2013 OSU Molecular Life Sciences
Interdisciplinary Graduate Programs Symposium

 

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Poster number 30 submitted by Lifei Tang

Contribution of the NOS1/CaMKII/RyR axis in the positive inotropic response to beta-adrenergic stimulation in ventricular myocytes

Lifei Tang (Department of Physiology and Cell Biology), Steve R Roof (Department of Physiology and Cell Biology), Xin Huang (Department of Physiology and Cell Biology), Peter J Mohler (The Dorothy M. Davis Heart and Lung Research Institute), Xander H Wehrens (Department of Molecular Physiology and Biophysics, Baylor College of Medicine), Mark T Ziolo (Department of Physiology and Cell Biology, The Dorothy M. Davis Heart and Lung Research Institute)

Abstract:
Stimulation of the beta-adrenergic (beta-AR) pathway leads to positive inotropy, and is the major regulator of heart function. Activation of neuronal nitric oxide synthase (NOS1) signaling has been reported to play an important role in the positive inotropy, but the molecular mechanisms of NOS1’s contribution are not well known. The purpose of this study is to delineate the NOS1 molecular pathway during beta-AR stimulation. Ventricular myocytes were isolated from wildtype (WT, C57Bl/6), NOS1 knockout (NOS1-/-), RyR knockin with CaMKII phosphorylation constitutively active (S2814D) mice. Ca2+ transients (Fluo-4) and cell shortening (edge detection) were simultaneously measured. 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. Interestingly, replenishment of NO (via the NO donor SNAP) to the NOS1-/- myocytes, increased beta-AR stimulated contraction, which was blocked by KN93. Our and others previous data have shown that NOS1 can directly activate CaMKII and that CaMKII phosphorylates RyR to increase SR Ca2+ release. Consistent with these data, beta-AR stimulated fractional release (an indicator of RyR activity) is decreased by KN93 in WT myocytes but not in NOS1-/- myocytes. Furthermore, in S2814D myocytes, RyR activity (SR Ca2+ leak-load relationship) was higher compared to WT, while acute NOS1 inhibition (SMLT) had no effect. As with contraction, beta-AR stimulated RyR activity in WT myocytes was blunted by NOS1 inhibition. In addition, we demonstrated that Akt activates NOS1 during beta-AR stimulation. Additional experiment excluded Epac as a potential mediator of RyR during beta-AR stimulation. These data suggest that during beta-AR stimulation, NOS1 is activated by Akt and contributes to positive inotropy via CaMKII-mediated RyR activation without involvement of the Epac pathway. Further study of this pathway is warranted since beta-AR stimulation, NOS1 and CAMKII expression/activity are increased in cardiac hypertrophy and heart failure.

References:
1. Ziolo MT, Kohr MJ, Wang H. Nitric oxide signaling and the regulation of myocardial function. J Mol Cell Cardiol. Nov 2008;45(5):625-632.
2. Wang H, Viatchenko-Karpinski S, Sun J, et al. Regulation of myocyte contraction via neuronal nitric oxide synthase: role of ryanodine receptor S-nitrosylation. J Physiol. Aug 1 2010;588(Pt 15):2905-2917.
3. Curran J, Hinton MJ, Rios E, et al. Beta-adrenergic enhancement of sarcoplasmic reticulum calcium leak in cardiac myocytes is mediated by calcium/calmodulin-dependent protein kinase. Circ Res. Feb 16 2007;100(3):391-398.

Keywords: NOS1, CaMKII, RyR