Melatonin Attenuates Pathological Cardiac Hypertrophy By Increasing MICU1 Expression

Objective:1.To determine the concrete role of mitochondrial MICU1 in pathological cardiac hypertrophy.2.To clarify whether[Ca²⁺]m overload inhibition is an intrinsic mechanism in MICU1's protection against cardiac hypertrophy.3.To investigate whether MICU1 and its effects on[Ca²⁺]m overload mediates melatonin's cardioprotection.Methods:The cardiac hypertrophy models were treated with chronic subcutaneous Ang-II infusion?1000 ng/kg/min?at a rate of 0.11?l/h by a mini-osmotic pump for 28 days.?1?The cardiac function was obtained by echocardiography;?2?the interstitial fibrosis was detected by Masson staining;?3?the size of heart was measured by H&E staining;?4?the expression of mitochondrial MICU1 in myocardial tissue was detected by Western blotting.In addition,the model of myocardial hypertrophy was established via incubation of NMVMs with Ang-II?200 nM?for 48 hours in vitro.?1?The cell surface area measurement were determined by?-actinin staining;?2?qRT-PCR was using to detect ANP,BNP,?-MHC and?-MHC expression level;?3?transmission electron microscopy was used to measure the mitochondrial morphologic alterations of mitochondria;?4?ATP content and the level of mitochondrial membrane potential were detected by ATP kit and JC-1 kit.?5?the[Ca²⁺]m concentrations were measured by atomic absorption flame spectroscopy and laser-scanning confocal microscope,respectively.Methods:1.With the methods of echocardiography,H&E staining,Masson staining,?-actinin staining,qRT-PCR and so on,the cardiac hypertrophy both in vivo and in vitro was successfully established.2.After 28 d chronic subcutaneous Ang-II infusion by a mini-osmotic pump,tissues were harvested from left ventricles.The total MICU1 protein level was significantly decreased.Meanwhile,western blotting also indicated that the mitochondrial MICU1 was inclined.3.To determine the role of mitochondrial MICU1 in pathological cardiac hypertrophy,we knocked down MICU1 in mice and NMVMs by MICU1-targeted siRNA.Echocardiography found that MICU1knockout significantly worsened the cardiac function,as evidenced by decreased LVEF,LVFS and increased LVPWd.HE staining showed that the absence of MICU1 enlarged hearts,increased the ratio of HW/BW and LW/BW.At the same time,the results of Masson staining also suggested that the loss of MICU1 significantly increases the level of fibrosis in the myocardium.In cell experiments,the decrease of MICU1 increased the average cross-sectional area of cells and increased the expression of ANP,BNP,?-MHC and decreased?-MHC.We further transfected the adenovirus to increase the expression of MICU1 in the heart tissue.The results showed that overexpression of MICU1could reverse the changes above and attenuated the cardiac hypertrophy both in vivo and vitro.4.MICU1 deletion in cardiomyocytes not only aggravated the mitochondrial damage induced by Ang-II?blurred crista and disintegrated membrane?,but also aggravated the inhibition of mitochondrial function?decreased ATP production and membrane potential?.[Ca²⁺]m overload is considered to be an important factor in myocardial injury.Mitochondrial MICU1 protect against[Ca²⁺]m overload in hypertrophic cardiomyocytes,determined by atomic flame and confocal laser confocal.However,overexpression of MICU1 maintained the mitochondrial morphologic integrity under hypertrophic conditions and significantly improved mitochondrial function.More importantly,the upregulation of MICU1 also alleviated[Ca²⁺]m overload.5.Melatonin intervention significantly increased the expression of mitochondrial MICU1 in myocardial tissue.However,the simultaneous intervention of luzindole?a nonspecific melatonin receptor blocker?and melatonin inhibited the expression of MICU1 in ischemic myocardium.These results suggested that melatonin increased the expression of MICU1 in a receptor-dependent way.In the condition of myocardial hypertrophy,melatonin reduced mitochondrial damage,improved cardiac function,and inhibited the fibrosis generation.However,the deletion of MICU1 eliminated the above myocardial protective effect of melatonin.Conclusion:1.The expression of mitochondrial MICU1 is inhibited in pathological cardiac hypertrophy.2.Mitochondrial MICU1 exerts cardioprotection on hypertrophy via the improved mitochondrial impairment,suppression of myocardial fibrosis and the recovery of cardiac function.3.Mitochondrial damage and[Ca²⁺]m overload are the key mechanisms of myocardial hypertrophy mediated by MICU1 deficiency.4.Melatonin can increase the expression of MICU1 in myocardium,and the latter is an essential factor for melatonin to exert myocardial protective effect.