Metformin Attenuates Slow-to-Fast Fiber Shift and Proteolysis Markers Increase in Rat Soleus after 7 Days of Rat Hindlimb Unloading

Muscle unloading leads to signaling alterations that cause muscle atrophy and weakness. The cellular energy sensor AMPK can regulate myofiber-type shift, calcium-dependent signaling and ubiquitin-proteasome system markers. We hypothesized that the prevention of p-AMPK downregulation during the first week of muscle unloading would impede atrophy development and the slow-to-fast shift of soleus muscle fibers, and the aim of the study was to test this hypothesis. Thirty-two male Wistar rats were randomly assigned to four groups: placebo control (C), control rats treated with metformin (C + M), 7 days of hindlimb suspension (HS) + placebo (7HS), and 7 days of HS + metformin administration (7HS + M). In the soleus of the 7HS rats, we detected a slow-to-fast fiber-type shift as well as a significant downregulation of MEF-2D and p300 in the nuclei. In the 7HS group, we also found decreases in p-ACC (AMPK target) protein level and in the expression of E3 ubiquitin ligases and p-CaMK II protein level vs. the C group. The 7-day metformin treatment for soleus muscle unloading (1) prevented slow-to-fast fiber-type shift; (2) counteracted changes in the p-ACC protein level; (3) hindered changes in the nuclear protein level of the slow myosin expression activators MEF-2D and p300, but did not affect NFATc1 signaling; and (4) attenuated the unloading-induced upregulation of MuRF-1, atrogin-1, ubiquitin and myostatin mRNA expression, but did not prevent soleus muscle atrophy. Thus, metformin treatment during muscle disuse could be useful to prevent the decrease in the percentage of slow-type fatigue-resistant muscle fibers

The Effect of SERCA Activation on Functional Characteristics and Signaling of Rat Soleus Muscle upon 7 Days of Unloading

Skeletal muscle abnormalities and atrophy during unloading are accompanied by the accumulation of excess calcium in the sarcoplasm. We hypothesized that calcium accumulation may occur, among other mechanisms, due to the inhibition of sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) activity. Consequently, the use of the SERCA activator will reduce the level of calcium in the sarcoplasm and prevent the negative consequences of muscle unloading. Wistar rats were randomly assigned into one of three groups (eight rats per group): control rats with placebo (C), 7 days of unloading/hindlimb suspension with placebo (7HS), and 7 days of unloading treated with SERCA activator CDN1163 (7HSC). After seven days of unloading the soleus muscle, the 7HS group displayed increased fatigue in the ex vivo test, a significant increase in the level of calcium-dependent CaMK II phosphorylation and the level of tropomyosin oxidation, as well as a decrease in the content of mitochondrial DNA and protein, slow-type myosin mRNA, and the percentage of slow-type muscle fibers. All of these changes were prevented in the 7HSC group. Moreover, treatment with CDN1163 blocked a decrease in the phosphorylation of p70S6k, an increase in eEF2 phosphorylation, and an increase in MuRF-1 mRNA expression. Nevertheless, there were no differences in the degree of fast and slow muscle fiber atrophy between the 7HS and 7HSC groups. Conclusion: SERCA activation during 7 days of unloading prevented an increase in soleus fatigue, the decrease of slow-type myosin, mitochondrial markers, and markers of calcium homeostasis but had no effect on muscle atrophy

Plantar Mechanical Stimulation Maintains Slow Myosin Expression in Disused Rat Soleus Muscle via NO-Dependent Signaling

It was observed that gravitational unloading during space missions and simulated microgravity in ground-based studies leads to both transformation of slow-twitch muscle fibers into fast-twitch fibers and to the elimination of support afferentation, leading to the “switching-off” of postural muscle motor units electrical activity. In recent years, plantar mechanical stimulation (PMS) has been found to maintain the neuromuscular activity of the hindlimb muscles. Nitric oxide (NO) was shown to be one of the mediators of muscle fiber activity, which can also promote slow-type myosin expression. We hypothesized that applying PMS during rat hindlimb unloading would lead to NO production upregulation and prevention of the unloading-induced slow-to-fast fiber-type shift in rat soleus muscles. To test this hypothesis, Wistar rats were hindlimb suspended and subjected to daily PMS, and one group of PMS-subjected animals was also treated with nitric oxide synthase inhibitor (L-NAME). We discovered that PMS led to sustained NO level in soleus muscles of the suspended animals, and NOS inhibitor administration blocked this effect, as well as the positive effects of PMS on myosin I and IIa mRNA transcription and slow-to-fast fiber-type ratio during rat hindlimb unloading. The results of the study indicate that NOS activity is necessary for the PMS-mediated prevention of slow-to-fast fiber-type shift and myosin I and IIa mRNA transcription decreases during rat hindlimb unloading

Rivaroxaban or aspirin for patent foramen ovale and embolic stroke of undetermined source: a prespecified subgroup analysis from the NAVIGATE ESUS trial

Background: Patent foramen ovale (PFO) is a contributor to embolic stroke of undetermined source (ESUS). Subgroup analyses from previous studies suggest that anticoagulation could reduce recurrent stroke compared with antiplatelet therapy. We hypothesised that anticoagulant treatment with rivaroxaban, an oral factor Xa inhibitor, would reduce the risk of recurrent ischaemic stroke compared with aspirin among patients with PFO enrolled in the NAVIGATE ESUS trial. Methods: NAVIGATE ESUS was a double-blinded, randomised, phase 3 trial done at 459 centres in 31 countries that assessed the efficacy and safety of rivaroxaban versus aspirin for secondary stroke prevention in patients with ESUS. For this prespecified subgroup analysis, cohorts with and without PFO were defined on the basis of transthoracic echocardiography (TTE) and transoesophageal echocardiography (TOE). The primary efficacy outcome was time to recurrent ischaemic stroke between treatment groups. The primary safety outcome was major bleeding, according to the criteria of the International Society of Thrombosis and Haemostasis. The primary analyses were based on the intention-to-treat population. Additionally, we did a systematic review and random-effects meta-analysis of studies in which patients with cryptogenic stroke and PFO were randomly assigned to receive anticoagulant or antiplatelet therapy. Findings: Between Dec 23, 2014, and Sept 20, 2017, 7213 participants were enrolled and assigned to receive rivaroxaban (n=3609) or aspirin (n=3604). Patients were followed up for a mean of 11 months because of early trial termination. PFO was reported as present in 534 (7·4%) patients on the basis of either TTE or TOE. Patients with PFO assigned to receive aspirin had a recurrent ischaemic stroke rate of 4·8 events per 100 person-years compared with 2·6 events per 100 person-years in those treated with rivaroxaban. Among patients with known PFO, there was insufficient evidence to support a difference in risk of recurrent ischaemic stroke between rivaroxaban and aspirin (hazard ratio [HR] 0·54; 95% CI 0·22–1·36), and the risk was similar for those without known PFO (1·06; 0·84–1·33; pinteraction=0·18). The risks of major bleeding with rivaroxaban versus aspirin were similar in patients with PFO detected (HR 2·05; 95% CI 0·51–8·18) and in those without PFO detected (HR 2·82; 95% CI 1·69–4·70; pinteraction=0·68). The random-effects meta-analysis combined data from NAVIGATE ESUS with data from two previous trials (PICSS and CLOSE) and yielded a summary odds ratio of 0·48 (95% CI 0·24–0·96; p=0·04) for ischaemic stroke in favour of anticoagulation, without evidence of heterogeneity. Interpretation: Among patients with ESUS who have PFO, anticoagulation might reduce the risk of recurrent stroke by about half, although substantial imprecision remains. Dedicated trials of anticoagulation versus antiplatelet therapy or PFO closure, or both, are warranted. Funding: Bayer and Janssen