Article Text

016 Distinctive role for protein phosphatases and protein kinases in the regulation of myocardial contraction by the neuronal nitric oxide synthase
  1. Y H Zhang,
  2. M H Zhang,
  3. C S Redwood,
  4. B Casadei
  1. Department of Cardiovascular Medicine, University of Oxford, Oxford, UK


Introduction Gene deletion of the neuronal nitric oxide synthase (nNOS) is associated with impaired relaxation and increased contraction both in vivo and in isolated left ventricular (LV) myocytes. Our recent work has shown that a reduction in protein kinase A-dependent phosphorylation of phospholamban, secondary to increased protein phosphatase (PP) activity, is responsible for the slower (Ca2+)i decay and myocyte relaxation that result from disrupting nNOS. The aim of the present study was to investigate the intracellular signalling underlying the nNOS-dependent regulation of myocardial contraction.

Methods and Results Cell shortening was significantly increased in LV myocytes isolated from nNOS−/− mice and in wild type myocytes (nNOS+/+) after acute nNOS inhibition with L-VNIO (100 μmol/L) both under field-stimulation (3 Hz, 35°C) and in voltage-clamped conditions (from −70 to +20 mV, 25 ms, 35°C). Changes in myofilament Ca2+ sensitivity did not contribute to the increase in contraction in nNOS-/− since the size of tetanic contraction (at 20 Hz) relative to the rise of (Ca2+)i in the presence of thapsigargin did not differ between groups. In contrast, the L-type Ca2+ current density (ICa) was significantly greater after nNOS inhibition/gene deletion. Inhibition of soluble guanylate cyclase with ODQ (10 μmol/l) or PKG with RP-8-Br-cGMP (100 μmol/l) increased cell shortening in wild type but not in nNOS−/− LV myocytes, indicating that cGMP-mediated regulation of cardiac function downstream of NO is abolished in the absence of nNOS. Intracellular perfusion of the PKA inhibitor PKI (1 μmol/l) significantly reduced contraction only in nNOS−/− myocytes, whereas PP2A inhibition with okadaic acid (OA, 10 nmol/l) increased cell shortening in nNOS+/+ LV myocytes but not in nNOS−/−. Interestingly, OA (10 nmol/l) increased ICa only in nNOS+/+ LV myocytes thereby abolished the difference in ICa between nNOS+/+ and nNOS−/−. Phophorylation of ICa (assessed using Pro-Q Diamond phosphoprotein gel staining with ICaα1.2 immunoprecipitation lysates) was significantly increased in nNOS−/− LV myocardium comparing to nNOS+/+. Inhibition of PP1 by intracellular dialysis of inhibitor-2 (500 nM) did not affect myocyte contraction in either group.

Summary and Conclusion Abolition of cGMP-mediated signalling together with increased PKA-mediated phosphorylation of L-type Ca2+ channels secondary to a local reduction in PP2A activity account for the increased myocardial contraction observed in the presence of nNOS disruption. Taken together with our aforementioned findings on the mechanisms underlying impaired relaxation in nNOS−/− mice, these data suggest that nNOS-derived NO regulates the activity of subcellular pools of PPs, and by doing so it directs PKA mediated phosphorylation to specific protein targets in the cardiomyocyte.

  • neuronal nitric oxide synthase
  • protein phosphatase
  • L-type Ca channel

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