Impaired regulation of neuronal nitric oxide synthase and heart rate during exercise in mice lacking one nNOS allele

Abstract

We tested the hypothesis that a single allele deletion of neuronal nitric oxide synthase (nNOS) would impair the neural control of heart rate following physical training, and that this phenotype could be restored following targeted gene transfer of nNOS. Voluntary wheel-running (+EX) in heterozygous nNOS knockout mice (nNOS(+/-), +EX; n= 52; peak performance 9.1 +/- 1.8 km day(-1)) was undertaken and compared to wild-type mice (n= 38; 9.5 +/- 0.8 km day(-1)). In anaesthetized wild-type mice, exercise increased phenylephrine-induced bradycardia by 67% (measured as heart rate change, in beats per minute, divided by the change in arterial blood pressure, in mmHg) or pulse interval response to phenylephrine by 52% (measured as interbeat interval change, in milliseconds, divided by the change in blood pressure). Heart rate changes or interbeat interval changes in response to right vagal nerve stimulation were also enhanced by exercise in wild-type atria (P < 0.05), whereas both in vivo and in vitro responses to exercise were absent in nNOS(+/-) mice. nNOS inhibition attenuated heart rate responses to vagal nerve stimulation in all atria (P < 0.05) and normalized the responses in wild-type, +EX with respect to wild-type with no exercise (-EX) atria. Atrial nNOS mRNA and protein were increased in wild-type, +EX compared to wild-type, -EX (P < 0.05), although exercise failed to have any effect in nNOS(+/-) atria. In vivo nNOS gene transfer using adenoviruses targeted to atrial ganglia enhanced choline acetyltransferase-nNOS co-localization (P < 0.05) and increased phenylephrine-induced bradycardia in vivo and heart rate responses to vagal nerve stimulation in vitro compared to gene transfer of enhanced green fluorescent protein (eGFP, P < 0.01). This difference was abolished by nNOS inhibition (P < 0.05). In conclusion, genomic regulation of NO bioavailability from nNOS in cardiac autonomic ganglia in response to training is dependent on both alleles of the gene. Although basal expression of nNOS is normal, polymorphisms of nNOS may interfere with neural regulation of heart rate following training. Targeted gene transfer of nNOS can restore this impairment.

Figure 1. Running distances and aortic eNOS protein levels in mice

A, daily running distances for wild-type (WT) and nNOS^+/− mice. B, Western Blot analysis showed elevated aortic eNOS protein levels in vessels from WT, +EX compared to WT, −EX and in vessels from nNOS^+/−, +EX compared to nNOS^+/−, −EX (*P < 0.01, unpaired t test).

Figure 2. nNOS mRNA and protein levels in atria

A, PCR-amplified nNOS mRNA expression revealed that nNOS mRNA was increased in WT, +EX atria with respect to WT, −EX (*P < 0.05, unpaired t test), but this effect was not evident in nNOS^+/− atria. B, Western Blot analysis showed elevated nNOS protein levels in WT, +EX atria compared to WT, −EX (*P < 0.05, unpaired t test), but no effect in nNOS^+/− atria.

Figure 3. Heart rate responses to vagal stimulation in vitro

The decrease in heart rate associated with vagal nerve stimulation (5 Hz) in isolated atria in vitro was enhanced in trained (+EX) wild-type atria with respect to untrained (−EX; *P < 0.05, unpaired t test). This effect was not evident in nNOS^+/− atria.

Figure 4. Immunohistochemical localization of nNOS in cholinergic ganglia

Confocal micrograph illustrates enhanced green fluorescence (far left, green panels) immunoreactivity against choline acetyltransferase (ChAT, middle, red panels) and nNOS (far right, green panels) in atria from nNOS^+/−, +EX mice pretreated with adenoviruses containing either eGFP (top row) or nNOS genes (bottom row). The panels show the presence of enhanced green fluorescence located with ChAT-positive cholinergic ganglia in an Ad.eGFP specimen with some neurones coexpressing nNOS. In the Ad.nNOS specimen, enhanced green fluorescence is not present, and coexpression of nNOS in cholinergic neurones is increased with respect to the Ad.eGFP group.

Figure 5. nNOS and eGFP protein levels in adenovirus-transfected atria

Western Blot analysis showed expression of nNOS (155 kDa) and eGFP (27 kDa) in wild-type, −EX and +EX; and nNOS^+/−, −EX and +EX atria treated with Ad.nNOS or Ad.eGFP adenovirus. Ad.eGFP treatment was successful in causing atria to express eGFP protein. Ad.nNOS significantly increased atrial expression of nNOS protein in all groups (*P < 0.001, unpaired t test).

Figure 6. Effects of nNOS gene transfer on in vivo phenylephrine-induced bradycardia and in vitro vagal-induced bradycardia

A, phenylephrine-induced bradycardia, expressed as change in interpulse interval (ms) per change in mean arterial blood pressure (mmHg), in response to pressor challenge was increased in anaesthetized nNOS^+/−, +EX mice treated with Ad.nNOS compared to treatment with Ad.eGFP adenovirus (*P < 0.05, unpaired t test). Ad.nNOS treatment failed to have a significant effect on phenylephrine-induced bradycardia in WT, +EX mice, although Ad.nNOS increased bradycardia in WT, −EX mice by 64% compared to Ad.eGFP (P < 0.05, unpaired t test; data not shown). B, bradycardia to vagal nerve stimulation (5 Hz) in isolated atria in vitro was enhanced in nNOS^+/−, +EX mice treated with Ad.nNOS compared to treatment with Ad.eGFP adenovirus (*P < 0.01, unpaired t test).