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Neuronal nitric oxide synthase gene disruption reduces the diastolic calcium leak from the ryanodine receptor: implications for excitation–contraction coupling in heart failure
  1. GBS Lim,
  2. MH Zhang,
  3. YH Zhang,
  4. B Casadei
  1. University of Oxford, Oxford, UK

Abstract

Increased Ca2+ leak from the ryanodine receptor (RyR2), possibly due to hyperphosphorylation of the RyR2 channel, has been proposed to deplete sarcoplasmic reticulum (SR) Ca2+ content and thus lead to impaired contractility in heart failure. Although nitric oxide (NO) and superoxide donors have been shown to modulate the open probability of reconstituted RyR2s, whether endogenous NO and superoxide production affect RyR2 function in intact cardiomyocytes remains uncertain. Using a method established by Shannon et al (2002) we measured diastolic RyR2 Ca2+ leak in left ventricular myocytes isolated from neuronal nitric oxide synthase (nNOS) knockout (KO) mice and their wild-type (WT) littermates.

As shown previously, (Ca2+)i transient amplitude and SR Ca2+ content were larger in KO myocytes at physiological (Ca2+)o. To investigate the relationship between SR Ca2+ load and RyR2 leak, we altered the former by varying (Ca2+)o. Increases in (Ca2+)o caused a larger rise in both SR Ca2+ content and (Ca2+)i transient amplitude in WT than in KO myocytes, suggesting that SR Ca2+ loading in KO myocytes may be impaired (in agreement with a reduction in phospholamban phosphorylation in these mice). When the cells were perfused with 4 mmol (Ca2+)o, the RyR2 leak was significantly larger in WT than KO myocytes (fura-2 fluorescence ratio F365/F380 0.240 ± 0.05 vs 0.124 ±0 .03; n  =  9, 10; p = 0.02). The lower RyR2 leak SR load relationship in the KO was associated with a decreased RyR2 phosphorylated fraction (serine 2809), consistent with the previously reported increase in protein phosphatase activity in the KO myocardium.

Lucigenin-enhanced chemiluminescence showed that superoxide production was higher in KO hearts at 2 mmol (Ca2+)o, but was decreased in both genotypes by higher (Ca2+)o. To investigate whether endogenous superoxide production modulated RyR2 function, myocytes were preincubated in, and perfused with, 100 Âμmol oxypurinol (to inhibit xanthine oxidoreductase) or 100 Âμmol apocynin (to inhibit NADPH oxidases). Neither inhibitor had a significant effect on the RyR2 leak – SR load relationship in either genotype.

In summary, our data indicate that (1) nNOS-derived NO increases RyR2 leak (possibly by increasing the channel phosphorylation), particularly at high SR Ca2+ loads; (2) Ca2+ mediated stimulation of nNOS activity in the myocardium may prevent SR Ca2+ overload through both a NO-mediated increase in RyR Ca2+ leak and the previously observed reduction in Ca2+ influx via the L-type Ca2+ channel. These findings suggest that nNOS overexpression in the failing myocardium may be an adaptive mechanism aimed at preventing myocardial oxidative damage and disruption of Ca2+ homeostasis.

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