Background: Cardiokines are a group of heart produced-secretomes that maintain cardiac function and modulate pathologic remodeling. CTRP9 is a newly identified cardiokine. However, the biological function of cardiac generated CTRP9 remains unclear.
Methods and Results: Mice were subjected to myocardial ischemia/reperfusion (MI/R) and gene interfering methods were used. Compare to vehicle, inhibition of cardiac CTRP9 via intra-myocardial siRNA injection significantly reduced left ventricular ejection fraction (LVEF, 28.6%, P<0.01), augmented infarct size (P<0.05) and increased apoptosis post-MI/R (P<0.05). In contrast, overexpression of CTRP9 via lentivirus reversely improved MI/R-induced cardiac dysfunction and apoptosis (P<0.05, respectively). Notably, cardiac CTRP9 overexpression up-regulated endoplasmic reticulum (ER) protein calreticulin (CRT) expression (P<0.01). Co-IP results demonstrated that cardiac CTRP9 interacted with CRT and confocal images revealed that CTRP9 colocalized with CRT in the cytoplasm of neonatal rat cardiomyocytes. Moreover, inhibition of CRT blunted anti-apoptotic actions of cardiac CTRP9, determined by LDH release, Caspase 3 activity and Bcl-2 to Bax ratio (P<0.01, respectively). These results suggests that cardiac CTRP9 augments CRT retrotranslocation from ER to cytoplasm, modulating CTRP9’s anti-apoptotic functions. Furthermore, cardiac CTRP9 significantly phosphorylated PKA at Thr187 (64.5%, P<0.01) and its downstream effector CREB (1.8-fold, P<0.01). While, inhibition of CRT blunted PKA and CREB phosphorylation (P<0.01). To further confirm the in vivo role of cardiac CTRP9, CTRP9 lentivirus was specifically injected into CTRP9 knockout (CTRP9KO) rat’s heart. Compared to WT, CTRP9-KO rats performed a further decrease of LVEF post-MI/R (P<0.01) while cardiac CTRP9 reversely enhanced cardiac function (P<0.01). Additionally, deletion of CTRP9 resulted in a decrease of cytosolic CRT expression, an interaction up-regulated by cardiac CTRP9 overexpression.
Conclusion: Collectively, our study demonstrated for the first time that cardiac generated CTRP9 protects against MI/R injury via the regulation of CRT retrotranslocation to inhibit cardiomyocytes apoptosis.