Molecular determinants of myofibrillar contractile function in the heart

Szilágyi, Szabolcs (orvos)
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Chronic heart failure (CHF) is a prominent public health problem in the developed societies still accompanied by unacceptably high morbidity and mortality. Our knowledge on the pathophysiological alterations at the molecular level in CHF is, however, still incomplete. Increasing number of CHF patients also claims for novel therapeutic approaches. Thus, the studies summarized in this thesis examined these two distinct aspects of heart failure. The central experimental method used throughout this work was the single cell force measurement in skinned cardiac myocytes. Following enzymatic or mechanical isolation of the myocytes their membranes were permeabilized with a detergent in order to allow precise control of both the intracellular medium composition and the sarcomere length. Skinned preparations were then attached to a highly sensitive force transducer and isometric contractures evoked by activating solutions with different calcium concentrations were recorded. A wide range of experimental methods from the molecular (e.g. SDS-PAGE, immunoblotting, PDE activity assays, real-time quantitative RT-PCR, etc.) to the whole organ (e.g. Langendorff-perfused heart, working-heart preparations) level completed the force measurements. First, we focused on the behaviour of the Frank-Starling relationship in heart failure. The Frank-Starling law describes a most characteristic internal regulatory mechanism of the cardiac muscle that is increased blood volume in the cardiac cavities results in augmented contractile force. In rat left ventricle (LV) when stretching the sarcomeres from 1.9 μm to 2.3 μm we proved positive correlation between titin based passive tension, stretch-induced increase in Ca2+ sensitivity and ventricular light chain 2 (VLC2) phosphorylation. The observed transmural gradient of both stretch sensitization and the accompanied increase in VLC2 phosphorylation was, however, blunted by the remodeling process in our rat model of CHF developed following myocardial infarction. Whether these alterations are compensatory changes or on the contrary, signs for decompensation of the myocardium remains to be elucidated. The second study presented here analyzed the involvement of the cardiac myosin binding protein C (cMyBP-C) in protein kinase A (PKA)-phosphorylation and SL-mediated changes in the myofibrillar Ca2+ sensitivity. Recent studies using a transgenic murine model that expressed the slow skeletal isoform of troponin I in the heart concluded that PKA induced reduction in Ca2+ sensitivity was due, solely, to phosphorylation of cardiac troponin I (cTnI), not cMyBP-C. Specific role of cMyBP-C in the determination of contractile properties was therefore studied in our cMyBP-C knock out murine model. The absence of cMyBP-C reduced stretch sensitization and almost abolished the PKA-induced decrease in Ca2+ sensitivity compared to wild type myofilaments. Thus, our results, in contrast with the aforementioned study, suggest that at least some of the PKA-phosphorylation mediated effects on myofibrillar Ca2+-sensitivity require cMyBP-C. Positive inotropic therapy represents an indispensable tool in the management of heart failure. Levosimendan, the first clinically approved Ca2+-sensitizer was shown to have both Ca2+ sensitising and phosphodiesterase (PDE) inhibitory potentials. In our further studies its mechanism of action in the heart was examined and compared with that of enoximone, a PDE III inhibitor. Up to 1 μM concentration levosimendan induced more potent positive inotropic response in isolated guinea pig hearts than enoximone. Izoenzyme selectivity (PDE III over PDE IV) of levosimendan proved to be higher than that of other PDE inhibitors, including enoximone. Furthermore, levosimendan inhibited only PDE III and not PDE IV at concentrations at which it increased myofibrillar Ca2+ sensitivity. Provided that dual inhibition of PDE III and PDE IV is a prerequisite of the increase in the intracellular cAMP concentration [cAMP]i our results support the hypothesis that in the clinically relevant concentration range levosimendan exerts positive inotropy through a Ca2+ sensitising mechanism. In contrast, up to the concentration of 10 μM enoximone failed to increase Ca2+ sensitivity. Thus it acts most likely via PDE inhibition with limited PDE III versus PDE IV selectivity and concomitant elevation in [cAMP]i. Interestingly, the clinically observed long-lasting beneficial hemodynamic effects of levosimendan are at least in part attributable to its biologically active metabolite OR-1896 that possesses a Ca2+ sensitising potential similar to that of levosimendan. In conclusion, our works furnished further evidences for the existence of transmural gradients of passive and active mechanical properties in the ventricular myocardium, revealed a new, exciting regulatory mechanism involved in the short-term regulation of stretch-sensitization and challenged the presumed exclusive role of cTnI in the Ca2+ desensitization of myofilaments induced by β-adrenergic stimulation. Comparison of levosimendan and enoximone firmed previous hypothesis that levosimendan acts predominantly as a Ca2+ sensitizer in the myocardium and characterization of OR-1896 suggested its significance as the mediator of long-term circulatory effects of levosimendan.