Expression of Cardiac Troponin‐T in Cardiac H9c2 Cells During Co‐Culture with Mesenchymal Stem Cells

Abstract

Previous studies have shown that intravenous injection of mouse bone marrow mesenchymal stem cells (MSC) attenuate the loss of function in the heart after coronary artery occlusion (Boomsma et al, Int J Cardiol 122:17 2007) and secrete paracrine factors that may be responsible for their beneficial effect (Boomsma and Geenen, PLoSONE 7:e3685, 2012). Mouse MSC were cultured in Mesencult + 20% serum supplement (Stem Cell Technologies) and rat cardiac H9c2 cells were cultured in DMEM + 10% FBS until confluent. MSC and H9c2 cells were then combined equally and plated in 6‐well dishes at a total concentration of 1.0 ×106 cells/well in DMEM + 10% FBS. After 2 days the media was changed to DMEM + 1% heat inactivated horse serum to promote myocyte differentiation. Control cultures containing H9c2 cells alone at the same cellular concentrations were treated similarly. After 1, 3, 5, and 7 days of culture in 1% serum RNA was isolated using RNAqueous 4 PCR (Ambion) and treated with DNAse. Cardiac troponin‐T (cTnT) was quantified in H9c2 cells by RT‐qPCR using an iScript cDNA Synthesis Kit and iQ SYBR Green Mastermix (BioRad). Relative expression was determined using the reference gene GAPDH; no‐RT and water controls were included. Species specific primers for rat cTnT and rat GAPDH were designed in order to separate out expression of cTnT in H9c2 cells (rat) from MSC (mouse). Mean and standard error was calculated for each group and significance was determined using Student's t‐test (p<0.05, n=5–6). Relative expression of cTnT in H9c2 cells was low in both co‐culture (0.422 ± 0.157) or alone (0.427 ± 0.065) after 1 day in 1% serum, but increased more than 10‐fold after 3–7 days in both co‐culture or alone. cTnT expression in H9c2 cells was significantly greater after 5 days in co‐culture with MSC (4.204 ± 0.524) compared to H9c2 cells cultured for 5 days alone (2.353 ± 0.510; p<0.05). Expression in H9c2 cells cultured alone continued to increase after 7 days (5.958 ± 1.179) such that there was no statistical difference from cells in co‐culture (3.918 ± 1.880). This study demonstrates that MSC accelerate cTnT expression in H9c2 cells and may partially explain the beneficial effect of MSC on cardiac function in vivo.Support or Funding InformationSupported by a VanderVelde Research Scholarship and a Summer Research Grant from Trinity Christian College.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.