Constitutive nitric oxide synthases (eNOS and nNOS) in heart, have been implicated to regulate myocardial Ca2+ handling, electrical activity and the contractility of cardiac myocytes. Recently, we have shown that nNOSa and/or nNOSμ are functionally expressed in the cytosol of left ventricular (LV) myocytes from rat hearts and nNOSa/μ are up-regulated in hypertension (HTN) whereas eNOS is decreased. In addition to, we revealed for the first time that nNOSß is expressed in the myofilament fraction of LV myocytes, whose expression was unaffected in HTN. In hypertensive atrial myocardium, eNOS and nNOS profiling are not demonstrated systematically.Design and Method:
1. Left atrium (LA) from sham Sprague-Dawley rats (12 weeks old, male) and HTN rats were used. HTN was induced by infusing Ang II subcutaneously using osmotic minipumps (Alzet model 2004, infusion rate 125 ng/min/kg) for 4 weeks. 2. Immunoblotting: cytosol and myofilament fractions of LA homogenates were separated by centrifugation at 8000 rpm for10 min 10times at 4°C. Specific antibodies targeting anti-nNOS, anti-eNOS, anti-GAPDH were used and secondary antibody were followed relevant primary antibodies.Results:
Our result showed that nNOSa/μ were expressed in cytosol fractions of LA and the expression was not different between sham and HTN. Interestingly, none of the nNOS splice variants (including nNOSa/μ and nNOSß) was detected in myofilament fraction of LA from either group. eNOS protein expression was not different between groups and among different fractions of LA. Incubation of LA with PA (100 mM, 1 hr) failed to affect total protein levels of eNOS or nNOS in cytosol fractions of LA from sham or HTN rats.Conclusions:
Expression profiles of eNOS and nNOS splice variants in LA are not different between sham and HTN. Furthermore, acute application of PA (1hr) did not affect the protein levels or the translocation of eNOS or nNOS splice variants in atrial myocardium.