P2586Cardiac progenitor cell exosome-associated periostin triggers reentry of cardiomyocytes into cell cycle

Abstract

Abstract Introduction Nanovesicles known as exosomes (Exo) from cardiac-derived progenitor cells (CPCs) are cardioprotective and improve cardiac function after myocardial infarction; however the mechanisms of benefit are incompletely understood, especially with respect to endogenous cardiomyocytes (CM) renewal. Periostin (POSTN), a secreted extracellular matrix protein, is emerging as a matricellular factor that can trigger CM proliferation. We have identified POSTN as a protein secreted by CPC and enriched in their exosomal fraction. Purpose We sought to determine whether Exo-CPC can induce proliferation of CM and to explore the role of exosomal POSTN in inducing reentry of CM into the cell cycle. Methods Exo were isolated from CPC condioned medium by density gradient ultracentrifugation. Fractions were analyzed by Western blotting for the presence of POSTN as well as specific Exo markers (TSG101, CD9). POSTN-depleted Exo (ExoCPC_SiPOSTN) were obtained by transfecting CPC with specific siRNA. Active DNA synthesis was assessed on primary cell culture of rat neonatal CM by EdU incorporation. H9C2 cardiomyocytic cells were used to assess by real-time RT-PCR the expression of downstream genes Hippo/Yes-associated protein (YAP) signaling pathway. Results Western blotting analysis allowed to specifically determining the presence of Exo markers and POSTN in the different fractions of secreted vesicles. Smaller fractions (f1-f3) have the highest amount of TSG101 and CD9 as well as POSTN, thus suggesting that CPC secrete POSTN associated with Exo. The silencing of POSTN in cells resulted in a 60% reduction of Exo-associated POSTN compared to naïve ExoCPC. ExoCPC but not ExoCPC_SiPOSTN, were able to increase phosporylation of AKT and ERK in H9C2 cells. YAP phosporylation and its degradation was decreased resulting in the activation of the downstream gene AurBKinase. By real-time PCR, AurBKinase expression was increased by 2.6 folds with ExoCPC and 1.5 folds with ExoCPC_SiPOSTN compared to cells not exposed to Exo. ExoCPC were able to increase 1.5 fold EdU incorporation in cardiac troponin-positive primary rat CM. ExoCPC_SiPSTN did not affect proliferation. Schematic figure Conclusion These results suggest that POSTN may promote cardiomyocyte proliferation through the direct activation of the AKT/ERK/Hippo-Yap pathway. Exosomes released by CPC are an important source of POSTN and may have a potential for promoting cardiac regeneration. Acknowledgement/Funding This work has been supported by The Swiss National Science Foundation under grant n° 310030_169194