Canstatin modulates L-type calcium channel activity in rat ventricular cardiomyocytes

Abstract

Excessive increase of cytosolic Ca2+ through the activation of L-type Ca2+ channels (LTCCs) via β adrenergic receptor induces apoptosis of cardiomyocytes. Canstatin, a cleaved fragment of collagen type IV α2 chain, is abundantly expressed in normal heart tissue. We previously reported that canstatin inhibits β adrenergic receptor-stimulated apoptosis in cardiomyoblasts. Here, we tested the hypothesis that canstatin regulates LTCCs activity in ventricular cardiomyocytes. Collagen type IV α2 chain (COL4A2) small interfering (si) RNA (for canstatin suppression) or control siRNA was injected via jugular vein in Wistar rats. Two days after the injection, electrocardiogram (ECG) was recorded and the left ventricular tissue was isolated using Langendorff apparatus. Immunofluorescence staining was performed to clarify the distribution of canstatin in cardiomyocytes. The knockdown efficiency was confirmed by Western blotting. The L-type Ca2+ channel current (ICaL) of ventricular cardiomyocyte was measured by a whole-cell patch clamp technique. In immunofluorescence staining, colocalization of canstatin and αv integrin was observed in the isolated ventricular cardiomyocytes. The ICaL of ventricular cardiomyocyte isolated from COL4A2 siRNA-injected rats was significantly enhanced compared with control siRNA-injected rats. Recombinant canstatin (250 ng/ml) significantly reversed it. ECG analysis showed that QT interval tended to be shortened and amplitude of T wave was significantly increased in the COL4A2 siRNA-injected rats. In summary, we for the first time clarified that suppressing canstatin expression increases the basal ICaL in ventricular cardiomyocytes. It is proposed that canstatin might play a role in the stabilization of cardiac function through the modulation of LTCC activity in cardiomyocytes.