Thursday, November 21, 2024
11/21/2024
PROJECT SUMMARY: Heart failure (HF) is a complex clinical syndrome and a predominant cause of mortality in adults. The overall objective of this renewal is to continue to elucidate the mechanism of action of the myosin binding protein-C (MyBP-C) family of proteins on cardiac function. MyBP-C proteins regulate contractile structure and function in both cardiac and skeletal muscles. The present proposal is focused on defining the role of one of the MyBP-C proteins, fast myosin binding protein-C (fMyBP-C), as a negative regulator of cardiac contractility. fMyBP-C is minimally expressed at baseline in the heart but is significantly increased in the cardiomyocytes during HF. In the pilot studies, a novel transgenic mouse that expresses fMyBP-C (fMyBP-CTg) specifically in cardiomyocytes and at levels similar to those observed in HF develops cardiac hypertrophy, decreased maximal force and reduced myofilament Ca2+ sensitivity. In contrast, homozygous fMyBP-C knockout (fMyBP-CKO) mice, when challenged with transverse aortic coarctation (TAC)-induced pressure overload, shows significantly improved cardiac function, compared to controls. Furthermore, in vitro studies revealed that fMyBP-C negatively modulates cardiomyocyte contractility by increasing super-relaxed state of myosin, actomyosin interactions and thin filament activation. However, the molecular mechanisms underlying the regulation of fMyBP-C and, in turn, its impact on sarcomere function, are completely unknown. On the basis of the preliminary findings, our central hypothesis is that fMyBP-C upregulation reduces cardiac function during HF by reducing actomyosin interactions, preventing thin and thick filament interactions and decreasing the rate of force generation, resulting in contractile dysfunction and HF. SPECIFIC AIM 1 will use a novel fMyBP-CTg mouse model to determine the sufficiency of fMyBP-C expression in the cardiomyocytes in reducing cardiac contractility, actomyosin interactions and cross- bridges and developing HF, compared to controls, including cMyBP-C transgenic and null mice. Using the fMyBP-CKO mice, SPECIFIC AIM 2 will define the necessity of fMyBP-C to exacerbate (TAC)-induced pressure overload HF using fMyBP-CKO mice, compared to controls. In the same aim, a novel Mybpc2- mScarlet (fluorescence) knock-in mouse model will be used to fate map fMyBP-C expression during TAC- induced pressure overload. Using these mouse models, SPECIFIC AIM 3 will elucidate the molecular mechanism(s) underlying reduced cardiac contractility using cardiomyocytes from the fMyBP-CTg and fMyBP-CKO mice, as well as recombinant proteins and chimeras of cMyBP-C/fMyBP-C, on various biophysical, biochemical and functional studies in vitro, compared to wild-type controls. Together, these studies will determine the necessity and sufficiency of fMyBP-C to negatively regulate inotropy and cause HF. The results will lead to the identification of potential therapeutic targets to treat HF.
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