Biphasic effect of SIN-1 is reliant upon cardiomyocyte contractile state

Abstract

Many studies have demonstrated a biphasic effect of peroxynitrite in the myocardium, but few studies have investigated this biphasic effect on β-adrenergic responsiveness and its dependence on contractile state. We have previously shown that high SIN-1 (source of peroxynitrite, 200 μmol/L) produced significant anti-adrenergic effects during maximal β-adrenergic stimulation in cardiomyocytes. In the current study, we hypothesize that the negative effects of high SIN-1 will be greatest during high contractile states, while the positive effects of low SIN-1 (10 μmol/L) will predominate during low contractility. Isolated murine cardiomyocytes were field stimulated at 1 Hz and [Ca 2+ ] i transients and shortening were recorded. Following submaximal ISO (β-adrenergic agonist, 0.01 μmol/L) stimulation, 200 μmol/L SIN-1 induced two distinct phenomena. Cardiomyocytes undergoing a large response to ISO showed a significant reduction in contractility, while cardiomyocytes exhibiting a modest response to ISO showed a further increase in contractility. Additionally, 10 μmol/L SIN-1 always increased contractility during low ISO stimulation, but had no effect during maximal ISO (1 μmol/L) stimulation. 10 μmol/L SIN-1 also increased basal contractility. Interestingly, SIN-1 only produced a contractile effect under one condition in phospholamban knockout cardiomyocytes, providing a potential mechanism for the biphasic effect of peroxynitrite. These results provide clear evidence for a biphasic effect of peroxynitrite, with high peroxynitrite modulating high levels of β-adrenergic responsiveness and low peroxynitrite regulating basal function and low levels of β-adrenergic stimulation.

Neuronal nitric oxide synthase signaling within cardiac myocytes targets phospholamban

“…These data are consistent with our previous study that showed that low levels of exogenous peroxynitrite (produced from SIN-1) increased basal Ca 2ϩ transient and shortening amplitude in murine myocytes. Interestingly, similar to NOS1 inhibition, the contractile effects of SIN-1 were absent in PLB Ϫ/Ϫ myocytes (22). However, it still remains to be determined whether NO itself or other reactive nitrogen species are also part of the NOS1 signaling pathway.…”

Cross-talk between lipid and protein carbonylation in a dynamic cardiomyocyte model of mild nitroxidative stress

“…Indeed, it was reported that 10 µmol/L SIN-1 would correspond to 3 nmol/L of peroxynitrite and thus represent physiological rather than pathological conditions [48]. Furthermore, the differences in the total (175% increase at 15 min) and protein-specific (significant increase at 30 min) carbonylation suggested that compared to proteins, lipids might be the primary targets of ROS/RNS.…”

“…In the presence of carbon dioxide (CO 2 ) peroxynitrite anion forms nitroso-peroxocarbonate (ONOOCO 2 - ), which in turn decomposes to • NO 2 , CO 3 - and NO 3 - [47]. We and others have previously demonstrated the complex nitroxidative effect of SIN-1 treatment in cell culture models [46], [48], [49], [50]. Here, free radical production in SIN-1-treated (10 µmol/L) CM was monitored by DCFDA assay (Fig.…”

Abnormal Ca2+Cycling in Failing Ventricular Myocytes: Role of NOS1-Mediated Nitroso-Redox Balance

“…But, similar to ROS, may also modulate the activity of various kinases and phosphatases to change the protein phosphorylation status. We have previously shown that a peroxynitrite donor (SIN-1) in a low dose was able to increase PLB Serine16 phosphorylation via activation of PKA (96,97). Surprisingly, the peroxynitrite-mediated increase in PKA activity occurred in the absence of cAMP, and most likely occurs via Snitrosylation (28).…”