Neuronal nitric oxide synthase is indispensable for the cardiac adaptive effects of exercise

Abstract

Exercise results in beneficial adaptations of the heart that can be directly observed at the ventricular myocyte level. However, the molecular mechanism(s) responsible for these adaptations are not well understood. Interestingly, signaling via neuronal nitric oxide synthase (NOS1) within myocytes results in similar effects as exercise. Thus, the objective was to define the role NOS1 plays in the exercise-induced beneficial contractile effects in myocytes. After an 8-week aerobic interval training program, exercise-trained (Ex) mice had higher VO(2max) and cardiac hypertrophy compared to sedentary (Sed) mice. Ventricular myocytes from Ex mice had increased NOS1 expression and nitric oxide production compared to myocytes from Sed mice. Remarkably, acute NOS1 inhibition normalized the enhanced contraction (shortening and Ca(2+) transients) in Ex myocytes to Sed levels. The NOS1 effect on contraction was mediated via greater Ca(2+) cycling that resulted from increased phospholamban phosphorylation. Intriguingly, a similar aerobic interval training program on NOS1 knockout mice failed to produce any beneficial cardiac adaptations (VO(2max), hypertrophy, and contraction). These data demonstrate that the beneficial cardiac adaptations observed after exercise training were mediated via enhanced NOS1 signaling. Therefore, it is likely that beneficial effects of exercise may be mimicked by the interventions that increase NOS1 signaling. This pathway may provide a potential novel therapeutic target in cardiac patients who are unable or unwilling to exercise.

Fig 1

Exercise increases myocyte NOS1 expression, nitric oxide (NO) production and enhances myocyte contraction, which is normalized by acute NOS1 inhibition. A) Summary data of NOS1 protein expression (A.U.–arbitrary units), n=12 mice/group. B) Summary data of NO production over a 15 minute time period (±acute NOS1 inhibition, SMLT, dashed), n=19-26 cells/3 hearts. C) Representative traces of Ca²⁺ transients and shortening. Summary data of Ca²⁺ transient amplitudes (D), Ca²⁺decline to 50% of its peak (RT₅₀)(E), shortening amplitudes (F), and relengthening RT₅₀ (G) in exercise (Ex-grey) and sedentary (Sed-black)myocytes (±acute NOS1 inhibition, SMLT, striped), n=39-42 cells/7-10 hearts, (H) Summary data of Ca²⁺ transient (top) and shortening (bottom) amplitudes to various concentrations of ISO, n=12-39 myocytes/4 hearts, *P< 0.05 vs corresponding Sed.

Fig 2

Exercise enhances SR Ca²⁺cycling and is normalized by acute NOS1 inhibition. Summary data of SR Ca²⁺ load (A) and SR Ca²⁺ fractional release (B), n=26-35 cells/11-12 hearts. C) Summary data of PLB Serine16 phosphorylation (n=6 hearts) in exercise (Ex-grey) and sedentary (Sed-black) myocytes (±acute NOS1 inhibition, SMLT, striped), *P< 0.05 vs corresponding Sed.

Fig 3

Protein phosphatase 1 and 2a inhibition (okadaic acid, OA) and PKA inhibition (PKI) abrogated the contractile difference between Sed and Ex myocytes. Summary data of Ca²⁺ transient amplitudes (A), Ca²⁺ transient RT₅₀ (B), shortening amplitudes and (C), relengthening RT₅₀ (D) in exercise (Ex-grey) and sedentary (Sed-black) myocytes. n=10-22 cells/4 hearts, *P< 0.05 vs corresponding Sed.

Fig 4

Negative effects of exercising on Ca²⁺handling in NOS1 deficient myocytes. Summary data of Ca²⁺ transient amplitudes (A), Ca²⁺ transient RT₅₀ (B), shortening amplitudes (C), relengthening RT₅₀ (D), SR Ca²⁺ load (E), and SR Ca²⁺ fractional release (F) in exercise (Ex-NOS1KO-grey) and sedentary (Sed-NOS1KO-black), n=22-42 cells/5-6 hearts, *P< 0.05 vs corresponding Sed.

Fig 5

Exercise in a trained canine model elicited similar results with NOS1 inhibition on myocyte contraction. Summary data of Ca²⁺ transient amplitudes (A), Ca²⁺ transient RT₅₀ (B), shortening amplitudes (C), relengthening RT₅₀ (D), SR Ca²⁺ load (E), and SR Ca²⁺ fractional release (F) in exercise (Ex-grey) and sedentary (Sed- black), (±acute NOS1 inhibition, SMLT, striped), n = 10-33 cells/3-4 hearts, *P< 0.05 vs corresponding Sed.

Fig 6

NOS1 and its downstream signaling targets have a greater effect with exercise. Summary data shown as change from control of Ca²⁺ transient amplitudes (A),Ca²⁺decline to 50% of its peak (RT₅₀)(B), shortening amplitudes (C), and relengthening RT₅₀ (D), SR Ca²⁺ load (E), and SR Ca²⁺ fractional release (F) in exercise (Ex-grey) and sedentary (Sed-black) myocytes, *P< 0.05 vs corresponding Sed.