Cardiac myosin binding protein c phosphorylation is cardioprotective

Abstract

Cardiac myosin binding protein C (cMyBP-C) has three phosphorylatable serines at its N terminus (Ser-273, Ser-282, and Ser-302), and the residues' phosphorylation states may alter thick filament structure and function. To examine the effects of cMyBP-C phosphorylation, we generated transgenic mice with cardiac-specific expression of a cMyBP-C in which the three phosphorylation sites were mutated to aspartic acid, mimicking constitutive phosphorylation (cMyBP-C AllP؉ ). The allele was bred into a cMyBP-C null background (cMyBP-C (t/t) ) to ensure the absence of endogenous dephosphorylated cMyBP-C. cMyBP-C AllP؉ was incorporated normally into the cardiac sarcomere and restored normal cardiac function in the null background. However, subtle changes in sarcomere ultrastructure, characterized by increased distances between the thick filaments, indicated that phosphomimetic cMyBP-C affects thick-thin filament relationships, and yeast two-hybrid data and pull-down studies both showed that charged residues in these positions effectively prevented interaction with the myosin heavy chain. Confirming the physiological relevance of these data, the cMyBP-C AllP؉:(t/t) hearts were resistant to ischemia-reperfusion injury. These data demonstrate that cMyBP-C phosphorylation functions in maintaining thick filament spacing and structure and can help protect the myocardium from ischemic injury.heart ͉ ischemia C ardiac myosin binding protein C (cMyBP-C) is localized to the sarcomere's thick filaments where it has structural and regulatory functions. MYBPC3 mutations account for 20-30% of all mutations linked to familial hypertrophic cardiomyopathy (1). cMyBP-C belongs to the intracellular Ig superfamily and is composed of Ig and fibronectin type-3 repeating domains (Fig. 1A). It is present not only in cardiac muscle, but also in skeletal muscle before the skeletal muscle-type isoforms are expressed, suggesting that the cardiac isoform is functional in early myofibrillogenesis and regenerating muscle (2, 3). cMyBP-C may modulate myosin assembly (4) and stabilize thick filaments (5). It binds titin via domains C8-C10 (6) and actin in the Pro-Alarich sequences between the C0 and C1 domains (7), which appear to be important for the precise arrangement of the actin-myosin filaments. Compared with the two skeletal muscle isoforms, the cardiac isoform contains an extra Ig domain at the N terminus (C0), an insertion of 28 residues within the C5 domain, and three potential phosphorylation sites that are substrates for cAMP-dependent PKA, Ca 2ϩ -calmodulin-activated kinase, and PKC (8). This region is located between the C1 and C2 domains of the N terminus, which binds to the subfragment 2 (S2) segment of myosin close to the lever arm domain (9-11), and this interaction may be dynamically regulated by the differential phosphorylation of cMyBP-C (12). cMyBP-C is the only thick filament protein that is differentially phosphorylated at multiple sites by the enzymes PKA, PKC, and Ca 2ϩ -calmodulin-activated kinase (13). Reconstitution studie...

Tropomyosin Dephosphorylation Results in Compensated Cardiac Hypertrophy

“…Post translational modifications, such as alterations in the phosphorylation status of sarcomeric and Z-disc proteins can result in altered cardiac contractility with progression to disease and death (11)(12)(13)(14). This is the first in vivo study investigating the functional role of cardiac ␣-Tm phosphorylation.…”

“…Taken together, these in vitro data suggest that altering phosphorylation status affects the ability of Tm to cooperatively activate the thin filament upon binding of Ca 2ϩ to troponin (Tn). In recent years in vivo studies performed on animal models indicate that changes in the phosphorylation status of sarcomeric proteins such as troponin I (TnI), myosin binding protein C (MyBPC), and the regulatory myosin light chain result in alterations in Ca 2ϩ sensitivity of the myofilament and changes in cardiac function and may play a role in the development of cardiac disease (11)(12)(13)(14)(15). Investigation of a dilated cardiomyopathy transgenic (TG) mouse model bearing a human ␣-Tm mutation (E54K) shows that phosphorylation levels of Tm decrease relative to non-transgenic (NTG) littermates (16,17).…”

Phosphorylation and calcium antagonistically tune myosin-binding protein C’s structure and function

“…1A) (9, 10). β-Adrenergic-stimulated phosphorylation of these serines is believed to enhance cardiac contractility (11,12), and a high level of phosphorylation appears to be critical to normal cardiac function, whereas dephosphorylation has been associated with heart failure (13)(14)(15). Although the mechanistic role of cMyBP-C phosphorylation in vivo remains unclear, it is known to reduce the extensibility of the M-domain (10,16), diminish the binding of cMyBP-C to actin (17,18) and to myosin S2 (19), and to tune cMyBP-C's ability to modulate actomyosin activity in vitro (20)(21)(22)(23)(24).…”

Top-down high-resolution mass spectrometry of cardiac myosin binding protein C revealed that truncation alters protein phosphorylation state

“…In this regard, cMyBP-C phosphorylation is significantly decreased during the development of heart failure and pathologic hypertrophy (10). More recently, it has been reported that cMyBP-C phosphorylation is cardioprotective when mouse hearts were subjected to ischemia and reperfusion injury (13). cMyBP-C is differentially phosphorylated at multiple sites by cAMP-dependent protein kinase A (PKA), protein kinase C (PKC) and Ca 2ϩ -calmodulin-activated kinase (10,14).…”