Clinical trials using adult stem cells to regenerate damaged heart tissue continue to this day 1,2 despite ongoing questions of efficacy and a lack of mechanistic understanding of the underlying biologic effect 3. The rationale for these cell therapy trials is derived from animal studies that show a modest but reproducible improvement in cardiac function in models of cardiac ischemic injury 4,5. Here we examined the mechanistic basis for cell therapy in mice after ischemia/ reperfusion (I/R) injury, and while heart function was enhanced, it was not associated with new cardiomyocyte production. Cell therapy improved heart function through an acute sterile immune response characterized by the temporal and regional induction of CCR2 + and CX3CR1 + macrophages. Intra-cardiac injection of 2 distinct types of adult stem cells, freeze/thaw-killed cells or a chemical inducer of the innate immune response similarly induced regional CCR2 + and CX3CR1 + macrophage accumulation and provided functional rejuvenation to the I/R-injured heart. This selective macrophage response altered cardiac fibroblast activity, reduced border zone extracellular matrix (ECM) content, and enhanced the mechanical properties of the injured area. Users may view, print, copy, and download text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use:

Abstract-The role of cardiac myosin binding protein-C (cMyBP-C) phosphorylation in cardiac physiology or pathophysiology is unclear. To investigate the status of cMyBP-C phosphorylation in vivo, we determined its phosphorylation state in stressed and unstressed mouse hearts. cMyBP-C phosphorylation is significantly decreased during the development of heart failure or pathologic hypertrophy. We then generated transgenic (TG) mice in which the phosphorylation sites of cMyBP-C were changed to nonphosphorylatable alanines (MyBP-C AllPϪ ). A TG line showing Ϸ40% replacement with MyBP-C AllPϪ showed no changes in morbidity or mortality but displayed depressed cardiac contractility, altered sarcomeric structure and upregulation of transcripts associated with a hypertrophic response. To explore the effect of complete replacement of endogenous cMyBP-C with MyBP-C AllPϪ , the mice were bred into the MyBP-C (t/t) background, in which less than 10% of normal levels of a truncated MyBP-C are present. Although MyBP-C AllPϪ was incorporated into the sarcomere and expressed at normal levels, the mutant protein could not rescue the MyBP-C (t/t) phenotype. The mice developed significant cardiac hypertrophy with myofibrillar disarray and fibrosis, similar to what was observed in the MyBP-C (t/t) animals. In contrast, when the MyBP-C (t/t) mice were bred to a TG line expressing normal MyBP-C (MyBP-C WT ), the MyBP-C (t/t) phenotype was rescued. These data suggest that cMyBP-C phosphorylation is essential for normal cardiac function. Key Words: mouse Ⅲ mouse mutants Ⅲ muscle Ⅲ muscle contraction Ⅲ myocardial contractility U nderstanding the structure/function relations for cardiac myosin binding protein-C (cMyBP-C) is clinically relevant, as cMyBP-C mutations are a widely recognized cause of familial hypertrophic cardiomyopathy. 1 Various cMyBP-C transgenic (TG) and gene-targeted mouse models have demonstrated the importance of the protein for long-term integrity of sarcomeric structure and for maintaining normal cardiac contractility. 2,3 Functional insight can be gained from appreciating the crucial structural differences between cMyBP-C and the skeletal isoform. Only the cardiac isoform contains an extra immunoglobulin domain at the N terminus (C0), an insertion of 28 residues within the C5 domain, and three phosphorylation sites (Ser273, that are substrates for cAMP-dependent protein kinase A (PKA), Ca 2ϩ -calmodulin-activated kinase and protein kinase C.In vivo, PKA-mediated phosphorylation of cMyBP-C is linked to modulation of cardiac contraction. 4 On adrenergic stimulation, PKA phosphorylates Ser273, -282, and -302, whereas protein kinase C phosphorylates only Ser273 and -302. 5 These residues, located near the N terminus of the protein, are of particular interest, as this region binds to the S2 segment of the myosin heavy chain (MHC), 6,7 which is close to the lever arm domain of myosin. It has been hypothesized that cMyBP-C/MHC interactions are dynamically regulated by the phosphorylation/dephosphorylation of cMyBP-C. 8...

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...

Heart failure is a leading cause of mortality in South Asians. However, its genetic etiology remains largely unknown1. Cardiomyopathies due to sarcomeric mutations are a major monogenic cause for heart failure (MIM600958). Here, we describe a deletion of 25 bp in the gene encoding cardiac myosin binding protein C (MYBPC3) that is associated with heritable cardiomyopathies and an increased risk of heart failure in Indian populations (initial study OR = 5.3 (95% CI = 2.3–13), P = 2 × 10−6; replication study OR = 8.59 (3.19–25.05), P = 3 × 10−8; combined OR = 6.99 (3.68–13.57), P = 4 × 10−11) and that disrupts cardiomyocyte structure in vitro. Its prevalence was found to be high (~4%) in populations of Indian subcontinental ancestry. The finding of a common risk factor implicated in South Asian subjects with cardiomyopathy will help in identifying and counseling individuals predisposed to cardiac diseases in this region.

During the past 5 years there has been an increasing body of literature describing the roles cardiac myosin binding protein C (cMyBP-C) phosphorylation play in regulating cardiac function and heart failure. cMyBP-C is a sarcomeric thick filament protein that interacts with titin, myosin and actin to regulate sarcomeric assembly, structure and function. Elucidating the function of cMyBP-C is clinically important because mutations in this protein have been linked to cardiomyopathy in more than sixty million people worldwide. One function of cMyBP-C is to regulate cross-bridge formation through dynamic phosphorylation by protein kinase A, protein kinase C and Ca(2+)-calmodulin-activated kinase II, suggesting that cMyBP-C phosphorylation serves as a highly coordinated point of contractile regulation. Moreover, dephosphorylation of cMyBP-C, which accelerates its degradation, has been shown to associate with the development of heart failure in mouse models and in humans. Strikingly, cMyBP-C phosphorylation presents a potential target for therapeutic development as protection against ischemic-reperfusion injury, which has been demonstrated in mouse hearts. Also, emerging evidence suggests that cMyBP-C has the potential to be used as a biomarker for diagnosing myocardial infarction. Although many aspects of cMyBP-C phosphorylation and function remain poorly understood, cMyBP-C and its phosphorylation states have significant promise as a target for therapy and for providing a better understanding of the mechanics of heart function during health and disease. In this review we discuss the most recent findings with respect to cMyBP-C phosphorylation and function and determine potential future directions to better understand the functional role of cMyBP-C and phosphorylation in sarcomeric structure, myocardial contractility and cardioprotection.