Neuronal Nitric Oxide Synthase Protects Against Myocardial Infarction-Induced Ventricular Arrhythmia and Mortality in Mice
Background— Neuronal nitric oxide synthase (nNOS) is expressed in cardiomyocytes and plays a role in regulating cardiac function and Ca2+ homeostasis. However, the role of nNOS in cardiac electrophysiology after myocardial infarction (MI) is unclear. We hypothesized that nNOS deficiency increases ventricular arrhythmia and mortality after MI.
Methods and Results— MI was induced in wild-type (WT) or nNOS−/− mice by ligation of the left coronary artery. Thirty-day mortality was significantly higher in nNOS−/− compared with WT mice. Additionally, nNOS−/− mice had impaired cardiac function 2 days after MI. Telemetric ECG monitoring showed that compared with WT, nNOS−/− mice had significantly more ventricular arrhythmias and were more likely to develop ventricular fibrillation after MI. Treatment with the L-type Ca2+ channel blocker verapamil reduced the incidence of arrhythmia and ventricular fibrillation in nNOS−/− mice after MI. To assess the role of nNOS in Ca2+ handling, patch-clamp and Ca2+ fluorescence techniques were used. Ca2+ transients and L-type Ca2+ currents were higher in nNOS−/− compared with WT cardiomyocytes. Additionally, nNOS−/− cardiomyocytes exhibited significantly higher systolic and diastolic Ca2+ over a range of pacing frequencies. Treatment with the NO donor S-nitroso N-acetyl-penicillamine decreased Ca2+ transients and L-type Ca2+ current in both nNOS−/− and WT cardiomyocytes. Furthermore, S-nitrosylation of Ca2+ handling proteins was significantly decreased in nNOS−/− myocardium after MI.
Conclusions— Deficiency in nNOS increases ventricular arrhythmia and mortality after MI in mice. The antiarrhythmic effect of nNOS involves inhibition of L-type Ca2+ channel activity and regulation of Ca2+ handling proteins via S-nitrosylation.
Received July 3, 2008; accepted July 2, 2009.
Neuronal nitric oxide (NO) synthase (nNOS) is a Ca2+-dependent enzyme initially identified in the brain and implicated in neurotransmission.1,2 Along with endothelial nitric oxide synthase (eNOS) and inducible NOS, nNOS belongs to a class of enzymes that produce NO through the conversion of l-arginine to l-citrulline.3 More recently, it has been demonstrated that nNOS is expressed in cardiomyocytes and localized to the sarcoplasmic reticulum.4 Interestingly, nNOS has been shown to be physically associated with the ryanodine receptor Ca2+ release channel (RyR2) and SERCA2.5,6 Under conditions in which nNOS is overexpressed in cardiomyocytes, it also binds to the L-type Ca2+ channel.6 Given the close association between nNOS and the proteins that are important in the regulation of Ca2+, it is not surprising that nNOS regulates intracellular Ca2+ ([Ca2+]i) levels in cardiomyocytes.6,7 Indeed, nNOS deletion is associated with increased Ca2+ transient amplitude and L-type Ca2+ currents (ICa,L), as well as reduced phospholamban phosphorylation and increased cardiomyocyte shortening.7–9 Additionally, nNOS-derived NO has been shown to inhibit myocardial responsiveness to β-adrenergic stimulation.8,10 Taken together, these observations indicate that nNOS plays an important physiological role in regulating Ca2+ cycling and cardiac function.11
Clinical Perspective on p 1354
Studies have found that nNOS expression and nNOS-derived NO production are increased in human and rat hearts during heart failure.5,12 However, the role of nNOS in heart failure is still not fully understood. Initial studies by Bendall et al13 showed that rats with myocardial infarction (MI)– induced heart failure exhibit increased myocardial sensitivity to β-adrenergic stimulation after pharmacological nNOS inhibition. In contrast, 2 recent studies demonstrated that cardiac contractile response to β-adrenergic stimulation was either decreased or unchanged in nNOS−/− mice after MI.14,15 Furthermore, mice deficient in nNOS exhibit accelerated left ventricular (LV) remodeling and increased oxidative stress after MI.14,15 Thus, it appears that the increases in nNOS expression during heart failure represent a beneficial, cardioprotective response. Indeed, deletion of nNOS has been associated with higher mortality after MI, which is not necessarily caused by heart failure.14,15
There is now emerging evidence that nNOS-derived NO production may play an important role in cardiomyocyte arrhythmogenesis.16 A recent report by Gonzalez et al16 found that a deficiency in nNOS was associated with impaired S-nitrosylation of the RyR2, which was associated with a proarrhythmic phenotype in isolated cardiomyocytes. Moreover, it has been suggested that NO may be capable of suppressing spontaneous Ca2+ release events in isolated rat cardiomyocytes.17 However, to the best of our knowledge, no study to date has examined the role of nNOS in arrhythmia in vivo.
The present study examined the role of nNOS in cardiac arrhythmia after MI. We hypothesized that nNOS deficiency would increase the incidence of cardiac arrhythmias and decrease survival after MI. Cardiac function, electrophysiology, and survival were studied in wild-type (WT) and nNOS−/− mice after MI. Ca2+ transient frequency response and ICa,L were determined in isolated cardiomyocytes. Furthermore, S-nitrosylation of Ca2+ handling proteins was measured in WT and nNOS−/− myocardium after MI. Our data demonstrated for the first time that deficiency in nNOS increases the incidence of ventricular arrhythmias, leading to higher mortality after MI.
WT and nNOS−/− mice of C57BL/6 background were purchased from The Jackson Laboratory (Bar Harbor, Me), and a breeding program was implemented at University of Western Ontario animal care facilities. Animals were provided water and food ad libitum and housed in a temperature- and humidity-controlled facility with 12-hour light/dark cycles. The investigations were approved by the institutional Animal Care Committee and conformed to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH publication No. 85–23).
To detect arrhythmia in mice after MI, ECGs were continuously recorded in freely moving animals with a telemetry system over a 10-day period. As a control, sham-operated mice were subjected to surgical anesthesia and their chests were opened, but they did not receive coronary artery ligation, and ECG was measured over 5 to 10 days.
ECG Monitoring During Myocardial Ischemia and Reperfusion
Short-term monitoring of ECG limb lead I during ischemia/reperfusion (I/R) was performed in anesthetized mice with needle electrodes inserted subcutaneously. ECG was recorded 5 minutes before and throughout the entire I/R protocol with Powerlab Chart 5.0 (AD Instruments, Colorado Springs, Colo).
S-Nitrosylation of Ca2+ Handling Proteins
To analyze S-nitrosylation of the L-type Ca2+ channels, RyR2, and SERCA2, a 1-mg protein lysate from isolated adult cardiomyocytes was incubated with an antibody targeted to the α1C subunit of the L-type Ca2+ channel (1:100, Santa Cruz Biotechnology, Santa Cruz, Calif), RyR2 (1:100, Affinity Bioreagents, Golden, Colo), or SERCA2 (1:200, Affinity Bioreagents). The immunocomplex was captured using protein A/G-agarose (Santa Cruz), and S-nitrosylation of immunoprecipitated subunits was directly measured by nonreducing Western blot analysis. Samples were then probed with an anti-S-nitrosocysteine antibody (1:2000, Sigma, Oakville, Ontario, Canada) and quantified with densitometry.
All data are expressed as mean±SEM. Statistical significance was assessed by 2-way ANOVA followed by the Bonferroni posthoc test with Prism 4.0 (GraphPad Software, La Jolla, Calif). Incidence of arrhythmia was analyzed with χ2 analysis, and survival was analyzed by the Kaplan-Meier method. Differences were considered significant at P<0.05.
For detailed methods relative to the induction of MI and I/R, hemodynamic measurements, measurement of apoptosis, superoxide anion (O2−) production, telemetry, ECG analysis, adult cardiomyocyte isolation, Ca2+ transients, and L-type Ca2+ current, see the online-only Data Supplement.
Mortality After MI
WT (n=74) and nNOS−/− (n=114) mice were subjected to MI or sham operation. Animals were excluded from analysis if perioperative death occurred within the first 24 hours after surgery. The remaining 64 WT and 82 nNOS−/− mice were included in the study, and their mortality was followed up for 30 days after surgery. After hemodynamic measurements were obtained, mice were killed. Although sham nNOS−/− mice had a lower ratio of LV weight to body weight than WT sham mice, there were no differences in age, body weight, ratio of LV weight to body weight, and infarct size between nNOS−/− and WT mice subjected to MI (Table 1). MI resulted in a significant increase in mortality in both nNOS−/− and WT mice compared with the sham operation groups (P<0.01; Figure 1). Furthermore, the 30-day survival was significantly lower in nNOS−/− mice (8 of 49, 16%) compared with WT mice (18 of 36, 50%; P<0.01; Figure 1).
To determine whether there were alterations in cardiac function in nNOS−/− mice, heart function was measured in subgroups at 2 and 30 days after MI with a Millar catheter. In sham mice, there was no significant difference between WT and nNOS−/− mice among any of the measured hemodynamic parameters (Table 2). Two days after MI, there was a significant reduction in systolic LV pressure, in the LV maximal rate of pressure development (dP/dt), and in the maximal rate of relaxation (−dP/dt) in WT and nNOS−/− mice. However, LV dP/dt and −dP/dt were both lower in nNOS−/− compared with WT mice. Additionally, 30 days after MI, systolic LV pressure, dP/dt, and −dP/dt also were lower in WT and nNOS−/− mice compared with their respective shams, whereas LV end-diastolic pressure was higher. However, there were no longer differences between surviving WT and nNOS−/− mice among any measured hemodynamic parameters (Table 2).
Superoxide Generation and Apoptosis
Oxidative stress and in particular O2− have previously been shown to cause impaired cardiac function in models of heart failure.18 These effects were associated with increases in apoptotic cell death.18 We measured myocardial O2− production and apoptosis in the peri-infarct region 2 days after MI. O2− production, measured by lucigenin assay, was significantly increased in both WT and nNOS−/− mice after MI (Figure 2A). However, the increases in O2− production were significantly higher in nNOS−/− compared with WT mice (Figure 2A). Furthermore, MI-induced apoptosis also was significantly higher in nNOS−/− compared with WT mice as measured by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) staining (Figure 2B) and caspase-3 activity (Figure 2C).
Ventricular Arrhythmia After MI
To determine whether nNOS deletion was associated with increased ventricular arrhythmia after MI, cardiac ECG was monitored through the use of telemetry in WT and nNOS−/− mice after MI for up to 10 days (Figure 3A). Although there was no change in heart rate among the treatment groups, nNOS−/− mice developed significantly more ventricular arrhythmias than their WT counterparts (Table 3). Additionally, nNOS−/− mice were significantly more likely to develop ventricular fibrillation (VF) in the hour preceding spontaneous death (Figure 3A and Table 3). Sham-operated mice displayed only a few isolated ventricular premature beats, and there were no differences between WT and nNOS−/− mice (Table 3).
nNOS deletion is associated with increased [Ca2+]i,7 and elevated [Ca2+]i has been shown to cause ventricular tachyarrhythmia.19 The L-type Ca2+ channel blocker verapamil was used to examine the role of Ca2+ in the increased arrhythmia seen in nNOS−/− mice. Verapamil treatment (0.1 mg/mL in drinking water), which was started 24 hours after MI, significantly reduced ventricular arrhythmias and the incidence of VF in nNOS−/− mice (Table 3).
Ventricular Arrhythmia During Myocardial I/R
The role of nNOS in ventricular arrhythmia also was examined in a mouse model of acute myocardial I/R. WT and nNOS−/− mice were subjected to I/R, and cardiac electric activity was monitored with a single-limb lead I ECG. Representative ECG tracings are shown in Figure 3B, and the incidence of arrhythmia is quantified in Table 4. Heart rate was similar across all treatment groups. However, the numbers of singlet and doublet ventricular premature beats and of ventricular tachycardias were significantly higher in nNOS−/− mice compared with their WT counterparts. Treatment with verapamil (0.02 mg/kg IP) after the start of ischemia significantly reduced the incidence of ventricular tachycardia and the number of ventricular premature beats in WT and nNOS−/− mice (Table 4). Thus, alterations in Ca2+ handling may contribute to the increased susceptibility to ventricular arrhythmia seen in these animals during I/R.
Intracellular Ca2+ Transients
To investigate Ca2+ handling mechanisms, we measured Ca2+ transients in fura-2-loaded ventricular myocytes isolated from WT and nNOS−/− mice. Peak [Ca2+]i was significantly higher in nNOS−/− compared with WT cardiomyocytes (Figure 4). Furthermore, pharmacological inhibition of nNOS with N5-(1-imino-3-butenyl)-l-ornithine (L-VNIO; 100 μmol/L) increased peak [Ca2+]i in WT but not nNOS−/− cardiomyocytes (Figure 4A through 4C). Conversely, treatment with the NO donor S-nitroso N-acetyl-penicillamine (SNAP; 10 μmol/L), significantly reduced peak [Ca2+]i in both WT and nNOS−/− cardiomyocytes (Figure 4D through 4F).
To determine whether the difference in Ca2+ transients is frequency dependent, WT and nNOS−/− cardiomyocytes were paced over a range of frequencies (0.5 to 4 Hz; Figure 5A through 5D). nNOS−/− cardiomyocytes exhibited consistently higher diastolic Ca2+ than WT cardiomyocytes (Figure 5A). Additionally, nNOS−/− cardiomyocytes exhibited significantly higher systolic Ca2+ levels at 0.5, 1, and 2 Hz (Figure 5B).
Patch-Clamp Recordings of L-Type Ca2+ Channel Currents
To determine whether the changes in the evoked Ca2+ transients were caused by regulation of Ca2+ current, we next used perforated patch-clamp recordings to characterize L-type Ca2+ current in isolated myocytes. Depolarization of cells elicited the characteristic L-type Ca2+ current, evident as a transient inward current, which was reversibly blocked by verapamil (1 μmol/L; 5 cells; not shown). The average access resistance in perforated patch configuration was 18±1 mol/LΩ (from 58 cells in 25 different cell preparations). Treatment of WT cardiomyocytes with the selective nNOS inhibitor L-VNIO (100 μmol/L) for 30 seconds caused a marked and consistent increase in the amplitude of the Ca2+ current (Figure 6A, left). The peak effect was evident within 20 seconds of the application of L-VNIO, and recovery occurred over several minutes. When cardiomyocytes isolated from nNOS−/− mice were studied, Ca2+ currents were significantly larger than those from WT mice (Figure 6A, right, and Figure 6B). Furthermore, treatment of the nNOS−/− cells with L-VNIO for comparable times did not result in a significant change in amplitude of the Ca2+ current, in contrast to that seen for WT cells (Figure 6A and 6B). To confirm the action of NO, we applied SNAP (10 μmol/L) during the recording of L-type Ca2+ current. In WT and nNOS−/− cardiomyocytes, Ca2+ currents were significantly inhibited by SNAP, which was reversible (Figure 7A and 7B).
S-Nitrosylation of Ca2+ Handling Proteins After MI
To determine whether nNOS-derived NO directly interacted with proteins involved in [Ca2+]i regulation, we measured S-nitrosylation of the α1C subunit of the L-type Ca2+ channel, RyR2, and SERCA2 in the LV myocardium 5 days after MI using immunoprecipitation followed by Western blotting. S-nitrosylation of all 3 proteins was detectable in WT and nNOS−/− myocardium (Figure 8). However, S-nitrosylation was significantly reduced in nNOS−/− compared with WT hearts (Figure 8), suggesting decreased S-nitrosylation of the L-type Ca2+ channel, RyR2, and SERCA2 in nNOS−/− mice after MI.
In the present study, we showed that nNOS deletion was associated with increased mortality, O2− production, and apoptosis in surviving mice and decreased contractility after MI. A novel finding from our study is that ventricular arrhythmias were higher in nNOS−/− than WT mice after MI. Deficiency in nNOS increased Ca2+ transient amplitude and L-type Ca2+ channel activity and increased diastolic Ca2+ levels in cardiomyocytes. S-nitrosylation of the L-type Ca2+ channel, RyR2, and SERCA2 in the myocardium was decreased after MI. These data suggest that nNOS has a beneficial role in the regulation of [Ca2+]i via S-nitrosylation of Ca2+ handling proteins, leading to reductions in ventricular arrhythmias and death after MI.
Two previous investigations have examined the role of nNOS after MI.14,15 In these studies, ventricular remodeling as measured by LV volume and myocyte size was increased in nNOS−/− mice. Although the inotropic response to dobutamine was attenuated, cardiac function, including LV dP/dt, LV end-diastolic pressure, and LV ejection fraction, was not significantly different between nNOS−/− and WT mice 4 weeks after MI.14,15 Consistent with these reports, we did not find a significant impairment in cardiac function between surviving nNOS−/− and WT mice 4 weeks after MI. However, survival was significantly decreased in nNOS−/− mice after MI. Because most animals died within a few days after MI, we also measured cardiac function 2 days after MI and showed that cardiac function was lower in nNOS−/− compared with WT mice. Lower cardiac function was associated with higher NAD(P)H oxidase-dependent O2− generation and myocardial apoptosis. Importantly, during measurements of cardiac function, more ventricular premature beats were observed in nNOS−/− mice, which led to our investigation of cardiac arrhythmia.
Perhaps the most striking observation from this study is that nNOS deletion was associated with more ventricular arrhythmias after MI. Mounting evidence suggests that nNOS is beneficial after MI.14,15 However, mechanisms responsible for the beneficial effects of nNOS are not fully understood. Recently, Gonzalez et al16 reported sarcoplasmic reticulum Ca2+ leak and an arrhythmogenic phenotype in isolated nNOS−/− cardiomyocytes. Our laboratory, to the best of our knowledge, provides the first in vivo evidence of increased ventricular arrhythmia in nNOS−/− mice. Deletion of nNOS resulted in higher incidences of ventricular tachycardia and/or VF after myocardial I/R and MI. Interestingly, nNOS−/− mice are known to have vastly accelerated mortality with aging (>10 months old) even in the absence of MI.20 The present study was done in mice 3 to 4 months of age, and no difference in arrhythmia was detected between sham-operated WT and nNOS−/− mice. It is possible that, with aging, nNOS−/− mice are more susceptible to arrhythmia, which may be responsible for increased mortality. Indeed, aging has been associated with an increased susceptibility to Ca2+ overload in rabbits and rats.21–23
nNOS deletion is associated with higher L-type Ca2+ channel activity, diastolic Ca2+ elevations, and higher [Ca2+]i in myocytes, which are known to cause ventricular arrhythmia.19 Therefore, we examined the effects of the L-type Ca2+ channel blocker verapamil on ventricular arrhythmias in nNOS−/− and WT mice during I/R and after MI. Although primarily used clinically for supraventricular arrhythmias, verapamil is also known to prevent VF caused by ischemia in patients with coronary artery disease and may reduce ventricular tachyarrhythmias associated with I/R.24,25 As expected, verapamil treatment significantly reduced I/R-induced ventricular tachyarrhythmia and reduced the incidence of VF after MI. These data suggest a role for altered Ca2+ handling in the increased cardiac arrhythmia in nNOS−/− mice after MI.
To further understand the role of nNOS in the regulation of [Ca2+]i, Ca2+ transients and L-type Ca2+ channel activity were determined. We found that pharmacological inhibition and genetic deletion of nNOS increased [Ca2+]i in isolated mouse cardiomyocytes. These observations are in agreement with the work of Sears et al,7 who also showed higher Ca2+ transient amplitude and L-type Ca2+ current in nNOS−/− cardiomyocytes. Although others have failed to observe alterations in Ca2+ transients in nNOS−/− mice,26,27 a recent report by Burkard and colleagues6 provided strong evidence of a role for nNOS in the regulation of [Ca2+]i in cardiomyocytes. In that study, the authors observed reductions in Ca2+ transient and L-type Ca2+ current amplitude in cardiomyocytes overexpressing nNOS. Similarly, we observed reductions in Ca2+ transient amplitude, as well as L-type Ca2+ current amplitude, after administration of the NO donor SNAP in both WT and nNOS−/− cardiomyocytes. These observations, coupled with the initial work of Sears et al,7 provide compelling evidence of a role for nNOS-derived NO production in the inhibition of the L-type Ca2+ channel and regulation of [Ca2+]i in cardiomyocytes. Interestingly, elevated [Ca2+]i such as that seen in nNOS−/− cardiomyocytes is thought to lead to Ca2+ overload, thereby triggering the initiation of ventricular arrhythmia.19 Thus, it is possible that the mechanism by which nNOS reduces ventricular arrhythmia is through this NO-dependent inhibition of the L-type Ca2+ channel and subsequent reduction in Ca2+ overload.
nNOS-derived NO also may play an important role in preventing diastolic Ca2+ leak in cardiomyocytes, particularly at higher pacing frequencies.16 In agreement with this, we found that diastolic Ca2+ was increased in nNOS−/− compared with WT cardiomyocytes when paced at higher frequencies. Diastolic Ca2+ leak is associated with spontaneous Ca2+ waves, which are capable of generating delayed afterdepolarizations.16,28 In fact, at higher pacing rates, this diastolic Ca2+ elevation appears to diminish contractile reserve and the peak [Ca2+]i levels because nNOS−/− cardiomyocytes are no longer significantly different from their WT counterparts.16 Thus, the arrhythmia seen in nNOS−/− mice may result from diastolic Ca2+ leak, in addition to Ca2+ overload.
S-nitrosylation of the L-type Ca2+ channel has recently been observed in the membrane fractions of mouse hearts, and S-nitrosylation of the L-type Ca2+ channel results in a decrease in activity of the channel.29 Moreover, deficiency in S-nitrosylation of RyR2 is associated with sarcoplasmic reticulum Ca2+ leak and arrhythmogenesis,16 whereas S-nitrosylation of SERCA2 appears to increase activity of the enzyme.29 In the present study, we demonstrated an important role for nNOS in the S-nitrosylation of [Ca2+]i handling proteins, including the L-type Ca2+ channel subunit α1C, RyR2, and SERCA2, in the LV myocardium after MI. S-nitrosylation of these proteins would prevent Ca2+ overload and subsequent ventricular arrhythmias. Indeed, nNOS is particularly suited to regulate Ca2+ because increases in [Ca2+]i also increase nNOS-derived NO production. This negative feedback via S-nitrosylation would, in turn, protect the cardiomyocyte from arrhythmogenesis. However, one cannot rule out potential effects of NO not attributable to S-nitrosylation. L-type Ca2+ channels, RyR2, and SERCA2 are subject to extensive redox regulation by reactive oxygen species.30,31 In the present study, we demonstrated that myocardial reactive oxygen species levels were increased in nNOS−/− mice, suggesting an antioxidant effect of nNOS.32 It is possible that increased reactive oxygen species production may contribute to the changes in Ca2+ seen in nNOS−/− cardiomyocytes. Moreover, NO also has been reported to modulate the L-type Ca2+ channel via cGMP.31,33 Thus, although S-nitrosylation is an important contributor to the altered Ca2+ handling and ventricular arrhythmia in nNOS−/− mice, the precise regulation of Ca2+ handling proteins is complex and may involve reactive oxygen species and other factors.
Finally, although this study has examined the role of nNOS-derived NO in the control of [Ca2+]i and ventricular arrhythmia, the contribution from eNOS cannot be excluded. This is particularly relevant given the residual S-nitrosylation of the L-type Ca2+ channel, RyR2, and SERCA2 seen even in the absence of nNOS. eNOS-derived NO has been reported to contribute to S-nitrosylation of the L-type Ca2+ channel in mouse hearts.34 However, Gonzalez et al16 did not find any changes in S-nitrosylation of the RyR2 in eNOS−/− cardiomyocytes, suggesting that eNOS is not involved in the S-nitrosylation of RyR2 under normal physiological conditions. In the failing myocardium, eNOS expression is upregulated.35 Whether increased eNOS expression after MI leads to S-nitrosylation of the RyR2 and inhibits ventricular arrhythmia requires further investigation.
Our results demonstrate that nNOS-derived NO is beneficial in mouse models of I/R and MI. Deficiency in nNOS decreases cardiac function and increases ventricular arrhythmias, leading to a higher mortality after MI. The finding that nNOS deletion increases ventricular arrhythmia suggests an antiarrhythmic mechanism of nNOS after MI.
Sources of Funding
This study was supported by grants from the Heart and Stroke Foundation of Ontario (T-6040 to Dr Feng) and the Canadian Institutes of Health Research (MOP-64395 to Dr Feng, MOP-10019 to Dr Sims). Dr Burger was supported by an Ontario Graduate Scholarship Award. Dr Feng is a Heart and Stroke Foundation of Ontario career investigator.
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Ventricular arrhythmia is a common cause of death after myocardial infarction (MI). Cardiomyocyte Ca2+ overload and diastolic Ca2+ leak are important mechanisms responsible for afterdepolarization and triggered activity leading to ventricular arrhythmia after MI. Emerging evidence suggests that neuronal nitric oxide synthase (nNOS) may inhibit arrhythmogenesis by maintaining intracellular Ca2+ homeostasis in cardiomyocytes. However, the role of nNOS in ventricular arrhythmia after MI in vivo is not clear. In the present study, we demonstrated that nNOS deletion was associated with increased mortality, reactive oxygen species production, and apoptosis after MI. A novel finding from our study is that ventricular arrhythmias were significantly higher in nNOS−/− compared with wild-type mice after MI. Deficiency in nNOS increased Ca2+ transient amplitude and L-type Ca2+ channel activity and increased diastolic Ca2+ levels in cardiomyocytes. Lack of nNOS decreased S-nitrosylation of the L-type Ca2+ channel, ryanodine receptor Ca2+ release channel, and SERCA2 in the myocardium after MI. Thus, nNOS has a beneficial role in Ca2+ homeostasis via S-nitrosylation of Ca2+ handling proteins, leading to reductions in ventricular arrhythmias and death after MI. Our study provides new insights into the protective role of nNOS in arrhythmogenesis and may have therapeutic implications after MI.
↵*Drs Burger and Lu contributed equally to this work.
The online-only Data Supplement is available with this article at http://circ.ahajournals.org/cgi/content/full/CIRCULATIONAHA.108.846402/DC1.