Cardiac Myocytes Expressing Disease-Causing Mutant Plakoglobin Exhibit Alterations in Mechanotransduction

Abstract

Introduction: Mechanotransduction is the process by which cells couple their external and internal environments by transducing mechanical stimuli into a biochemical response. While this process is responsible for regulating cell fate through gene expression (i.e. - c-myc, c-fos, etc.), it remains incompletely characterized in cardiac myocytes. Our previous work demonstrated that primary neonatal rat ventricular myocytes (NRVM) respond to mechanical shear stress via junctional remodeling of plakoglobin (JUP) and N-cadherin (NC). Here, we study myocyte mechanotransduction by investigating ERK phosphorylation, c-myc & c-fos gene regulation; we also investigate the effects of inhibiting primary cilia in NRVM, one potential mechanism for transduction of mechanical stimuli.Methods: NRVM were transfected with adenoviruses to express either of two different mutations in JUP (2057del2 & S39_K40insS) which cause arrhythmogenic right ventricular cardiomyopathy (ARVC). Primary cilia were inhibited by transfecting NRVM with siRNA against intraflagellar transport protein 88 (IFT88). NRVM were sheared under oscillatory flow in a parallel-plate shear chamber at physiologically-relevant shear stresses (forces in the plane of the cell layer that mimic those in contracting myocardium; 0.06 Pascal). Samples were subsequently stained and imaged on an Olympus IX-81 confocal microscope using quantitative confocal microscopy. Immunoblotting was performed on NRVM lysates separated by PAGE.Conclusions: We demonstrate that ERK phosphorylation, and c-myc & c-fos regulation are unaffected by mutant JUP expression. Our previous results indicate that cardiac myocytes are responsive to shear stimuli, and undergo junctional remodeling of both NC and JUP under oscillatory shear, a phenomenon absent in cells expressing ARVC-causing mutant JUP. Interestingly, knockdown of IFT88 does not have a dramatic effect on junctional remodeling; these results suggest that the role of primary cilia in cardiomyocyte shear sensing is varied and does not affect all shear-dependent pathways.