The phenotypic switch underlying the differentiation of cardiac fibroblasts into hypersecretory myofibroblasts is critical for cardiac remodeling following myocardial infarction. Myofibroblasts facilitate wound repair in the myocardium by secreting and organizing extracellular matrix (ECM) during the wound healing process. However, the molecular mechanisms involved in myofibroblast differentiation are not well known. TGF-β has been shown to promote differentiation and this, combined with the robust mechanical environment in the heart, lead us to hypothesize that the mechanotransduction and TGF-β signaling pathways play active roles in the differentiation of cardiac fibroblasts to myofibroblasts. Here, we show that the mechanosensitve ion channel TRPV4 is required for TGF-β1-induced differentiation of cardiac fibroblasts into myofibroblasts. We found that the TRPV4-specific antagonist AB159908 and siRNA knockdown of TRPV4 significantly inhibited TGFβ1-induced differentiation as measured by incorporation of α-SMA into stress fibers. Further, we found that TGF-β1-induced myofibroblast differentiation was dependent on ECM stiffness, a response that was attenuated by TRPV4 blockade. Finally, TGF-β1 treated fibroblasts exhibited enhanced TRPV4 expression and TRPV4-mediated calcium influx compared to untreated controls. Taken together these results suggest for the first time that the mechanosensitive ion channel, TRPV4, regulates cardiac fibroblast differentiation to myofibroblasts by integrating signals from TGF-β1 and mechanical factors.

Rationale We previously reported that type VI collagen deposition increases in the infarcted myocardium in vivo. To date, a specific role for this non-fibrillar collagen has not been explored in the setting of myocardial infarction (MI). Objective To determine whether deletion of type VI collagen in an in vivo model of post-MI wound healing would alter cardiac function and remodeling in the days to weeks after injury. Methods and Results Wild type (WT) and Col6a1-/- mice were subjected to MI followed by serial echocardiographic and histological assessments. At 8 weeks post-MI, infarct size was significantly reduced, ejection fraction was significantly preserved (43.9 ± 3.3% vs. 29.1 ± 4.3% for WT) and left ventricular (LV) chamber dilation was attenuated in the Col6a1-/- MI group (25.8 ± 7.9% increase vs. 62.6 ± 16.5% for WT). The improvement in cardiac remodeling was evident as early as 10 days post-MI in the Col6a1-/- mice. Myocyte apoptosis within the infarcted zones was initially greater in the Col6a1-/- group 3 days post-MI but by day 14 this was significantly reduced. Collagen deposition was also reduced in the infarcted and remote areas of the Col6a1-/- hearts. The reductions in chronic myocyte apoptosis and fibrosis are critical events leading to improved long-term remodeling and functional outcomes. Conclusions These unexpected results demonstrate for the first time that deletion of type VI collagen in this knockout model plays a critical protective role following MI by limiting infarct size, chronic apoptosis, aberrant remodeling and fibrosis leading to preservation of cardiac function.

During heightened cardiac work, O2 consumption by the heart benefits energy production via mitochondria. However, some electrons leak from the respiratory chain and yield superoxide, which is rapidly metabolized into H2O2 by SOD2. To understand the systemic effects of the metabolic dilator, H2O2, we studied mice with cardiac-specific SOD2 overexpression (SOD2-tg), which increases the H2O2 produced by cardiac mitochondria. Contrast echocardiography was employed to evaluate cardiac function, indicating that SOD2-tg had a significantly greater ejection fraction and a lower mean arterial pressure (MAP) that was partially normalized by intravenous injection of catalase. Norepinephrine-mediated myocardial blood flow (MBF) was significantly enhanced in SOD2-tg mice. Coupling of MBF to the double product (Heart Rate × MAP) was increased in SOD2-tg mice, indicating that the metabolic dilator, “spilled” over, inducing systemic vasodilation. The hypothesis that SOD2 overexpression effectively enhances mitochondrial function was further evaluated. Mitochondria of SOD2-tg mice had a decreased state 3 oxygen consumption rate, but maintained the same ATP production flux under the basal and L-NAME treatment conditions, indicating a higher bioenergetic efficiency. SOD2-tg mitochondria produced less superoxide, and had lower redox activity in converting cyclic hydroxylamine to stable nitroxide, and a lower GSSG concentration. EPR analysis of the isolated mitochondria showed a significant decrease in semiquinones at the SOD2-tg Qi site. These results support a more reductive physiological setting in the SOD2-tg murine heart. Cardiac mitochondria exhibited no significant differences in the respiratory control index between WT and SOD2-tg. We conclude that SOD2 overexpression in myocytes enhances mitochondrial function and metabolic vasodilation, leading to a phenotype of supernormal cardiac function.

Diabetes is an independent risk factor of cardiovascular disease that can eventually cause cardiomyopathy and heart failure. Cardiac fibroblasts (CF) are the critical mediators of physiological and pathological cardiac remodeling, however, the effects of hyperglycemia on cardiac fibroblast function and differentiation is not well known. Here, we performed a comprehensive investigation on the effects of hyperglycemia on cardiac fibroblasts and show that hyperglycemia enhances cardiac fibroblast function and differentiation. We found that high glucose treatment increased collagen I, III and VI gene expression in rat adult cardiac fibroblasts. Interestingly, hyperglycemia increased CF migration and proliferation which is augmented by collagen I and III. Surprisingly, we found that short term hyperglycemia transiently inhibited ERK1/2 activation but increased AKT phosphorylation. Finally, high glucose treatment increased spontaneous differentiation of cardiac fibroblasts to myofibroblasts with increasing passage compared to low glucose. Taken together, these findings suggest that hyperglycemia induces cardiac fibrosis by modulating collagen expression, migration, proliferation and differentiation of cardiac fibroblasts.