In healthy cardiac muscle a delicate equilibrium exists between its mechanic and energetic properties and, a perturbation of this balance is related to several pathological conditions i.e. hypertrophic cardiomyopathy (HCM). Since the origin of the coupling between mechanic and energetic events during cardiac sarcomere contraction resides in the rates governing CB turn over, myofibril mechanic experiments associated with mechanic and energetic experiments in skinned muscle strips, represent unique tools to dissect the physiology and pathophysiology of sarcomere dynamics and energetics. Based on a simple two-state model of the CB cycle, myofibril isometric relaxation kinetics (slow kREL) represents the apparent forward rate with which CBs leave force generating states (gapp) under isometric conditions and correlates with the energy cost of tension generation (ATPase/tension ratio); in short slow kREL~gapp~tension cost. In the first part of this thesis, by combining kinetic experiments in isolated myofibrils and mechanical and energetic measurements in multicellular cardiac strips, we provided direct evidence for a positive linear correlation that exists between myofibril slow kREL and tension cost both measured in preparations from the same cardiac sample. This correlation remained true among different types of cardiac muscles with different ATPase activities and also when CB kinetics were altered by cardiomyopathy-related mutations. Sarcomeric mutations associated to HCM, a primary cardiac disorder caused by mutations in genes encoding sarcomeric proteins, have been often found to accelerate CB turnover rate and increase the energy cost of myocardial contraction. Here we reviewed data showing that faster cross-bridge detachment results in a proportional increase in the energetic cost of tension generation in heart samples from both HCM patients and mouse models of the disease. In the second part of this work, we directly investigated if an energetic impairment is associated with a missense HCM-mutation in MYBPC3, the gene coding for cardiac myosin-binding protein-C (cMyBP-C). Mutations in MYBPC3 are the most common cause of hypertrophic cardiomyopathy (HCM). In the present work we show by haplotype analysis that the highly penetrant missense mutation E258K-cMyBP-C is a founder mutation in Tuscany. A comprehensive clinical characterization of the Tuscan E258K cohort is provided and three representative subjects who underwent myectomy (and show an approximately 30% cMyBP-C haploinsufficiency) were selected for in vitro studies. In ventricular E258K myofibrils compared to donors, the rate of tension generation following maximal Ca2+ activation (kACT) and isometric relaxation (slow kREL) were faster, suggesting faster cross-bridge detachment and increased energy cost of tension generation. Direct energetic measurements were performed in permeabilized multicellular preparations and, to avoid artificial results related to myocardial structural changes, a tissue clearing procedure combined with a novel 3D cytoarchitecture analysis were developed to determine cardiomyocyte orientation across and along the multicellular strips at single cell level. ATP consumption and isometric active tension (modulated by Ca2+ activation) were simultaneously measured and analyzed in a correlative manner with the structural data. This novel multimodal approach allowed us to demonstrate that an HCM-related missense cMyBP-C mutation primarily impairs sarcomere energetics in human myocardium.

Vitale, G. (2020). Sarcomere mechanics and energetics: a close interplay in the physiology and pathophysiology of cardiac muscle.

Sarcomere mechanics and energetics: a close interplay in the physiology and pathophysiology of cardiac muscle

Vitale Giulia
2020-01-01

Abstract

In healthy cardiac muscle a delicate equilibrium exists between its mechanic and energetic properties and, a perturbation of this balance is related to several pathological conditions i.e. hypertrophic cardiomyopathy (HCM). Since the origin of the coupling between mechanic and energetic events during cardiac sarcomere contraction resides in the rates governing CB turn over, myofibril mechanic experiments associated with mechanic and energetic experiments in skinned muscle strips, represent unique tools to dissect the physiology and pathophysiology of sarcomere dynamics and energetics. Based on a simple two-state model of the CB cycle, myofibril isometric relaxation kinetics (slow kREL) represents the apparent forward rate with which CBs leave force generating states (gapp) under isometric conditions and correlates with the energy cost of tension generation (ATPase/tension ratio); in short slow kREL~gapp~tension cost. In the first part of this thesis, by combining kinetic experiments in isolated myofibrils and mechanical and energetic measurements in multicellular cardiac strips, we provided direct evidence for a positive linear correlation that exists between myofibril slow kREL and tension cost both measured in preparations from the same cardiac sample. This correlation remained true among different types of cardiac muscles with different ATPase activities and also when CB kinetics were altered by cardiomyopathy-related mutations. Sarcomeric mutations associated to HCM, a primary cardiac disorder caused by mutations in genes encoding sarcomeric proteins, have been often found to accelerate CB turnover rate and increase the energy cost of myocardial contraction. Here we reviewed data showing that faster cross-bridge detachment results in a proportional increase in the energetic cost of tension generation in heart samples from both HCM patients and mouse models of the disease. In the second part of this work, we directly investigated if an energetic impairment is associated with a missense HCM-mutation in MYBPC3, the gene coding for cardiac myosin-binding protein-C (cMyBP-C). Mutations in MYBPC3 are the most common cause of hypertrophic cardiomyopathy (HCM). In the present work we show by haplotype analysis that the highly penetrant missense mutation E258K-cMyBP-C is a founder mutation in Tuscany. A comprehensive clinical characterization of the Tuscan E258K cohort is provided and three representative subjects who underwent myectomy (and show an approximately 30% cMyBP-C haploinsufficiency) were selected for in vitro studies. In ventricular E258K myofibrils compared to donors, the rate of tension generation following maximal Ca2+ activation (kACT) and isometric relaxation (slow kREL) were faster, suggesting faster cross-bridge detachment and increased energy cost of tension generation. Direct energetic measurements were performed in permeabilized multicellular preparations and, to avoid artificial results related to myocardial structural changes, a tissue clearing procedure combined with a novel 3D cytoarchitecture analysis were developed to determine cardiomyocyte orientation across and along the multicellular strips at single cell level. ATP consumption and isometric active tension (modulated by Ca2+ activation) were simultaneously measured and analyzed in a correlative manner with the structural data. This novel multimodal approach allowed us to demonstrate that an HCM-related missense cMyBP-C mutation primarily impairs sarcomere energetics in human myocardium.
2020
POGGESI, CORRADO
Vitale, G. (2020). Sarcomere mechanics and energetics: a close interplay in the physiology and pathophysiology of cardiac muscle.
Vitale, Giulia
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11365/1107290
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