Loss of nNOS Sarcolemmal Localization and Activity During Hindlimb Unloading is Mitigated by Losartan Administration

Abstract

Spaceflight and immobilization lead to weakening and atrophy of antigravity skeletal muscles. Recently we observed that angiotensin II type 1 receptor (AT1R) blockade partially prevented the decrease in muscle fiber cross sectional area (CSA) and the type I to II fiber type shift that occurs in solei during 7 days of hindlimb unloading. Recent studies reveal that translocation of neuronal nitric oxide synthase (nNOS) from the sarcolemma to the sarcoplasm is a causal event to atrophy during disuse. PURPOSE: Determine whether the AT1R blocker Losartan prevents the translocation of nNOS from the sarcolemma to the sarcoplasm in soleus muscle fibers during 7 days of hindlimb unloading. METHODS: Rats were divided into 4 groups: ambulatory control (CON), ambulatory + Losartan (40 mg/kg/day) (CON-L), 7 days hindlimb unloaded (HU), and HU + Losartan (HU-L). Solei were collected and analyzed. Localization of nNOS was assessed through immunohistochemistry. We measured protein content and localization via subcellular fractionation and immunoblotting. nNOS activity was observed through NADPH-diaphorase histochemistry. RESULTS: Hindlimb unloading decreased sarcolemmal-localized nNOS compared to CON muscles, an effect ameliorated by Losartan. Subcellular fractionization and immunoblotting revealed significantly elevated levels of sarcoplasmic nNOS in the HU group compared to CON (p < 0.05). Sarcoplasmic nNOS content in HU-L was not significantly different from CON. nNOS activity in the CON solei displayed activity primarily near the sarcolemma, while sarcoplasmic nNOS activity and lack of sarcolemmal activity was detected in HU muscle fibers. Losartan administration during hindlimb unloading partially preserved nNOS activity at the sarcolemma. CON-L findings were no different than CON findings in any of the experiments. CONCLUSIONS: AT1R blockade with Losartan during 7 days of hindlimb unloading aided in the retention of sarcolemmal nNOS, which partially maintained sarcolemmal nNOS activity, thus, limiting the reduction in fiber CSA and the fiber type shift that occurs during disuse-induced atrophy. Supported by NASA (NNX13AE45G), Predoctoral Space Physiology Grant from ACSM / NASA, and the Sydney and J.L. Huffines Institute.