Calpain-2核转位调节腹主动脉缩窄大鼠肥大心肌细胞凋亡易感性的机制

It will be impossible to terminate apoptotic pathway if the mitochondria-dependent death cascades initiate. We therefore hypothesized that to reduce the apoptotic susceptibility in hypertrophic cardiomyocytes is the key procedure to prevent from heart failure. Our lab has shown that the extent of hypertrophy in cardiomyocytes was positively related to the apoptotic susceptibility. Compelling evidence suggested that nuclear translocation of calpain-2, a Ca2+-dependent cysteine protease, might be associated with the enhanced apoptotic susceptibility of hypertrophic cardiomyocytes in transverse aortic constriction (TAC) rats. The experiments here will be undertaken (1)to determine whether the nuclear translocation of activated calpain-2 occurs consequent to enhanced cytoplasmic Ca2+ and ROS in cardiomyocytes of TAC rat, (2)to clarify that the promoted myocardial noradrenaline release from cardiac sympathetic nerve terminals, in association with the increased function of calcium transport of nucleoplasmic reticulum, can further induce a higher level of calpain-2 activation in nuclei of hypertrophic cardiomyocytes, (3)to elucidate that the activated calpain-2 in nucleolus cleaves CaMK IIδB and induces downregulation of Bcl-2, therefore the apoptotic susceptibility will be promoted via reduced inhibition of mitochondria-dependent apoptotic pathway, and (4)to confirm that the apoptotic susceptibility will not increase under apoptotic stimuli in calpain-2 siRNA-transfected cardiomyocytes of TAC rat. These data will support a critical role for calpain-2 in apoptotic susceptibility and provide a potential therapeutic target during the transition from cardiac hypertrophy to heart failure.

细胞一旦启动线粒体凋亡级联反应，则难以阻止其进程，故防治应置于早期环节- - 降低凋亡易感性。我们证实心肌细胞随肥大程度加大，其凋亡易感性增加；还发现肥大心肌细胞钙蛋白酶calpain-2向核转位，可能介导凋亡易感性增加。因此，本项目拟用腹主动脉缩窄高血压(TAC)大鼠模型：1)证明TAC大鼠肥大心肌细胞内Ca2+和ROS浓度升高，激活calpain-2向核转位；2)探明TAC大鼠肥厚心肌局部去甲肾上腺素浓度升高，激活核质网钙转运系统，使核Ca2+浓度升高，提高核转位calpain-2活性；3)阐明核内calpain-2降解核CaMK IIδB，进而下调Bcl-2表达，减弱对线粒体凋亡通路的抑制，使心肌细胞凋亡易感性增加；4)确认抑制calpain-2核转位可恢复肥大心肌凋亡易感性至对照水平，并证明calpain-2核转位为凋亡易感性增加的关键环节。以便为转变心衰的防治策略提供实验依据。

心肌细胞凋亡在肥厚心肌转向心衰中扮演着重要角色，在代偿性心肌肥厚早期，肥大心肌细胞在血管紧张素Ⅱ（AngⅡ）的作用下凋亡率会升高，即肥大心肌细胞凋亡易感性升高，但其具体机制尚不清楚。. 本研究重要结果包括（1）腹主动脉缩窄（TAC）16周大鼠心肌出现肥厚，凋亡易感性明显增加。TAC大鼠心脏室间隔和后壁明显肥厚，短轴缩短率及射血分数增加，心脏呈现代偿性肥厚，而其细胞凋亡率并未明显升高；AngⅡ作用后，TAC组凋亡率明显高于对照组；钙蛋白酶（calpain）抑制剂则能明显抑制其凋亡诱导作用。（2）肥大心肌细胞内Ca2+浓度升高。在对照组，核内与胞浆内的Ca2+浓度无差异，AngⅡ可明显提高CON组中核内的Ca2+浓度；在TAC组，胞浆与核内Ca2+浓度都明显高于CON组，AngⅡ使细胞内的Ca2+浓度进一步升高。（3）肥大心肌细胞中calpain-2活性升高，向细胞核转位明显。 Calpain-1和calpastatin主要分布于胞浆。Calpain-2除分布于胞浆外，在核膜周围或核内有少量分布，AngⅡ作用后，核内分布有增多的趋势；在TAC组，核转位的calpain-2明显增多。将胞浆蛋白与核蛋白分离，检测其中calpains的变化，发现calpain-1和calpastatin主要分布于细胞浆组分，而calpain-2在AngⅡ作用的TAC组核内表达明显增多，不但calpain-2的整体活性增强，而且核内calpain-2大部分是被激活的calpain-2，核内没有calpastatin的抑制作用，calpain-2的活性更高。（4）核转位的calpain-2激活后降解CaMKⅡδB。随着核内calpain-2的增多和活性的增强，CaMKⅡδB的表达减少。TAC组经AngⅡ作用后，核内的calpain-2与CaMKⅡδB的相互作用增加，提示CaMKⅡδB可能是激活的calpain-2的底物蛋白。（5）Bcl-2表达减少，线粒体凋亡通路更易被激活。TAC组大鼠心脏在AngⅡ的作用下Bcl-2的mRNA和蛋白表达水平明显减少；AngⅡ作用使Bcl-2的表达进一步降低，线粒体膜电位降低，Cyto C释放增多，激活线粒体凋亡通路。. 本研究阐述了calpain-2核转位介导肥大心肌细胞凋亡易感性增加的分子机制，为治疗和预防心肌肥厚向心衰转化提供了实验证据。