Nuclear Translocation Of Calpain-2Mediates Apoptosis In Hypertrophic Cardiomyocytes Of Transverse Aortic Constriction Rat

IntroductionDespite advances in treatment, cardiovascular disease is still the top cause ofmorbidity and mortality in the world. Left ventricular hypertrophy （LVH）represents both an adaptive response to increased cardiac workload and aprecursor state of heart failure. Accumulated evidence has indicated thatcardiomyocyte death by apoptosis contributes a lot to the transition from LVH toheart failure （HF）. Moreover, apoptosis accurs in early stage of LVH and thehypertrophic cardiomyocytes exhibits higher susceptibility to apoptosisstimulated by angiotensinâ…¡ （AngⅡ）. The mechanisms underlying the enhancedsusceptibility to apoptosis of hypertrophic cardiomyocytes have not beencompletely elucidated yet. In our previous experiments, we have showed thatcalpain-2translocated to the cardiomyocyte nucleus in tail-suspended rat heartand might mediate apoptosis. Similarly, we have obtained some evidence thatcalpain-2translocates into nuclei in hypertrophic cardiomyocytes of transverseaortic constriction rat （TAC）. Therefore, we hypothesized that nucleartranslocation of calpain-2might induce apoptosis of hypertrophic cardiomyocytes.Aims1Assessment of the left ventricular geometry and function in the transverseaortic constriction rat.2Elucidation of the impact of Angâ…¡on apoptotic rate of CON and TACcardiomyocytes.3Observation on the electrically stimulated calcium transient incardiomyocytes.4Observation on the changes of expression, activity and location of calpain-2in TAC cardiomyocytes.5Observation on the expression of CaMKâ…¡ B in cardiomyocyte nuclei.6Observation on the expression of Bcl-2, mitochondrial membrane potentialand cytochrome C release.MethodsIn the present study, a rat model of chronic pressure overload induced bytransverse aortic constriction surgery was applied. Echocardiograms wereperformed on anesthetized animals to assess the changes in left ventriculargeometry and function after16weeks of surgery. Two-dimensionalechocardiography in the parasternal long-axis views of the LV and M-modemeasurements at the level of the papillary muscle were obtained. To evaluate thecardiomyocyte apoptosis, the TUNEL staining was performed with the use offluorescein-dUTP. Calcium transient of intracellular Ca²⁺concentration ([Ca²⁺])was observed in the cardiomyocyte labeled by Fluo3/AM using a confocalmicroscope in the line-scan mode under488nm wavelength of excitation.Immunofluorescent cytochemistry and confocal analysis was carried out tovalidate the location of calpains, cytochrome c release, etc. Western blot was done to analyze the expression of calpain-1, calpain-2, calpastatin, Bcl-2andCaMKâ…¡ B. Co-immunoprecipitation was applied to validate the relationshipbetween calpain-2and CaMKâ…¡ B. The mRNA of bcl-2was examined byReal-Time PCR in the left ventricular myocardium. JC-1was used to estimate thechanges of mitochondrial membrane potential （Δψm） and cytochrome C releasewas assessed based on the absence or presence of punctuate fluorescent staining,indicating the presence of cytochrome c inside the mitochondria.Results1Transverse aortic constriction rats progressively developed concentricLVH and were susceptible to apoptosis compared with control rats.Echocardiography revealed that the left ventricular wall thickness of TAChearts was markedly higher than that measured in CON hearts after16weeks ofsurgical operation. The shortening fraction （FS） and ejection fraction （EF） ofTAC hearts was significantly higher than its control group, indicating that thecardiac function of TAC rats was greatly enhanced. The number ofTUNEL-positive nuclei nuclei was increased in cardiomyocytes of TAC rats. Inparticular, the number of TUNEL-positive nuclei was significantly increased inTAC with Angâ…¡ treatment compared with CON without Angâ…¡ treatment.Calpain inhibitor, PD150606, can effectively inhibit the impact of Angâ…¡ onhypertrophic cardiomyocytes.2Calcium concentration in hypertrophic cardiomyocytes was significantlyincreased. Angâ…¡caused a marked increase of Ca²⁺concentration in nuclei.Line-scan imaging of a ventricular myocyte during an electrically stimulatedcalcium transient indicated that before exposure to Angâ…¡, the intranuclear [Ca²⁺]was essentially identical to the cytoplasmic [Ca²⁺] in the control group, while theintranuclear [Ca²⁺] was markedly higher than the cytoplasmic [Ca²⁺] in the hypertrophic cardiomyocytes. Exposed to10nmol/L Angâ…¡, the intranuclear[Ca²⁺] was significantly higher than the cytoplasmic [Ca²⁺] in the control group;both the intranuclear [Ca²⁺] and the cytoplasmic [Ca²⁺] of the hypertrophicmyocytes were increased greatly compared with before, implying Ang â…¡increased nuclear calcium transients selectively without elevating cytoplasmiccalcium transients in control myocytes, but Angâ…¡increased both of them inhypertrophic myocytes.3Calpain-2activity was increased in hypertrophy heart and the activatedcalpain-2was translocated to the cardiomyocyte nucleus.Calpain-1and calpastatin were located in the cytoplasm and calpain-2waslocated in the cytoplasm as well as in the nuclei according to theimmunofluorescent cytochemistry and confocal analysis of isolatedcardiomyocytes. In particular, the nuclear translocation of calpain-2increased inthe hypertrophic myocytes. Western blot validated that the expression of total andautolyzed calpain-2increased in isolated nuclei of myocytes in TAC rats underAngâ…¡ treatment, indicating that the calpain-2activity increased in the TAC rats.The nuclear calpastatin was not detectable by Western blot in both TAC andcontrol groups.4Activated calpain-2degraded CaMKⅡδB.The nuclear expression of CaMKâ…¡ B decreased as the nuclear expressionand activity of calpain-2increased in TAC hearts with Angâ…¡ treatment.Co-immunoprecipitation verified Ang â…¡ enhanced the interaction betweenactivated calpain-2and CaMKâ…¡ B. CaMKâ…¡ B may be a substrating protein ofnuclear calpain-2.5Bcl-2gene expression was inhibited and mitochondria-dependent deathcascades was initiated in TAC heart stimulated by Angâ…¡. Real-Time PCR showed the mRNA of bcl-2was significantly decreased inthe TAC rats with Angâ…¡treatment. Similarly, the expression of Bcl-2protein wasmarkedly reduced by Angâ…¡ treatment in TAC rats. Angâ…¡ induced a strongermitochondrial depolarization and cytochrome C release in the hypertrophicmyocytes compared with those in the control group.ConclusionThe above data have suggested that in response to chronic pressure overload,hypertrophic cardiomyocytes apoptotic susceptibility is greatly increased.Calpain-2is activated and translocates to the nucleus in the TAC heart. Angâ…¡stimulation increases the intranuclear [Ca²⁺], thus further activates nuclearcalpain-2without the endogenous inhibitor of calpastatin and then degradesCaMKâ…¡ B. Consequently, expression of antiapoptotic gene bcl-2decreases,predisposing hypertrophic cardiomyocytes to apoptosis.