Nebivolol, a potent cardioselective β1-receptor blocker with additional antioxidative and nitric oxide-releasing properties, reduced systemic and cardiac oxidative stress and associated cardiac fibrosis in SHRs fed a HSD. The decrease in oxidative stress was accompanied by reduced cardiac Ang II immunostaining potentially reflecting the effect of nebivolol on ACE/ACE2, the two critical enzymes involved in Ang II formation and metabolism, respectively. The beneficial effects of nebivolol on cardiac oxidative stress were not associated with changes in arterial pressure, cardiac function, and cardiac nitric oxide metabolites.
It is important to note that the current study confirmed our previous findings that nebivolol did not affect salt-related increase in arterial pressure [12
]. These data underscore the view that dietary salt excess attenuates the anti-hypertensive properties of drug therapies [22
]. In our previous study nebivolol did not facilitate systemic vascular nitric oxide availability in salt-loaded SHRs [12
]. We extended these findings by now demonstrating that nebivolol, in concert with its antioxidative properties, reduced systemic oxidative stress. Thus, our results suggest that exacerbation of hypertension in SHRs fed HSD is not related to an increase in systemic oxidative stress or decreased nitric oxide availability due to increased nitric oxide scavenge by reactive oxygen species (ROS). Although increased systemic ROS production was coupled with development of hypertension [9
], other studies suggest that vascular oxidative stress does not determine blood pressure regulation [26
]. Further studies are necessary to elucidate the exact mechanisms underlying a salt-related blood pressure elevation in hypertensive patients. The data reported in this study do not negate a beneficial effect of this drug in hypertension, not aggravated by salt, since potent anti-hypertensive effects have been reported in SHRs even after treatment withdrawal [28
]. Additional studies showed that nebivolol improved arterial structure coupled with improved endothelium-dependent relaxation [28
]. Vasodilatory properties of nebivolol are mainly related to its ability to stimulate endothelial-dependent nitric oxide release shown not only in experimental animals [11
] but also in healthy volunteers [30
] and hypertensive patients [31
Activation of β1-adrenergic receptors is one of the blood pressure-independent mechanisms contributing to cardiac hypertrophy and fibrosis [32
]. To the best of our knowledge, this is the first study to show that nebivolol effectively reduced salt-related cardiac fibrosis independent of its effect on blood pressure. Nebivolol may have also exerted its antifibrotic effects by reducing salt-related cardiac oxidative stress reflected in pronounced 3-NT staining. It has been shown that gp91phox
is critical for development of cardiac fibrosis in response to either pressure overload or RAS stimulation [34
]. Moreover, left-ventricular failure in Dahl salt-sensitive rats was associated with increased ROS production and p22phox
up-regulation that was corrected with both AT1
receptor antagonist and tempol [36
]. We also previously found an increased gp91phox
protein in the kidney of SHRs fed HSD [12
]. However, in the present study, gene expression of the two critical components of NADPH oxidase, p22phox
, was not altered with high dietary salt intake. Further studies are warranted to explore additional potential sources for ROS generation in response to dietary salt excess in SHRs such as xanthine oxidase, uncoupled nitric oxide synthase, mitochondria, and cytochrome P-460 enzymes [37
]. Similarly, nebivolol did not affect the gene expression of these two NADPH oxidase subunits; however, its antioxidant properties could have arisen from its intrinsic direct superoxide scavenger properties [11
β1-adrenergic receptor blockers have been used clinically for a long time in the treatment of different cardiovascular diseases, including hypertension as well as impaired renal and cardiac function [38
]. Their beneficial effects are linked not only to their ability to decrease enhanced sympathetic drive but also to reduce renin release [41
]. In the present study, we report that the beneficial antifibrotic and antioxidative effects of nebivolol in SHRs fed a HSD were associated with not only suppression of PRC but also with reduction in the cardiac expression of Ang II. It is well accepted that increased Ang II activity is linked to development of cardiac hypertrophy and fibrosis as well as dysfunction. Moreover, the reduction in cardiac Ang II content in nebivolol-treated SHRs fed HSD may be seen in the context of nebivolol’s ability to reduce the elevated ACE/ACE2 activity ratio observed in untreated rats. ACE has been involved not only in Ang II formation but also the metabolism of the antiproliferative and vasodilator actions of Ang-(1–7) [42
], whereas ACE2 preferentially cleaves Ang II to form Ang-(1–7) [45
]. Moreover, decreased cardiac Ang-(1–7) was associated with pronounced left-ventricular remodeling in SHRs fed HSD [18
]. Thus, the imbalance between ACE/ACE2 may have favored Ang II over Ang-(1–7) in salt-loaded rats contributing to the specific phenotype observed in this group. Consistent with a multilevel interaction between sympathetic and RAS systems [46
], we recently showed that nebivolol prevented the salt-induced reduction in Ang-(1–7) receptor mas
along with improvement in renal blood flow, urinary protein excretion, and collagen deposition in salt-loaded SHRs [12
]. On the contrary, Ang-(1–7)-producing fusion protein in the heart attenuated isoproterenol-induced cardiac fibrosis [47
]. Future studies will explore in more details the contribution of counterbalancing Ang-(1–7) to the cardiac beneficial effects of nebivolol and the potential underlying mechanisms.
We also examined the expression of a novel angiotensin peptide precursor, Ang-(1–12) [21
], in an attempt to further characterize the cardiac RAS under the condition of dietary salt excess and/or nebivolol treatment. Neither dietary salt excess nor nebivolol treatment altered the Ang-(1–12) level in the heart of SHRs. Since Ang II formation from Ang-(1–12) occurs via a nonrenin pathway in both the heart [49
] and the systemic circulation [17
], the current finding of unchanged Ang-(1–12) expression in the presence of PRC changes are in keeping with previous observations. Nagata et al
] recently reported that cardiac level of Ang-(1–12) did not change after exposure of normotensive rats to diets with different sodium content. They showed that increased plasma renin activity in response to low-salt diet was not accompanied with changes in Ang I, Ang II, and Ang-(1–12) in the heart. It is therefore possible that the absence of changes in the expression of cardiac Ang-(1–12) in response to either dietary sodium or the combination of dietary sodium and β1-receptor blockade reflects that this substrate is regulated by mechanisms that are independent of both renin and the deleterious effects of salt excess.
Finally, a growing body of evidence argues for favorable effects of nebivolol on left-ventricular dysfunction [13
]. However, despite its beneficial effects on cardiac fibrosis and oxidative stress in SHRs fed a HSD in the present study, nebivolol did not improve impaired ventricular diastolic function nor did it reduce left-ventricular mass. Failure to improve cardiac function and left-ventricular hypertrophy may be related to the combined effects of salt-loading and hypertension since in the Zucker rat, a model of obesity and hypertension, nebivolol reduces myocardial structural maladaptive changes and improves diastolic relaxation in concert with improvements in insulin sensitivity and endothelial nitric oxide synthase activation [13
]. Although increased left-ventricular wall thickness in both untreated and nebivolol-treated rats may have enhanced myocardial oxygen demand compromising coronary circulation and left-ventricular relaxation, decreased oxidative stress in rats treated with β1-adrenergic receptor blocker argues against compromised myocardial oxygen supply in these rats.
Consistent with this notion, decreased heart rate by nebivolol treatment would reduce oxygen consumption while prolonging diastolic time and in this way improve coronary hemodynamics since coronary flow occurs mainly through this phase of the cardiac cycle [54
]. Thus, other mechanism of still impaired relaxation in nebivolol-treated rats should be explored. In addition to alteration in myofilament properties and cytoskeletal architecture, alteration in calcium handling has been linked to diastolic dysfunction in hypertrophied LV [55
] and Ang II has an important contributory role [56
]. Importantly, AT1
receptor antagonism under similar experimental conditions beneficially affected both left-ventricular hypertrophy and fibrosis improving left-ventricular dysfunction and coronary hemodynamic impairment, yet similarly to nebivolol, it did not prevent salt-induced rise in arterial pressure [6
]. It is quite possible that, whereas nebivolol reduced Ang II in the heart of salt-loaded rats to the level comparable to the control rats, even that level of Ang II in response to an increase in salt intake might be inappropriately high.
In summary, our data suggest that nebivolol, in a blood pressure-independent manner, ameliorated cardiac oxidative stress and associated fibrosis but not left-ventricular dysfunction in salt-loaded SHRs. The beneficial effects of nebivolol may be attributed, at least in part, to the decreased ACE/ACE2 ratio and consequent reduction of cardiac Ang II levels.