Abstract 5534: Reversal of the Cardiac Dysfunction in Neuronal Nitric Oxide Synthase Knockout Myocytes by the Nitric Oxide Donor SNAP
Neuronal nitric oxide synthase (nNOS, NOS1) signaling has positive inotropic and lusitropic effects in cardiac myocytes. Knockout of NOS1 results in decreased myocyte Ca2+ transient and cell shortening amplitudes. This resulting dysfunction is due to, in part, decreased protein S-nitrosylation. Thus, we hypothesize that the nitric oxide donor SNAP, which S-nitrosylates proteins, will have a greater effect in NOS1 knockout (NOS1−/−) myocytes, resulting in a reversal of the cardiac dysfunction. Ventricular myocytes were isolated from NOS1−/− and wildtype (WT) mice. We also acutely inhibited NOS1 with S-metyl-L-thiocitrulline (SMLT) in WT myocytes. Ca2+ transients (Fluo-4 AM) and cell shortening (edge detection) were simultaneously measured. As we have previously shown, NOS1 knockout and inhibition resulted in decreased Ca2+ transient and cell shortening amplitudes. SNAP (1 μM) significantly increased the Ca2+ transient amplitude in all three groups (P<0.05 vs without SNAP). SNAP increased the Ca2+ transient amplitude in WT myocytes by 42±14%. However, SNAP had a much greater effect in NOS1−/− myocytes (88±27%) and WT+SMLT myocytes (118±19%). In fact, SNAP now normalized Ca2+ transient amplitudes in NOS1−/− and WT+SMLT myocytes (P=NS vs WT). Similar effects were observed in myocyte shortening amplitudes. Part of the contractile dysfunction may be due to abnormal ryanodine receptor (RyR) activity. An indirect method to measure RyR activity is measuring the fractional Ca2+ release (Ca2+ transient amplitude/SR Ca2+ load). We found that NOS1 knockout or inhibition leads to a reduced fractional Ca2+ release (P<0.05 vs WT), indicative of reduced RyR activity. SNAP slightly increased fractional Ca2+ release in WT myocytes (8±9%), but this was not significant. SNAP increased fractional release in NOS1−/− and WT+SMLT myocytes (P<0.05 vs without SNAP), resulting a greater increase (38±17%, and 56±18% respectively). It is known that SNAP is able to S-nitrosylate RyR and that NOS1−/− myocytes have reduced S-nitrosylation levels. Thus, our data indicate that the contractile dysfunction with NOS1 knockout or inhibition is partly due to reduced S-nitrosylation of RyR and can be reversed by increasing S-nitrosylation levels.
This research has received full or partial funding support from the American Heart Association, AHA Great Rivers Affiliate (Delaware, Kentucky, Ohio, Pennsylvania & West Virginia).