The Role Of Calpains On The Cardiomyocyte Growth

BackgroundHeart failure(HF) is one of the important causes of cardiovascular disease(CVD) mortality. The transition from compensatory myocardial hypertrophy induced by pressure overload to HF has seriously constraint the effectiveness of the prevention and treatment for CVD. It would contribute to our understanding for the mechanism of the transition to explore the model of myofibrils addition in parallel. Accumulated evidence has revealed that intercalated disc(ICD) plays an important role in this process. The proposal of weaving hypothesis and transitional junction has explained the mechanism of sarcomere addition in series; however, the manner how to add a myofibril laterally is still not clarified. The proteins that consist of the myofibrils are in a dynamic balance between constant synthesis and degradation and both of which can be activated by increased cardiac workload. Calpain is a calcium-dependent neutral protease and calpastatin is its endogenous inhibitor. It has been shown that calpains are involved in the renewal of myofibril proteins and the remodeling of the cytoskeleton, and their protein expression level is increased during cardiomyocyte hypertrophy. Thus, they would also play an important role in the physiological hypertrophy of cardiomyocyte. During the postnatal development of rats, cardiomyocyte becomes longer and wider along with the increases in cardiac volume- and pressure-load. Therefore, the present study took the process of rat growth after birth as a model to explore the pattern of myofibril addition in parallel and the role of calpain on ICD remodeling during the heart development.AimsBy means of cardiomyocyte physiological hypertrophy during rat postnatal development, this study aimed to establish the model of myofibril addition in parallel and to explore the possible role of calpain and calpastatin in the remodeling of ICD and the addition of sarcomere.MethodThe body weight and heart weight of rats were weighed on the postnatal 1st, 2nd, 3rd, 4th, 5th, 6th, 7th, 9th, 11 th, 14 th, 28 th and 90 th days(P1, P2, P3, P4, P5, P6, P7, P9, P11, P14, P28, and P90), respectively. The cardiomyocyte proliferation rate during postnatal development was calculated by labeling nuclei with Ki67 and by identifying the source of nuclei with wheat germ agglutinin(WGA). The time window of observation was selected during rapid cardiac growth. The ultrastructures of myocardial development were qualitatively and quantitatively analyzed, and then, the lateral assembly model of new myofibrils was proposed. Afterwards, the expression level of N-cadherin(N-cad), connexion 43(CX43), calpain-1, calpain-2 and calpastatin was detected by Western blots and the localization of N-cad, desmoglein-2(DSG-2), calpain-2 and calpastatin was observed by immunohistochemistry. The migrating pattern of N-cadherin, DSG-2, CX43, calpain-2 and calpastatin was analyzed during the ICD remodeling.Result1. A novel model of myofibril and ICD assembly in parallel.Cardiomyocyte proliferation rate decreased by 6.76% ± 1.14% with Ki67 staining on the P7. Combining the data of rat body weight and heart weight, we selected the time window of observation from the P7 to the P28. The P90 was used as a control point of adult heart.The frequency distribution of myofibril width suggests that there was the fundamental unit in width in the myofibril which was called primary myofibril. Primary myofibril was largely located under plasma membrane. The percentages of primary myofibril under plasma membrane were 48.53% ± 0.93% on the P7, 49.69% ± 2.51% on the P14, 36.84% ± 0.27% on the P28, and 4.86% ± 0.12% on the P90, respectively. The percentages of primary myofibril rapidly decreased on the P90. The above results suggest that primary myofibril under membrane might be the newly formed myofibril, and then primary myofibrils fused to mature myofibrils.There were three kinds of ICDs in rat myocardium during postnatal development. The parallel ICD showed desmosome-like or fascia adheren-like structures, parallel with the longitudinal axis of cardiomyocyte, and no attachment of myofibrils. The oblique ICD was attached with primary myofibrils. The primary transverse ICD was similar with the mature ICD, but had one plica. The mature transverse ICD has plicae and was attached with the myofibrils. The mature ICD had an average width of 0.2 ~ 0.5 μm and a maximal width of 2 μm. There was a positive linear correlation between myofibril width and the plica number of fascia adherens. The percentage of parallel ICD decreased gradually, but the percentage of transverse ICD increased gradually from the P7 to P90. The percentage of oblique ICD kept a constant level from the P7 to P28, but decreased sharply on the P90. These results indicate that parallel ICD transited to oblique ICD and further to transverse ICD during the postnatal development of rat heart.According to the above results, we proposed a novel model of the lateral assembly of myofibrils during physiological cardiac hypertrophy. The parallel ICD occurred at the Z-disc or transitional junction between two neighboring cardiomyocytes. Primary myofibrils attached to parallel ICD. The pulling force of primary myofibrils transited parallel ICD to oblique ICD. Oblique ICD developed to the primary transverse ICD by structure remodeling in order to stabilize the transmission of contraction force. Finally, the primary transverse ICD and primary myofibrils were widened by repeating the preceding process or the pre-existing ICD was widened.2. Dynamic alteration of transmembrane proteins of ICD during postnatal development of rat heart.The expression of N-cadherin increased gradually during postnatal development of heart. The expression of CX43 reached a peak on the P14, and then decreased to a stable level on the P90. N-cad, DSG-2 and CX43 located all around plasma membrane on the P1, then accumulated to the longitudinal ends of cardiomyocytes, indicating preliminary formation of the ICD on the P7. The localization of three proteins at the ICD increased progressively, but their lateral localization showed an inverse trend from the P14 to P90. However, CX43 still kept a certain amount of lateral localization even on the P90 as compared with N-cad and DSG-2. Quantitative colocalization of proteins was analyzed by the stereological method. Total percentage of colocalization of N-Cad with DSG2 was 33.5% on the P1, and increased to 38.6% on the P7, 9.4% in ICD and 29.2% in lateral side. Total percentage of colocalization of N-cad with DSG-2 increased to 65.7% on the P90, ICD colocalization increasing to 60.5% and lateral colocalization decreasing to 5.2%. Total percentage of colocalization of N-cad with CX43 increased from 10.3% on the P1 to 37.1% on the P90, and only ICD colocalization increased, but lateral colocalization kept about 5%. The colocalization pattern of DSG-2 with CX43 was similar to that of N-cad with CX43. Total percentage of colocalization of N-cad with DSG-2 was higher than those of N-cad or DSG-2 with CX43. The above results suggest that the formation of mechanical junctions at the ICD of cardiomyocyte is prior to that of electrochemistry junctions during postnatal development. In other words, cardiomyocyte growth needs a stable mechanical environment at first.3. The effects of calpain system on ICD remodeling during postnatal development of the heart.Immunohistochemistry and immunogold electron microscopy images showed that calpain-2 and calpastatin located at the ICD of cardiomyocytes. The expression of calpastatin increased progressively, but the expression of calpain-2 decreased gradually in the myocardium during postnatal development. The expression of calpain-2 or calpastatin on the P90 was significantly lower or higher than that on the P1. The localization of calpastatin at the longitudinal ends of cardiomyocytes was earlier than N-cad and DSG-2, but calpain-2 was later than N-cad and DSG-2. The percentage of calpastatin colocalized with N-cad or DSG-2 increased progressively, but the percentage of calpain-2 colocalized with N-cad decreased continuously in the myocardium during postnatal development. As calpastatin is the endogenous inhibitor of calpain-2, these results suggest that the assembly of N-cad and DSG-2 at ICDs needs a stable chemistry environment provided by calpastatin. Calpain-2 may play a role in the structure remodeling of ICDs.ConclusionThe above data suggest that primary myofibril would be added at the site of oblique ICD; primary myofibrils fused to become mature myofibrils,in the meanwhile the oblique ICD developed to mature transverse ICD during physiological hypertrophy of cardiomyocytes. The molecular mechanism underlying this process may be as follows: calpastatin accumulated firstly at the longitudinal ends of cardiomyocyte to provide a stable chemistry environment, and then primary ICDs were assembled by N-cad and DSG-2. Calpain-2 may be involved in the structure remodeling of ICDs in order that primary ICDs developed to mature ICDs. The ICD proteins that located around cell membrane would be degraded by calpain-2 and fulfilled the physiological hypertrophy of cardiomyocytes.