Specific Role of Neuronal Nitric-oxide Synthase when Tethered to the Plasma Membrane Calcium Pump in Regulating the β-Adrenergic Signal in the Myocardium*
- Tamer M. A. Mohamed12,
- Delvac Oceandy2,
- Sukhpal Prehar,
- Nasser Alatwi,
- Zeinab Hegab,
- Florence M. Baudoin,
- Adam Pickard,
- Aly O. Zaki,
- Raja Nadif,
- Elizabeth J. Cartwright3 and
- Ludwig Neyses4
- Department of Cardiology, University of Manchester and Biomedical Research Centre, Central Manchester NHS Foundation Trust, Manchester, Lancashire M13 9PT, United Kingdom
- 4 To whom correspondence should be addressed: 1.302 Stopford Bldg., University of Manchester, Oxford Rd., Manchester M13 9PT, United Kingdom. Tel.: 44-161-2766631; Fax: 44-161-2755669; E-mail: Ludwig.Neyses{at}cmft.nhs.uk.
Abstract
The cardiac neuronal nitric-oxide synthase (nNOS) has been described as a modulator of cardiac contractility. We have demonstrated previously that isoform 4b of the sarcolemmal calcium pump (PMCA4b) binds to nNOS in the heart and that this complex regulates β-adrenergic signal transmission in vivo. Here, we investigated whether the nNOS-PMCA4b complex serves as a specific signaling modulator in the heart. PMCA4b transgenic mice (PMCA4b-TG) showed a significant reduction in nNOS and total NOS activities as well as in cGMP levels in the heart compared with their wild type (WT) littermates. In contrast, PMCA4b-TG hearts showed an elevation in cAMP levels compared with the WT. Adult cardiomyocytes isolated from PMCA4b-TG mice demonstrated a 3-fold increase in Ser16 phospholamban (PLB) phosphorylation as well as Ser22 and Ser23 cardiac troponin I (cTnI) phosphorylation at base line compared with the WT. In addition, the relative induction of PLB phosphorylation and cTnI phosphorylation following isoproterenol treatment was severely reduced in PMCA4b-TG myocytes, explaining the blunted physiological response to the β-adrenergic stimulation. In keeping with the data from the transgenic animals, neonatal rat cardiomyocytes overexpressing PMCA4b showed a significant reduction in nitric oxide and cGMP levels. This was accompanied by an increase in cAMP levels, which led to an increase in both PLB and cTnI phosphorylation at base line. Elevated cAMP levels were likely due to the modulation of cardiac phosphodiesterase, which determined the balance between cGMP and cAMP following PMCA4b overexpression. In conclusion, these results showed that the nNOS-PMCA4b complex regulates contractility via cAMP and phosphorylation of both PLB and cTnI.
Footnotes
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↵5 The abbreviations used are: nNOS, neuronal nitric-oxide synthase; TG, transgenic; PLB, phospholamban; cTnI, cardiac troponin I; NRCM, neonatal rat cardiomyocyte(s); sGC, soluble guanylyl cyclase; PDE, phosphodiesterase(s); PKA, protein kinase A; SMLT, S-methyl-l-thiocitrulline; DAF-FM, 4-amino-5-methylamino-2,7-difluorescein diacetate.
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↵* This work was supported in part by Medical Research Council Programme Grant G0500025 (to L. N.) and British Heart Foundation Project Grant PG-05-082 (to D. O., E. J. C., and L. N.). This work was also supported by Manchester Biomedical Research Centre, which is funded by the National Institute for Health Research.
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The online version of this article (available at http://www.jbc.org) contains supplemental “Methods,” Figs. 1 and 2, “Discussion,” and Refs. 1–14.
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↵1 Recipient of an Egyptian government postgraduate research scholarship (Biochemistry Department, Faculty of Pharmacy, Zagazig University, Zagazig, Egypt).
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↵2 Both authors contributed equally to this work.
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↵3 Co-senior author.
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- Received December 3, 2008.
- Revision received March 4, 2009.
- The American Society for Biochemistry and Molecular Biology, Inc.
