Background: Mechanical forces are critically important for the hypertrophic growth in the overloaded myocardium. Hypertrophic response of cardiac myocytes is characterized by an increase in cell size, an accumulation of total protein, enhanced sarcomeric reorganization and complex changes in cardiac gene expression. However, the mechanical stretch triggered gene expression changes in cardiac myocytes are not fully known.
Methods: We performed the first genome-wide time series study of mechanical stretch induced gene expression changes in cultured neonatal rat ventricular myocytes (NRVM)s. Cyclic mechanical stretch for 4, 12, 24 and 48 hours were applied to NRVM cultures with a Flexercell Strain Unit. Gene expression and microRNA profiling were done by using Affymetrix and miRCURYTM LNA arrays, respectively. Ingenuity pathway analysis (IPA) was used to identify functional interaction networks among differentially expressed genes.
Results: Mechanical stretching of NRVMs resulted in 205, 579, 737, 621 and 1542 differentially expressed (>2-fold, p<0.05) genes in response to 1, 4 12, 24 and 48 hours of cyclic mechanical stretch. Cellular functions related to cellular growth and proliferation were activated throughout the study course. In acute phase (1-12 hours) all the significantly modulated pathways were activated, such as proinflammatory interleukin-signaling pathways. In late phase response (48 hours), majority of signaling pathways were repressed. NRF2-mediated-signaling was the pathway having the greatest activation (significant p-value, z-score >2) after 12 hours of stretching. The analysis showed a total of 8 and 12 miRNAs were significantly altered by 1-12 and 24-48 hours of mechanical stretch, respectively. We identified potential mRNA-miRNA interactions by performing a correlation analysis between selected miRNAs and mRNA expression data sets, revealing let-7a, let-7c and let-7f miRNAs as important regulators of stretch activated hypertrophic gene response.
Conclusion: By analyzing the comprehensive time course of mechanical stretch-activated gene expression changes in an in vitro model of cultured NRVM, we identified NRF2 and let-7 family of miRNA as upstream regulators of stretch-induced hypertrophic gene response.