Diesterified nitrone rescues nitroso-redox levels and increases myocyte contraction via increased SR Ca(2+) handling

Abstract

Nitric oxide (NO) and superoxide (O(2) (-)) are important cardiac signaling molecules that regulate myocyte contraction. For appropriate regulation, NO and O(2) (.-) must exist at defined levels. Unfortunately, the NO and O(2) (.-) 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 O(2) (.-) 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/O(2) (.-) imbalance, and incubated with EMEPO. EMEPO significantly reduced O(2) (.-) (lucigenin-enhanced chemiluminescence) and elevated NO (DAF-FM diacetate) levels in NOS1(-/-) myocytes. Furthermore, EMEPO increased NOS1(-/-) myocyte basal contraction (Ca(2+) 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 Ca(2+) 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 O(2) (.-) levels may have therapeutic potential in the treatment of various cardiomyopathies.

Figure 1. Structure of DMPO and EMEPO.

Figure 2. EMEPO decreases O₂ ^.− levels and increases NO levels in NOS1^−/− myocytes.

A: Summary data (mean±s.e.m.) of O₂ ^.− levels in WT and NOS1^−/− myocytes. * P<0.05 NOS1^−/− vs. WT and NOS1^−/−+EMEPO. n = 3–4 hearts/group. B: Summary data (mean±s.e.m.) of NO levels in NOS1^−/− and WT myocytes (±EMEPO). * P<0.05 vs. -EMEPO. n = 13–20 myocytes per group.

Figure 3. EMEPO increases contraction in NOS1^−/− myocytes with no effect in WT myocytes.

A: Individual, steady-state cell shortening (top) and Ca²⁺ transient (bottom) traces measured in NOS1^−/− (CONT, black) and EMEPO incubated NOS1^−/− (EMEPO, gray) myocytes. B: Summary data (mean±s.e.m.) of the effects of EMEPO and DMPO on shortening (left) and Ca²⁺ transient (right) amplitudes. C: Summary data (mean±s.e.m.) of the effects of EMEPO and DMPO on rate of relaxation (left) and [Ca²⁺]_i decline (right) measured as time to 50% relaxation (RT₅₀). ^* P<0.05 vs. control. n = 6 cells/3 hearts for NOS1^−/−, n = 18 cells/3 hearts for NOS1^−/−+EMEPO, and n = 21 cells/3 hearts for NOS1^−/−+DMPO, n = 25 cells/5 hearts for WT, n = 22 cells/3 hearts for WT+EMEPO.

Figure 4. EMEPO increases FFR and potentiates the β-AR response in NOS1^−/− myocytes.

A: Summary data (mean±s.e.m.) of the effect of EMEPO in NOS1^−/− and WT myocytes at various stimulation frequencies. ^* P<0.05 vs. NOS1^−/− -EMEPO. n = 37 cells/5 hearts for WT+EMEPO and 37cells/6 hearts for NOS1^−/−+EMEPO (gray). n = 5–8 myocytes/3 hearts for WT and NOS1^−/− - EMEPO. B: Summary data (mean±s.e.m.) of the effect of EMEPO on β-AR stimulated Ca²⁺ transient amplitudes in NOS1^­/− and WT myocytes. ^* P<0.05 NOS1^−/− +ISO vs NOS1^−/− +ISO/EMEPO. n = 13 cells/5 hearts for NOS1^−/− +ISO, n = 10 cells/4 heart for NOS1^−/− +ISO/EMEPO, n = 16 cells/6 hearts for WT +ISO, n = 22 cells/6 hearts for WT +ISO/EMEPO.

Figure 5. Larger increase in contraction with EMEPO compared to a superoxide scavenger or NO donor (SNAP) in NOS1^−/− myocytes.

Summary data (mean±s.e.m.) of the effects of MENO, SNAP, and EMEPO on shortening (A) and Ca²⁺ transient (B) amplitudes. ^* P<0.05 EMEPO vs. MENO and SNAP. n = 17 cells/5 hearts for NOS1^−/− +MENO, n = 25 cells/5 hearts NOS1^−/− +SNAP, and n = 18 cells/3 hearts for NOS1^−/− +EMEPO.

Figure 6. EMEPO increases RyR activity and PLB Serine₁₆ phosphorylation in NOS1^−/− myocytes.

A: Plot of the SR Ca²⁺ leak/load relationship in NOS1^−/− and WT myocytes. n = 10 cells/6 hearts for NOS1^−/−, and n = 19 cells/6 hearts NOS1^−/− +EMEPO, n = 9 cells/4 hearts for WT, n = 16 cells/5 hearts for WT +EMEPO. B: Summary data (mean±s.e.m.) of EMEPO’s effect on Serine₁₆ phosphorylation (normalized to total PLB) in NOS1^−/− hearts. n = 4 hearts for NOS1^−/− and n = 5 hearts for NOS1^−/− +EMEPO. ^* P<0.05 vs. control.

Figure 7. EMEPO increases contraction in a canine post-infarction model.

A: Individual, steady-state cell shortening (top) and Ca²⁺ transient (bottom) traces measured in post-MI canine cardiac myocytes with and without EMEPO incubation. B: Summary data (mean±s.e.m.) of the effect of EMEPO on shortening (left) and Ca²⁺ transient (right) amplitudes C: Summary data (mean±s.e.m.) of the effect of EMEPO on rate of relaxation (left) and [Ca²⁺]_i decline (right) measured as time to 50% relaxation (RT₅₀). * P<0.05 vs. -EMEPO. n = 14–20 myocytes/2 hearts per group.