Biochemistry of Cardiac Muscle

Karel Kotaska

Cardiac cells

Cardiac cells

normal state

reversible damageto the cell

Celldeath

-clustered membrane integrins are coupled to the Z-disk of the sarcomere proteins including calsarcin 1, MLP and titin cap (T-cap) -These proteins couple the input from the integrin to the contractile (thick and thin) filaments by interacting with -actinin, titin, actin and other proteins.

-Ca2+ interacts with troponin C, resulting in a conformational change in troponin I. This, in turn, releases -tropomyosin from its position, in which it prevents actin from binding to myosin.

- The result is the formation of force-generating crossbridges.

-Thin-filament regulatory proteins (namely troponin T, troponin C, troponin I, myosin-binding protein C and -tropomyosin) and titin can be post-translationally regulated by protein kinases and/or phosphatasesPDE5 is present in the Z-disk and might regulate local pools of cGMP, which could then activate PKG.

- This, in turn, could reduce the sensitivity of myofilaments to Ca2+ and thereby depress contraction. In the failing heart, the post-translational modification of titin seems to be an important mechanism leading to contractile dysfunction.

Contraction in cardiomyocyte

Transport of Ca during contraction

1. The entry of Ca2+ to a cardiomyocyte through L-type Ca2+ channels stimulates the release of Ca2+ from the sarcoplasmic reticulum through RYR2, leading to activation of myofilaments (in the sarcomere).

2. Resting Ca2+ concentrations are restored mainly through re-uptake of Ca2+ into the sarcoplasmic reticulum, by the Ca2+-uptake pump SERCA2A, which is regulated by phospholamban (PLN). Ca2+ is also removed from the cell through the sodium (Na+)/Ca2+ exchanger.

3. The binding of -adrenergic agonists to -ARs results

in the activation of PKA, which leads to the phosphorylation of the L-type Ca2+ channel, RYR2, PLN and sarcomere proteins

4. This process increases both cellular contraction and relaxation, through the delivery of more Ca2+ to the myofilaments (increasing contraction) and the improved re-uptake of Ca2+ by the sarcoplasmic reticulum and desensitization of the myofilaments to Ca2+ (increasing relaxation).

5. In addition to Ca2+ entry through L-type Ca2+ channels, Ca2+ release through RYR2 is also modulated by the interaction of RYR2 with calstabin 2.

• 1. The initial event is membrane depolarization, which occurs with ion entry through connexin channels from a neighbouring cardiomyocyte (right) followed by opening of voltage-gated Na+ channels and Na+ entry (top).

• 2. The resultant rapid depolarization of the membrane inactivates Na+ channels and opens both K+ channels and Ca2+ channels. Entry of Ca2+ into the cell triggers the release of Ca2+ from the sarcoplasmic reticulum through the ryanodine channel.

• 3. Ca2+ then binds to the troponin complex and activates the contractile apparatus. Cellular relaxation occurs on removal of Ca2+ from the cytosol by the Ca2+-uptake pumps of the sarcoplasmic reticulum and by Na+/Ca2+ exchange with the extracellular fluid. Intracellular Na+ homeostasis is achieved by the Na+/K+ pump.

• 4. The molecular components that are required for

normal electrophysiological activity, contractile function and cell–cell adhesion all need to be positioned correctly within the cell and anchored to each other and the cytoskeleton.

Cardiovascular stress

• Stress stimuli are transduced by many intracellular signalling pathways and ultimately result in changes in cardiomyocyte function and growth.

• Stress stimuli include nitric oxide, neurohormones (such as natriuretic peptides and angiotensin II, the latter of which binds to Gq- or G11-protein-coupled receptors), neurotransmitters (such as catecholamines, which bind to -adrenergic receptors (-ARs)), cytokines and growth factors.

• After cell-surface receptors bind to these ligands, the signal is transmitted to protein kinases, which in turn activate signalling nodes (where many pathways converge).

• These nodes include calcium (Ca2+)/calmodulin-dependent kinase II (CAMKII), Akt, glycogen synthase kinase 3 (GSK3) and cyclic GMP (cGMP)-dependent protein kinase (PKG).

AC, adenylyl cyclase; AKAP1, PKA anchor protein 1; ANP, atrial natriuretic peptide; cAMP, cyclic AMP; DAG, diacylglycerol; FOXO, forkhead-box O proteins; HDAC, histone deacetylase; INPP5F, inositol polyphosphate-5-phosphatase F; InsP3, inositol-1,4,5-trisphosphate; JAK, Janus kinase; MAPK, mitogen-activated protein kinase; MEF2, myocyte enhancer factor 2; mRNA, messenger RNA; miRNA, microRNA; NFAT, nuclear factor of activated T cells; NKX2-5, NK2 transcription factor related, locus 5; NO, nitric oxide; pGC, particulate guanylyl cyclase; PICOT, PKC-interacting cousin of thioredoxin; PI(3)K-, phosphatidylinositol 3-OH-kinase-; PKA, cAMP-dependent protein kinase; PKC, protein kinase C; PDE5, phosphodiesterase 5; PKD, protein kinase D; PLC, phospholipase C; PtdIns(4,5)P2, phosphatidylinositol-4,5-bisphosphate; PTEN, phosphatase and tensin homologue; RCAN1, regulator of calcineurin 1; ROCK, Rho-associated, coiled-coil-containing protein kinase; sGC, soluble guanylyl cyclase; SRF, serum response factor; STAT, signal transducer and activator of transcription.

Myocardium metabolic pathway

Energy of the heart

• The heart consumes more energy than any other organ and it is the greatest oxygen-consuming organ in the body, around 8–15 ml O2 min/100 g heart, with the capacity to increase up to 70 ml under exercise conditions.

• Everyday the heart beats about 100,000 times, pumps approximately 10 tons of blood through the body,

• cycles about 6 kg of adenosine-triphosphate (ATP) (20–30 times its own weight)

Heart failure

depressed PKA activity, reduced Ca2+ re-uptake by the sarcoplasmic reticulum, increased Ca2+ extrusion through the Na+/Ca2+ exchanger, and increased RYR2 phosphorylation and calstabin-2 dissociation.

increased activation of Gq/11-protein-coupled receptor signalling, which in turn increases PKC- activity. PKC- then blocks activity of the phosphatase inhibitor I-1, thereby increasing the activation of the serine/threonine phosphatase PP1.

This further reduces PLN phosphorylation and depresses both cellular contraction and relaxation, by preventing re-uptake of Ca2+ by the sarcoplasmic reticulum. Activation of Gq/11-protein-coupled receptors also increases the amount of InsP3 generated. InsP3 interacts with receptors (InsP3R) in the sarcoplasmic-reticulum membrane to stimulate Ca2+ release.

InsP3 also enters the nucleus, where it interacts with InsP3R, leading to Ca2+-mediated activation of intranuclear CAMKII. This, in turn, activates PKD, resulting in the phosphorylation of HDAC and its subsequent nuclear export and thereby altering transcriptional regulation.

Pools of intracellular Ca2+ also activate cytosolic calmodulin–CAMKII, resulting in the activation of NFAT. Activated NFAT then translocates to the nucleus, where it is involved in transcriptional regulation.

Heart ischaemia and failure

Cardiac Muscle and Ischemia

• Coronary artery occlusion → ischemia → significant change in cell structure, chemistry and function

– loss of contractile function – arrhythmias– cell death

• The decrease of the ATP / ADP, the accumulation of AMP, inorganic phosphate, metabolic products are removed (lactate).

• The rapid decline in creatine phosphate - creatine kinase reaction is only short-term mechanism to compensate for reduced ATP production in mitochondria

Cardiac Muscle and Ischemia

• Even mild ischemia reduces the concentration of ATP and creatine phosphate, increases the level of inorganic phosphate → activation of glycolysis (glucose needed from the bloodstream into the heart cells) → increase in the concentration of pyruvate → conversion by LDH to lactate.

• Prolonged ischemia - the accumulation of substrates (lactate, NADH and H+) → inhibition of glycolysis at the level of phosphofructokinase and glyceraldehyde-3-dehydrogenase.

Heart ischemia

Laboratory markers of myocardial Laboratory markers of myocardial necrosisnecrosis

MyoglobinMyoglobin

Elevation Elevation 2 hours after onset , maximum after 6 - 9 hours 2 hours after onset , maximum after 6 - 9 hours and after 36 hours back in reference interval and after 36 hours back in reference interval

False positive results – muscle injuries (manual False positive results – muscle injuries (manual resuscitation, impaired renal filtration)resuscitation, impaired renal filtration)

Important negative predictive value (between 2-12 hour) – Important negative predictive value (between 2-12 hour) –

(60% NPV 3 hrs after onset of ischemia , 90% NPV 4 hrs after) – (60% NPV 3 hrs after onset of ischemia , 90% NPV 4 hrs after) – exclusion of acute coronary lesionexclusion of acute coronary lesion

Reference valuesReference values0 - 15 yrs: 15 - 50 μg/l,Males: 15 - 150 years: 23 - 72 μg/l, Females: 15 - 150 yrs: 19 - 51 μg/l

Creatinkinase and isoenzymesCreatinkinase and isoenzymes CreatinkinaseCreatinkinase

Elevation Elevation 4-6 hours after onset of ischemia4-6 hours after onset of ischemia none specificnone specific ethnics, gender, musculatureethnics, gender, musculature

Creatinkinase isoenzyme – MB

Enzymatic activity (CK-MB)Enzymatic activity (CK-MB) or or mass concentration (CK-MBmass concentration (CK-MBmassmass)) CardiospecificCardiospecific enzyme, enzyme, retrospective finding of amount of necrosis tissue. retrospective finding of amount of necrosis tissue. Elevation 4-6 hours after onset of ischemia, Elevation 4-6 hours after onset of ischemia, elevation during 24 – 36 hours, normalisation during 5 dayselevation during 24 – 36 hours, normalisation during 5 days detection of reinfarct in early stage after first AIMdetection of reinfarct in early stage after first AIM

Reference valuesReference values Males15 - 99 yrs: 0 - 7,20 μg/l

Females15 - 99 yrs: 0 - 3,4 μg/l

Creatinkinase and isoenzymesCreatinkinase and isoenzymesElevated plasma CKMB Elevated plasma CKMB

- AIM , myocarditis- AIM , myocarditis        - Muscular dystrophia Duchenne- Muscular dystrophia Duchenne        - malignant hyperthermia- malignant hyperthermia        - polymyozitis and dermatomyositis- polymyozitis and dermatomyositis        - Rey syndroma- Rey syndroma        - Rheumatoid arthritis with high concentration of RF- Rheumatoid arthritis with high concentration of RF

None elevated plasma CK MBNone elevated plasma CK MB

- AP - AP - myxedema - myxedema - intramuscular injections, - intramuscular injections, - ictus, pericvarditis, pneumonia - ictus, pericvarditis, pneumonia - lung embolia, epilepsy - lung embolia, epilepsy

Reference values

CK MB

15 - 60 years: 0 - 0,42 μkat/l

Cardiac troponinsCardiac troponinsOnly under pathological condition, very small concentrationsOnly under pathological condition, very small concentrations

Troponin TTroponin T

Elevation of TnT in 4 – 6 hours, elevation during 10 days - 2 weeks ,Elevation of TnT in 4 – 6 hours, elevation during 10 days - 2 weeks ,

Troponin ITroponin I

6 hodin after onset of ischemia with 7 – 10 days duration6 hodin after onset of ischemia with 7 – 10 days duration

Myocardial infarction

- SAP – none - UAP – ↑ cTnI (necrosis)

-Elevation of cTn correlate with necrosis

- Reinfarct – 7-14 days after AIM asi u 17% cases, cTnI, Myoglobine and CK-MB

Reference values and diagnostic cut offs are assay dependent

Cardiac TroponinsCardiac TroponinsAdvantages:

• Cardiospecifity • High diagnostic sensitivity• Elevation of concentration - result of myocardial necrosis• Elevation of concentration correlated with large of

myocarfdial necrosis• Long time elevation of concentation• Fast analytical methods• Changes in concentrations are inevitable for changing

medical therapy• Evaluation of spontaneous and therapeutic thrombolysis• Short and long time risk stratification

Cardiac troponinsCardiac troponins

Disadvantages

• Relatively late elevation after the start of necrosis (3 hours)

• Limited use in evaluation reperfusion, reinfarct and large of necrosis

• Analytical variability (different methods – different results)

Dynamic of changes in cardiac Dynamic of changes in cardiac biomarkersbiomarkers

Dynamic of changes in cardiac Dynamic of changes in cardiac biomarkersbiomarkers

Reactive oxygen and cardiovascular disease

Reactive oxygen and cardiovascular disease

Reactive oxygen and cardiovascular disease

Renin angiotensin aldosterone system

Physiology of renin receptor

Function of AT receptors

Regulation of RAS

Natriuretic peptides

Function of NP

Natriuretic peptides as endogenous vasodiators