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

Christopher J Traynham,Ira O Racoma,Mark T Ziolo,Robert A Prosak,Steve R Roof,Yongbin Han,Xin Huang,George E Billman,Shang-U Kim,Dominic J Catalano,Lifei Tang,Frederick A Villamena,Honglan Wang,Serge Viatchenko-Karpinski,Hsiang-Ting Ho,Sandor Gyorke,Marcello Rota

doi:10.1371/journal.pone.0052005

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.

Highlights

Despite recent advances in treatment strategies, heart failure (HF) is a growing epidemic that still presents with poor clinical prognosis
Previous studies have shown that O2.2 levels are increased and NOS1 activity is decreased in NOS12/2 myocytes [25,26,27,28]
NOS12/2 myocytes had significantly higher O2.2 levels compared to WT myocytes (22.468.4 vs.1.260.6 RLU, P,0.05, Figure 2A), which was decreased with EMEPO (1.160.4 RLU, P,0.05 vs. +EMEPO, Figure 2A)

Summary

Introduction

Despite recent advances in treatment strategies, heart failure (HF) is a growing epidemic that still presents with poor clinical prognosis. Therapies have been developed to target superoxide (O2.2) or nitric oxide (NO) [1]. For both of these signaling molecules to appropriately regulate myocyte contraction, they must exist at defined levels [2]. In a clinical trial, the XO inhibitor oxypurinol did not lead to clinical benefits in HF patients [9]. This type of therapy may not have been beneficial since reducing O2.2 levels by itself will not restore the altered nitroso levels because there are changes in NO bioavailability [10]. A therapy is needed that will restore both O2.2 and NO levels

Methods

Results

Conclusion