We demonstrated that cardiomyocytic apoptosis, collagen deposition in the surviving myocardium after MI and LV end-diastolic diameter were decreased; While LV weight / body weight ratio and LVEF were increased with C3G knockout lentiviral treatment via down-regulation of pro-survival molecules including C3G, p-ERK1/2 and Bcl-2, and up-regulation of pro-apoptotic Bax expression level in the surviving myocardium. However, in our study, knockout of C3G was discovered to delete C3G and diminish cellular proliferation in vitro cardiac myocytic and fibroblastic cell lines respectively via down-regulation of p-ERK1/2 and Bcl-2, and up-regulation of Bax protein expression. Knockdown of pro-survival C3G in myocardium may mitigate surviving myocardial remodeling after MI, possibly via down-regulation of p-ERK1/2, Bcl-2 and up-regulation of Bax in vivo cardiac myocytes and fibroblasts in the surviving myocardium.
With the reparative fibrosis of infarcted myocardium, the remodeling of the surviving myocardium in the border and remote zones around the infarcted lesion is associated with detrimental processes of LV function and poor prognosis. Terminally differentiated cardiomyocyte in vivo makes up the most (≈ 90%) of cardiac mass, although it is usually unable to proliferate. Cardiac fibroblast constitutes the majority (≈ 70%) of cells in normal cardiac muscle, because it can proliferate [1]. Therefore, the over-proliferated cardiac fibroblasts are very important for the progressive myocardial remodeling after MI [1].
The surviving myocardial remodeling results from global activation of sympathetic nervous and renin-angiotensin-aldosterone systems (RAAS) in the compensatory responses induced by MI [11, 12]. After MI, the activated autonomic nervous system and RAAS have been proved to promote cell survival both in vivo cardiomyocytes and cardiac fibroblasts via up-regulation of pro-survival gene expression [13, 14]. The over-activation of sympathetic nervous system and RAAS result in ongoing cardiac fibroblast proliferation, myocardial fibrosis, and consequently increase the apoptosis in vivo cardiomyocytes, leading to the progressive surviving myocardial remodeling after MI [1].
In contrast, experimental studies have confirmed that globally non-cardiac selective blockage of the activated sympathetic nervous system and RAAS is able to relieve apoptosis of cardiomyocytes and other harmful process in the surviving myocardium after MI [15], although inhibition of the activated autonomic nervous system and RAAS is considered to result in apoptosis in vivo cardiomyocytes and cardiac fibroblasts via up-regulation of pro-apoptotic gene expression [15].
A previous study has proved that cardiomyocyte-specific knockout of a pro-apoptotic gene attenuated myocardial remodeling induced by pressure overload or MI [2]; However, systemic knockout of the same pro-apoptotic gene can augment LV remodeling process [2].
Integrin pathway links extracellular matrix substances to cytoskeleton in cells, and senses mechanical stretch, and translates it into intracellular biochemical signaling [3, 16]. Cardiomyocyte-specific deletion of pro-survival integrin β1 subunit and its downstream molecules such as FAK may increase apoptosis in vivo cardiomyocytes and aggravate cardiomyopathy induced by MI, isoproterenol or pressure overload [4, 5]. Knockout of cardiomyocyte-specific pro-survival FAK or integrin-linked kinase (ILK) even spontaneously results in dilated cardiomyopathy with reduced LV function [17, 18]. However, a previous study has demonstrated that globally systemic inhibition of pro-survival FAK attenuated cardiomyopathy and preserved cardiac function in post-MI [19]. Therefore, knockdown of the integrin pathway pro-survival component C3G in myocardium may have a protective effect against myocardial remodeling after MI.
Pro-survival C3G protein is a family member of guanine nucleotide exchange factor in the integrin pathway. After stimulation by signals through integrins, C3G binds with N-terminal Src homology 3 domain of its upstream adaptor CrkL, and subsequently activates its downstream GTPases such as Rac1 [20]. On the basis of existing evidences, pro-survival C3G can inhibit cell apoptosis in vitro cardiomyocytes via up-regulation of its downstream pro-survival AKT, p-ERK1/2, Bcl-2 and down-regulation of pro-apoptotic Bax [7, 9]. In addition, our current experimental study has replenished that knockout of pro-survival C3G by a specific knockout lentivirus can promote cell apoptosis in vitro cardiac fibroblastic cell line via down-regulation of p-ERK1/2, Bcl-2, and up-regulation of Bax. A previous study has confirmed that pro-survival expression of C3G, p-ERK1/2 and Bcl-2 were up-regulated, while pro-apoptotic Bax expression was down-regulated in the surviving myocardium after MI [8]. These molecular expression changes possibly resulted from the global activation of sympathetic nervous system and RAAS induced by MI. However, for the first time, we have documented that knockdown of pro-survival C3G in myocardium mitigated cardiomyocytic apoptosis, myocardial fibrosis and remodeling around the infarcted lesion, possibly via down-regulation of pro-survival p-ERK1/2, Bcl-2, and up-regulation of pro-apoptotic Bax mainly in vivo cardiac fibroblasts in the surviving myocardium.
The underlying mechanisms for the mitigation of the surviving myocardial remodeling after MI by knockdown of pro-survival C3G in myocardium are as follows: Cardiac fibroblast in vivo may play a major role in cell survival in vivo cardiomyocytes after MI [1]; While the proliferative cardiac fibroblasts in vivo may be more susceptible to the treatment with C3G knockdown lentivirus than non-proliferative cardiomyocytes in vivo. Knockdown of pro-survival C3G may mainly depress the proliferation of cardiac fibroblasts in the myocardium, and alleviate myocardial fibrosis, and consequently result in inhibition of cardiomyocytic apoptosis and myocardial remodeling after MI, although knockdown of C3G may lead to cellular apoptosis both in vivo cardiac fibroblasts and myocytes in the surviving myocardium. Appropriate-intensity transient knockdown of pro-survival C3G, which may mainly inhibit cardiac fibroblast proliferation, could be developed as a novel potential therapeutic measure to relieve the surviving myocardial remodeling, fibrosis, ischemic cardiomyopathy and cardiac dysfunction. Moreover, we have used experimental results in vitro H9C2 cardiac myoblasts to interpret mechanisms for cardiac structural phenotypic changes in vivo. As considering that the H9C2 cell has some different phenotypes from adult primary cardiomyocyte, the conclusions made in this study should be taken cautiously.
In this study, the recombinant lentivirus can knockout C3G in vitro cardiac myocytic and fibroblastic cell lines. However, it can not knockout all of C3G in the myocardium, because C3G knockout lentivirus was administered just transiently in the myocardium. Nevertheless, it can knockdown part of C3G in the myocardium and positively affect the myocardial remodeling. The effects of permanently systemic C3G knockout on surviving myocardial remodeling after MI are still unclear. The role of pro-survival C3G in myocardial remodeling remains to be redefined in the future via establishment of animal transgenic model with systemic knockout of C3G.