Cardiac myocyte neuronal nitric oxide synthase. New therapeutic target in heart failure?

Although nitric oxide-dependent regulation of contractile function is altered in the diseased and failing heart, several aspects of nitric oxide (NO) signalling in the myocardium remain poorly understood. Some apparently contrasting findings may have arisen from the use of non-isoform-specific inhibitors of NO synthase isoforms (NOS) as compared to the use of mouse models genetically deficient or overexpressing the NOS thought to be responsible for the increase in NO production in heart failure (mainly NOS2 and NOS3). In recent years, identification of the neuronal NOS (NOS1) isoform in cardiac myocytes and the recognition of the importance of its subcellular localisation have greatly advanced the understanding of the critical role of NOS1-derived NO in the control of myocardial contractility both in the normal and failing heart. The challenge is now to confirm these emerging findings on the critical role of NOS1-derived NO in human cardiac physiology and hopefully translate them into therapy.     

Sub-cellular targeting of constitutive NOS in health and disease

Constitutive nitric oxide synthases (NOSs) are ubiquitous enzymes that play a pivotal role in the regulation of myocardial function in health and disease. The discovery of both a neuronal NOS (nNOS) and an endothelial NOS (eNOS) isoform in the myocardium and the availability of genetically modified mice with selective eNOS or nNOS gene deletion have been of crucial importance for understanding the role of constitutive nitric oxide (NO) production in the myocardium. eNOS and nNOS are homologous in structure and utilize the same co-factors and substrates; however, they differ in their subcellular localization, regulation, and downstream signaling, all of which may account for their distinct effects on excitation-contraction coupling. In particular, eNOS-derived NO has been reported to increase left ventricular (LV) compliance, attenuate beta-adrenergic inotropy and enhance parasympathetic/muscarinic responses, and mediate the negative inotropic response to β3 adrenoreceptor stimulation via cGMP-dependent signaling. Conversely, nNOS-derived NO regulates basal myocardial inotropy and relaxation by inhibiting the sarcolemmal Ca(2+) current (I(Ca)) and promoting protein kinase A-dependent phospholamban (PLN) phosphorylation, independent of cGMP. By inhibiting the activity of myocardial oxidase systems, nNOS regulates the redox state of the myocardium and contributes to maintain eNOS "coupled" activity. After myocardial infarction, up-regulation of myocardial nNOS attenuates adverse remodeling and prevents arrhythmias whereas uncoupled eNOS activity in murine models of left ventricular pressure overload accelerates the progress towards heart failure. Here we review the evidence in support of the idea that NOS subcellular localization, mode of activation, and downstream signaling account for the diverse and highly specialized actions of NO in the heart. This article is part of a Special Issue entitled "Local Signaling in Myocytes".     

Caveolins and the regulation of endothelial nitric oxide synthase in the heart

Virtually all cell types within the myocardium express caveolae, where cell-specific isoforms of caveolin both maintain the structural organisation of these cholesterol-rich of the plasmalemma and serve as scaffolds for the dynamic constitution of "signalosomes", or hubs concentrating numerous transmembrane signaling proteins and their effectors. Analysis of the phenotype of mice with genetic deletion or overexpression of specific caveolin isoforms has provided key evidence for the importance of caveolins and caveolae in several aspects of the cardiovascular biology, including vascular contractility, lipid metabolism, angiogenesis, or the control of cardiac hypertrophy. Among specific protein-protein interactions involving caveolins in cardiac tissue, these genetic models unequivocally confirmed the functional importance of the dynamic association of the endothelial isoform of nitric oxide synthase (eNOS) for its post-translational regulation in endothelial cells and cardiac myocytes, which bears on the enzyme's capacity to modulate nitric oxide (NO)-dependent endothelial function, angiogenesis, and excitation-contraction coupling. We will review the current understanding of this regulation of eNOS (and potentially other NOS isoforms) through protein-protein interactions involving several G-protein-coupled receptors and other allosteric modulators in the context of emerging paradigms on the regulation of cardiac function by NO.     

Arginase modulates myocardial contractility by a nitric oxide synthase 1-dependent mechanism

Cardiac myocytes contain two constitutive NO synthase (NOS) isoforms with distinct spatial locations, which allows for isoform-specific regulation. One regulatory mechanism for NOS is substrate (l-arginine) bioavailability. We tested the hypothesis that arginase (Arg), which metabolizes l-arginine, constrains NOS activity in the cardiac myocyte in an isoform-specific manner. Arg activity was detected in both rat heart homogenates and isolated myocytes. Although both Arg I and II mRNA and protein were present in whole heart, Arg II alone was found in isolated myocytes. Arg inhibition with S-(2-boronoethyl)-l-cysteine (BEC) augmented Ca(2+)-dependent NOS activity and NO production in myocytes, which did not depend on extracellular l-arginine. Arg II coimmunoprecipited with NOS1 but not NOS3. Isolation of myocyte mitochondrial fractions in combination with immuno-electron microscopy demonstrates that Arg II is confined primarily to the mitochondria. Because NOS1 positively modulates myocardial contractility, we determined whether Arg inhibition would increase basal myocardial contractility. Consistent with our hypothesis, Arg inhibition increased basal contractility in isolated myocytes by a NOS-dependent mechanism. Both the Arg inhibitors N-hydroxy-nor-l-arginine and BEC dose-dependently increased basal contractility in rat myocytes, which was inhibited by both nonspecific and NOS1-specific NOS inhibitors N(G)-nitro-l-arginine methyl ester and S-methyl-l-thiocitrulline, respectively. Also, BEC increased contractility in isolated myocytes from WT and NOS3 but not NOS1 knockout mice. We conclude that mitochondrial Arg II negatively regulates NOS1 activity, most likely by limiting substrate availability in its microdomain. These findings have implications for therapy in pathophysiologic states such as aging and heart failure in which myocardial NO signaling is disrupted.     

Cardiomyocyte-specific overexpression of nitric oxide synthase 3 improves left ventricular performance and reduces compensatory hypertrophy after myocardial infarction

Nitric oxide (NO) is an important modulator of cardiac performance and left ventricular (LV) remodeling after myocardial infarction (MI). We tested the effect of cardiomyocyte-restricted overexpression of one NO synthase isoform, NOS3, on LV remodeling after MI in mice. LV structure and function before and after permanent LAD coronary artery ligation were compared in transgenic mice with cardiomyocyte-restricted NOS3 overexpression (NOS3-TG) and their wild-type littermates (WT). Before MI, systemic hemodynamic measurements, echocardiographic assessment of LV fractional shortening (FS), heart weight, and myocyte width (as assessed histologically) did not differ in NOS3-TG and WT mice. The inotropic response to graded doses of isoproterenol was significantly reduced in NOS3-TG mice. One week after LAD ligation, the infarcted fraction of the LV did not differ in WT and NOS3-TG mice (34+/-4% versus 36+/-12%, respectively). Four weeks after MI, however, end-systolic LVID was greater, and fractional shortening and maximum and minimum rates of LV pressure development were less in WT than in NOS3-TG mice. LV weight/body weight ratio was greater in WT than in NOS3-TG mice (5.3+/-0.2 versus 4.6+/-0.5 mg/g; P<0.01). Myocyte width in noninfarcted myocardium was greater in WT than in NOS3-TG mice (18.8+/-2.0 versus 16.6+/-1.6 microm; P<0.05), whereas fibrosis in noninfarcted myocardium was similar in both genotypes. Cardiomyocyte-restricted overexpression of NOS3 limits LV dysfunction and remodeling after MI, in part by decreasing myocyte hypertrophy in noninfarcted myocardium.     

Heterogeneous basal expression of nitric oxide synthase and superoxide dismutase isoforms in mammalian heart : implications for mechanisms governing indirect and direct nitric oxide-related effects.

The basal expression patterns of NO synthase (NOS; endothelial [eNOS], neuronal [nNOS], and cytokine-inducible [iNOS]) and superoxide dismutase (SOD; extracellular membrane bound [ECSOD], MnSOD, and CuZnSOD) isoforms in ferret heart (tissue sections and isolated myocytes) were determined by immunofluorescent localization. We demonstrate the following for the first time in the mammalian heart: (1) heterogeneous expression patterns of the 3 NOS and 3 SOD isoforms among different tissue and myocyte types; (2) colocalization of eNOS and ECSOD at both the tissue and myocyte levels; (3) a significant gradient of eNOS and ECSOD expression across the left ventricular (LV) wall, with both enzymes being highly expressed and colocalized in LV epicardial myocytes but markedly reduced in LV endocardial myocytes; and (4) specific subcellular localization patterns of eNOS and the 3 SOD isoforms. In particular, eNOS and ECSOD are demonstrated (electron and confocal microscopy) to be specifically localized to the sarcolemma of ventricular myocytes. Similar heterogeneous eNOS and ECSOD expression patterns were also obtained in human LV tissue sections, underscoring the general importance of these novel findings. Our data suggest a strong functional correlation between the activities of sarcolemmally localized myocyte eNOS and ECSOD in governing NO*/O(2-) interactions and suggest that NO-related modulatory effects on cardiac myocyte protein and/or ion channel function may be significantly more complex than is presently believed.     

Sca-1~+心肌干细胞的研究进展

干细胞抗原1(Sca-1)是干细胞的一种重要表面标记物,体内许多干细胞都表达Sca-1。同样,心肌干细胞也表达Sca-1。目前研究发现,Sca-1+心肌干细胞能够分化为心肌细胞,对心肌梗死后心室重构与心肌再生具有明显作用,能够促进心脏的修复。因此,Sca-1+心肌干细胞有可能使心肌梗死的临床治疗取得实质性的进展。现综述了Sca-1+心肌干细胞的来源、分化、作用等方面。     

Reduction of myocardial infarct size by inhibition of inducible nitric oxide synthase

The inducible nitric oxide synthase isoform (iNOS) is upregulated by cytokines and endotoxins in many types of cells, including cardiac myocytes. Nitric oxide (NO) induced by cytokines can be cytotoxic, and has been implicated in the pathophysiology of myocardial infarction, cardiomyopathy, and septic shock. To examine the role of iNOS in the ischemic myocardium, we studied: 1) the time course of expression of iNOS mRNA after myocardial infarction (MI) in male Sprague-Dawley rat hearts and expression of iNOS protein in the infarcted region; 2) whether hypoxia in vitro is a potential mediator of the induction of iNOS mRNA; and 3) whether inhibition of iNOS by two different selective inhibitors (aminoguanidine and S-methylisothiourea sulfate) in vivo influences infarct size. Myocardial infarction was induced by ligation of the left anterior descending coronary artery (LAD), and tissue was collected at selected times thereafter from both ligated and sham-operated rats. iNOS mRNA was induced in the infarcted region of the left ventricle for 7 days; iNOS protein was also detected in the infarcted area. We next tested whether hypoxia would induce iNOS in vitro. In cultured neonatal ventricular myocytes, iNOS mRNA was slightly induced by 6 to 24 h of hypoxia; however, iNOS protein was only detected when the cytokine interleukin-1β was present. To study whether iNOS activity contributed to myocardial damage (eg, infarct size), we administered the first dose of the NOS inhibitors 24 h before LAD occlusion and then a second dose after surgery. Inhibition of iNOS activity with aminoguanidine reduced infarct size by 20% but had no effect on infiltration by neutrophils, whereas the more selective inhibitor S-methylisothiourea sulfate reduced infarct size by 41%. These data suggest that NO derived from the iNOS isoform contributes to some of the myocardial injury following MI, possibly by causing myocardial cell death in areas bordering the ischemic region of the heart.     

Control of cardiac muscle cell function by an endogenous nitric oxide signaling system.

Nitric oxide (NO) synthesized from L-arginine is a ubiquitous intracellular chemical messenger and is involved in signal transduction in diverse mammalian cells, including vascular endothelium and neuronal tissues. The role of the NO-signaling pathway in the direct modulation of cardiac function is less well characterized. In this report, the effects of inhibitors of NO synthase (NOS) were examined in isolated neonatal and adult rat ventricular myocytes exposed to either muscarinic or adrenergic agonists. Carbachol (10 microM) caused a 91% inhibition of the spontaneous beating rate of cultured neonatal rat cardiac myocytes. N omega-monomethyl-L-arginine, an L-arginine analog that inhibits NOS, and methylene blue, an inhibitor of NO, blocked the negative chronotropic effect of carbachol but had no effect on the basal beating rate of these cells. The inhibition by N omega-monomethyl-L-arginine of the negative chronotropic effect of carbachol was reversed by adding excess L-arginine. The negative chronotropic effect of carbachol was also mimicked by analogs of cGMP, a second messenger implicated in mediating the action of NO in other cell types. Production of NO could be detected directly in carbachol-stimulated neonatal myocytes by using a reporter cell bioassay. The regulation of adrenergic responsiveness by the NO signaling system was also documented in studies of adult cardiac myocyte contractility. The NOS inhibitor N omega-nitro-L-arginine significantly increased the inotropic effect of the beta-adrenergic agonist isoproterenol on electrically stimulated adult rat ventricular myocytes, whereas this inhibitor had no effect on basal contractility. Inhibition of NO production by N omega-monomethyl-L-arginine in these cells, as measured by reporter cell bioassay, was also reversible with excess L-arginine. Thus, the physiologic response of isolated neonatal and adult ventricular myocytes to both muscarinic cholinergic and beta-adrenergic stimulation is mediated, at least in part, by products of an endogenous NOS.     

Cell transplantation and cardiac recovery:myocardial regeneration from endogenous cardiac progenitor cells

Following the bone marrow or cardiac derived progenitor cells transplantation,improved left ventricular(LV) function,decreased LV remodeling, and decreased fibrosis of non-infarcted LV regions,and in some cases,the reduction of infarct scar size have been reported to occur in animal mycardial infarction(MI) models.In clinical trials, stem cell transplantation has also been associated with significant,but modest improvements of LV functional parameters.These beneficial effects do occur although in many animal studies there is often very low long term engraftment or transdifferentiation of transplanted cells into myocytes and vascular cells.Importantly,paracrine signals generated by the implanted progenitor cells seem to play an important role in limiting or reversing myocardial damage as- sociated with acute MI.Paracrine signaling effects include increased myocardial vascularization and reduced apoptosis of native cardiomyocytes;these responses are most prominent in peri-myocardial infarction (MI) boarder zone(BZ) of the heart.Although much data supports the possibility that engrafted progenitor cells can mobilize endogenous cardiac progenitor cells(CPC) to the cardiac injury site and also stimulate them to propagate and transd-ifferentiate into cardiomyocytes and vascular cells, this concept remains controversial.We and others have reported evidences supporting the view that endogenous CPC can be stimulated to differentiate and partially replace cardiomyocytes destroyed during an MI.Data from our laboratory will be reviewed.     

Cardiomyocyte GTP Cyclohydrolase 1 and Tetrahydrobiopterin Increase NOS1 Activity and Accelerate Myocardial Relaxation

Rationale: Tetrahydrobiopterin (BH4) is an essential cofactor of nitric oxide synthases (NOS). Oral BH4 supplementation preserves cardiac function in animal models of cardiac disease; however, the mechanisms underlying these findings are not completely understood. Objective: To study the effect of myocardial transgenic overexpression of the rate-limiting enzyme in BH4 biosynthesis, GTP cyclohydrolase 1 (GCH1), on NOS activity, myocardial function, and Ca(2+) handling. Methods and Results: GCH1overexpression significantly increased the biopterins level in left ventricular (LV) myocytes but not in the nonmyocyte component of the LV myocardium or in plasma. The ratio between BH4 and its oxidized products was lower in mGCH1-Tg, indicating that a large proportion of the myocardial biopterin pool was oxidized; nevertheless, myocardial NOS1 activity was increased in mGCH1-Tg, and superoxide release was significantly reduced. Isolated hearts and field-stimulated LV myocytes (3 Hz, 35°C) overexpressing GCH1 showed a faster relaxation and a PKA-mediated increase in the PLB Ser(16) phosphorylated fraction and in the rate of decay of the [Ca(2+)](i) transient. RyR2 S-nitrosylation and diastolic Ca(2+) leak were larger in mGCH1-Tg and I(Ca) density was lower; nevertheless the amplitude of the [Ca(2+)](i) transient and contraction did not differ between genotypes, because of an increase in the SR fractional release of Ca(2+) in mGCH1-Tg myocytes. Xanthine oxidoreductase inhibition abolished the difference in superoxide production but did not affect myocardial function in either group. By contrast, NOS1 inhibition abolished the differences in I(Ca) density, Ser(16) PLB phosphorylation, [Ca(2+)](i) decay, and myocardial relaxation between genotypes. Conclusions: Myocardial GCH1 activity and intracellular BH4 are a limiting factor for constitutive NOS1 and SERCA2A activity in the healthy myocardium. Our findings suggest that GCH1 may be a valuable target for the treatment of LV diastolic dysfunction.     

17 Beta-estradiol stimulates expression of endothelial and inducible NO synthase in rat myocardium in-vitro and in-vivo

OBJECTIVES: NO production has been attributed to play a major role in cardiac diseases such as cardiac hypertrophy and cardiac remodeling after myocardial infarction which display significant gender-based differences. Therefore we assessed the effect of 17 beta-estradiol (E2) on estrogen receptor (ER) alpha and beta and endothelial and inducible NO synthase in neonatal and adult rat cardiomyocytes. METHODS: The presence of ER alpha and ER beta was demonstrated by immunofluorescence and western blot analysis as well as the expression pattern of inducible NO synthase (iNOS) and endothelial NOS (eNOS) in isolated cardiomyocytes from neonatal and adult rats. Furthermore, regulation of myocardial iNOS and eNOS expression by estrogen was evaluated in the myocardium from ovariectomized or sham-operated adult Wistar-Kyoto rats. RESULTS: Incubation with E2 led to translocalization of the ER into the nucleus and increased receptor protein expression. E2 stimulated expression of iNOS and eNOS in both neonatal and adult cardiac myocytes. Coincubation with the pure anti-estrogen ICI 182,780 inhibited upregulation of ER and NOS expression. In ovariectomized rats myocardial iNOS and eNOS protein levels were significantly lower compared to sham-operated female animals. CONCLUSION: Taken together, these results show that E2 stimulates the expression of iNOS/eNOS in neonatal and adult cardiomyocytes in-vivo and in-vitro. These novel findings provide a potential mechanism of how estrogen may modulate NOS expression and NO formation in the myocardium.     

Involvement of inducible nitric oxide synthase in the inflammatory process of myocardial infarction

Inducible nitric oxide synthase (iNOS), which catalyzes the reaction of -arginine to -citrulline and nitric oxide (NO), plays an important role in immune-mediated cardiac disorders. The present report summarizes and discusses findings on the induction of NOS in myocardial infarction of rabbits. iNOS was significantly increased in infarcted myocardium 48 h after coronary artery ligation. The effect persisted for 14 days and declined thereafter. Immunohistochemical localization revealed macrophages as a major source of iNOS expression; iNOS expression was also present in infarcted human myocardium. Increased iNOS activity appeared to be related to the induction of apoptosis in infiltrating macrophages and cardiomyocytes. Moreover, preferential inhibition of iNOS by S-methylisothiourea sulfate (SMT) resulted in significant improvement of left ventricular performance and increased regional myocardial blood flow. These findings suggest that selective inhibition of iNOS activity may provide a therapeutic strategy in cardiac disorders such as myocardial infarction.     

Ranolazine Treatment for Myocardial Infarction? Effects on the Development of Necrosis,Left Ventricular Function and Arrhythmias in Experimental Models

Ranolazine, an inhibitor of the late current of the cardiac action potential (late I_Na), is a well established clinical treatment for chronic angina. The late I_Na in cardiac myocytes also plays an important role in the pathophysiology of acute myocardial ischemia and reperfusion, and thus is a potential therapeutic target to ameliorate consequences of myocardial infarction. In experimental animal models, ranolazine has been shown to reduce myocardial infarct size, improve left ventricular function, decrease ischemia/reperfusion-induced arrhythmias and improve outcome in heart failure. Here we focus specifically on data from in vivo animal studies of myocardial ischemia and reperfusion.     

Imbalance between xanthine oxidase and nitric oxide synthase signaling pathways underlies mechanoenergetic uncoupling in the failing heart

Inhibition of xanthine oxidase (XO) in failing hearts improves cardiac efficiency by an unknown mechanism. We hypothesized that this energetic effect is due to reduced oxidative stress and critically depends on nitric oxide synthase (NOS) activity, reflecting a balance between generation of nitric oxide (NO) and reactive oxygen species. In dogs with pacing-induced heart failure (HF), ascorbate (1000 mg) mimicked the beneficial energetic effects of allopurinol, increasing both contractility and efficiency, suggesting an antioxidant mechanism. Allopurinol had no additive effect beyond that of ascorbate. Crosstalk between XO and NOS signaling was assessed. NOS inhibition with N(G)-monomethyl-L-arginine (L-NMMA; 20 mg/kg) had no effect on basal contractility or efficiency in HF, but prevented the +26.2+/-3.5% and +66.5+/-17% enhancements of contractility and efficiency, respectively, observed with allopurinol alone. Similarly, improvements in contractility and energetics due to ascorbate were also inhibited by L-NMMA. Because of the observed NOS-XO crosstalk, we predicted that in normal hearts NOS inhibition would uncover a depression of energetics caused by XO activity. In normal conscious dogs, L-NMMA increased myocardial oxygen consumption (MVO2) while lowering left ventricular external work, reducing efficiency by 31.1+/-3.8% (P<0.005). Lowered efficiency was reversed by XO inhibition (allopurinol, 200 mg) or by ascorbate without affecting cardiac load or systemic hemodynamics. Single-cell immunofluorescence detected XO protein in cardiac myocytes that was enhanced in HF, consistent with autocrine signaling. These data show that both NOS and XO signaling systems participate in the regulation of myocardial mechanical efficiency and that upregulation of XO relative to NOS contributes to mechanoenergetic uncoupling in heart failure.     

Compensatory hypertrophy induced by ventricular cardiomyocyte-specific COX-2 expression in mice

Cyclooxygenase-2 (COX-2) is an important mediator of inflammation in stress and disease states. Recent attention has focused on the role of COX-2 in human heart failure and diseases owing to the finding that highly specific COX-2 inhibitors (i.e., Vioxx) increased the risk of myocardial infarction and stroke in chronic users. However, the specific impact of COX-2 expression in the intact heart remains to be determined. We report here the development of a transgenic mouse model, using a loxP-Cre approach, which displays robust COX-2 overexpression and subsequent prostaglandin synthesis specifically in ventricular myocytes. Histological, functional, and molecular analyses showed that ventricular myocyte specific COX-2 overexpression led to cardiac hypertrophy and fetal gene marker activation, but with preserved cardiac function. Therefore, specific induction of COX-2 and prostaglandin in vivo is sufficient to induce compensated hypertrophy and molecular remodeling.     

Attenuation of myocardial ischemia/reperfusion injury in mice with myocyte-specific overexpression of endothelial nitric oxide synthase

OBJECTIVE: The role of nitric oxide (NO) in myocardial ischemia/reperfusion injury remains controversial as both NO donors and NO synthase (NOS) inhibitors have shown to be protective. We generated transgenic (TG) mice that overexpress endothelial NOS (eNOS) exclusively in cardiac myocytes to determine the effects of high cardiac NO levels on ischemia/reperfusion injury and cellular Ca(2+) homeostasis. Wild-type (WT) mice served as controls. METHODS: Hearts were perfused in vitro and subjected to 20 min of total no-flow ischemia and 30 min of reperfusion (n=5 per group). Left ventricular function, cGMP levels and intracellular Ca(2+) transients (Ca(2+)(i)) were determined. RESULTS: Left ventricular pressure was reduced (maximum, -33%) and basal cardiac cGMP was increased (twofold) in TG hearts, and the changes were reversed by NOS blockade with N(G)-nitro-L-arginine methyl ester (L-NAME). Relative to baseline, recovery of reperfusion contractile function was significantly better in hearts from TG (98%) than WT (51%) mice, and L-NAME abolished this effect. Heart rate and coronary perfusion pressure were not different between groups. Systolic and diastolic Ca(2+)(i) concentrations were similar in WT and TG hearts, but Ca(2+)(i) overload during early reperfusion tended to be less in TG hearts. Kinetic analysis of pressure curves and Ca(2+)(i) transients revealed a faster left ventricular diastolic relaxation and abbreviated aequorin light signals in TG hearts at baseline and during reperfusion. CONCLUSIONS: High levels of NO/cGMP strongly protect against ischemia/reperfusion injury, the protection is largely independent of changes in Ca(2+)(i) modulation, but relates to reduced preischemic performance. Myocyte-specific NO augmentation may aid in studies of the (patho)physiological roles of cardiac-derived NO.     

Conditional neuronal nitric oxide synthase overexpression impairs myocardial contractility

The role of the neuronal NO synthase (nNOS or NOS1) enzyme in the control of cardiac function still remains unclear. Results from nNOS(-/-) mice or from pharmacological inhibition of nNOS are contradictory and do not pay tribute to the fact that probably spatial confinement of the nNOS enzyme is of major importance. We hypothesize that the close proximity of nNOS and certain effector molecules like L-type Ca(2+)-channels has an impact on myocardial contractility. To test this, we generated a new transgenic mouse model allowing conditional, myocardial specific nNOS overexpression. Western blot analysis of transgenic nNOS overexpression showed a 6-fold increase in nNOS protein expression compared with noninduced littermates (n=12; P<0.01). Measuring of total NOS activity by conversion of [(3)H]-l-arginine to [(3)H]-l-citrulline showed a 30% increase in nNOS overexpressing mice (n=18; P<0.05). After a 2 week induction, nNOS overexpression mice showed reduced myocardial contractility. In vivo examinations of the nNOS overexpressing mice revealed a 17+/-3% decrease of +dp/dt(max) compared with noninduced mice (P<0.05). Likewise, ejection fraction was reduced significantly (42% versus 65%; n=15; P<0.05). Interestingly, coimmunoprecipitation experiments indicated interaction of nNOS with SR Ca(2+)ATPase and additionally with L-type Ca(2+)- channels in nNOS overexpressing animals. Accordingly, in adult isolated cardiac myocytes, I(Ca,L) density was significantly decreased in the nNOS overexpressing cells. Intracellular Ca(2+)-transients and fractional shortening in cardiomyocytes were also clearly impaired in nNOS overexpressing mice versus noninduced littermates. In conclusion, conditional myocardial specific overexpression of nNOS in a transgenic animal model reduced myocardial contractility. We suggest that nNOS might suppress the function of L-type Ca(2+)-channels and in turn reduces Ca(2+)-transients which accounts for the negative inotropic effect.     

Reduced phospholamban phosphorylation is associated with impaired relaxation in left ventricular myocytes from neuronal NO synthase-deficient mice

Stimulation of nitric oxide (NO) release from the coronary endothelium facilitates myocardial relaxation via a cGMP-dependent reduction in myofilament Ca2+ sensitivity. Recent evidence suggests that NO released by a neuronal NO synthase (nNOS) in the myocardium can also hasten left ventricular relaxation; however, the mechanism underlying these findings is uncertain. Here we show that both relaxation (TR50) and the rate of [Ca2+]i transient decay (tau) are significantly prolonged in field-stimulated or voltage-clamped left ventricular myocytes from nNOS-/- mice and in wild-type myocytes (nNOS+/+) after acute nNOS inhibition. Disabling the sarcoplasmic reticulum abolished the differences in TR50 and tau, suggesting that impaired sarcoplasmic reticulum Ca2+ reuptake may account for the slower relaxation in nNOS-/- mice. In line with these findings, disruption of nNOS (but not of endothelial NOS) decreased phospholamban phosphorylation (P-Ser16 PLN), whereas nNOS inhibition had no effect on TR50 or tau in PLN-/- myocytes. Inhibition of cGMP signaling had no effect on relaxation in either group whereas protein kinase A inhibition abolished the difference in relaxation and PLN phosphorylation by decreasing P-Ser16 PLN and prolonging TR50 in nNOS+/+ myocytes. Conversely, inhibition of type 1 or 2A protein phosphatases shortened TR50 and increased P-Ser16 PLN in nNOS-/- but not in nNOS+/+ myocytes, in agreement with data showing increased protein phosphatase activity in nNOS-/- hearts. Taken together, our findings identify a novel mechanism by which myocardial nNOS promotes left ventricular relaxation by regulating the protein kinase A-mediated phosphorylation of PLN and the rate of sarcoplasmic reticulum Ca2+ reuptake via a cGMP-independent effect on protein phosphatase activity.