Diesterified Nitrone Rescues Nitroso-Redox Levels and Increases Myocyte Contraction Via Increased SR Ca2+ Handling

Abstract

Nitric oxide (NO) and superoxide (O2-) are important cardiac signaling molecules that regulate myocyte contraction. For appropriate regulation, NO and O2.- must exist at defined levels. Unfortunately, the NO and O2.- levels are altered in many cardiomyopathies (heart failure, ischemia, hypertrophy, etc.) leading to contractile dysfunction and adverse remodeling. Hence, rescuing the nitroso-redox levels is a potential therapeutic strategy. Nitrone spin traps have been shown to scavenge O2.- while releasing NO as a reaction byproduct; and we synthesized a novel, cell permeable nitrone, 2-2-3,4-dihydro-2H-pyrrole 1-oxide (EMEPO). We hypothesized that EMEPO would improve contractile function in myocytes with altered nitroso-redox levels. Ventricular myocytes were isolated from wildtype (C57Bl/6) and NOS1 knockout (NOS1-/-) mice, a known model of NO/O2.- imbalance, and incubated with EMEPO. EMEPO significantly reduced O2.- (lucigenin-enhanced chemiluminescence) and elevated NO (DAF-FM diacetate) levels in NOS1-/- myocytes. Furthermore, EMEPO increased NOS1-/- myocyte basal contraction (Ca2+ transients, Fluo-4AM; shortening, video-edge detection), the force-frequency response and the contractile response to β-adrenergic stimulation. EMEPO had no effect in wildtype myocytes. EMEPO also increased ryanodine receptor activity (sarcoplasmic reticulum Ca2+ leak/load relationship) and phospholamban Serine16 phosphorylation (Western blot). We also repeated our functional experiments in a canine post-myocardial infarction model and observed similar results to those seen in NOS1-/- myocytes. In conclusion, EMEPO improved contractile function in myocytes experiencing an imbalance of their nitroso-redox levels. The concurrent restoration of NO and O2.- levels may have therapeutic potential in the treatment of various cardiomyopathies. © 2012 Traynham et al.