β3-Adrenoreceptor Stimulation Protects against Myocardial Infarction Injury <i>via</i> eNOS and nNOS Activation

<div><p>β3-adrenergic receptor (AR) and the downstream signaling, nitric oxide synthase (NOS) isoforms, have been emerged as novel modulators of heart function and even potential therapeutic targets for cardiovascular diseases. However, it is not known whether β3-AR plays cardioprotective effects against myocardial infarction (MI) injury. Therefore, the present study was designed to determine the effects of β3-AR on MI injury and to elucidate the underlying mechanism. MI model was constructed by left anterior descending (LAD) artery ligation. Animals were administrated with β3-AR agonist BRL37344 (BRL) or β3-AR inhibitor SR59230A (SR) respectively at 0.1 mg/kg/hour one day after MI operation. The scar area, cardiac function and the apoptosis of myocardial were assessed by Masson's trichrome stain, echocardiography and TUNEL assay respectively. Western blot analysis was performed to elucidate the expressions of target proteins. β3-AR activation with BRL administration significantly attenuated fibrosis and decreased scar area after MI. Moreover, BRL also preserved heart function, and reduced the apoptosis of cardiomyocyte induced by MI. Furthermore, BRL treatment altered the phosphorylation status of endothelial NOS (eNOS) and increased the expression of neuronal NOS (nNOS). These results suggested that β3-AR stimulation has a substantial effect on recovery of heart function. In addition, the activations of both eNOS and nNOS may be associated with the cardiac protective effects of β3-AR.</p></div

Effect of BRL and SR on LV dilation and LV systolic function after MI.

<p>(A) Representative M-mode echocardiography images were taken at the level of the papillary muscle where left ventricular diameters can be measured. Quantification of left ventricular end diastolic diameter (LVEDd) (B), end systolic diameter (LVESd) (C), left ventricular ejection fraction (EF) (D) and fractional shortening (FS) (E) 4 weeks after MI. (*<i>p</i><0.05 <i>vs</i>. MI.)</p

β3-AR stimulation altered the phosphorylation status of eNOS and increased the expression of nNOS.

<p>(A) Representative immunoblots of <i>p</i>-eNOS (Ser1177/Thr495/Ser114/Ser633) and total eNOS in sham, MI, MI+BRL and MI+SR groups. (B) Representative immunoblots of <i>p</i>-nNOS (Ser1417/Ser847), total nNOS, <i>p</i>-iNOS, iNOS and β-actin in all groups. Semiquantitative analysis of the expressions of eNOS, nNOS, iNOS(C), <i>p</i>-eNOS (Ser1177/Thr495/Ser114/Ser633) (D), <i>p</i>-nNOS(Ser1417/Ser847) (E) and <i>p</i>-iNOS (Tyr151) (F) (*<i>p</i><0.05).</p

Effect of BRL and SR on left ventricular fibrosis induced by MI.

<p>(A) Representative Masson's trichrome staining revealed left ventricular fibrosis 4 weeks after MI (magnification: 4x). (B)Histological examination of fibrosis by Masson's trichrome staining in the border zone. Red indicates viable myocardium; blue indicates fibrosis due to infarction damage. Scale bar represents 50 µm. (C) Quantitative analysis of the scar area (*<i>p</i><0.05).</p

Effect of BRL and SR on the expressions of β-AR subtypes.

<p>(A) Representative immunoblots of β1-AR, β2-AR and β3-AR in sham, MI, MI+BRL and MI+SR groups. (B) Semiquantitative analysis of the expressions of β1-AR, β2-AR and β3-AR (*<i>p</i><0.05).</p

β3-AR stimulation decreased cardiomyocyte apoptosis.

<p>(A) Representative photographs of TUNEL-stained heart sections from sham group, MI group, MI+BRL group and MI+SR group. Apoptotic nuclei were identified as TUNEL positive (green fluorescent). Myocardium was stained using a monoclonal antibody against Troponin I (red fluorescent) and total nuclei was stained by DAPI (blue fluorescent). Scale bar represents 50 µm. (B) Apoptotic cells were quantified by apoptotic index (AI) which was termed as the percentage of apoptotic cells. (C) BRL administration also significantly decreased caspase-3 activity compared with MI group and MI+SR group (*<i>p</i><0.05).</p