Chronic exercise downregulates myocardial myoglobin and attenuates nitrite reductase capacity during ischemia-reperfusion

Abstract

The infarct sparing effects of exercise are evident following both long-term and short-term training regimens. Here we compared the infarct-lowering effects of nitrite therapy, voluntary exercise, and the combination of both following myocardial ischemia-reperfusion (MI/R) injury. We also compared the degree to which each strategy increased cardiac nitrite levels, as well as the effects of each strategy on the nitrite reductase activity of the heart. Mice subjected to voluntary wheel running (VE) for 4weeks displayed an 18% reduction in infarct size when compared to sedentary mice, whereas mice administered nitrite therapy (25mg/L in drinking water) showed a 53% decrease. However, the combination of VE and nitrite exhibited no further protection than VE alone. Although the VE and nitrite therapy mice showed similar nitrite levels in the heart, cardiac nitrite reductase activity was significantly reduced in the VE mice. Additionally, the cardiac protein expression of myoglobin, a known nitrite reductase, was also reduced after VE. Further studies revealed that cardiac NFAT activity was lower after VE due to a decrease in calcineurin activity and an increase in GSK3β activity. These data suggest that VE downregulates cardiac myoglobin levels by inhibiting calcineurin/NFAT signaling. Additionally, these results suggest that the modest infarct sparing effects of VE are the result of a decrease in the hearts ability to reduce nitrite to nitric oxide during MI/R.

Keywords: Calcineurin/NFAT signaling; Exercise; Myocardial ischemia–reperfusion; Myoglobin; Nitric oxide; Nitrite.

Fig. 1. Nitrite Supplementation Does not Enhance the Cardioprotective Effects of Exercise Training

Mice were housed in cages fitted with running wheels and allowed to exercise voluntarily for 4 weeks (VE). Control mice (sedentary, SED) were housed in cages without running wheels for the same durations as the VE mice. Groups of both SED and VE were also administered oral nitrite therapy (NO₂, 25 mg/L in drinking water; NO₂ and VE+NO₂). (A) Representative mid-ventricular photomicrographs of hearts from each of the groups showing the degree of infarction following 45 minutes of left coronary artery occlusion and 24 hours or reperfusion. (B) Myocardial area-at-risk (AAR) as a percentage (%) of total left ventricle (LV) and infarct size (INF) as a percentage of area-at-risk (AAR) and (C) circulating Troponin-I levels (ng/mL) in each of the groups. (D) Ejection fraction (%) was calculated in separate groups of mice using echocardiography images at baseline and 7 days following myocardial ischemia (POST). Values are means ± S.E.M. Numbers inside the bars are the number of animals investigated. *p<0.05, **p<0.01, and ***p<0.001 vs. SED or Baseline.

Fig. 2. Exercise Training and Nitrite Supplementation Increase the Steady-State Levels of Nitrite and Nitrosothiols

Steady-state levels of (A) plasma nitrite, (B) plasma nitrosothiols (RXNO), (C) heart nitrite, and (D) heart RXNO and nitrosyl-heme (NO-Heme) from SED, NO₂, VE, and VE+NO₂ mice. (E) Nitrite reductase activity from the hearts of SED, NO₂, VE, and VE+NO₂ mice. (F) Myocardial area-at-risk (AAR) as a percentage (%) of total left ventricle (LV) and infarct size (INF) as a percentage of area-at-risk (AAR) and (F) circulating Troponin-I levels (ng/mL) from SED and VE mice treated with the NO donor, DETANO, or vehicle at the time of reperfusion. Values are means ± S.E.M. *p<0.05, **p<0.01, and ***p<0.001 vs. SED or SED Veh.

Fig. 3. Exercise Training Decreases the Expression of Cardiac Myoglobin

(A) Gene expression and (B) representative immunoblots and densitometric analysis of myoglobin from the hearts of SED, NO₂, VE, and VE+NO₂ mice. (C) Nitrite reductase activity from the hearts of SED, NO₂, VE, and VE+NO₂ mice in the presence and absence of myoglobin (25 μM). Values are means ± S.E.M. *p<0.05 and ***p<0.001 vs. SED.

Fig. 4. Exercise Training Decreases the Nuclear Expression and Activity of NFAT

(A–C) Representative immunoblots and densitometric analysis of nuclear NFATc2 and NFATc4 from the hearts of SED, NO₂, VE, and VE+NO₂ mice. (D) Cardiac luciferase activity from the hearts of NFAT-Luciferase reporter mice divided into SED, NO₂, VE, and VE+NO₂ groups. Values are means ± S.E.M. *p<0.05 and **p<0.01 vs. SED.

Fig. 5. Exercise Training Increases the Expression of Calsarcin-1

(A) Representative immunoblots and densitometric analysis of calcineurin Aβ (CnAβ) and calsarcin-1 from the hearts of SED, NO₂, VE, and VE+NO₂ mice. (B) Representative immunoblots and densitometric analysis of the interaction of CnAβ with calsarcin-1 from the hearts of SED, NO₂, VE, and VE+NO₂ mice. For these experiments, heart homogenates were immunoprecipitated with a CnAβ antibody. The samples were then subjected to standard Western blot techniques and the membranes probed with CnAβ and calsarcin-1 antibodies to reveal the interaction. (C) Calcinuerin activity (pmoles/mg) from the hearts of SED, NO₂, VE, and VE+NO₂ mice. (D) Representative immunoblots and densitometric analysis of myoglobin from the hearts of Wild-type (WT), CnAβ deficient (CnAβ^−/), NFATc4 deficient (NFATc4^−/−), NFATc2 deficient (NFATc2^−/−), and NFATc2/NFATc4 double deficient (NFATc2^−/−/c4^−/−) mice. Numbers inside the bars are the number of animals investigated. Values are means ± S.E.M. *p<0.05, **p<0.01, and ***p<0.001 vs. SED or WT.

Fig. 6. Exercise Training Suppresses GSK3

β Inactivation. (A) Representative immunoblots and densitometric analysis of (B) total GSK3β, (C) phosphorylated GSK3β at tyrosine residue 216 (GSK3β-P^Tyr216) and phosphorylated GSK3β at serine residue 9 (GSK3β-P^Ser9) in nuclear preparations from the hearts of SED, NO₂, VE, and VE+NO₂ mice. (D) GSK3β activity (pmol/min/mg) in nuclear fractions from the hearts of SED, NO₂, VE, and VE+NO₂ mice. Values are means ± S.E.M. *p<0.05, **p<0.01, and ***p<0.001 vs. SED.