The Role Of Calpains In Intercalated Disc Remodeling

【Background】Cardiovascular diseases are the top killer all around the world. Cardiac remodeling plays a critical role in transition from compensate remodeling to uncompensated remodeling, that is heart failure. And intervention of heart remodeling is a key principle in clinical treatment of heart diseases and an important direction in basic research of cardiovascular physiology and pathophysiology. The intercalated disc(ICD) is the interconnection at the ends of two cardiomyocytes, a complex structure essential for mechanical, electrical and other signaling communication between the myocytes and, hence, heart function and growth. ICD remodeling is an integrated participant during heart remodeling. However, the mechanism of ICD remodeling is still unknown. Calcium overloading is a common pathophysiological phenomenon in various cardiomyopathies. Calpain is a neutral protease family dependent on the free Ca2+ concentration in cytoplasm, and calpastatin is the endogenous inhibitor of calpain. Calpain/calpastatin takes part in numerous physiological and pathophysiological processes mediated by Ca2+. Previous studies in our lab indicated that calpain-2 and calpastatin might locate at ICDs and there was little research on the role of calpain-2 and calpastatin in ICD remodeling. Therefore, we hypothesized that calpain-2 and calpastatin might participate in ICD remodeling during cardiac adaptation in tail-suspended rats, ischemia-reperfusion injury hearts and TAC induced cardiac hypertrophy. 【Objectives】(1) Observation of calpain-2 and calpastatin distribution in cardiomyocytes.(2) Comparison the distribution alteration of calpain-2 of calpastion in CON, SUS, I-R, and TAC hearts.(3) Observation ICD morphology change during cardiac adaptation in SUS, I-R, and TAC rat models.(4) Exploration of the substrates and function of calpains in ICD remodeling. 【Methods】In the present study, we applied immunohistochemistry(IHC), immunofluorescence(IF), pre-embedding and post-embedding immuno-electron microscopy to elucidate the distribution of calpain-2 and calpastatin in cardiac frozen sections as well as enzymatic isolated single cardiomyocytes. Laser scanning confocal microscopy was performed to observe the fine location of calpain-2 and calpastatin in cardiac tissues and cardiomyocytes. Three rat models were applied to induce different kinds of heart remodeling, those were, 4-week tail-suspended rats to simulate microgravity, in vitro ischemia-reperfusion injury hearts and transverse abdominal aortic constriction induced hypertension rats. Transmission electron microscopy(TEM) was used to observe the ultrastructural morphology of ICD during cardiac remodeling. To explore the roles of calpain-2 and calpastatin at ICDs, in vitro cultured neonatal rat cardiomyocytes were treated with ionophore A23187 to enhance the activity of calpains and PD150606 to inhibit calpain activity. The Calpain GloTM Protease assay was modified to test the activity of calpain in CON, A23187 treated and A23187 plus PD150606 treated neonatal rat cardiomyocytes. N-cadherin and CX-43, as potential substrates of calpains, were observed by immunofluorescence. 【Results】(1) There were calpain-2 and calpastatin distributed at the ICDs.IHC and IF revealed that calpain-2 in cardiomyocytes was distributed in a striated pattern and a moderate amount of calpain-2 was aggregated at the ICDs. Double immunostaining of calpastatin and N-cadherin/CX-43 demonstrated that calpastatin was distributed in a diffused pattern in the cytoplasm and a large amount of calpastatin was aggregated at the ICDs. Pre-embedding and post-embedding immune-electron microscopy verified the ultrastructural location of calpain-2 and calpastatin, showing that calpain-2 was located at Z-discs and the ICDs, and calpastatin was distributed randomly in the cytoplasm and aggregated at the ICDs, which was consistent with the distribution characteristics reflected by IHC and IF.(2) In SUS hearts, expression and distribution of calpastatin showed no significant difference, and calpain-2 demonstrated significant translocation into the nuclei.The soleus was significantly atrophied after 4-week tail-suspension, but the heart weight to body weight ratio in SUS showed no significant difference compared with that of the CON hearts. Western blot demonstrated that the total expression of calpain-2 and calpastatin didn’t change in SUS hearts compared with CON hearts. IF revealed that calpastatin distribution at the ICDs in SUS hearts showed no significant difference from the CON hearts, and calpain-2 was obviously translocated into the nuclei, which may enhance the apoptosis propensity of SUS cardiomyocytes.(3) Expression of calpastatin and N-cadherin in infarct zone was significantly decreased in I-R hearts.In I-R hearts, CX-43 and p-CX-43 were degraded in infarct zone and maintained intact in remote zone, indicating the border zone after I-R injury. Calpastatin expression and distribution significantly decreased by the side of infarct zone, and didn’t change by the side of remote zone. So did N-cadherin expression. TEM showed the ICDs of the infarct cardiomyocytes were disrupted and the end of myofibrils was detached from the membrane of ICDs, resulting in sarcomere contracture and cell death.(4) ICD remodeling was significantly increased after TAC surgery and calpain-2 was increased at the ICDs in TAC cardiomyocytes.TEM showed the ultrastructure change of ICDs in TAC hearts. The amplitude of the ICDs was increased even 4 h after TAC surgery, and continued till 12 weeks after TAC surgery. New sarcomeres were inserted into the myofibril between the ICD and the end of myofibril. IHC of calpain-2 showed that calpain-2 was increased at the ICDs in TAC hearts. IF of calpastatin showed no significant change in CON and TAC hearts.(5) N-cadherin was degraded by increasing calpain activity in cultured neonatal rat cardiomyocytes.Calpain activity was increased in cultured neonatal rat cardiomyocytes after ionophore A23187 stimulation, and PD150606 could significantly, but not completely, inhibit calpain activity induced by A23187. The fluorescence intensity of myofibrils labeled by phalloidin was significantly decreased in A23187 treated cardiomyocytes. N-cadherin was significantly degraded after calpain activation, but CX-43 expression and distribution showed no significant change. PD150606 could significantly inhibit the degradation of N-cadherin in A23187 treated cardiomyocytes. 【Conclusion】In the present study, distribution of calpain-2 and calpastatin at the ICDs is verified by IHC, IF and immuno-electron microscopy. Calpain-2 and calpastatin may take part in the ICD remodeling and sarcomerogenesis during TAC surgery induced cardiac hypertrophy and I-R injury. In sarcomerogenesis, calpain-2 may be involved in degradation of old structural proteins and calpastatin may be necessary to maintain the homeostasis in favor of new protein synthesis and assembly. When calpain is activated and lacks calpastatin inhibition, calpain will degrade N-cadherin, thus disrupts the connections between myofibrils and the ICDs.