Daniel Gamu

Co-Expression of SERCA Isoforms, Phospholamban and Sarcolipin in Human Skeletal Muscle Fibers

Sarcolipin (SLN) and phospholamban (PLN) inhibit the activity of sarco(endo)plasmic reticulum Ca2+-ATPases (SERCAs) by reducing their apparent affinity for Ca2+. A ternary complex between SLN, PLN, and SERCAs results in super-inhibition of SERCA activity. Analysis of skeletal muscle homogenate has limited our current understanding of whether SLN and PLN regulate SERCA1a, SERCA2a, or both in skeletal muscle and whether SLN and PLN are co-expressed in skeletal muscle fibers. Biopsies from human vastus lateralis were analyzed through single fiber Western blotting and immunohisto/fluorescence staining to circumvent this limitation. With a newly generated SLN antibody, we report for the first time that SLN protein is present in human skeletal muscle. Addition of the SLN antibody (50 µg) to vastus lateralis homogenates increased the apparent Ca2+ affinity of SERCA (K Ca, pCa units) (-Ab, 5.85 ± 0.02 vs. +Ab, 5.95 ± 0.02) and maximal SERCA activity (μmol/g protein/min) (-Ab, 122 ± 6.4 vs. +Ab, 159 ± 11) demonstrating a functional interaction between SLN and SERCAs in human vastus lateralis. Specifically, our results suggest that although SLN and PLN may preferentially regulate SERCA1a, and SERCA2a, respectively, physiologically they both may regulate either SERCA isoform. Furthermore, we show that SLN and PLN co-immunoprecipitate in human vastus lateralis homogenate and are simultaneously expressed in 81% of the fibers analyzed with Western blotting which implies that super-inhibition of SERCA may exist in human skeletal muscle. Finally, we demonstrate unequivocally that mouse soleus contains PLN protein suggesting that super-inhibition of SERCA may also be important physiologically in rodent skeletal muscle.

Sarcolipin trumps β-adrenergic receptor signaling as the favored mechanism for muscle-based diet-induced thermogenesis

Sarcolipin (SLN) regulates muscle‐based nonshivering thermogenesis and is up‐regulated with high‐fat feeding (HFF). To investigate whether other muscle‐based thermogenic systems compensate for a lack of Sln and to firmly establish SLN as a mediator of diet‐induced thermogenesis (DIT), we measured muscle and whole‐body energy expenditure in chow‐ and high‐fat‐fed Sln–/– and wild‐type (WT) mice. Following HFF, resting muscle metabolic rate (VO2, μl/g/s) was increased similarly in WT (0.28±0.02 vs. 0.31 ±0.03) and Sln–/– (0.23±0.03 vs. 0.35±0.02) mice due to increased sympathetic nervous system activation in Sln–/– mice; however, whole‐body metabolic rate (VO2, ml/kg/h) was lower in Sln–/– compared with WT mice following HFF but only during periods when the mice were active in their cages (WT, 2894±87 vs. Sln–/–, 2708±61). Treatment with the β‐adrenergic receptor (β‐AR) antagonist propranolol during HFF completely prevented muscle‐based DIT in Sln–/– mice; however, it had no effect in WT mice, resulting in greater differences in whole‐body metabolic rate and diet‐induced weight gain. Our results suggest that β‐AR signaling partially compensates for a lack of SLN to activate muscle‐based DIT, but SLN is the primary and more effective mediator.—Bombardier, E., Smith, I. C., Gamu, D., Fajardo, V. A., Vigna, C., Sayer, R. A., Gupta, S. C., Bal, N. C., Periasamy, M., Tupling, A. R., Sarcolipin trumps β‐adrenergic receptor signaling as the favored mechanism for muscle‐based diet‐induced thermogenesis. FASEB J. 27, 3871–3878 (2013). www.fasebj.org

Sarcolipin provides a novel muscle-based mechanism for adaptive thermogenesis.

The sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) transports Ca2+ into the sarcoplasmic reticulum lumen and contributes significantly to skeletal muscle metabolic rate. Sarcolipin (SLN) has been shown recently to uncouple Ca2+ transport from adenosine triphosphate hydrolysis by SERCA. We have hypothesized that SLN provides a novel mechanism of adaptive thermogenesis within skeletal muscle and protects against diet-induced obesity.

Phospholamban overexpression in mice causes a centronuclear myopathy-like phenotype.

ABSTRACT Centronuclear myopathy (CNM) is a congenital myopathy that is histopathologically characterized by centrally located nuclei, central aggregation of oxidative activity, and type I fiber predominance and hypotrophy. Here, we obtained commercially available mice overexpressing phospholamban (PlnOE), a well-known inhibitor of sarco(endo)plasmic reticulum Ca2+-ATPases (SERCAs), in their slow-twitch type I skeletal muscle fibers to determine the effects on SERCA function. As expected with a 6- to 7-fold overexpression of phospholamban, SERCA dysfunction was evident in PlnOE muscles, with marked reductions in rates of Ca2+ uptake, maximal ATPase activity and the apparent affinity of SERCA for Ca2+. However, our most significant discovery was that the soleus and gluteus minimus muscles from the PlnOE mice displayed overt signs of myopathy: they histopathologically resembled human CNM, with centrally located nuclei, central aggregation of oxidative activity, type I fiber predominance and hypotrophy, progressive fibrosis and muscle weakness. This phenotype is associated with significant upregulation of muscle sarcolipin and dynamin 2, increased Ca2+-activated proteolysis, oxidative stress and protein nitrosylation. Moreover, in our assessment of muscle biopsies from three human CNM patients, we found a significant 53% reduction in SERCA activity and increases in both total and monomeric PLN content compared with five healthy subjects, thereby justifying future studies with more CNM patients. Altogether, our results suggest that the commercially available PlnOE mouse phenotypically resembles human CNM and could be used as a model to test potential mechanisms and therapeutic strategies. To date, there is no cure for CNM and our results suggest that targeting SERCA function, which has already been shown to be an effective therapeutic target for murine muscular dystrophy and human cardiomyopathy, might represent a novel therapeutic strategy to combat CNM. Summary: Phospholamban overexpression in mouse slow-twitch muscle impairs SERCA function and causes histopathological features associated with human centronuclear myopathy.

Beetroot juice increases human muscle force without changing Ca2+-handling proteins

Dietary inorganic nitrate (NO3−) supplementation improves skeletal muscle (SkM) contractile efficiency, and although rodent literature has suggested improvements in calcium handling or redox modifications as likely explanations, the direct mechanism of action in humans remains unknown. Purpose This study aimed to examine the effects of 7 d of beetroot juice (BRJ) supplementation on SkM contractile characteristics and function. Methods Recreationally active males (n = 8) underwent transcutaneous electrical muscle stimulation of the vastus lateralis for the evaluation of contractile characteristics before and after 7 d of BRJ supplementation (280 mL·d−1, ~26 mmol NO3−). An additional group of individuals (n = 8) followed the same supplementation protocol but underwent SkM biopsies pre- and post-supplementation for the determination of proteins associated with calcium handling via Western blotting, and the ratio of reduced/oxidized glutathione (GSH:GSSG), an indicator of cellular redox state, via high-performance liquid chromatography (HPLC). Results After supplementation, there was no change in maximal voluntary force production (602 ± 50 vs 596 ± 56 N) or electrically induced tetanic contractions. By contrast, force production was increased at 10 Hz electrical stimulation (41.1% ± 2.3% vs 37.6% ± 2.4% of peak force, P < 0.05), as was peak twitch tension (164.0 ± 12.5 vs 136.5 ± 7.2 N, P < 0.01) and maximal rates of force development and relaxation (3582.8 ± 382.3 vs 2575.7 ± 196.2 and −2752.4 ± 423.9 vs −2104.4 ± 249.0 N·s−1, respectively, P < 0.05). Despite these measurements implicating a change in calcium handling, the content of associated proteins (SERCA1a, SERCA2a, dihydropyradine receptor, ryanodine receptor, and calsequestrin) and the GSH:GSSG ratio were unaltered by BRJ. Conclusion BRJ supplementation increases force production at low-stimulation frequencies; however, in human SkM, this is independent of changes in redox stress or the expression of protein targets associated with calcium handling.

Sarcolipin knockout mice fed a high-fat diet exhibit altered indices of adipose tissue inflammation and remodeling

To investigate indices of adipose tissue inflammation and remodeling in high‐fat diet (HFD) sarcolipin‐knockout (SLN−/−) mice. SLN regulates muscle‐based nonshivering thermogenesis and is up‐regulated with HFD. SLN−/− mice develop greater diet‐induced obesity and glucose intolerance. This is accompanied by increases in circulating catecholamines and fatty acids. Catecholamines and fatty acids play a role in the pathology of adipose tissue inflammation.

Persistence of diet-induced obesity despite access to voluntary activity in mice lacking sarcolipin

Several rodent models of obesity have been shown to develop excessive adiposity only when voluntary cage ambulation is restricted. We have previously shown that mice lacking the sarco(endo)plasmic reticulum Ca2+‐ATPase pump regulatory protein sarcolipin (Sln−/−), an uncoupler of Ca2+ uptake, develop excessive diet‐induced obesity under standard housing conditions. However, it is unclear whether this phenotype is due, in part, to the sedentary housing environment in which these animals are kept. To address this, we allowed wild‐type and Sln−/− animals ad libitum access to voluntary wheel running while consuming a standard chow or high‐fat diet for 8 weeks. During this period, wheel revolutions were monitored along with weekly mass gain. Postdiet glucose tolerance and visceral adiposity were also taken. The volume of wheel running completed was similar between genotype, regardless of diet. Although voluntary activity reduced mass gain relative to sedentary controls within each diet (P < 0.05), visceral adiposity was surprisingly unaltered with activity. However, Sln−/− mice developed excessive obesity (P < 0.05) and glucose intolerance (P < 0.05) with high‐fat feeding relative to wild‐type controls. These findings indicate that the excessive diet‐induced obese phenotype previously observed in Sln−/− mice is not the result of severely restricted daily ambulation, but in fact the inability to recruit uncoupling of the Ca2+‐ATPase pump.

Sarcolipin deletion exacerbates soleus muscle atrophy and weakness in phospholamban overexpressing mice

Sarcolipin (SLN) and phospholamban (PLN) are two small proteins that regulate the sarco(endo)plasmic reticulum Ca2+-ATPase pumps. In a recent study, we discovered that Pln overexpression (PlnOE) in slow-twitch type I skeletal muscle fibers drastically impaired SERCA function and caused a centronuclear myopathy-like phenotype, severe muscle atrophy and weakness, and an 8 to 9-fold upregulation of SLN protein in the soleus muscles. Here, we sought to determine the physiological role of SLN upregulation, and based on its role as a SERCA inhibitor, we hypothesized that it would represent a maladaptive response that contributes to the SERCA dysfunction and the overall myopathy observed in the PlnOE mice. To this end, we crossed Sln-null (SlnKO) mice with PlnOE mice to generate a PlnOE/SlnKO mouse colony and assessed SERCA function, CNM pathology, in vitro contractility, muscle mass, calcineurin signaling, daily activity and food intake, and proteolytic enzyme activity. Our results indicate that genetic deletion of Sln did not improve SERCA function nor rescue the CNM phenotype, but did result in exacerbated muscle atrophy and weakness, due to a failure to induce type II fiber compensatory hypertrophy and a reduction in total myofiber count. Mechanistically, our findings suggest that impaired calcineurin activation and resultant decreased expression of stabilin-2, and/or impaired autophagic signaling could be involved. Future studies should examine these possibilities. In conclusion, our study demonstrates the importance of SLN upregulation in combating muscle myopathy in the PlnOE mice, and since SLN is upregulated across several myopathies, our findings may reveal SLN as a novel and universal therapeutic target.

Muscle RANK is a key regulator of Ca2+ storage, SERCA activity, and function of fast-twitch skeletal muscles.

Receptor-activator of nuclear factor-κB (RANK), its ligand RANKL, and the soluble decoy receptor osteoprotegerin are the key regulators of osteoclast differentiation and bone remodeling. Here we show that RANK is also expressed in fully differentiated myotubes and skeletal muscle. Muscle RANK deletion has inotropic effects in denervated, but not in sham, extensor digitorum longus (EDL) muscles preventing the loss of maximum specific force while promoting muscle atrophy, fatigability, and increased proportion of fast-twitch fibers. In denervated EDL muscles, RANK deletion markedly increased stromal interaction molecule 1 content, a Ca(2+)sensor, and altered activity of the sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA) modulating Ca(2+)storage. Muscle RANK deletion had no significant effects on the sham or denervated slow-twitch soleus muscles. These data identify a novel role for RANK as a key regulator of Ca(2+)storage and SERCA activity, ultimately affecting denervated skeletal muscle function.

Fattening the role of Ca 2+ cycling in adaptive thermogenesis

A new study shows that deleting uncoupling protein 1 activates Ca2+ cycling thermogenesis within beige fat, protecting mice against cold-induced hypothermia and dysglycemia following diet-induced obesity.