Canstatin suppresses isoproterenol-induced cardiac hypertrophy through inhibition of calcineurin/nuclear factor of activated T-cells pathway in rats

Pathological cardiac hypertrophy associated with cardiac dysfunction is an independent risk factor for arrhythmia, myocardial infarction and sudden death. Canstatin, a C-terminal fragment of type IV collagen alpha 2 chain, is abundantly expressed in normal heart tissue. We previously demonstrated that canstatin inhibits isoproterenol (ISO)-induced dephosphorylation of nuclear factor of activated T-cells (NFAT)c4, which plays an important role in cardiac hypertrophy, in differentiated H9c2 cardiomyoblasts. Thus, we investigated whether in vivo canstatin administration prevents ISO-induced cardiac hypertrophy through the inhibition of NFATc4 pathway. Rats were subcutaneously injected with ISO (5 mg/kg) or saline (Cont) for 7 days. Simultaneously, recombinant mouse canstatin (20 mu g/kg) or vehicle was intraperitoneally administered. After left ventricular wall thickness and cardiac function were measured by echocardiography, the hearts were isolated and left ventricular weight (LVW) was weighed. Azan staining was performed to measure cross-sectional diameter of cardiomyocytes. Activity of calcineurin, which dephosphorylates NFATc4, was measured by calcineurin phosphatase activity assay. Immunohistochemical staining was performed to evaluate nuclear translocation of NFATc4. Intracellular Ca2+ concentration in neonatal rat cardiomyocytes (NRCMs) was measured by using a calcium indicator. Canstatin significantly inhibited ISO-induced increase of LVW, left ventricular posterior wall thickness at end-diastole and diameter of cardiomyocytes. Canstatin significantly inhibited ISO-induced activation of calcineurin, nuclear translocation of NFATc4, increased mRNA expression of beta-myosin heavy chain and a-skeletal actin, and intracellular Ca2+ rise in NRCMs. In summary, we for the first time demonstrated that canstatin administration suppresses ISO-induced cardiac hypertrophy possibly through the blockade of calcineurin/NFATc4 pathway in rats.