| The tissue kallikrein-related peptidase family (KLK) is a group of trypsin-andchymotrypsin-like serine proteases. KLK1is identified in heart and has cardioprotectiveand anti-hypertrophic effects. KLK1acts mainly on low-molecular weight kininogen toproduce kinin peptides, which exert cardioprotective effects mainly via kinin B2R.However,whether other KLK family members play a role in the cardioprotection andhypertrophy remains unknown. Our previous study has found KLK8overexpressionincreased release of bradykinin in cardiomyocytes and attenuated H/R-induced injurymainly via kinin B2R in culture rat neonatal cardiomyocytes. In addition, we found thatKLK8overexpression induces hypertrophy via serine protease-dependent, B1R/B2R-independent pathway, however critical mechanism involved in this effect still remainsunknown.As serine proteases, KLKs hydrolyzed a series of protein substrate generatedbiological effects. Besides kininogens, a variety of substrates can be degraded by KLKfamily serine proteases. For instance, KLKs are known to degrade extracellular matrix(ECM), pro-epidermal growth factor (EGF) and Protease-Activated Receptor (PAR).Both EGF and PAR have been known to mediate biological functions in myocardialcells, however, whether KLK8induce cardiac hypertrophy via EGF and PAR dependentis still unknown. In our study, we firstly identify the changes of KLK8expression inheart tissue of rat hypertrophy model. Then, using intracardial injection of adenovirusKLK8expression vector, we investigated whether KLK induced myocardialhypertrophy in vivo. Finally, in the primary cultured myocardial cells, we furtherexplored whether EGF or PAR was involved in KLK8-induced myocardial hypertrophy.Content and result:1. KLK8is expressed in myocardiumUsing immunohistochemisry technique, we first confirmed that the KLK8wasexpressed in rat heart tissues.2. KLK8expression in rat heart tissue of cardiac hypertrophymodelReal time PCR results show that the SHR and TAC rat, cardiac hypertrophymarkers ANP and Myh7mRNA expression levels were significantly increasedcompared with the control group. In SHR rats, elevated KLK8expressions were further confirmed by2-foldincreased mRNA,1.7-fold increased of protein as compared with WKY rats. In TACrats, elevated KLK8expressions were further confirmed by2.5-fold increased mRNA,2-fold increased of protein as compared with Sham rats. The results showed that KLK8expression was significantly increased in the myocardial tissue of rats with cardiachypertrophy3. Local cardiac overexpression of KLK8inducemyocardial hypertrophyIn the SHR and TAC myocardial hypertrophy modle, compared with the controlgroup, expression level of KLK8were significantly increased, suggesting that KLK8may be involved in the process of cardiac hypertrophy. So we constructed adenovirusKLK8, and further investigated the effects of KLK8overexpression on myocardialremodeling and function.1. Intracardiac injection of adenovirus KLK8(Ad-KLK8) into left ventricularanterior wall led to significant increases in KLK8mRNA and protein expression.2. Ad-KLK8injection led to significant increases in cardiac ANP and Myh7mRNA expression.3. Masson staining showed Ad-KLK8injection led to no significant change infibrosis level compared with adenovirus vector (Ad-vector) injection group;immunohistochemical results showed that Ad-klk8injection led to significant increasein rat myocardium cross sectional area.4. Echocardiogram results showed that compared with the injection vectoradenovirus (Ad-vector) group, the left ventricular injection of Ad-KLK8led tosignificant increases in rats systolic and diastolic anterior wall thickness (LVAWs,LVAWd), ejection fraction(%EF) and fractional shortening (%FS); the left ventricularinjection of Ad-KLK8led to significant decreases in left ventricular systolic anddiastolic the end diameter (LVIDs, LVIDd), left ventricular systolic and diastolicend volume of (LVs, LVs); the left ventricular injection of Ad-KLK8led to no obviouschange in left ventricular systolic and diastolic posterior wall (LVPWs, LVPWd) andthe ratio of heart weight and body weight of rats (HW/BW).These results suggest that, cardiac overexpression of KLK8could significantlyinduce myocardial hypertrophy, leading to enhancement of cardiac ejection function. 4. Both EGF and PARs are involved in the pro-hypertrophiceffects of KLK8in primary cultured neonatal cardiomyocytes.(1) The hypertrophic effect of KLK8is dependent on EGF signaling.We then investigated the molecular mechanisms responsible for KLK8inductionof hypertrophy. At first, we determined whether the hypertrophic effect of KLK8isthrough its proteolytic activity. Two serine protease inhibitors, antipain and ZnSO4were used to block the proteolytic activity of KLK8as described previously. We foundthat antipain and ZnSO4entirely blocked the hypertrophic effects of KLK8.KLKs have the ability to hydrolyze a variety of substrates such as pro-epidermalgrowth factor (pro-EGF). It is well known that EGF induces hypertrophy in various celltypes. Thus, we investigated whether the hypertrophic effect of KLK8was throughreleasing EGF. It was found that overexpression of KLK8significantly increased theEGF level in the culture media. This effect was completely abolished by serine proteaseinhibitors antipain and ZnSO4. However, neither pharmacological antagonists norsiRNAs of kinin B1and B2receptors affected EGF level induced by KLK8overexpression.To confirm EGF is involved in hypertrophic effect of KLK8, the effects of theantagonist of EGF receptor and siRNA targeting EGF receptor (EGFR) on KLK8-induced hypertrophy were then investigated. We found that EGF receptor antagonistblocked hypertrophic effects of KLK8. In addition, we also used EGFR siRNA, whichresulted in70%reduction of EGFR. It was found that knockdown of EGFR alsosignificantly attenuated KLK8-induced hypertrophy. The Ad-KLK8transfection of cultured neonatal rat myocardial cells, results showed that KLK8overexpression can lead to cardiac myocyte hypertrophy marker ANP and Myh7expression of mRNA increased significantly, while the content of total protein inmyocardial cells and cell size were all significantly increased, indicating that KLK8may induce cardiomyocyte hypertrophy. At the same time, the overexpression of KLK8can significantly increase the content of EGF in the culture supernatants, and the effectof KLK8can be partially reversed by serine inhibitor protease activity of antipain andZnSO4. In addition, EGF receptor antagonist (AG1478) and small interfering RNA(EGFR siRNA)could significantly reverse KLK8induced cardiac myocyte hypertrophyeffect.These results showed that: KLK8may in part by promoting EGF generate,activation of EGF receptors and to induce the hypertrophy of cardiac myocytes in vitro.(2) The hypertrophic effect of KLK8is dependent on PAR activation PARs is a G protein coupled receptors, including PAR1, PAR2and PAR4arewidely distributed in the cardiovascular system, some scholars also found in vascularsmooth muscle cells, plasma kallikrein can activate PAR1/2by does not depend on thebradykinin receptors, followed activation of MAPKs (ERK1/2and JNK) pathway.We found that PAR1antagonist (RWJ56110) and PAR2antagonist (FSLLRY-NH2)blocked hypertrophic effects of KLK8. In addition, we also used PAR1siRNA andPAR2siRNA, which resulted in about80%reduction of PAR1and PAR2, respectively.It was found that knockdown of PAR1or PAR2also significantly attenuatedKLK8-induced hypertrophy. These results showed that: KLK8may in part by activatingPAR1/2, and then induce the hypertrophy of cardiac myocytes in vitro.Conclusion：The research shown, KLK8expression increased significantly in myocardiumhypertrophy rats; intracardiac injection of Ad-KLK8can lead to cardiac hypertrophyand enhancement of left ventricular function; Both EGF and PARs are involved in thepro-hypertrophic effects of KLK8in primary cultured neonatal cardiomyocytes. |
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