Calpains have been implicated in heart diseases. While calpain-1 has been detrimental to the heart, the role of calpain-2 in cardiac pathology remains controversial. In this study we investigated whether sustained over-expression of calpain-2 had any adverse effects on the heart and the underlying mechanisms. Double transgenic mice (Tg-Capn2/tTA) were generated, which express human CAPN2 restricted to cardiomyocytes. The mice were subjected to echocardiography at age 3, 6, 8 and 12 months, and their heart tissues and sera were collected for analyses. We showed that transgenic mice over-expressing calpain-2 restricted to cardiomyocytes had normal heart function with no evidence of cardiac pathological remodeling at age 3 months. However, they exhibited features of dilated cardiomyopathy including increased heart size, enlarged heart chambers and heart dysfunction from age 8 months; histological analysis revealed loss of cardiomyocytes replaced by myocardial fibrosis and cardiomyocyte hypertrophy in transgenic mice from age 8 months. These cardiac alterations closely correlated with aberrant autophagy evidenced by significantly increased LC3BII and p62 protein levels and accumulation of autophagosomes in the hearts of transgenic mice. Notably, injection of 3-methyladenine, a well-established inhibitor of autophagy (30 mg/kg, i.p. once every 3 days starting from age 6 months for 2 months) prevented aberrant autophagy, attenuated myocardial injury and improved heart function in the transgenic mice. In cultured cardiomyocytes, over-expression of calpain-2 blocked autophagic flux by impairing lysosomal function. Furthermore, over-expression of calpain-2 resulted in lower levels of junctophilin-2 protein in the heart of transgenic mice and in cultured cardiomyocytes, which was attenuated by 3-methyladenine. In addition, blockade of autophagic flux by bafilomycin A (100 nM) induced a reduction of junctophilin-2 protein in cardiomyocytes. In summary, transgenic over-expression of calpain-2 induces age-dependent dilated cardiomyopathy in mice, which may be mediated through aberrant autophagy and a reduction of junctophilin-2. Thus, a sustained increase in calpain-2 may be detrimental to the heart.

钙蛋白酶与心脏病有关。虽然 calpain-1 对心脏有害，但 calpain-2 在心脏病理学中的作用仍存在争议。在这项研究中，我们调查了 calpain-2 的持续过度表达是否对心脏及其潜在机制有任何不利影响。产生了双转基因小鼠（Tg-Capn2/tTA），其表达仅限于心肌细胞的人CAPN2。在3、6、8和12个月时对小鼠进行超声心动图检查，并收集它们的心脏组织和血清进行分析。我们发现，仅限于心肌细胞过度表达 calpain-2 的转基因小鼠在 3 个月大时具有正常的心脏功能，没有证据显示心脏病理重塑。然而，他们从 8 个月起就表现出扩张型心肌病的特征，包括心脏体积增大、心室扩大和心功能不全；组织学分析显示，8个月大的转基因小鼠中，心肌细胞丧失，取而代之的是心肌纤维化和心肌细胞肥大。这些心脏改变与异常自噬密切相关，转基因小鼠心脏中 LC3BII 和 p62 蛋白水平显着增加以及自噬体积累证明了这一点。值得注意的是，注射 3-甲基腺嘌呤（一种成熟的自噬抑制剂）（30 mg/kg，从 6 个月大起每 3 天腹腔注射一次，持续 2 个月）可防止转基因小鼠的异常自噬，减轻心肌损伤并改善心脏功能。在培养的心肌细胞中，calpain-2 的过度表达通过损害溶酶体功能来阻断自噬流。此外，calpain-2 的过度表达导致转基因小鼠心脏和培养的心肌细胞中 junctophilin-2 蛋白水平较低，而 3-甲基腺嘌呤可减弱这种水平。此外，巴弗洛霉素 A (100 nM) 阻断自噬流会导致心肌细胞中 junctophilin-2 蛋白减少。总之，calpain-2 的转基因过度表达会诱导小鼠年龄依赖性扩张型心肌病，这可能是通过异常自噬和 junctophilin-2 的减少介导的。因此，calpain-2 的持续增加可能对心脏有害。