Cardiac myosin binding protein C regulates postnatal myocyte cytokinesis

Homozygous cardiac myosin binding protein C-deficient (Mybpc^t/t) mice develop dramatic cardiac dilation shortly after birth; heart size increases almost twofold. We have investigated the mechanism of cardiac enlargement in these hearts. Throughout embryogenesis myocytes undergo cell division while maintaining the capacity to pump blood by rapidly disassembling and reforming myofibrillar components of the sarcomere throughout cell cycle progression. Shortly after birth, myocyte cell division ceases. Cardiac MYBPC is a thick filament protein that regulates sarcomere organization and rigidity. We demonstrate that many Mybpc^t/t myocytes undergo an additional round of cell division within 10 d postbirth compared with their wild-type counterparts, leading to increased numbers of mononuclear myocytes. Short-hairpin RNA knockdown of Mybpc3 mRNA in wild-type mice similarly extended the postnatal window of myocyte proliferation. However, adult Mybpc^t/t myocytes are unable to fully regenerate the myocardium after injury. MYBPC has unexpected inhibitory functions during postnatal myocyte cytokinesis and cell cycle progression. We suggest that human patients with homozygous MYBPC3-null mutations develop dilated cardiomyopathy, coupled with myocyte hyperplasia (increased cell number), as observed in Mybpc^t/t mice. Human patients, with heterozygous truncating MYBPC3 mutations, like mice with similar mutations, have hypertrophic cardiomyopathy. However, the mechanism leading to hypertrophic cardiomyopathy in heterozygous MYBPC3^+/− individuals is myocyte hypertrophy (increased cell size), whereas the mechanism leading to cardiac dilation in homozygous Mybpc3^−/− mice is primarily myocyte hyperplasia.Dilated cardiomyopathy (DCM) leads to heart failure and is a leading cause of morbidity and mortality (1, 2). DCM is generally diagnosed as left ventricular (LV) dilation with associated reduction in cardiac contraction measured as impaired fractional shortening (3). Hearts from affected individuals frequently demonstrate myocyte elongation, myocyte death, and fibrosis, in addition to LV dilation. DCM results from a variety of environmental factors, such as viral infection and alcohol abuse, as well as from mutations in a number of genes including titin, lamin A/C, cardiac actin, cardiac myosin heavy chain, and phospholamban (reviewed in refs. 4–6). Whether all of these DCM-inducing factors activate the same or different cellular pathways to produce similar clinical features remains uncertain. The mechanisms by which mutations in the cardiac myosin binding protein C (MYBPC3) gene and other sarcomere protein genes lead to cardiac dilatation are under investigation.MYBPC is a thick filament accessory protein component of the striated muscle sarcomere A band that constitutes 2–4% of the myofibril (discussed in ref. 7). Although there are four Mybpc genes in the mammalian genome, only cardiac Mybpc (Mybpc3) is expressed in embryonic, neonatal, and adult hearts (8, 9). Cardiac MYBPC interacts with at least four sarcomere components: myosin heavy chain, actin, myosin light chain 2, and titin (10–12). More than 400 cardiac MYBPC3 gene mutations have been identified in patients as a cause of hypertrophic cardiomyopathy (HCM), an autosomal dominant disorder resulting from defective sarcomeres (for reviews, see refs. 12, 13). Due to an ancient founder mutation, 4% of the population of India carries a truncating MYBPC3 mutation (14, 15). The majority of cardiac MYBPC3 mutations are predicted to encode truncated proteins that lack portions of either the carboxyl myosin and/or titin binding domains (7, 13). These truncating MYBPC3 mutations are thought to cause cardiac hypertrophy by inducing myocyte hypertrophy (increased cell size), rather than myocyte hyperplasia.We and other researchers have created mice that carry a mutant cardiac Mybpc3 gene to create murine HCM models (16–18). Heterozygous mice, designated Mybpc^t/+, like humans bearing the same mutation, develop adult onset HCM. Homozygous MYBPC3 mutations are a much rarer cause of human DCM than autosomal dominant mutations in other sarcomere protein genes. However, homozygous Mybpc^t/t mice that express two mutant alleles and no wild-type cardiac Mybpc3 develop LV dilation by 3 d postbirth and have all of the features of DCM, including LV chamber dilation, albeit mildly impaired fractional shortening (16). Unlike most humans with DCM, homozygous mutant cardiac Mybpc^t/t mice have normal survival despite their cardiac disease. Other homozygous null cardiac Mybpc3 mice develop an identical phenotype (7, 17, 18). Hence, for the studies described here, we assume that the phenotype of the Mybpc^t/t mice is due to lack of MYBPC protein, rather than to small amounts of truncated protein. Recently, two groups have demonstrated that delivery of MYBPC to Mybpc3-null hearts restores cardiac function and morphology (19, 20). Here, we have begun to dissect the mechanism by which homozygous Mybpc^t/t hearts develop DCM.Because Mybpc^t/t mice begin LV dilation within a few days postbirth (16), we hypothesized that this reflected abnormal development of neonatal myocytes. During fetal and early perinatal development in wild-type hearts, cardiomyocytes divide rapidly, producing hyperplastic cardiac growth (21). However, at 10 d postbirth, cardiomyocytes cease to divide and all subsequent increases in myocardial mass result from myocyte hypertrophy (22). Despite the importance of this phenomenon, little is known about the molecular basis for the transition from hyperplasic to hypertrophic-based myocardial growth. We hypothesized that abnormal cardiomyocyte growth, either hyperplastic or hypertrophic, in the perinatal period accounted for the LV dilation of Mybpc^t/t mouse hearts. To address this question, we have counted and measured cardiomyocytes from Mybpc^t/t and wild-type mice. We have also studied the consequences of reducing MYBPC levels by injecting Mybpc3-specific shRNA at birth. Neonatal cardiomyocytes lacking cardiac MYBPC, due to Mybpc3-specific shRNA knockdown, undergo an additional round of cytokinesis. We conclude that dramatic reductions in the amount of cardiac MYBPC leads to aberrant cell cycle regulation at the G1/S checkpoint, resulting in at least one extra round of myocyte division and DCM.