Nitric oxide inhibits TIMP‐3 expression in mouse neonatal cardiomyocytes via S‐nitrosylation

Extracellular matrix (ECM) remodeling is mediated by a family of zinc‐dependent endopeptidases called matrix metalloproteinases (MMPs) and their endogenous inhibitors, the tissue inhibitors of metalloproteinases (TIMPs). Detrimental remodeling of the myocardial ECM plays a critical role in the progression of heart failure. TIMP‐3 levels have previously been shown to be significantly reduced in patients with heart failure by unknown mechanisms. We hypothesize that nitric oxide (NO) reduces TIMP‐3 levels during heart failure through S‐nitrosylation of its transcription factors. Treatment of wild‐type (WT) neonatal mouse cardiomyocytes with an endothelial nitric oxide synthase adenovirus (Ad‐eNOS) resulted in a 1.7‐fold decline in TIMP‐3 expression (P<0.05) as compared to cardiomyocytes infected with the control Ad‐GFP. Furthermore, treatment of WT cardiomyocytes with the NOS inhibitor N ω ‐nitro‐L‐arginine methyl ester (L‐NAME) showed a time‐dependent significant increase in TIMP‐3 levels over 6 hours. In addition, TIMP‐3 transcript levels were 1.7‐fold greater (P<0.05) in eNOS−/− as compared to WT cardiomyocytes. To determine a possible mechanism of action for NO, cells were infected with Ad‐eNOS, and Ad‐GFP in the presence of dithiothreitol (DTT), a reducing agent that is known to reverse NO‐induced S‐nitrosylation of various transcription factors. DTT treatment reversed the effect of Ad‐eNOS on TIMP‐3 expression and resulted in an increase in TIMP‐3 levels as compared to cells treated with Ad‐eNOS alone. These results demonstrate that NO inhibits TIMP‐3 expression in cardiomyocytes likely through S‐nitrosylation.