Brittany C. Collins

Estradiol modulates myosin regulatory light chain phosphorylation and contractility in skeletal muscle of female mice

Impairment of skeletal muscle function has been associated with changes in ovarian hormones, especially estradiol. To elucidate mechanisms of estradiol on skeletal muscle strength, the hormones effects on phosphorylation of the myosin regulatory light chain (pRLC) and muscle contractility were investigated, hypothesizing an estradiol-specific beneficial impact. In a skeletal muscle cell line, C2C12, pRLC was increased by 17β-estradiol (E2) in a concentration-dependent manner. In skeletal muscles of C57BL/6 mice that were E2 deficient via ovariectomy (OVX), pRLC was lower than that from ovary-intact, sham-operated mice (Sham). The reduced pRLC in OVX muscle was reversed by in vivo E2 treatment. Posttetanic potentiation (PTP) of muscle from OVX mice was low compared with that from Sham mice, and this decrement was reversed by acute E2 treatment, demonstrating physiological consequence. Western blot of those muscles revealed that low PTP corresponded with low pRLC and higher PTP with greater pRLC. We aimed to elucidate signaling pathways affecting E2-mediated pRLC using a kinase inhibitor library and C2C12 cells as well as a specific myosin light chain kinase inhibitor in muscles. PI3K/Akt, MAPK, and CamKII were identified as candidate kinases sensitive to E2 in terms of phosphorylating RLC. Applying siRNA strategy in C2C12 cells, pRLC triggered by E2 was found to be mediated by estrogen receptor-β and the G protein-coupled estrogen receptor. Together, these results provide evidence that E2 modulates myosin pRLC in skeletal muscle and is one mechanism by which this hormone can affect muscle contractility in females.

Deletion of estrogen receptor alpha in skeletal muscle results in impaired contractility in female mice

Estradiol deficiency in females can result in skeletal muscle strength loss, and treatment with estradiol mitigates the loss. There are three primary estrogen receptors (ERs), and estradiol elicits effects through these receptors in various tissues. Ubiquitous ERα-knockout mice exhibit numerous biological disorders, but little is known regarding the specific role of ERα in skeletal muscle contractile function. The purpose of this study was to determine the impact of skeletal muscle-specific ERα deletion on contractile function, hypothesizing that ERα is a main receptor through which estradiol affects muscle strength in females. Deletion of ERα specifically in skeletal muscle (skmERαKO) did not affect body mass compared with wild-type littermates (skmERαWT) until 26 wk of age, at which time body mass of skmERαKO mice began to increase disproportionally. Overall, skmERαKO mice had low strength demonstrated in multiple muscles and by several contractile parameters. Isolated extensor digitorum longus muscles from skmERαKO mice produced 16% less eccentric and 16-26% less submaximal and maximal isometric force, and isolated soleus muscles were more fatigable, with impaired force recovery relative to skmERαWT mice. In vivo maximal torque productions by plantarflexors and dorsiflexors were 16% and 12% lower in skmERαKO than skmERαWT mice, and skmERαKO muscles had low phosphorylation of myosin regulatory light chain. Plantarflexors also generated 21-32% less power, submaximal isometric and peak concentric torques. Data support the hypothesis that ablation of ERα in skeletal muscle results in muscle weakness, suggesting that the beneficial effects of estradiol on muscle strength are receptor mediated through ERα. NEW & NOTEWORTHY We comprehensively measured in vitro and in vivo skeletal muscle contractility in female estrogen receptor α (ERα) skeletal muscle-specific knockout mice and report that force generation is impaired across multiple parameters. These results support the hypothesis that a primary mechanism through which estradiol elicits its effects on strength is mediated by ERα. Evidence is presented that estradiol signaling through ERα appears to modulate force at the molecular level via posttranslational modifications of myosin regulatory light chain.

Influence of Xpand Nitric Oxide Reactor, L-Arginine Alpha-Ketoglutarate, and Caffeine Supplementation on Calf Muscle Re-Oxygenation During and after Acute Resistance Exercise

Xpand Nitric Oxide Reactor is a ‘cocktail’ supplement proposed to improve skeletal muscle blood flow via arginine’s effect on nitric oxide synthesis and vasodilation. Two other major ingredients, caffeine and creatine, cause vasoconstriction, which could potentially counteract the proposed hemodynamic effects of arginine. The purpose of this study was to examine the influence of Xpand Nitric Oxide Reactor on muscle re-oxygenation after resistance exercise compared to supplementation with constituent ingredients L-arginine alpha-ketoglutarate and caffeine. Nine recreationally active men (21±1y) performed 3 sets of 20 repetitions of seated single-leg calf raise at 60% 1-RM with 3 min rests. The same calf raise exercise was performed following 4 separate supplementation conditions: L-arginine alpha-ketoglutarate (AAKG), caffeine (CAFF), Xpand Nitric Oxide Reactor (XPAND), and placebo (PLAC). Soleus muscle re-oxygenation time was measured before, during, and immediately after exercise using near infrared spectroscopy. Supplementation with XPAND (0.43±0.03), AAKG (0.34±0.02), and CAFF (0.45±0.05) did not significantly affect muscle re-oxygenation halftime (minutes) compared to placebo (0.35±0.04). An arginine containing ‘cocktail’ supplement did not affect skeletal muscle re-oxygenation after resistance exercise, possibly due to a wash-out effect caused by the multiple ingredients.