Chris Vigna

ATP Consumption by Sarcoplasmic Reticulum Ca2+ Pumps Accounts for 40-50% of Resting Metabolic Rate in Mouse Fast and Slow Twitch Skeletal Muscle

The main purpose of this study was to directly quantify the relative contribution of Ca2+ cycling to resting metabolic rate in mouse fast (extensor digitorum longus, EDL) and slow (soleus) twitch skeletal muscle. Resting oxygen consumption of isolated muscles (VO2, µL/g wet weight/s) measured polarographically at 30°C was ~20% higher (P<0.05) in soleus (0.326 ± 0.022) than in EDL (0.261 ± 0.020). In order to quantify the specific contribution of Ca2+ cycling to resting metabolic rate, the concentration of MgCl2 in the bath was increased to 10 mM to block Ca2+ release through the ryanodine receptor, thus eliminating a major source of Ca2+ leak from the sarcoplasmic reticulum (SR), and thereby indirectly inhibiting the activity of the sarco(endo) plasmic reticulum Ca2+-ATPases (SERCAs). The relative (%) reduction in muscle VO2 in response to 10 mM MgCl2 was similar between soleus (48.0±3.7) and EDL (42.4±3.2). Using a different approach, we attempted to directly inhibit SERCA ATPase activity in stretched EDL and soleus muscles (1.42x optimum length) using the specific SERCA inhibitor cyclopiazonic acid (CPA, up to 160 µM), but were unsuccessful in removing the energetic cost of Ca2+ cycling in resting isolated muscles. The results of the MgCl2 experiments indicate that ATP consumption by SERCAs is responsible for 40–50% of resting metabolic rate in both mouse fast- and slow-twitch muscles at 30°C, or 12–15% of whole body resting VO2. Thus, SERCA pumps in skeletal muscle could represent an important control point for energy balance regulation and a potential target for metabolic alterations to oppose obesity.

Enhanced Ca2+ transport and muscle relaxation in skeletal muscle from sarcolipin-null mice

Sarcolipin (SLN) inhibits sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA) pumps. To evaluate the physiological significance of SLN in skeletal muscle, we compared muscle contractility and SERCA activity between Sln-null and wild-type mice. SLN protein expression in wild-type mice was abundant in soleus and red gastrocnemius (RG), low in extensor digitorum longus (EDL), and absent from white gastrocnemius (WG). SERCA activity rates were increased in soleus and RG, but not in EDL or WG, from Sln-null muscles, compared with wild type. No differences were seen between wild-type and Sln-null EDL muscles in force-frequency curves or maximum rates of force development (+dF/dt). Maximum relaxation rates (-dF/dt) of EDL were higher in Sln-null than wild type across a range of submaximal stimulation frequencies, but not during a twitch or peak tetanic contraction. For soleus, no differences were seen between wild type and Sln-null in peak tetanic force or +dF/dt; however, force-frequency curves showed that peak force during a twitch and 10-Hz contraction was lower in Sln-null. Changes in the soleus force-frequency curve corresponded with faster rates of force relaxation at nearly all stimulation frequencies in Sln-null compared with wild type. Repeated tetanic stimulation of soleus caused increased (-dF/dt) in wild type, but not in Sln-null. No compensatory responses were detected in analysis of other Ca(2+) regulatory proteins using Western blotting and immunohistochemistry or myosin heavy chain expression using immunofluorescence. These results show that 1) SLN regulates Ca(2+)-ATPase activity thereby regulating contractile kinetics in at least some skeletal muscles, 2) the functional significance of SLN is graded to the endogenous SLN expression level, and 3) SLN inhibitory effects on SERCA function are relieved in response to repeated contractions thus enhancing relaxation rates.

Effects of ovarian sex hormones and downhill running on fiber-type-specific HSP70 expression in rat soleus

This study examined the influence of the ovarian sex hormones, estrogen and progesterone, on the fiber-type-specific response of the inducible 70-kDa heat shock protein (HSP70) to damaging exercise in rat soleus. Ovariectomized female rats were divided into three treatment groups (n = 16 per group): sham (S), progesterone (P; 25 mg pellet), and estrogen (E; 0.25 mg pellet). Each treatment group was divided into control and exercised groups. After 8 days of sham or hormone treatment, animals ran downhill intermittently for 90 min (17 m/min, -13.5 degrees grade) on a treadmill, and soleus muscles were removed 24 h postexercise. HSP70 expression was assessed in whole muscle homogenates by Western blotting and in individual muscle fiber types by immunohistochemical analysis of serial cross sections of soleus samples. Comparisons between control groups showed that HSP70 expression in soleus was increased (P < 0.05) in E compared with both S and P. No difference (P > 0.05) was observed between S and P. Following downhill running, HSP70 content in soleus was increased (P < 0.05) compared with control in S and P, but not (P > 0.05) in E. As a result, soleus HSP70 content following downhill running was not different (P > 0.05) between any of the treatment groups. Under all conditions, HSP70 content was higher in type I vs. type II fibers, and the effects of both estrogen and exercise on HSP70 expression in soleus were also more pronounced in type I vs. type II fibers. These results demonstrate that 1) estrogen regulates HSP70 expression in skeletal muscle, increasing basal HSP70 expression and preventing further increases in HSP70 in response to exercise; 2) progesterone is not involved in the regulation of HSP70 expression in skeletal muscle; and 3) the effects of estrogen and exercise on HSP70 expression in skeletal muscle are fiber type specific.

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

Ablation of sarcolipin decreases the energy requirements for Ca2+ transport by sarco(endo)plasmic reticulum Ca2+-ATPases in resting skeletal muscle.

The purpose of this study was to examine the effects of sarcolipin (SLN) on sarco(endo) plasmic reticulum Ca2+‐ATPase (SERCA pump) energetics in vivo and resting skeletal muscle metabolic rate. Using SLN knockout (Sln −/−) mice we show that SLN ablation increases the transport stoichiometry of SERCA pumps (Ca2+ uptake/Ca2+‐ATPase activity) and decreases the relative contribution of SERCA pumps to resting oxygen consumption (VO 2) in soleus without affecting soleus or whole body VO 2. These data suggest that the lower energy requirements for Ca2+ cycling in resting skeletal muscle of Sln −/− mice do not impact significantly either skeletal muscle or whole body metabolic rate.

ATP consumption by sarcoplasmic reticulum Ca2+ pumps accounts for 50% of resting metabolic rate in mouse fast and slow twitch skeletal muscle

In this study, we aimed to directly quantify the relative contribution of Ca(2+) cycling to resting metabolic rate in mouse fast-twitch (extensor digitorum longus, EDL) and slow-twitch (soleus) skeletal muscle. Resting oxygen consumption of isolated muscles (Vo(2), microl.g wet wt(-1).s(-1)) measured polarographically at 30 degrees C was approximately 25% higher in soleus (0.61 +/- .03) than in EDL (0.46 +/- .03). To quantify the specific contribution of Ca(2+) cycling to resting metabolic rate, cyclopiazonic acid (CPA), a highly specific inhibitor of sarco(endo)plasmic reticulum Ca(2+) ATPases (SERCAs), was added to the bath at different concentrations (1, 5, 10, and 15 microM). There was a concentration-dependent effect of CPA on Vo(2), with increasing CPA concentrations up to 10 microM resulting in progressively greater reductions in muscle Vo(2). There were no differences between 10 and 15 microM CPA, indicating that 10 microM CPA induces maximal inhibition of SERCAs in isolated muscle preparations. Relative reduction in muscle Vo(2) in response to CPA was nearly identical in EDL (1 microM, 10.6 +/- 3.0%; 5 microM, 33.2 +/- 3.4%; 10 microM, 49.2 +/- 2.9%; 15 microM, 50.9 +/- 2.1%) and soleus (1 microM, 11.2 +/- 1.5%; 5 microM, 37.7 +/- 2.4%; 10 microM, 50.0 +/- 1.3%; 15 microM, 49.9 +/- 1.6%). The results indicate that ATP consumption by SERCAs is responsible for approximately 50% of resting metabolic rate in both mouse fast- and slow-twitch muscles at 30 degrees C. Thus SERCA pumps in skeletal muscle could represent an important control point for energy balance regulation and a potential target for metabolic alterations to oppose obesity.

Initiating treadmill training in late middle age offers modest adaptations in Ca2+ handling but enhances oxidative damage in senescent rat skeletal muscle

Aging skeletal muscle shows an increased time to peak force and relaxation and a decreased specific force, all of which could relate to changes in muscle Ca(2+) handling. The purpose of this study was to determine if Ca(2+)-handling protein content and function are decreased in senescent gastrocnemius muscle and if initiating a training program in late middle age (LMA, 29 mo old) could improve function in senescent (34- to 36-mo-old) muscle. LMA male Fischer 344 x Brown-Norway rats underwent 5-7 mo of treadmill training. Aging resulted in a decrease in maximal sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA) activity and a decrease in Ca(2+) release rate but no change in Ca(2+) uptake rate. Efficiency of the Ca(2+) pump was increased with age, as was the content of SERCA2a. Training caused a further increase in SERCA2a content. Aging also caused an increase in protein carbonyl and reactive nitrogen species damage accumulation, and both further increased with training. Consistent with the increase in oxidative damage, heat shock protein 70 content was increased with age and further increased with training. Together, these results suggest that while initiating exercise training in LMA augments the age-related increase in expression of heat shock protein 70 and the more efficient SERCA2a isoform, it did not prevent the decrease in SERCA activity and exacerbated oxidative damage in senescent gastrocnemius muscle.

Cardiac calcium pump inactivation and nitrosylation in senescent rat myocardium are not attenuated by long-term treadmill training.

The senescent heart has decreased systolic and diastolic functions, both of which could be related to alterations in cardiac sarcoplasmic reticulum (SR) calcium (Ca(2+)) handling. The purpose of this study was to determine if SR protein content and rates of Ca(2+) release and uptake and ATPase activity are lower in the senescent (34-36 mo) Fisher 344×Brown-Norway F1 hybrid rat heart and if a long-term exercise training program could maintain SR function. Late middle aged (29 mo) male rats underwent 5-7 mo of treadmill training. Aging resulted in a decrease in SERCA activity and modest decrease in the rate of Ca(2+) uptake but no change in Ca(2+) release rate. SERCA2a content was not decreased with age but nitrotyrosine accumulation was increased and Ser16 phosphorylated phospholamban (PLN) was decreased. Ryanodine receptor content was not decreased with age but dihydropyridine receptor content was decreased in the senescent heart. Treadmill training had no significant effect on any of the SR properties or protein contents in the senescent rat heart. These results suggest that decreases in Ca(2+) uptake and SERCA activity in the senescent F344BN rat heart are due to increased SERCA2a damage from nitrotyrosine accumulation and inhibition by PLN and that exercise training initiated at late middle age is unable to prevent these age-related changes in cardiac SR function.

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.