Kallikrein-related peptidase 8 is expressed in myocardium and induces cardiac hypertrophy
Xiaoyan Zhu，Xin Ni
The tissue kallikrein-related peptidase family (KLK) is a group of trypsin- and chymotrypsin-like serine proteases that share a similar homology to parent tissue kallikrein (KLK1). KLK1 is identified in heart and has anti-hypertrophic effects. However, whether other KLK family members play a role in regulating cardiac function remains unknown. In the present study, we demonstrated for the first time that KLK8 was expressed in myocardium. KLK8 expression was upregulated in left ventricle of cardiac hypertrophy models. Both intra-cardiac adenovirus-mediated and transgenic-mediated KLK8 overexpression led to cardiac hypertrophy in vivo. In primary neonatal rat cardiomyocytes, KLK8 knockdown inhibited phenylephrine (PE)-induced cardiomyocyte hypertrophy, whereas KLK8 overexpression promoted cardiomyocyte hypertrophy via a serine protease activity-dependent but kinin receptor-independent pathway. KLK8 overexpression increased epidermal growth factor (EGF) production, which was blocked by the inhibitors of serine protease. EGF receptor (EGFR) antagonist and EGFR knockdown reversed the hypertrophy induced by KLK8 overexpression. KLK8-induced cardiomyocyte hypertrophy was also significantly decreased by blocking the protease-activated receptor 1 (PAR1) or PAR2 pathway. Our data suggest that KLK8 may promote cardiomyocyte hypertrophy through EGF signaling- and PARs-dependent but a kinin receptor-independent pathway. It is implied that different KLK family members can subtly regulate cardiac function and remodeling.As shown in Fig. 1A, immunoreactive KLK8 was localized in ventricular myocytes of rat heart using a primary antibody that recognizes KLK8. KLK8 Immunoreactivity was abolished when the antibody was preabsorbed with excess peptide, thereby confirming the specificity of the antibody.We then investigated the expression pattern of KLK8 in hypertrophic hearts. Compared to the sham-operated rats, 8-week TAC rats showed significant left ventricular hypertrophy as evidenced by 1.5-fold increased heart weight to body weight (HW/BW) ratio, 2.3-fold increased ANP mRNA expression, 2.4-fold increased ANP mRNA expression, and 1.9-fold increased Myh7 mRNA expression (Fig. 1B). The mRNA and protein levels of KLK8 were significantly increased in the hearts subjected to TAC compared with Sham group (Fig. 1C, P < 0.05).It is known that hypertension could result in left ventricular hypertrophy. In spontaneously hypertensive rats (SHR), hypertrophy changes were confirmed by 3.7-fold increased ANP, 2.7-fold increased of BNP, and 2.1-fold increased Myh7 mRNA expression as compared with Wister Kyoto (WKY) rats (Fig. 1D). Myocardium KLK8 expression was also higher in SHR rats compared with WKY rats (Fig. 1E, P < 0.01).Given that KLK1 is also involved in cardiac hypertrophy, the expression of KLK1 in hypertrophic hearts was also examined. As shown in Fig. 1F,G, KLK1 mRNA and protein levels were decreased in myocardium of TAC rats compared with controls. However, there is no significant change in KLK1 mRNA and protein levels in SHR hearts compared with WKY hearts.Male Sprague-Dawley rats (8-week-old), SHR rats (16-week-old) and WKY rats (16-week-old) were obtained from Shanghai SLAC Laboratory Animal Co (Shanghai, China) and housed at controlled room temperature with free access to food and water under a natural day/night cycle. All animal protocols were approved by the Ethical Committee of Experimental Animals of Second Military Medical University, and confirmed to the principal in the Guide for the Care and Use of Laboratory Animals published by the United States National Institute of Health (NIH publication No. 85-23, revised 1996).The authors thank Jian-Guo Jia and Rui Chen (Shanghai Institute of Cardiovascular Diseases, Zhongshan Hospital of Fudan University) for their technical assistance of Echocardiography analysis. This work was supported by Major State Basic Research Program of China (No. 2013CB967404 to Xin Ni), National Natural Science Foundation of China (No. 31171120 & No. 31271270 to Xiao-Yan Zhu and No. 31571227 to Jianqiang Lu) and New Outstanding Young Scholar Program of Shanghai Health Bureau (XYQ2011045 to Xiao-Yan Zhu).Author Contributions X.N. and X.Z. conceived and designed the study. B.C. (Buqing Cao), Q.Y. and W.Z. performed all the experiments. Q.Y., Z.T., B.C. (Binhai Cong), J.L. and J.D. prepared the figures. X.N. and X.Z. wrote the manuscript. All authors have reviewed the manuscript.