Cardiac cholinergic NO-cGMP signaling following acute myocardial infarction and nNOS gene transfer

Abstract

Myocardial infarction (MI) is associated with oxidative stress, which may cause cardiac autonomic impairment. We tested the hypothesis that acute MI disrupts cardiac cholinergic signaling by impairing nitric oxide (NO)-cGMP modulation of acetylcholine (ACh) release and whether the restoration of this pathway following cardiac neuronal NO synthase (nNOS) gene transfer had any bearing on the neural phenotype. Guinea pigs underwent four ligature coronary artery surgery (n = 50) under general anesthesia to induce MI or sham surgery (n = 32). In a separate group, at the time of MI surgery, adenovirus encoding nNOS (n = 29) or enhanced green fluorescent protein (eGFP; n = 30) was injected directly into the right atria, where the postganglionic cholinergic neurons reside. In vitro-evoked right atrial [3H]ACh release, right atrial NOS activity, and cGMP levels were measured at 3 days. Post-MI 24% of guinea pigs died compared with 9% in the sham-operated group. Evoked right atrial [3H]ACh release was significantly (P < 0.05) decreased in the MI group as was NOS activity and cGMP levels. Tetrahydrobiopterin levels were not significantly different between the sham and MI groups. Infarct sizes between gene-transferred groups were not significantly different. The nNOS transduced group had significantly increased right atrial [3H]ACh release, right atrial NOS activity, cGMP levels, and decreased cAMP levels. Fourteen percent of the nNOS transduced animals died compared with 31% mortality in the MI + eGFP group at 3 days. In conclusion, cardiac nNOS gene transfer partially restores the defective NO-cGMP cholinergic pathway post-MI, which was associated with a trend of improved survival at 3 days.

Fig. 1.

i: bright light microscopy image (20×) of cholinergic ganglia residing within the right atria wall. ii: representative slide showing Sirius red collagen staining of sham-operated guinea pig left ventricle. iii: Sirius red staining of myocardial infarction (MI) model guinea pig left ventricle. iv: significant difference between sham (n = 6) and MI (n = 6) left ventricle free wall collagen staining (**P < 0.001; unpaired t-test).

Fig. 2.

i: nitric oxide synthase (NOS) activity measured in right atria sham and MI guinea pigs showing a significant decrease in the MI group (sham, n = 9 vs. MI, n = 9; **P < 0.01, unpaired Mann-Whitney's U exact test). ii: cGMP levels in the right atria were significantly reduced in the MI animals (sham, n = 10, P < 0.01 vs. MI, n = 10, †P < 0.01; unpaired t-test).

Fig. 3.

i: means ± SE of raw data (uncorrected for total count) plot from naïve guinea pigs (n = 5) demonstrating consistency of ³H ACh release from the right atrium following 5 Hz epicardial stimulation at 30 (S1) and 76 (S2) min. ii: representative raw data trace of right atrial ³H ACh release with epicardial stimulation pre- (S1) and post- (S2) addition of neuronal NOS (nNOS) inhibitor (4S)-N-(4-amino-5-[aminoethyl]aminopentyl)-N′-nitroguanidine (NNNG) from a sham-operated guinea pig. iii: representative raw data plots of right atrial ³H ACh release with epicardial stimulation pre- (S1) and post- (S2) addition of nNOS inhibitor NNNG from a MI guinea pig. iv: MI significantly attenuated ³H ACh release poststimulation (sham, n = 6 vs. MI, n = 6; ††P < 0.01, 2-factor ANOVA independent factor comparison). NNNG caused a significant decrease in ³H ACh release in both the sham and MI guinea pigs (**P < 0.01; 2-factor ANOVA paired comparisons).

Fig. 4.

Ganglia from cytomegalovirus (CMV) adenovirus (Ad) enhanced green fluorescent protein (eGFP; i) or nNOS (ii) gene transferred guinea pig atria at ×20 magnification. Ganglia are viewed with bright field microscopy (Ganglia), Texas red choline acetyl-transferease immunofluorescence (ChAT), overlaid images demonstrating colocalization (ChAT:nNOS), and fluoroscein nNOS immunofluorescence. All images had brightness and contrast equally enhanced by 50% and 40%, respectively.

Fig. 5.

i: embedded bitmap of Western blot for nNOS showing standard (STD), eGFP (E), and nNOS (N) transduced right atria. ii: Western blot analysis showing increased nNOS protein in the Ad.nNOS- compared with the Ad.eGFP-transduced MI guinea pigs (n = 6 in each group; **P < 0.01, unpaired Mann-Whitney's U exact test). iii: right atrial NOS activity was increased in the MI + nNOS guinea pigs compared with the MI + eGFP group (MI + eGFP, n = 11 vs. MI + nNOS, n = 11; ††P < 0.01; unpaired Mann-Whitney's U exact test). iv: cGMP levels were increased in the right atria of MI + nNOS compared with MI + eGFP guinea pigs (MI + eGFP, n = 15 vs. MI + nNOS, n = 13; **P < 0.01; unpaired Mann-Whitney's U exact test). v: right atrial cAMP levels were lower in the nNOS- compared with the eGFP-transduced group (MI + eGFP, n = 15 vs. MI + nNOS, n = 13; *P < 0.05; 1-tailed, independent t-test for unequal variances). MM, molecular mass.

Fig. 6.

i: representative raw data plot of right atria ³H ACh release with epicardial stimulation pre- (S1) and post- (S2) addition of nNOS inhibitor NNNG in MI + eGFP guinea pig. ii: representative raw data plot of right atria ³H ACh release with epicardial stimulation pre- (S1) and post- (S2) addition of NNNG in MI + nNOS guinea pig. iii: nNOS gene transfer significantly increased right atrial ³H ACh release with epicardial stimulation at 3 days post-MI (n = 6) compared with that of eGFP (n = 6)-transduced animals (*P < 0.05; unpaired Mann-Whitney's U exact test). Inhibition of nNOS caused a significant decrease in ³H ACh release in the MI + eGFP and MI + nNOS groups (**P < 0.05; Wilcoxon's signed ranks exact test). AMI, acute MI.

Fig. 7.

i: survival graph for guinea pigs postsham (n = 32) or infarct (n = 50) surgery showing more deaths in the MI group up to experimental endpoint at 72 h. There was significantly more mortality in the MI group [*P < 0.05; Kaplan-Meier Log-Rank (Mantel Cox)]. Survival graphs, from either all-cause (ii) or unexplained death (iii), of guinea pigs post-infarct surgery with Ad.eGFP (n = 30) or Ad.nNOS (n = 29) transduction up to experimental end point at 72 h. There was significantly improved survival from 6 h (dotted line), when viral transduction of proteins encoded with the CMV promoter have first been shown to express in vivo, to end point in the nNOS compared with the eGFP group in both the all-cause [*P < 0.05; Kaplan-Meier Log-Rank (Mantel Cox)] and unexplained (*P < 0.05; Fisher's exact test) death groups.