scholarly journals Neuronal nitric oxide synthase signaling within cardiac myocytes targets phospholamban

2008 ◽  
Vol 294 (6) ◽  
pp. C1566-C1575 ◽  
Author(s):  
Honglan Wang ◽  
Mark J. Kohr ◽  
Christopher J. Traynham ◽  
Debra G. Wheeler ◽  
Paul M. L. Janssen ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Nitric Oxide Synthase ◽  
Neuronal Nitric Oxide Synthase ◽  
Force Frequency ◽  
Adrenergic Stimulation ◽  
Cell Shortening ◽  
Mediated Effects ◽  
Intracellular Ca ◽  
Force Frequency Response ◽  
Oxide Synthase

Studies have shown that neuronal nitric oxide synthase (nNOS, NOS1) knockout mice (NOS1−/−) have increased or decreased contractility, but consistently have found a slowed rate of intracellular Ca2+ ([Ca2+]i) decline and relengthening. Contraction and [Ca2+]i decline are determined by many factors, one of which is phospholamban (PLB). The purpose of this study is to determine the involvement of PLB in the NOS1-mediated effects. Force-frequency experiments were performed in trabeculae isolated from NOS1−/− and wild-type (WT) mice. We also simultaneously measured Ca2+ transients (Fluo-4) and cell shortening (edge detection) in myocytes isolated from WT, NOS1−/−, and PLB−/− mice. NOS1−/− trabeculae had a blunted force-frequency response and prolonged relaxation. We observed similar effects in myocytes with NOS1 knockout or specific NOS1 inhibition with S-methyl-l-thiocitrulline (SMLT) in WT myocytes (i.e., decreased Ca2+ transient and cell shortening amplitudes and prolonged decline of [Ca2+]i). Alternatively, NOS1 inhibition with SMLT in PLB−/− myocytes had no effect. Acute inhibition of NOS1 with SMLT in WT myocytes also decreased basal PLB serine16 phosphorylation. Furthermore, there was a decreased SR Ca2+ load with NOS1 knockout or inhibition, which is consistent with the negative contractile effects. Perfusion with FeTPPS (peroxynitrite decomposition catalyst) mimicked the effects of NOS1 knockout or inhibition. β-Adrenergic stimulation restored the slowed [Ca2+]i decline in NOS1−/− myocytes, but a blunted contraction remained, suggesting additional protein target(s). In summary, NOS1 inhibition or knockout leads to decreased contraction and slowed [Ca2+]i decline, and this effect is absent in PLB−/− myocytes. Thus NOS1 signaling modulates PLB serine16 phosphorylation, in part, via peroxynitrite.

Abstract 1333: Modulation Of Cardiac Beta-adrenergic Responsiveness By The Neuronal Nitric Oxide Synthase/Sarcolemmal Calcium Pump Complex

Circulation ◽  
2007 ◽  
Vol 116 (suppl_16) ◽  
Author(s):  
Tamer M Mohamed ◽  
Delvac Oceandy ◽  
Nasser Alatwi ◽  
Florence Baudoin ◽  
Elizabeth J Cartwright ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Nitric Oxide Synthase ◽  
Neuronal Nitric Oxide Synthase ◽  
Neonatal Rat ◽  
No Production ◽  
Adrenergic Stimulation ◽  
Rat Cardiomyocytes ◽  
Adrenergic Response ◽  
Oxide Synthase

The pivotal role of neuronal nitric oxide synthase (nNOS) in regulating cardiac function has only recently been unveiled. Notably, others have shown that responsiveness to β-adrenergic stimulation is dependent on nNOS activity. In a cellular model, we showed that the Ca 2+ /calmodulin-dependent nNOS activity is reduced by overexpression of isoform 4b of the plasma membrane Ca 2+ /Calmodulin-dependent Ca 2+ -pump (PMCA4b), which binds to nNOS. We demonstrated that PMCA4b overexpression in the heart reduced β-adrenergic responsiveness in vivo via an nNOS dependent mechanism (Oceandy et al, Circulation 2007). Here we investigated the cellular mechanisms of the regulation of the β-adrenergic response by PMCA4b. We used an adenoviral system to overexpress PMCA4b (PMCA4b cells) or LacZ (control, C) in neonatal rat cardiomyocytes. PMCA4b cells showed an 18±5% and 24±5% reduction in nitric oxide (DAF-FM fluorescence) and cGMP levels, respectively (n=6, p<0.05 each) compared to C demonstrating the regulation of NO production by the PMCA4b in this system. Since nNOS has been shown to regulate phospholamban (PLB) phosphorylation, we examined phosphorylation of PLB at Ser16. PMCA4b cells showed a significant increase in Ser16-PLB at baseline (66±17%, p<0.05) compared to C. As a result of increased baseline Ser16-PLB in PMCA4b cells, β-adrenergic stimulation of PMCA4b cells using 2μM isoproter-enol (IP) showed reduced relative induction in Ser16-PLB (23±10% vs. 78±19% in C; n=5, p<0.05). Further analysis in adult cardiomyocytes isolated from our PMCA4b transgenic mice (PMCA4b TG) demonstrated that PMCA4b TG showed 3-fold higher Ser16-PLB phosphorylation at baseline compared to wild type (WT) myocytes and the relative response following β-adrenergic stimulation was significantly reduced (1.2±0.2 fold induction after IP treatment in PMCA4b TG, vs. 3.1±0.7 in WT, n=5, p<0.05). Thus, PMCA4b regulates NO production from nNOS, which in turn modulates cGMP levels and PLB phosphorylation. These findings provide mechanistic insight into the regulation of the β-adrenergic response in the heart by PMCA4b and place this Ca 2+ -pump upstream of the recently described pathway linking nNOS and Ser16-PLB phosphorylation and downstream of the β-adrenergic receptor(s).

Abstract 852: Conditional Overexpression Of Neuronal Nitric Oxide Synthase Is Cardioprotective In Ischemia-reperfusion

Circulation ◽  
2007 ◽  
Vol 116 (suppl_16) ◽  
Author(s):  
Natalie Burkard ◽  
Adam Rokita ◽  
Susann Kaufmann ◽  
Matthias Hallhuber ◽  
Claudia Gebhardt ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Nitric Oxide Synthase ◽  
Myocardial Contractility ◽  
Ischemia Reperfusion ◽  
Neuronal Nitric Oxide Synthase ◽  
Reduced Ejection Fraction ◽  
Mild Reduction ◽  
Intracellular Ca ◽  
Oxide Synthase

Introduction: We previously demonstrated that conditional overexpression of the neuronal nitric oxide synthase (nNOS, NOS1) inhibited L-type Ca 2+ -channels. We now hypothesize that nNOS overexpression has an impact on myocardial contractility and acts cardioprotective after ischemia-reperfusion. Methods and results: We assessed cardiac function in the newly established transgenic mouse model with conditional, myocardial nNOS overexpression. NOS-activity (22 ± 1.5 vs. 29 ± 1μM /sec, n=18, p<0.05) was significantly enhanced after nNOS overexpression. Co-immunoprecipitation experiments indicated interaction of nNOS with SR Ca 2+ ATPase and additionally with L-type Ca 2+ -channels in nNOS overexpressing animals. I ca,L (reduction of 40±6 rel. %, n=12, p<0.05) as well as intracellular Ca 2+ -transients and fractional shortening in cardiomyocytes were clearly impaired in nNOS overexpressing mice (3.0 ± 0.4F/F 0 vs. 2.2 ± 0.2F/F 0 , n=13, p<0.005 and 7.7 ± 1.3% vs. 3.8 ± 0.5%, n=13, p<0.05). In vivo examinations of the nNOS overexpressing mice showed a decrease of +dp/dt max (reduction for 52 ± 17%, n=12, p<0.05) as well as a reduced ejection fraction (43±5% vs. 63±9%, n=12, p<0.05). Ischemia-reperfusion experiments showed a cardio-protective effect of nNOS overexpression (30 min post-ischemia, LVDP 20±6 in non-induced animals vs. 60±11 mmHg in nNOS overexpressing animals, n=6, p<0.05). Discussion: In summary, we demonstrated that under basline conditions, conditional transgenic overexpression of nNOS resulted in a mild reduction of myocardial contractility, mainly due to inhibition of the L-type Ca 2+ -channel. In contrast, under pathophysiological conditions (i.e. ischemia-reperfusion) nNOS overexpression acts cardioprotective. These effects might be caused by a reduction of myocardial Ca 2+ -overload after reperfusion.

Abstract P076: Essential Role of Neuronal Nitric Oxide Synthase in Ca 2+ /Calmodulin-Dependent Protein Kinase II Activation in Cardiac Myocytes During β-Adrenergic Stimulation

2011 ◽  
Vol 109 (suppl_1) ◽  
Author(s):  
Lifei Tang ◽  
Steve Roof ◽  
Mark Ziolo
Keyword(s):  
Nitric Oxide ◽  
Protein Kinase ◽  
Nitric Oxide Synthase ◽  
Neuronal Nitric Oxide Synthase ◽  
Dependent Protein Kinase ◽  
Positive Inotropy ◽  
Cell Shortening ◽  
Protein Kinase Ii ◽  
Dependent Protein ◽  
Oxide Synthase

RATIONALE: Stimulation of the beta-adrenergic (beta-AR) pathway leads to positive inotropy, and is the major regulator of heart function. In addition to the traditional PKA pathway, activation of Ca 2+ /calmodulin-dependent protein kinase II (CaMKII) and neuronal nitric oxide synthase (NOS1) signaling also play important roles in the positive inotropy by modulating ryanodine receptor (RyR) activity. OBJECTIVE: The upstream activators of CaMKII during beta-AR stimulation are not well defined. The purpose of this study is to investigate if there is any cross-talk between the CaMKII and NOS1 signaling pathways. METHODS AND RESULTS: Myocytes were isolated from wildtype (WT, C57Bl/6) and NOS1 −/− mice. Ca 2+ transients (Fluo-4) and cell shortening (edge detection) were simultaneously measured. RyR activity was measured using the SR Ca 2+ leak/load relationship. CaMKII was acutely inhibited by KN93. In WT myocytes, KN93 decreased beta-AR stimulated contraction (Ca2+ transients (Fluo-4) and cell shortening). In NOS1 −/− myocytes, beta-AR stimulated contraction was blunted compared to WT, and KN93 had no further effect on contraction. Furthermore, beta-AR stimulated RyR activity was blunted in NOS1 −/− compared to WT myocytes. As with contraction, KN93 decreased beta-AR stimulated RyR activity in WT myocytes, but had no effect in NOS1 −/− myocytes. CONCLUSION: These data suggest that NOS1 is required for CaMKII-mediated RyR activation which contributes to positive inotropy during beta-AR stimulation. Further study of this pathway is warranted since CAMKII expression and activity are increased in cardiac hypertrophy and heart failure. A better understanding of the NOS1/CaMKII pathway during beta-AR stimulation has beneficial therapeutic potential for heart diseases.

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