Abstract
AimsIn healthy hearts, the neuronal nitric oxide synthase (nNOS) is predominantly localized to the sarcoplasmic reticulum (SR), where it regulates the ryanodine receptor Ca2+ release channel (RyR2) and phospholamban (PLB) phosphorylation, and to a lesser extent to the sarcolemmal membrane where it inhibits the L-type Ca2+ current (ICa). However, in failing hearts, impaired relaxation and depressed inotropy are associated with a larger proportion of nNOS being localized to the sarcolemmal membrane. Whether there is a causal relationship between altered myocardial function and subcellular localization of nNOS remains to be assessed.Methods and resultsAdenoviruses (AdV) encoding for a human nNOS.eGFP fusion protein or eGFP were injected into the left ventricle (LV) of nNOS−/− mice. nNOS.eGFP localized to the sarcolemmal and t-tubular membrane and immunoprecipitated with syntrophin and caveolin-3 but not with RyR2. Myocardial transduction of nNOS.eGFP resulted in a significantly increased NOS activity (10-fold, P < 0.01), a 20% increase in myocardial tetrahydrobiopterin (BH4) (P < 0.05), and a 30% reduction in superoxide production (P < 0.001). LV myocytes transduced with nNOS.eGFP showed a significantly lower basal and β-adrenergic stimulated ICa, [Ca2+]i transient amplitude and cell shortening (vs. eGFP). All differences between groups were abolished after NOS inhibition. In contrast, nNOS.eGFP had no effect on RyR nitrosylation, PLB phosphorylation or the rate of myocardial relaxation and [Ca2+]i decay.ConclusionOur findings indicate that nNOS-mediated regulation of myocardial excitation–contraction (E–C) coupling is exquisitely dependent on nNOS subcellular localization and suggests a partially adaptive role for sarcolemmal nNOS in the human failing myocardium.
Highlights
IntroductionA ‘neuronal’ isoform of nitric oxide synthase (nNOS or NOS1) in the mammalian myocardium has been reported to regulate cardiac inotropy and relaxation by decreasing inhibits the L-type Ca2þ current (ICa),[1,2] increasing ryanodine receptor Ca2þ release channel (RyR2) open probability[3,4] and promoting PLB phosphorylation.[5,6] neuronal nitric oxide synthase (nNOS)-derived NO has been shown to inhibit myocardial superoxide production,[4,7,8,9] preserve left ventricle (LV) diastolic function[4,10] and protect against adverse LV remodelling and arrhythmias after myocardial infarction.[2,11,12] In healthy hearts, nNOS is predominantly located to the sarcoplasmic reticulum (SR),[13] where it co-localizes with the RyR2,14 and to a lesser extent to the VC The Author 2017
Our findings indicate that neuronal nitric oxide synthase (nNOS)-mediated regulation of myocardial excitation–contraction (E–C) coupling is exquisitely dependent on nNOS subcellular localization and suggests a partially adaptive role for sarcolemmal nNOS
It has been suggested that rearrangement in nNOS subcellular localization in the presence of heart failure may play an important role in the downstream effects of NO on cardiac function; if that were the case, the sarcolemmal localization of nNOS in failing hearts would be expected to result in a reduction in PLB phosphorylation, contributing to impaired relaxation and depressed contractility secondary to a reduction in Ca2þ influx via inhibits the L-type Ca2þ current (ICa)
Summary
A ‘neuronal’ isoform of nitric oxide synthase (nNOS or NOS1) in the mammalian myocardium has been reported to regulate cardiac inotropy and relaxation by decreasing ICa,[1,2] increasing ryanodine receptor Ca2þ release channel (RyR2) open probability[3,4] and promoting PLB phosphorylation.[5,6] nNOS-derived NO has been shown to inhibit myocardial superoxide production,[4,7,8,9] preserve left ventricle (LV) diastolic function[4,10] and protect against adverse LV remodelling and arrhythmias after myocardial infarction.[2,11,12] In healthy hearts, nNOS is predominantly located to the sarcoplasmic reticulum (SR),[13] where it co-localizes with the RyR2,14 and to a lesser extent to the VC The Author 2017. Sarcolemmal membrane.[15,16,17] in the presence of heart failure or ischemia/reperfusion injury, myocardial nNOS is upregulated and preferentially located to the sarcolemmal membrane where it binds to caveolin-3 (Cav-3).[18,19,20,21] It has been suggested that rearrangement in nNOS subcellular localization in the presence of heart failure may play an important role in the downstream effects of NO on cardiac function; if that were the case, the sarcolemmal localization of nNOS in failing hearts would be expected to result in a reduction in PLB phosphorylation, contributing to impaired relaxation and depressed contractility secondary to a reduction in Ca2þ influx via ICa. the diffusibility of NO and the close proximity of the ion channels and transporters involved in excitation–contraction (E–C) coupling may mitigate against the importance of nNOS subcellular localization.
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