

ing

()

30

60

DCM DCMHET

Ejec

tion

frac

tion

()

(b)

500

1000

WT DCM DCMHET

lowast

lowast

Nppa

Gap

dh(

leve

ls)

WT DCM DCMHET

Acta

1 G

apdh

( le

vels)

50

100

500

1000

WT DCM DCMHET

Myh

7 G

apdh

( le

vels)

lowast

(c)

Figure 4 Continued


WT DCM DCMHET

50

100

lowast

lowast

Atp2

a2 G

apdh

( le

vels)

dagger

(d)

DCM

DCM

DCM

DCM

HET

DCM

HET

DCM

HET

SERCA2a

sactin

50

100

DCM DCMHETSE

RCA2

a s

actin

( le

vels)

+

(e)







50

100

Surv

ival

( le

vels)

HCM HCMHET

(1416)

(315)

(a)

HCM HCMHET

(b)

4

8

HCM HCMHET

HW

BW

ratio

(mg

g)

dagger

(c)

4

2

HCM HCMHET

dagger

VW

BW

ratio

(mg

g)

(d)

100

200

HCM HCMHET

dagger

Nppa

Gap

dh(

leve

ls)

(e)

100

200

300

HCM HCMHET

dagger

Myh

7 G

apdh

( le

vels)

(f)

50

150

100

HCM HCMHET

dagger

Acta

1 G

apdh

( le

vels)

(g)

100

200

HCM HCMHET

dagger

Ctgf

Gap

dh(

leve

ls)

(h)



dagger

lowast

lowast

50

100

WT HCM HCMHET

Atp2

a2 G

apdh

( le

vels)

(a)

WT HCM HCMHET

daggerlowast

50

100

Gap

dh(

leve

ls)Pl

n

(b)

HCM

HCM

HET

HCM

HCM

HET

HCM

HCM

HET

SERCA2a

PLN

sactin

(c)

dagger

50

100

HCM HCMHET

SERC

A2

a s

actin

( le

vels)

(d)

Atp2

b4(P

mca

4) G

apdh

( le

vels)

lowast

50

100

WT HCM HCMHET

lowast

(e)

Figure 6 Continued


Atp2

b1(P

mca

1) G

apdh

( le

vels)

dagger

WT HCM HCMHET

50

100

(f)

WT HCM HCMHET

Ora

i1 G

apdh

( le

vels)

dagger

50

100

150

lowast

(g)

WT HCM HCMHET

Stim

1 G

apdh

( le

vels)

50

100

150

dagger

lowast

(h)

HCM

HCM

HET

HCM

HCM

HET

HCM

HCM

HET

sactin

PP1-C

PP2A-C

CnA

(i)

Figure 6 Continued


dagger50

100

PP1

-C s

actin

( le

vels)

HCM HCMHET

(j)

lowast

lowast

100

Rcan

1 G

apdh

( le

vels)

300

200

WT HCM HCMHET

(k)

Rcan

2 G

apdh

( le

vels)

dagger

50

100

WT HCM HCMHET

lowast

(l)






WT HCM

Hsp

90b1

(Grp94

) G

apdh

( le

vels)

100

50

100

50

Hsp

a4(B

iP)

Gap

dh(

leve

ls)

WT HCM

(a)

50

100

150

Eif2

ak3

(PER

K) G

apdh

( le

vels)

lowast

WT HCM

(b)

lowast

Ddi

t3(C

HO

P) G

apdh

( le

vels)

WT HCM

50

100

150

(c)

lowast

Casp

12 G

apdh

( le

vels)

WT HCM

50

150

100

(d)

50

150

100

lowast

Hax

1 G

apdh

( le

vels)

WT HCM

(e)

50

100

Hsp

a4(B

iP)

Gap

dh(

leve

ls)


50

100

Hsp

90b1

(Grp94

) G

apdh

( le

vels)

(f)

HCM HCMHET

Eif2

ak3

(PER

K) G

apdh

( le

vels)

50

100

(g)

Figure 7 Continued


HCM HCMHET

50

150

100

dagger

Ddi

t3(C

HO

P) G

apdh

( le

vels)

(h)

HCM HCMHET

dagger

50

100

Casp

12 G

apdh

( le

vels)

(i)

HCM HCMHET

dagger

50

100

Hax

1 G

apdh

( le

vels)

(j)







50

100

Ppar120574

Gap

dh(

leve

ls)

daggerlowast

WT HCM HCMHET

(a)

Fabp

3 G

apdh

( le

vels)

WT HCM HCMHET

50

100

daggerlowast

lowast

(b)

WT HCM HCMHET

50

100

daggerlowast

lowast

Cpt1

b G

apdh

( le

vels)

(c)

WT HCM HCMHET

50

100

daggerlowast

Acac

b G

apdh

( le

vels)

(d)

WT HCM HCMHET

50

100

daggerlowast

Acox

1 G

apdh

( le

vels)

(e)

WT HCM HCMHET

50

100

daggerlowast

lowast

Slc2

a4(G

LUT4

) G

apdh

( le

vels)

(f)









4 Discussion


















Acknowledgments


References

















































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















250

280

310

340H

R (b

pm)

0 1 2 4 8 16 32

Dobutamine (nggmin)

(a)

40

55

70

MA

P (m

mH

g)

0 1 2 4 8 16 32

Dobutamine (nggmin)

(b)

65

85

Systo

lic L

VP

(mm

Hg)

0 1 2 4 8 16 32

Dobutamine (nggmin)

(c)

9k

5k

13k

+dPdt

max

(mm

Hg

s)

0 1 2 4 8 16 32

Dobutamine (nggmin)

(d)

0 1 2 4 8 16 32

Dobutamine (nggmin)

HETWild-type

11k

8k

5k

dPdt40

(mm

Hg

s)

(e)

0 1 2 4 8 16 32

Dobutamine (nggmin)

minus8k

minus6k

minus4k

minusdPdt

max

(mm

Hg

s)

HETWild-type

(f)



2

4

6

DCM DCMHET

HW

BW

ratio

(mg

g)4

8

DCM DCMHET

HW

TL

ratio

(mg

mm

)

(a)

10

20

DCM DCMHET

Frac

tiona

l sho

rten

ing

()

30

60

DCM DCMHET

Ejec

tion

frac

tion

()

(b)

500

1000

WT DCM DCMHET

lowast

lowast

Nppa

Gap

dh(

leve

ls)

WT DCM DCMHET

Acta

1 G

apdh

( le

vels)

50

100

500

1000

WT DCM DCMHET

Myh

7 G

apdh

( le

vels)

lowast

(c)

Figure 4 Continued


WT DCM DCMHET

50

100

lowast

lowast

Atp2

a2 G

apdh

( le

vels)

dagger

(d)

DCM

DCM

DCM

DCM

HET

DCM

HET

DCM

HET

SERCA2a

sactin

50

100

DCM DCMHETSE

RCA2

a s

actin

( le

vels)

+

(e)







50

100

Surv

ival

( le

vels)

HCM HCMHET

(1416)

(315)

(a)

HCM HCMHET

(b)

4

8

HCM HCMHET

HW

BW

ratio

(mg

g)

dagger

(c)

4

2

HCM HCMHET

dagger

VW

BW

ratio

(mg

g)

(d)

100

200

HCM HCMHET

dagger

Nppa

Gap

dh(

leve

ls)

(e)

100

200

300

HCM HCMHET

dagger

Myh

7 G

apdh

( le

vels)

(f)

50

150

100

HCM HCMHET

dagger

Acta

1 G

apdh

( le

vels)

(g)

100

200

HCM HCMHET

dagger

Ctgf

Gap

dh(

leve

ls)

(h)



dagger

lowast

lowast

50

100

WT HCM HCMHET

Atp2

a2 G

apdh

( le

vels)

(a)

WT HCM HCMHET

daggerlowast

50

100

Gap

dh(

leve

ls)Pl

n

(b)

HCM

HCM

HET

HCM

HCM

HET

HCM

HCM

HET

SERCA2a

PLN

sactin

(c)

dagger

50

100

HCM HCMHET

SERC

A2

a s

actin

( le

vels)

(d)

Atp2

b4(P

mca

4) G

apdh

( le

vels)

lowast

50

100

WT HCM HCMHET

lowast

(e)

Figure 6 Continued


Atp2

b1(P

mca

1) G

apdh

( le

vels)

dagger

WT HCM HCMHET

50

100

(f)

WT HCM HCMHET

Ora

i1 G

apdh

( le

vels)

dagger

50

100

150

lowast

(g)

WT HCM HCMHET

Stim

1 G

apdh

( le

vels)

50

100

150

dagger

lowast

(h)

HCM

HCM

HET

HCM

HCM

HET

HCM

HCM

HET

sactin

PP1-C

PP2A-C

CnA

(i)

Figure 6 Continued


dagger50

100

PP1

-C s

actin

( le

vels)

HCM HCMHET

(j)

lowast

lowast

100

Rcan

1 G

apdh

( le

vels)

300

200

WT HCM HCMHET

(k)

Rcan

2 G

apdh

( le

vels)

dagger

50

100

WT HCM HCMHET

lowast

(l)






WT HCM

Hsp

90b1

(Grp94

) G

apdh

( le

vels)

100

50

100

50

Hsp

a4(B

iP)

Gap

dh(

leve

ls)

WT HCM

(a)

50

100

150

Eif2

ak3

(PER

K) G

apdh

( le

vels)

lowast

WT HCM

(b)

lowast

Ddi

t3(C

HO

P) G

apdh

( le

vels)

WT HCM

50

100

150

(c)

lowast

Casp

12 G

apdh

( le

vels)

WT HCM

50

150

100

(d)

50

150

100

lowast

Hax

1 G

apdh

( le

vels)

WT HCM

(e)

50

100

Hsp

a4(B

iP)

Gap

dh(

leve

ls)


50

100

Hsp

90b1

(Grp94

) G

apdh

( le

vels)

(f)

HCM HCMHET

Eif2

ak3

(PER

K) G

apdh

( le

vels)

50

100

(g)

Figure 7 Continued


HCM HCMHET

50

150

100

dagger

Ddi

t3(C

HO

P) G

apdh

( le

vels)

(h)

HCM HCMHET

dagger

50

100

Casp

12 G

apdh

( le

vels)

(i)

HCM HCMHET

dagger

50

100

Hax

1 G

apdh

( le

vels)

(j)







50

100

Ppar120574

Gap

dh(

leve

ls)

daggerlowast

WT HCM HCMHET

(a)

Fabp

3 G

apdh

( le

vels)

WT HCM HCMHET

50

100

daggerlowast

lowast

(b)

WT HCM HCMHET

50

100

daggerlowast

lowast

Cpt1

b G

apdh

( le

vels)

(c)

WT HCM HCMHET

50

100

daggerlowast

Acac

b G

apdh

( le

vels)

(d)

WT HCM HCMHET

50

100

daggerlowast

Acox

1 G

apdh

( le

vels)

(e)

WT HCM HCMHET

50

100

daggerlowast

lowast

Slc2

a4(G

LUT4

) G

apdh

( le

vels)

(f)









4 Discussion


















Acknowledgments


References

















































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















2

4

6

DCM DCMHET

HW

BW

ratio

(mg

g)4

8

DCM DCMHET

HW

TL

ratio

(mg

mm

)

(a)

10

20

DCM DCMHET

Frac

tiona

l sho

rten

ing

()

30

60

DCM DCMHET

Ejec

tion

frac

tion

()

(b)

500

1000

WT DCM DCMHET

lowast

lowast

Nppa

Gap

dh(

leve

ls)

WT DCM DCMHET

Acta

1 G

apdh

( le

vels)

50

100

500

1000

WT DCM DCMHET

Myh

7 G

apdh

( le

vels)

lowast

(c)

Figure 4 Continued


WT DCM DCMHET

50

100

lowast

lowast

Atp2

a2 G

apdh

( le

vels)

dagger

(d)

DCM

DCM

DCM

DCM

HET

DCM

HET

DCM

HET

SERCA2a

sactin

50

100

DCM DCMHETSE

RCA2

a s

actin

( le

vels)

+

(e)







50

100

Surv

ival

( le

vels)

HCM HCMHET

(1416)

(315)

(a)

HCM HCMHET

(b)

4

8

HCM HCMHET

HW

BW

ratio

(mg

g)

dagger

(c)

4

2

HCM HCMHET

dagger

VW

BW

ratio

(mg

g)

(d)

100

200

HCM HCMHET

dagger

Nppa

Gap

dh(

leve

ls)

(e)

100

200

300

HCM HCMHET

dagger

Myh

7 G

apdh

( le

vels)

(f)

50

150

100

HCM HCMHET

dagger

Acta

1 G

apdh

( le

vels)

(g)

100

200

HCM HCMHET

dagger

Ctgf

Gap

dh(

leve

ls)

(h)



dagger

lowast

lowast

50

100

WT HCM HCMHET

Atp2

a2 G

apdh

( le

vels)

(a)

WT HCM HCMHET

daggerlowast

50

100

Gap

dh(

leve

ls)Pl

n

(b)

HCM

HCM

HET

HCM

HCM

HET

HCM

HCM

HET

SERCA2a

PLN

sactin

(c)

dagger

50

100

HCM HCMHET

SERC

A2

a s

actin

( le

vels)

(d)

Atp2

b4(P

mca

4) G

apdh

( le

vels)

lowast

50

100

WT HCM HCMHET

lowast

(e)

Figure 6 Continued


Atp2

b1(P

mca

1) G

apdh

( le

vels)

dagger

WT HCM HCMHET

50

100

(f)

WT HCM HCMHET

Ora

i1 G

apdh

( le

vels)

dagger

50

100

150

lowast

(g)

WT HCM HCMHET

Stim

1 G

apdh

( le

vels)

50

100

150

dagger

lowast

(h)

HCM

HCM

HET

HCM

HCM

HET

HCM

HCM

HET

sactin

PP1-C

PP2A-C

CnA

(i)

Figure 6 Continued


dagger50

100

PP1

-C s

actin

( le

vels)

HCM HCMHET

(j)

lowast

lowast

100

Rcan

1 G

apdh

( le

vels)

300

200

WT HCM HCMHET

(k)

Rcan

2 G

apdh

( le

vels)

dagger

50

100

WT HCM HCMHET

lowast

(l)






WT HCM

Hsp

90b1

(Grp94

) G

apdh

( le

vels)

100

50

100

50

Hsp

a4(B

iP)

Gap

dh(

leve

ls)

WT HCM

(a)

50

100

150

Eif2

ak3

(PER

K) G

apdh

( le

vels)

lowast

WT HCM

(b)

lowast

Ddi

t3(C

HO

P) G

apdh

( le

vels)

WT HCM

50

100

150

(c)

lowast

Casp

12 G

apdh

( le

vels)

WT HCM

50

150

100

(d)

50

150

100

lowast

Hax

1 G

apdh

( le

vels)

WT HCM

(e)

50

100

Hsp

a4(B

iP)

Gap

dh(

leve

ls)


50

100

Hsp

90b1

(Grp94

) G

apdh

( le

vels)

(f)

HCM HCMHET

Eif2

ak3

(PER

K) G

apdh

( le

vels)

50

100

(g)

Figure 7 Continued


HCM HCMHET

50

150

100

dagger

Ddi

t3(C

HO

P) G

apdh

( le

vels)

(h)

HCM HCMHET

dagger

50

100

Casp

12 G

apdh

( le

vels)

(i)

HCM HCMHET

dagger

50

100

Hax

1 G

apdh

( le

vels)

(j)







50

100

Ppar120574

Gap

dh(

leve

ls)

daggerlowast

WT HCM HCMHET

(a)

Fabp

3 G

apdh

( le

vels)

WT HCM HCMHET

50

100

daggerlowast

lowast

(b)

WT HCM HCMHET

50

100

daggerlowast

lowast

Cpt1

b G

apdh

( le

vels)

(c)

WT HCM HCMHET

50

100

daggerlowast

Acac

b G

apdh

( le

vels)

(d)

WT HCM HCMHET

50

100

daggerlowast

Acox

1 G

apdh

( le

vels)

(e)

WT HCM HCMHET

50

100

daggerlowast

lowast

Slc2

a4(G

LUT4

) G

apdh

( le

vels)

(f)









4 Discussion


















Acknowledgments


References

















































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















WT DCM DCMHET

50

100

lowast

lowast

Atp2

a2 G

apdh

( le

vels)

dagger

(d)

DCM

DCM

DCM

DCM

HET

DCM

HET

DCM

HET

SERCA2a

sactin

50

100

DCM DCMHETSE

RCA2

a s

actin

( le

vels)

+

(e)







50

100

Surv

ival

( le

vels)

HCM HCMHET

(1416)

(315)

(a)

HCM HCMHET

(b)

4

8

HCM HCMHET

HW

BW

ratio

(mg

g)

dagger

(c)

4

2

HCM HCMHET

dagger

VW

BW

ratio

(mg

g)

(d)

100

200

HCM HCMHET

dagger

Nppa

Gap

dh(

leve

ls)

(e)

100

200

300

HCM HCMHET

dagger

Myh

7 G

apdh

( le

vels)

(f)

50

150

100

HCM HCMHET

dagger

Acta

1 G

apdh

( le

vels)

(g)

100

200

HCM HCMHET

dagger

Ctgf

Gap

dh(

leve

ls)

(h)



dagger

lowast

lowast

50

100

WT HCM HCMHET

Atp2

a2 G

apdh

( le

vels)

(a)

WT HCM HCMHET

daggerlowast

50

100

Gap

dh(

leve

ls)Pl

n

(b)

HCM

HCM

HET

HCM

HCM

HET

HCM

HCM

HET

SERCA2a

PLN

sactin

(c)

dagger

50

100

HCM HCMHET

SERC

A2

a s

actin

( le

vels)

(d)

Atp2

b4(P

mca

4) G

apdh

( le

vels)

lowast

50

100

WT HCM HCMHET

lowast

(e)

Figure 6 Continued


Atp2

b1(P

mca

1) G

apdh

( le

vels)

dagger

WT HCM HCMHET

50

100

(f)

WT HCM HCMHET

Ora

i1 G

apdh

( le

vels)

dagger

50

100

150

lowast

(g)

WT HCM HCMHET

Stim

1 G

apdh

( le

vels)

50

100

150

dagger

lowast

(h)

HCM

HCM

HET

HCM

HCM

HET

HCM

HCM

HET

sactin

PP1-C

PP2A-C

CnA

(i)

Figure 6 Continued


dagger50

100

PP1

-C s

actin

( le

vels)

HCM HCMHET

(j)

lowast

lowast

100

Rcan

1 G

apdh

( le

vels)

300

200

WT HCM HCMHET

(k)

Rcan

2 G

apdh

( le

vels)

dagger

50

100

WT HCM HCMHET

lowast

(l)






WT HCM

Hsp

90b1

(Grp94

) G

apdh

( le

vels)

100

50

100

50

Hsp

a4(B

iP)

Gap

dh(

leve

ls)

WT HCM

(a)

50

100

150

Eif2

ak3

(PER

K) G

apdh

( le

vels)

lowast

WT HCM

(b)

lowast

Ddi

t3(C

HO

P) G

apdh

( le

vels)

WT HCM

50

100

150

(c)

lowast

Casp

12 G

apdh

( le

vels)

WT HCM

50

150

100

(d)

50

150

100

lowast

Hax

1 G

apdh

( le

vels)

WT HCM

(e)

50

100

Hsp

a4(B

iP)

Gap

dh(

leve

ls)


50

100

Hsp

90b1

(Grp94

) G

apdh

( le

vels)

(f)

HCM HCMHET

Eif2

ak3

(PER

K) G

apdh

( le

vels)

50

100

(g)

Figure 7 Continued


HCM HCMHET

50

150

100

dagger

Ddi

t3(C

HO

P) G

apdh

( le

vels)

(h)

HCM HCMHET

dagger

50

100

Casp

12 G

apdh

( le

vels)

(i)

HCM HCMHET

dagger

50

100

Hax

1 G

apdh

( le

vels)

(j)







50

100

Ppar120574

Gap

dh(

leve

ls)

daggerlowast

WT HCM HCMHET

(a)

Fabp

3 G

apdh

( le

vels)

WT HCM HCMHET

50

100

daggerlowast

lowast

(b)

WT HCM HCMHET

50

100

daggerlowast

lowast

Cpt1

b G

apdh

( le

vels)

(c)

WT HCM HCMHET

50

100

daggerlowast

Acac

b G

apdh

( le

vels)

(d)

WT HCM HCMHET

50

100

daggerlowast

Acox

1 G

apdh

( le

vels)

(e)

WT HCM HCMHET

50

100

daggerlowast

lowast

Slc2

a4(G

LUT4

) G

apdh

( le

vels)

(f)









4 Discussion


















Acknowledgments


References

















































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















50

100

Surv

ival

( le

vels)

HCM HCMHET

(1416)

(315)

(a)

HCM HCMHET

(b)

4

8

HCM HCMHET

HW

BW

ratio

(mg

g)

dagger

(c)

4

2

HCM HCMHET

dagger

VW

BW

ratio

(mg

g)

(d)

100

200

HCM HCMHET

dagger

Nppa

Gap

dh(

leve

ls)

(e)

100

200

300

HCM HCMHET

dagger

Myh

7 G

apdh

( le

vels)

(f)

50

150

100

HCM HCMHET

dagger

Acta

1 G

apdh

( le

vels)

(g)

100

200

HCM HCMHET

dagger

Ctgf

Gap

dh(

leve

ls)

(h)



dagger

lowast

lowast

50

100

WT HCM HCMHET

Atp2

a2 G

apdh

( le

vels)

(a)

WT HCM HCMHET

daggerlowast

50

100

Gap

dh(

leve

ls)Pl

n

(b)

HCM

HCM

HET

HCM

HCM

HET

HCM

HCM

HET

SERCA2a

PLN

sactin

(c)

dagger

50

100

HCM HCMHET

SERC

A2

a s

actin

( le

vels)

(d)

Atp2

b4(P

mca

4) G

apdh

( le

vels)

lowast

50

100

WT HCM HCMHET

lowast

(e)

Figure 6 Continued


Atp2

b1(P

mca

1) G

apdh

( le

vels)

dagger

WT HCM HCMHET

50

100

(f)

WT HCM HCMHET

Ora

i1 G

apdh

( le

vels)

dagger

50

100

150

lowast

(g)

WT HCM HCMHET

Stim

1 G

apdh

( le

vels)

50

100

150

dagger

lowast

(h)

HCM

HCM

HET

HCM

HCM

HET

HCM

HCM

HET

sactin

PP1-C

PP2A-C

CnA

(i)

Figure 6 Continued


dagger50

100

PP1

-C s

actin

( le

vels)

HCM HCMHET

(j)

lowast

lowast

100

Rcan

1 G

apdh

( le

vels)

300

200

WT HCM HCMHET

(k)

Rcan

2 G

apdh

( le

vels)

dagger

50

100

WT HCM HCMHET

lowast

(l)






WT HCM

Hsp

90b1

(Grp94

) G

apdh

( le

vels)

100

50

100

50

Hsp

a4(B

iP)

Gap

dh(

leve

ls)

WT HCM

(a)

50

100

150

Eif2

ak3

(PER

K) G

apdh

( le

vels)

lowast

WT HCM

(b)

lowast

Ddi

t3(C

HO

P) G

apdh

( le

vels)

WT HCM

50

100

150

(c)

lowast

Casp

12 G

apdh

( le

vels)

WT HCM

50

150

100

(d)

50

150

100

lowast

Hax

1 G

apdh

( le

vels)

WT HCM

(e)

50

100

Hsp

a4(B

iP)

Gap

dh(

leve

ls)


50

100

Hsp

90b1

(Grp94

) G

apdh

( le

vels)

(f)

HCM HCMHET

Eif2

ak3

(PER

K) G

apdh

( le

vels)

50

100

(g)

Figure 7 Continued


HCM HCMHET

50

150

100

dagger

Ddi

t3(C

HO

P) G

apdh

( le

vels)

(h)

HCM HCMHET

dagger

50

100

Casp

12 G

apdh

( le

vels)

(i)

HCM HCMHET

dagger

50

100

Hax

1 G

apdh

( le

vels)

(j)







50

100

Ppar120574

Gap

dh(

leve

ls)

daggerlowast

WT HCM HCMHET

(a)

Fabp

3 G

apdh

( le

vels)

WT HCM HCMHET

50

100

daggerlowast

lowast

(b)

WT HCM HCMHET

50

100

daggerlowast

lowast

Cpt1

b G

apdh

( le

vels)

(c)

WT HCM HCMHET

50

100

daggerlowast

Acac

b G

apdh

( le

vels)

(d)

WT HCM HCMHET

50

100

daggerlowast

Acox

1 G

apdh

( le

vels)

(e)

WT HCM HCMHET

50

100

daggerlowast

lowast

Slc2

a4(G

LUT4

) G

apdh

( le

vels)

(f)









4 Discussion


















Acknowledgments


References

















































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















dagger

lowast

lowast

50

100

WT HCM HCMHET

Atp2

a2 G

apdh

( le

vels)

(a)

WT HCM HCMHET

daggerlowast

50

100

Gap

dh(

leve

ls)Pl

n

(b)

HCM

HCM

HET

HCM

HCM

HET

HCM

HCM

HET

SERCA2a

PLN

sactin

(c)

dagger

50

100

HCM HCMHET

SERC

A2

a s

actin

( le

vels)

(d)

Atp2

b4(P

mca

4) G

apdh

( le

vels)

lowast

50

100

WT HCM HCMHET

lowast

(e)

Figure 6 Continued


Atp2

b1(P

mca

1) G

apdh

( le

vels)

dagger

WT HCM HCMHET

50

100

(f)

WT HCM HCMHET

Ora

i1 G

apdh

( le

vels)

dagger

50

100

150

lowast

(g)

WT HCM HCMHET

Stim

1 G

apdh

( le

vels)

50

100

150

dagger

lowast

(h)

HCM

HCM

HET

HCM

HCM

HET

HCM

HCM

HET

sactin

PP1-C

PP2A-C

CnA

(i)

Figure 6 Continued


dagger50

100

PP1

-C s

actin

( le

vels)

HCM HCMHET

(j)

lowast

lowast

100

Rcan

1 G

apdh

( le

vels)

300

200

WT HCM HCMHET

(k)

Rcan

2 G

apdh

( le

vels)

dagger

50

100

WT HCM HCMHET

lowast

(l)






WT HCM

Hsp

90b1

(Grp94

) G

apdh

( le

vels)

100

50

100

50

Hsp

a4(B

iP)

Gap

dh(

leve

ls)

WT HCM

(a)

50

100

150

Eif2

ak3

(PER

K) G

apdh

( le

vels)

lowast

WT HCM

(b)

lowast

Ddi

t3(C

HO

P) G

apdh

( le

vels)

WT HCM

50

100

150

(c)

lowast

Casp

12 G

apdh

( le

vels)

WT HCM

50

150

100

(d)

50

150

100

lowast

Hax

1 G

apdh

( le

vels)

WT HCM

(e)

50

100

Hsp

a4(B

iP)

Gap

dh(

leve

ls)


50

100

Hsp

90b1

(Grp94

) G

apdh

( le

vels)

(f)

HCM HCMHET

Eif2

ak3

(PER

K) G

apdh

( le

vels)

50

100

(g)

Figure 7 Continued


HCM HCMHET

50

150

100

dagger

Ddi

t3(C

HO

P) G

apdh

( le

vels)

(h)

HCM HCMHET

dagger

50

100

Casp

12 G

apdh

( le

vels)

(i)

HCM HCMHET

dagger

50

100

Hax

1 G

apdh

( le

vels)

(j)







50

100

Ppar120574

Gap

dh(

leve

ls)

daggerlowast

WT HCM HCMHET

(a)

Fabp

3 G

apdh

( le

vels)

WT HCM HCMHET

50

100

daggerlowast

lowast

(b)

WT HCM HCMHET

50

100

daggerlowast

lowast

Cpt1

b G

apdh

( le

vels)

(c)

WT HCM HCMHET

50

100

daggerlowast

Acac

b G

apdh

( le

vels)

(d)

WT HCM HCMHET

50

100

daggerlowast

Acox

1 G

apdh

( le

vels)

(e)

WT HCM HCMHET

50

100

daggerlowast

lowast

Slc2

a4(G

LUT4

) G

apdh

( le

vels)

(f)









4 Discussion


















Acknowledgments


References

















































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Atp2

b1(P

mca

1) G

apdh

( le

vels)

dagger

WT HCM HCMHET

50

100

(f)

WT HCM HCMHET

Ora

i1 G

apdh

( le

vels)

dagger

50

100

150

lowast

(g)

WT HCM HCMHET

Stim

1 G

apdh

( le

vels)

50

100

150

dagger

lowast

(h)

HCM

HCM

HET

HCM

HCM

HET

HCM

HCM

HET

sactin

PP1-C

PP2A-C

CnA

(i)

Figure 6 Continued


dagger50

100

PP1

-C s

actin

( le

vels)

HCM HCMHET

(j)

lowast

lowast

100

Rcan

1 G

apdh

( le

vels)

300

200

WT HCM HCMHET

(k)

Rcan

2 G

apdh

( le

vels)

dagger

50

100

WT HCM HCMHET

lowast

(l)






WT HCM

Hsp

90b1

(Grp94

) G

apdh

( le

vels)

100

50

100

50

Hsp

a4(B

iP)

Gap

dh(

leve

ls)

WT HCM

(a)

50

100

150

Eif2

ak3

(PER

K) G

apdh

( le

vels)

lowast

WT HCM

(b)

lowast

Ddi

t3(C

HO

P) G

apdh

( le

vels)

WT HCM

50

100

150

(c)

lowast

Casp

12 G

apdh

( le

vels)

WT HCM

50

150

100

(d)

50

150

100

lowast

Hax

1 G

apdh

( le

vels)

WT HCM

(e)

50

100

Hsp

a4(B

iP)

Gap

dh(

leve

ls)


50

100

Hsp

90b1

(Grp94

) G

apdh

( le

vels)

(f)

HCM HCMHET

Eif2

ak3

(PER

K) G

apdh

( le

vels)

50

100

(g)

Figure 7 Continued


HCM HCMHET

50

150

100

dagger

Ddi

t3(C

HO

P) G

apdh

( le

vels)

(h)

HCM HCMHET

dagger

50

100

Casp

12 G

apdh

( le

vels)

(i)

HCM HCMHET

dagger

50

100

Hax

1 G

apdh

( le

vels)

(j)







50

100

Ppar120574

Gap

dh(

leve

ls)

daggerlowast

WT HCM HCMHET

(a)

Fabp

3 G

apdh

( le

vels)

WT HCM HCMHET

50

100

daggerlowast

lowast

(b)

WT HCM HCMHET

50

100

daggerlowast

lowast

Cpt1

b G

apdh

( le

vels)

(c)

WT HCM HCMHET

50

100

daggerlowast

Acac

b G

apdh

( le

vels)

(d)

WT HCM HCMHET

50

100

daggerlowast

Acox

1 G

apdh

( le

vels)

(e)

WT HCM HCMHET

50

100

daggerlowast

lowast

Slc2

a4(G

LUT4

) G

apdh

( le

vels)

(f)









4 Discussion


















Acknowledgments


References

















































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















dagger50

100

PP1

-C s

actin

( le

vels)

HCM HCMHET

(j)

lowast

lowast

100

Rcan

1 G

apdh

( le

vels)

300

200

WT HCM HCMHET

(k)

Rcan

2 G

apdh

( le

vels)

dagger

50

100

WT HCM HCMHET

lowast

(l)






WT HCM

Hsp

90b1

(Grp94

) G

apdh

( le

vels)

100

50

100

50

Hsp

a4(B

iP)

Gap

dh(

leve

ls)

WT HCM

(a)

50

100

150

Eif2

ak3

(PER

K) G

apdh

( le

vels)

lowast

WT HCM

(b)

lowast

Ddi

t3(C

HO

P) G

apdh

( le

vels)

WT HCM

50

100

150

(c)

lowast

Casp

12 G

apdh

( le

vels)

WT HCM

50

150

100

(d)

50

150

100

lowast

Hax

1 G

apdh

( le

vels)

WT HCM

(e)

50

100

Hsp

a4(B

iP)

Gap

dh(

leve

ls)


50

100

Hsp

90b1

(Grp94

) G

apdh

( le

vels)

(f)

HCM HCMHET

Eif2

ak3

(PER

K) G

apdh

( le

vels)

50

100

(g)

Figure 7 Continued


HCM HCMHET

50

150

100

dagger

Ddi

t3(C

HO

P) G

apdh

( le

vels)

(h)

HCM HCMHET

dagger

50

100

Casp

12 G

apdh

( le

vels)

(i)

HCM HCMHET

dagger

50

100

Hax

1 G

apdh

( le

vels)

(j)







50

100

Ppar120574

Gap

dh(

leve

ls)

daggerlowast

WT HCM HCMHET

(a)

Fabp

3 G

apdh

( le

vels)

WT HCM HCMHET

50

100

daggerlowast

lowast

(b)

WT HCM HCMHET

50

100

daggerlowast

lowast

Cpt1

b G

apdh

( le

vels)

(c)

WT HCM HCMHET

50

100

daggerlowast

Acac

b G

apdh

( le

vels)

(d)

WT HCM HCMHET

50

100

daggerlowast

Acox

1 G

apdh

( le

vels)

(e)

WT HCM HCMHET

50

100

daggerlowast

lowast

Slc2

a4(G

LUT4

) G

apdh

( le

vels)

(f)









4 Discussion


















Acknowledgments


References

















































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















WT HCM

Hsp

90b1

(Grp94

) G

apdh

( le

vels)

100

50

100

50

Hsp

a4(B

iP)

Gap

dh(

leve

ls)

WT HCM

(a)

50

100

150

Eif2

ak3

(PER

K) G

apdh

( le

vels)

lowast

WT HCM

(b)

lowast

Ddi

t3(C

HO

P) G

apdh

( le

vels)

WT HCM

50

100

150

(c)

lowast

Casp

12 G

apdh

( le

vels)

WT HCM

50

150

100

(d)

50

150

100

lowast

Hax

1 G

apdh

( le

vels)

WT HCM

(e)

50

100

Hsp

a4(B

iP)

Gap

dh(

leve

ls)


50

100

Hsp

90b1

(Grp94

) G

apdh

( le

vels)

(f)

HCM HCMHET

Eif2

ak3

(PER

K) G

apdh

( le

vels)

50

100

(g)

Figure 7 Continued


HCM HCMHET

50

150

100

dagger

Ddi

t3(C

HO

P) G

apdh

( le

vels)

(h)

HCM HCMHET

dagger

50

100

Casp

12 G

apdh

( le

vels)

(i)

HCM HCMHET

dagger

50

100

Hax

1 G

apdh

( le

vels)

(j)







50

100

Ppar120574

Gap

dh(

leve

ls)

daggerlowast

WT HCM HCMHET

(a)

Fabp

3 G

apdh

( le

vels)

WT HCM HCMHET

50

100

daggerlowast

lowast

(b)

WT HCM HCMHET

50

100

daggerlowast

lowast

Cpt1

b G

apdh

( le

vels)

(c)

WT HCM HCMHET

50

100

daggerlowast

Acac

b G

apdh

( le

vels)

(d)

WT HCM HCMHET

50

100

daggerlowast

Acox

1 G

apdh

( le

vels)

(e)

WT HCM HCMHET

50

100

daggerlowast

lowast

Slc2

a4(G

LUT4

) G

apdh

( le

vels)

(f)









4 Discussion


















Acknowledgments


References

















































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















HCM HCMHET

50

150

100

dagger

Ddi

t3(C

HO

P) G

apdh

( le

vels)

(h)

HCM HCMHET

dagger

50

100

Casp

12 G

apdh

( le

vels)

(i)

HCM HCMHET

dagger

50

100

Hax

1 G

apdh

( le

vels)

(j)







50

100

Ppar120574

Gap

dh(

leve

ls)

daggerlowast

WT HCM HCMHET

(a)

Fabp

3 G

apdh

( le

vels)

WT HCM HCMHET

50

100

daggerlowast

lowast

(b)

WT HCM HCMHET

50

100

daggerlowast

lowast

Cpt1

b G

apdh

( le

vels)

(c)

WT HCM HCMHET

50

100

daggerlowast

Acac

b G

apdh

( le

vels)

(d)

WT HCM HCMHET

50

100

daggerlowast

Acox

1 G

apdh

( le

vels)

(e)

WT HCM HCMHET

50

100

daggerlowast

lowast

Slc2

a4(G

LUT4

) G

apdh

( le

vels)

(f)









4 Discussion


















Acknowledgments


References

















































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















50

100

Ppar120574

Gap

dh(

leve

ls)

daggerlowast

WT HCM HCMHET

(a)

Fabp

3 G

apdh

( le

vels)

WT HCM HCMHET

50

100

daggerlowast

lowast

(b)

WT HCM HCMHET

50

100

daggerlowast

lowast

Cpt1

b G

apdh

( le

vels)

(c)

WT HCM HCMHET

50

100

daggerlowast

Acac

b G

apdh

( le

vels)

(d)

WT HCM HCMHET

50

100

daggerlowast

Acox

1 G

apdh

( le

vels)

(e)

WT HCM HCMHET

50

100

daggerlowast

lowast

Slc2

a4(G

LUT4

) G

apdh

( le

vels)

(f)









4 Discussion


















Acknowledgments


References

















































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of























4 Discussion


















Acknowledgments


References

















































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of




























Acknowledgments


References

















































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of




















Acknowledgments


References

















































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of











































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of











































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





















of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















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