This study shows the effects of a single 1-h exposure to pGz, on the expression of various NOS isoforms. We confirmed that pGz increased NO production, as indicated by increased nitrosylated proteins, and upregulated eNOS and nNOS, both protein and mRNA, in heart. The effects persisted for at least 24 h after cessation of the stimulus. pGz did not affect iNOS protein levels. Additionally, pGz also increased phosphorylation of eNOS and Akt (active forms), without change in either total Akt or MAP kinase pathway.
eNOS upregulation is known to occur in both isolated vessels, and endothelial cell cultures after exposure to pulsatile shear stress [28
]. Periodic acceleration imposes low amplitude additional pulsation on the intrinsic pulsations produced by the beating heart, and induces pulsatile shear stress both in vivo
and in vitro
]. Physical exercise also increases pulsatile shear stress and increases eNOS expression. This pathway may be an important component of cardioprotective conferred by exercise [30
]. Hambrecht et al. [34
] recently demonstrated that eNOS mRNA levels increase by 97%, 36 h after exercise, similar to our results of 75% increase 24 h after pGz treatment. Therefore, pGz may afford equivalent cardioprotection possibly through a similar pathway to exercise training.
Two signal transduction pathways, Akt pathway and MAPK (ERK1/2) pathway were examined in this study. pGz increased Akt phosphorylation, without effecting total Akt, or total and phosphorylated ERK1/2. Shear stress has been shown to increases eNOS phosphorylation through Akt pathway [35
]. Therefore, we postulate that pGz-induced eNOS phosphorylation is at least in part mediated through Akt pathway by increasing pulsatile shear stress. However, the exact mechanism how pGz upregulates the expression of eNOS and nNOS needs to be further elucidated.
nNOS is emerging as an important regulator of cardiac contractility. Hare and co-workers [6
] found nNOS immunoprecipitated with the ryanodine receptor of sarcoplasmic reticulum in mouse heart homogenates, suggesting a role in the regulation of calcium handling. nNOS has been demonstrated to regulate excitation–contraction coupling in the heart [6
]. Additionally, nNOS derived NO changes the cardiac autonomic balance with enhanced vagal activity and reduced β-adrenergic responsiveness [39
]. nNOS deficient animals appear to have compromised vagal control of heart rate, impaired diastole [40
], and increased mortality after experimental myocardial infarction [43
]. Adams et al. [44
] reported that pigs with selective inhibition of nNOS before global ischemia/reperfusion do not have return of spontaneous circulation (ROSC) whereas 75% of control group have ROSC. In a myocardial infarction model, Dawson [45
] and Saraiva [43
] showed that nNOS knock-out mice developed more pronounced left ventricular dilatation and dysfunction compared to wild type. Taken together these data suggest that nNOS is of vital importance to cardiovascular function in normal and diseased hearts. We investigated the localization of nNOS in the heart using immunohistochemical staining. nNOS is present exclusively in the cardiomyocytes and pGz increased its expression. The latter might account for the improved cardiac function during post-resuscitation when pGz is employed as a CPR method or when used in delayed post-conditioning tactic [20
A limitation to the present study relates to the analysis of heart tissue homogenates for the expression of NOS isoforms. Since heart homogenates also contain endothelial cells from vascular and endothelial lining of the heart and cardiomyocytes it is possible that eNOS expression could be primarily localized in endothelial cells. In contrast we found that nNOS was localized exclusively in the cardiomyocytes and pGz increased its expression.
In conclusion, this study provides a molecular mechanism for the effects of a single exposure of pGz on NO production and its respective NOS isoforms. A single pGz exposure produces a robust change in cardiac NOS isoform phenotype. Whether or not additional exposures to pGz can result in greater output and thus a more robust phenotype, remains to be determined. The clinical applicability of such a noninvasive method extends from preconditioning prior to a planned ischemia reperfusion event (cardiac surgery, revascularization and others) to post-conditioning strategies.