Neuronal nitric oxide synthase modulation of intracellular Ca2+ handling overrides fatty acid potentiation of cardiac inotropy in hypertensive rats.

Cardiac neuronal nitric oxide synthase (nNOS) is an important molecule that regulates intracellular Ca2+ homeostasis and contractility of healthy and diseased hearts. Here, we examined the effects of nNOS on fatty acid (FA) regulation of left ventricular (LV) myocyte contraction in sham and angiotensin II (Ang II)-induced hypertensive (HTN) rats. Our results showed that palmitic acid (PA, 100 µM) increased the amplitudes of sarcomere shortening and intracellular ATP in sham but not in HTN despite oxygen consumption rate (OCR) was increased by PA in both groups. Carnitine palmitoyltransferase-1 inhibitor, etomoxir (ETO), reduced OCR and ATP with PA in sham and HTN but prevented PA potentiation of sarcomere shortening only in sham. PA increased nNOS-derived NO only in HTN. Inhibition of nNOS with S-methyl-L-thiocitrulline (SMTC) prevented PA-induced OCR and restored PA potentiation of myocyte contraction in HTN. Mechanistically, PA increased intracellular Ca2+ transient ([Ca2+]i) without changing Ca2+ influx via L-type Ca2+ channel (I-LTCC) and reduced myofilament Ca2+ sensitivity in sham. nNOS inhibition increased [Ca2+]i, I-LTCC and reduced myofilament Ca2+ sensitivity prior to PA supplementation; as such, normalized PA increment of [Ca2+]i. In HTN, PA reduced I-LTCC without affecting [Ca2+]i or myofilament Ca2+ sensitivity. However, PA increased I-LTCC, [Ca2+]i and reduced myofilament Ca2+ sensitivity following nNOS inhibition. Myocardial FA oxidation (18F-fluoro-6-thia-heptadecanoic acid, 18F-FTHA) was comparable between groups, but nNOS inhibition increased it only in HTN. Collectively, PA increases myocyte contraction through stimulating [Ca2+]i and mitochondrial activity in healthy hearts. PA-dependent cardiac inotropy was limited by nNOS in HTN, predominantly due to its modulatory effect on [Ca2+]i handling.

ROS and endothelial nitric oxide synthase (eNOS)-dependent trafficking of angiotensin II type 2 receptor begets neuronal NOS in cardiac myocytes.

Angiotensin II (Ang II), a potent precursor of hypertrophy and heart failure, upregulates neuronal nitric oxide synthase (nNOS or NOS1) in the myocardium. Here, we investigate the involvement of type 1 and 2 angiotensin receptors (AT1R and AT2R) and molecular mechanisms mediating Ang II-upregulation of nNOS. Our results showed that pre-treatment of left ventricular (LV) myocytes with antagonists of AT1R or AT2R (losartan, PD123319) and ROS scavengers (apocynin, tiron or PEG-catalase) blocked Ang II-upregulation of nNOS. Surface biotinylation or immunocytochemistry experiments demonstrated that AT1R expression in plasma membrane was progressively decreased (internalization), whereas AT2R was increased (membrane trafficking) by Ang II. Inhibition of AT1R or ROS scavengers prevented Ang II-induced translocation of AT2R to plasma membrane, suggesting an alignment of AT1R-ROS-AT2R. Furthermore, Ang II increased eNOS-Ser(1177) but decreased eNOS-Thr(495), indicating concomitant activation of eNOS. Intriguingly, ROS scavengers but not AT2R antagonist prevented Ang II-activation of eNOS. NOS inhibitor (L-NG-Nitroarginine Methyl Ester, L-NAME) or eNOS gene deletion (eNOS(-/-)) abolished Ang II-induced membrane trafficking of AT2R, nNOS protein expression and activity. Mechanistically, S-nitrosation of AT2R was increased by sodium nitroprusside (SNP), a NO donor. Site-specific mutagenesis analysis reveals that C-terminal cysteine 349 in AT2R is essential in AT2R translocation to plasma membrane. Taken together, we demonstrate, for the first time, that Ang II upregulates nNOS protein expression and activity via AT1R/ROS/eNOS-dependent S-nitrosation and membrane translocation of AT2R. Our results suggest a novel crosstalk between AT1R and AT2R in regulating nNOS via eNOS in the myocardium under pathogenic stimuli.

Molecular mechanisms of neuronal nitric oxide synthase in cardiac function and pathophysiology.

Neuronal nitric oxide synthase (nNOS or NOS1) is the major endogenous source of myocardial nitric oxide (NO), which facilitates cardiac relaxation and modulates contraction. In the healthy heart it regulates intracellular Ca(2+), signalling pathways and oxidative homeostasis and is upregulated from early phases upon pathogenic insult. nNOS plays pivotal roles in protecting the myocardium from increased oxidative stress, systolic/diastolic dysfunction, adverse structural remodelling and arrhythmias in the failing heart. Here, we show that the downstream target proteins of nNOS and underlying post-transcriptional modifications are shifted during disease progression from Ca(2+)-handling proteins [e.g. PKA-dependent phospholamban phosphorylation (PLN-Ser(16))] in the healthy heart to cGMP/PKG-dependent PLN-Ser(16) with acute angiotensin II (Ang II) treatment. In early hypertension, nNOS-derived NO is involved in increases of cGMP/PKG-dependent troponin I (TnI-Ser(23/24)) and cardiac myosin binding protein C (cMBP-C-Ser(273)). However, nNOS-derived NO is shown to increase S-nitrosylation of various Ca(2+)-handling proteins in failing myocardium. The spatial compartmentation of nNOS and its translocation for diverse binding partners in the diseased heart or various nNOS splicing variants and regulation in response to pathological stress may be responsible for varied underlying mechanisms and functions. In this review, we endeavour to outline recent advances in knowledge of the molecular mechanisms mediating the functions of nNOS in the myocardium in both normal and diseased hearts. Insights into nNOS gene regulation in various tissues are discussed. Overall, nNOS is an important cardiac protector in the diseased heart. The dynamic localization and various mediating mechanisms of nNOS ensure that it is able to regulate functions effectively in the heart under stress.

Low K⁺ current in arterial myocytes with impaired K⁺-vasodilation and its recovery by exercise in hypertensive rats.

K(+) channels determine the plasma membrane potential of vascular myocytes, influencing arterial tone. In many types of arteries, a moderate increase in [K(+)]e induces vasorelaxation by augmenting the inwardly rectifying K(+) channel current (I Kir). K(+)-vasodilation matches regional tissue activity and O2 supply. In chronic hypertension (HT), small arteries and arterioles undergo various changes; however, ion channel remodeling is poorly understood. Here, we investigated whether K(+) channels and K(+)-induced vasodilation are affected in deep femoral (DFA) and cerebral artery (CA) myocytes of angiotensin II-induced hypertensive rats (Ang-HT). Additionally, we tested whether regular exercise training (ET) restores HT-associated changes in K(+) channel activity. In Ang-HT, both the voltage-gated K(+) channel current (I Kv) and I Kir were decreased in DFA and CA myocytes, and were effectively restored and further increased by combined ET for 2 weeks (HT-ET). Consistently, K(+)-vasodilation of the DFA was impaired in Ang-HT, and recovered in HT-ET. Interestingly, ET did not reverse the decreased K(+)-vasodilation of CA. CA myocytes from the Ang-HT and HT-ET groups demonstrated, apart from K(+) channel changes, an increase in nonselective cationic current (I NSC). In contrast, DFA myocytes exhibited decreased I NSC in both the Ang-HT and HT-ET groups. Taken together, the decreased K(+) conductance in Ang-HT rats and its recovery by ET suggest increased peripheral arterial resistance in HT and the anti-hypertensive effects of ET, respectively. In addition, the common upregulation of I NSC in the CA in the Ang-HT and HT-ET groups might imply a protective adaptation preventing excessive cerebral blood flow under HT and strenuous exercise.

Myofilament Ca2+ desensitization mediates positive lusitropic effect of neuronal nitric oxide synthase in left ventricular myocytes from murine hypertensive heart.

Neuronal nitric oxide synthase (NOS1 or nNOS) exerts negative inotropic and positive lusitropic effects through Ca(2+) handling processes in cardiac myocytes from healthy hearts. However, underlying mechanisms of NOS1 in diseased hearts remain unclear. The present study aims to investigate this question in angiotensin II (Ang II)-induced hypertensive rat hearts (HP). Our results showed that the systolic function of left ventricle (LV) was reduced and diastolic function was unaltered (echocardiographic assessment) in HP compared to those in shams. In isolated LV myocytes, contraction was unchanged but peak [Ca(2+)]i transient was increased in HP. Concomitantly, relaxation and time constant of [Ca(2+)]i decay (tau) were faster and the phosphorylated fraction of phospholamban (PLN-Ser(16)/PLN) was greater. NOS1 protein expression and activity were increased in LV myocyte homogenates from HP. Surprisingly, inhibition of NOS1 did not affect contraction but reduced peak [Ca(2+)]i transient; prevented faster relaxation without affecting the tau of [Ca(2+)]i transient or PLN-Ser(16)/PLN in HP, suggesting myofilament Ca(2+) desensitization by NOS1. Indeed, relaxation phase of the sarcomere length-[Ca(2+)]i relationship of LV myocytes shifted to the right and increased [Ca(2+)]i for 50% of sarcomere shortening (EC50) in HP. Phosphorylations of cardiac myosin binding protein-C (cMyBP-C(282) and cMyBP-C(273)) were increased and cardiac troponin I (cTnI(23/24)) was reduced in HP. Importantly, NOS1 or PKG inhibition reduced cMyBP-C(273) and cTnI(23/24) and reversed myofilament Ca(2+) sensitivity. These results reveal that NOS1 is up-regulated in LV myocytes from HP and exerts positive lusitropic effect by modulating myofilament Ca(2+) sensitivity through phosphorylation of key regulators in sarcomere.

Neuronal nitric oxide synthase is up-regulated by angiotensin II and attenuates NADPH oxidase activity and facilitates relaxation in murine left ventricular myocytes.

Angiotensin II (Ang II) is critical in myocardial pathogenesis, mostly via stimulating NADPH oxidase. Neuronal nitric oxide synthase (nNOS) has recently been shown to play important roles in modulating myocardial oxidative stress and contractility. Here, we examine whether nNOS is regulated by Ang II and affects NADPH oxidase production of intracellular reactive oxygen species (ROS(i)) and contractile function in left ventricular (LV) myocytes. Our results showed that Ang II induced biphasic effects on ROS(i) and LV myocyte relaxation (TR(50)) without affecting the amplitude of sarcomere shortening and L-type Ca(2+) current density: TR(50) was prolonged at 30 min but was shortened after 3h (or after Ang II treatment in vivo). Correspondingly, ROS(i) was increased, followed by a reduction to control level. Quantitative RT-PCR and immunoblotting experiments showed that Ang II (3h) increased the mRNA and protein expression of nNOS and increased NO production (nitrite assay) in LV myocyte homogenates, suggesting that nNOS activity may be enhanced and involved in mediating the effects of Ang II. Indeed, n(omega)-nitro-l-arginine methyl ester (l-NAME) or a selective nNOS inhibitor, S-methyl-l-thiocitrulline (SMTC) increased NADPH oxidase production of superoxide/ROS(i) and abolished faster myocyte relaxation induced by Ang II. The positive lusitropic effect of Ang II was not mediated by PKA-, CaMKII-dependent signaling or peroxynitrite. Conversely, inhibition of cGMP/PKG pathway abolished the Ang II-induced faster relaxation by reducing phospholamban (PLN) Ser(16) phosphorylation. Taken together, these results clearly demonstrate that myocardial nNOS is up-regulated by Ang II and functions as an early adaptive mechanism to attenuate NADPH oxidase activity and facilitate myocardial relaxation.

S-nitrosylation of transglutaminase 2 impairs fatty acid-stimulated contraction in hypertensive cardiomyocytes.

The myocardium in hypertensive heart exhibits decreased fatty acid utilization and contractile dysfunction, leading to cardiac failure. However, the causal relationship between metabolic remodeling and cardiomyocyte contractility remains unestablished. Transglutaminase 2 (TG2) has been known to promote ATP production through the regulation of mitochondrial function. In this study, we investigated the involvement of TG2 in cardiomyocyte contraction under fatty acid supplementation. Using TG2 inhibitor and TG2-deficient mice, we demonstrated that fatty acid supplementation activated TG2 and increased ATP level and contractility of cardiac myocyte from the normal heart. By contrast, in cardiac myocytes from angiotensin-II-treated rats and mice, the effects of fatty acid supplementation on TG2 activity, ATP level, and myocyte contraction were abolished. We found that TG2 was inhibited by S-nitrosylation and its level increased in hypertensive myocytes. Treatment with inhibitor for neuronal NOS restored fatty acid-induced increase of TG2 activity and myocyte contraction. Moreover, intracellular Ca2+ levels were increased by fatty acid supplementation in both normal and hypertensive myocytes, showing that S-nitrosylation of TG2 but not alteration of intracellular Ca2+ levels is responsible for contractile dysfunction. These results indicate that TG2 plays a critical role in the regulation of myocyte contractility by promoting fatty acid metabolism and provide a novel target for preventing contractile dysfunction in heart with high workload.

Synthesis of Novel 8-alkylamino-5, 6-dihydro-4H-benzo[f] [1,2,4]triazolo [4,3-a]azepines as Anticonvulsant Agents.

A series of new 8-alkylamino-5, 6-dihydro-4H-benzo[f][1,2,4]triazolo [4,3-a]azepine derivatives were synthesized and screened for their anticonvulsant activities by the maximal electroshock (MES) test, subcutaneous pentylenetetrazol (scPTZ) test, and their neurotoxicity was evaluated by the rotarod neurotoxicity test. The results of these tests showed that 8-heptylamino-5,6- dihydro-4H-benzo[f][1,2,4] triazolo[4,3-a]azepine (7g) was the most promising compound, with median effective dose (ED50) of 19.0 mg/kg, and protective index (PI) value of 14.8 in the MES test, which is much higher than the PI value of the prototype antiepileptic drug carbamazepine (PI = 8.1), phenytoin (PI = 6.9), phenobarbital (PI = 3.2), and sodium valproate (PI = 1.6). The possible structure-activity relationship was discussed.

Modulation of L-type Ca²âº channel activity by neuronal nitric oxide synthase and myofilament Ca²âº sensitivity in cardiac myocytes from hypertensive rat.

Neuronal nitric oxide synthase (nNOS) is important in cardiac protection in diseased heart. Recently, we have reported that nNOS is associated with myofilament Ca(2+) desensitization in cardiac myocytes from hypertensive rats. So far, the effect of myofilament Ca(2+) desensitization or nNOS on L-type Ca(2+) channel activity (I(Ca)) in cardiac myocyte is unclear. Here, we examined nNOS regulation of I(Ca) in left ventricular (LV) myocytes from sham and angiotensin II (Ang II)-induced hypertensive rats. Our results showed that basal I(Ca) was not different between sham and hypertension (from -60 to +40 mV, 0.1 Hz). S-methyl-L-thiocitrulline (SMTC), a selective nNOS inhibitor, increased peak I(Ca) similarly in both groups. However, chelation of intracellular Ca(2+) [Ca(2+)]i with BAPTA increased I(Ca) and abolished SMTC-augmentation of I(Ca) only in hypertension. Myofilament Ca(2+) desensitization with butanedione monoxime (BDM), a myosin ATPase inhibitor, decreased I(Ca) in both groups but to a greater extent in hypertension. Intracellular BAPTA or nNOS inhibition reinstated I(Ca) in the presence of BDM to the basal level, suggesting Ca(2+)-dependent inactivation of I(Ca) by nNOS and greater vulnerability in hypertension. Increasing stimulation frequencies (2, 4 and 8 Hz) attenuated myofilament Ca(2+) sensitivity in sham and reduced peak ICa in both groups. Nevertheless, SMTC or BAPTA exerted no effect on I(Ca) at high frequencies in either group. These results suggest that nNOS attenuates I(Ca) via Ca(2+)-dependent mechanism and the vulnerability is greater in hypertension subject to myofilament Ca(2+) desensitization. nNOS or [Ca(2+)]i does not affect I(Ca) at high stimulation frequencies. The results were recapitulated with computer simulation.

Exercise training increases inwardly rectifying K(+) current and augments K(+)-mediated vasodilatation in deep femoral artery of rats.

AIMS: A moderate increase in extracellular [K(+)] ([K(+)](e)) induces relaxation of small arteries by activating inwardly rectifying K(+) current (I(Kir)). The K(+)-induced vasodilatation is an important mechanism for exercise-induced hyperaemia in skeletal muscle. We investigated whether I(Kir) and K(+)-induced vasodilatation are enhanced in deep femoral arteries (DFAs) from exercise-trained rats (ET rats; treadmill running for 20 min at 20 m/min, 3 days/week for 2 weeks). The effects of exercise training on K(+)-induced vasodilatation and I(Kir) were also investigated in cerebral (CA) and mesenteric arteries. METHODS AND RESULTS: The K(+)-induced vasodilatation of DFAs and the density of I(Kir) and voltage-gated K(+) current (I(Kv)) were increased in DFA myocytes of ET rats. The myogenic tone of the DFA was unchanged by exercise. Although similar functional up-regulations of I(Kir) and I(Kv) were observed in CA myocytes, the K(+)-induced vasodilatation was not increased in the CA of ET rats. Interestingly, concomitant to the increases in I(Kir) and I(Kv), background Na(+) conductance was also increased in the CA myocytes. However, such an effect was not observed in DFA myocytes from ET rats. Neither I(Kir) nor K(+)-induced vasodilatation was observed in mesenteric arteries of ET rats. CONCLUSION: The present study provides evidence that regular exercise up-regulates I(Kir) in DFA and CA myocytes. Although the increase in I(Kir) was observed in two types of arteries, augmentation of K(+)-induced relaxation was observed only in the DFA of ET rats, possibly due to the increased Na(+) conductance in CA myocytes. The increases in I(Kir) and K(+)-induced vasodilatation of the arteries of skeletal muscle suggest novel mechanisms of improved exercise hyperaemia with physical training.