Eric Bombardier

Sarcolipin is a newly identified regulator of muscle-based thermogenesis in mammals

The role of skeletal muscle in nonshivering thermogenesis (NST) is not well understood. Here we show that sarcolipin (Sln), a newly identified regulator of the sarco/endoplasmic reticulum Ca2+-ATPase (Serca) pump, is necessary for muscle-based thermogenesis. When challenged to acute cold (4 °C), Sln−/− mice were not able to maintain their core body temperature (37 °C) and developed hypothermia. Surgical ablation of brown adipose tissue and functional knockdown of Ucp1 allowed us to highlight the role of muscle in NST. Overexpression of Sln in the Sln-null background fully restored muscle-based thermogenesis, suggesting that Sln is the basis for Serca-mediated heat production. We show that ryanodine receptor 1 (Ryr1)-mediated Ca2+ leak is an important mechanism for Serca-activated heat generation. Here we present data to suggest that Sln can continue to interact with Serca in the presence of Ca2+, which can promote uncoupling of the Serca pump and cause futile cycling. We further show that loss of Sln predisposes mice to diet-induced obesity, which suggests that Sln-mediated NST is recruited during metabolic overload. These data collectively suggest that SLN is an important mediator of muscle thermogenesis and whole-body energy metabolism.

Initial aerobic power does not alter muscle metabolic adaptations to short-term training

To investigate the hypothesis that training-induced increases in muscle mitochondrial potential are not obligatory to metabolic adaptations observed during submaximal exercise, regardless of peak aerobic power (V˙o 2 peak) of the subjects, a short-term training study was utilized. Two groups of untrained male subjects ( n = 7/group), one with a high (HI) and the other with a low (LO)V˙o 2 peak(means ± SE; 51.4 ± 0.90 vs. 41.0 ± 1.3 ml ⋅ kg-1 ⋅ min-1; P< 0.05), cycled for 2 h/day at 66-69% ofV˙o 2 peak for 6 days. Muscle tissue was extracted from vastus lateralis at 0, 3, and 30 min of standardized cycle exercise before training (0 days) and after 3 and 6 days of training and analyzed for metabolic and enzymatic changes. During exercise after 3 days of training in the combined HI + LO group, higher ( P < 0.05) concentrations (mmol/kg dry wt) of phosphocreatine (40.5 ± 3.4 vs. 52.2 ± 4.2) and lower ( P < 0.05) concentrations of Pi (61.5 ± 4.4 vs. 53.3 ± 4.4), inosine monophosphate (0.520 ± 0.19 vs. 0.151 ± 0.05), and lactate (37.9 ± 5.5 vs. 22.8 ± 4.8) were observed. These changes were also accompanied by reduced levels of calculated free ADP, AMP, and Pi. All adaptations were fully expressed by 3 min of exercise and by 3 days of training and were independent of initialV˙o 2 peak levels. Moreover, maximal activity of citrate synthase, a measure of mitochondrial capacity, was only increased with 6 days of training (5.71 ± 0.29 vs. 7.18 ± 0.37 mol ⋅ kg protein-1 ⋅ h-1; P < 0.05). These results demonstrate that metabolic adaptations to prolonged exercise occur within the first 3 days of training and during the non-steady-state period. Moreover, neither time course nor magnitude of metabolic adaptations appears to depend on increases in mitochondrial potential or on initial aerobic power.

Ca2+ dysregulation in Ryr1I4895T/wt mice causes congenital myopathy with progressive formation of minicores, cores, and nemaline rods

Ryr1I4895T/wt (IT/+) mice express a knockin mutation corresponding to the human I4898T EC-uncoupling mutation in the type 1 ryanodine receptor/Ca2+ release channel (RyR1), which causes a severe form of central core disease (CCD). IT/+ mice exhibit a slowly progressive congenital myopathy, with neonatal respiratory stress, skeletal muscle weakness, impaired mobility, dorsal kyphosis, and hind limb paralysis. Lesions observed in myofibers from diseased mice undergo age-dependent transformation from minicores to cores and nemaline rods. Early ultrastructural abnormalities include sarcomeric misalignment, Z-line streaming, focal loss of cross-striations, and myofibrillar splitting and intermingling that may arise from defective myofibrillogenesis. However, manifestation of the disease phenotype is highly variable on a Sv129 genomic background. Quantitative RT-PCR shows an equimolar ratio of WT and mutant Ryr1 transcripts within IT/+ myofibers and total RyR1 protein expression levels are normal. We propose a unifying theory in which the cause of core formation lies in functional heterogeneity among RyR1 tetramers. Random combinations of normal and either leaky or EC-uncoupled RyR subunits would lead to spatial differences in Ca2+ transients; the resulting heterogeneity of contraction among myofibrils would lead to focal, irreversible tearing and shearing, which would, over time, enlarge to form minicores, cores, and nemaline rods. The IT/+ mouse line is proposed to be a valid model of RyR1-related congenital myopathy, offering high potential for elucidation of the pathogenesis of skeletal muscle disorders arising from impaired EC coupling.

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.

Organization of metabolic pathways in vastus lateralis of patients with chronic obstructive pulmonary disease

The objective of this study was to determine whether patients with chronic obstructive lung disease (COPD) display differences in organization of the metabolic pathways and segments involved in energy supply compared with healthy control subjects. Metabolic pathway potential, based on the measurement of the maximal activity (V(max)) of representative enzymes, was assessed in tissue extracted from the vastus lateralis in seven patients with COPD (age 67 +/- 4 yr; FEV(1)/FVC = 44 +/- 3%, where FEV(1) is forced expiratory volume in 1 s and FVC is forced vital capacity; means +/- SE) and nine healthy age-matched controls (age 68 +/- 2 yr; FEV(1)/FVC = 75 +/- 2%). Compared with control, the COPD patients displayed lower (P < 0.05) V(max) (mol.kg protein(-1).h(-1)) for cytochrome c oxidase (COX; 21.2 +/- 2.0 vs. 28.7 +/- 2.2) and 3-hydroxyacyl-CoA dehydrogenase (HADH; 2.54 +/- 0.14 vs. 3.74 +/- 0.12) but not citrate synthase (CS; 2.20 +/- 0.16 vs. 3.19 +/- 0.5). While no differences between groups were observed in V(max) for creatine phosphokinase, phosphorylase (PHOSPH), phosphofructokinase (PFK), pyruvate kinase, and lactate dehydrogenase, hexokinase (HEX) was elevated in COPD (P < 0.05). Enzyme activity ratios were higher (P < 0.05) for HEX/CS, HEX/COX, PHOSPH/HADH and PFK/HADH in COPD compared with control. It is concluded that COPD patients exhibit a reduced potential for both the electron transport system and fat oxidation and an increased potential for glucose phosphorylation while the potential for glycogenolysis and glycolysis remains normal. A comparison of enzyme ratios indicated greater potentials for glucose phosphorylation relative to the citric acid cycle and the electron transport chain and glycogenolysis and glycolysis relative to beta-oxidation.

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.

DJ-1 links muscle ROS production with metabolic reprogramming and systemic energy homeostasis in mice

Reactive oxygen species (ROS) have been linked to a wide variety of pathologies, including obesity and diabetes, but ROS also act as endogenous signalling molecules, regulating numerous biological processes. DJ-1 is one of the most evolutionarily conserved proteins across species, and mutations in DJ-1 have been linked to some cases of Parkinsons disease. Here we show that DJ-1 maintains cellular metabolic homeostasis via modulating ROS levels in murine skeletal muscles, revealing a role of DJ-1 in maintaining efficient fuel utilization. We demonstrate that, in the absence of DJ-1, ROS uncouple mitochondrial respiration and activate AMP-activated protein kinase, which triggers Warburg-like metabolic reprogramming in muscle cells. Accordingly, DJ-1 knockout mice exhibit higher energy expenditure and are protected from obesity, insulin resistance and diabetes in the setting of fuel surplus. Our data suggest that promoting mitochondrial uncoupling may be a potential strategy for the treatment of obesity-associated metabolic disorders.

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.

Quadriceps metabolism during constant workrate cycling exercise in chronic obstructive pulmonary disease

Impaired resting metabolism in peripheral muscles potentially contributes to exercise intolerance in chronic obstructive pulmonary disease (COPD). This study investigated the cytosolic energy metabolism of the quadriceps, from glycogen degradation to lactate accumulation, in exercising patients with COPD, in comparison to healthy controls. We measured, in 12 patients with COPD and 10 control subjects, resting and post-cycling exercise quadriceps levels of 1) energy substrates and end products of glycolysis (glycogen, glucose, pyruvate, and lactate) and intermediate markers of glycolysis (glucose-6-phosphate, glucose-1-phosphate, fructose-6-phosphate) and 2) the activity of key enzymes involved in the regulation of glycolysis (phosphofructokinase, lactate dehydrogenase). Exercise intensity (P < 0.01), duration (P = 0.049), and total work (P < 0.01) were reduced in patients with COPD. The variations in energy substrates and end products of glycolysis after cycling exercise were of similar magnitude in patients with COPD and controls. Glucose-6-phosphate (P = 0.036) and fructose-6-phosphate (P = 0.042) were significantly elevated in patients with COPD after exercise. Phosphofructokinase (P < 0.01) and lactate dehydrogenase (P = 0.02) activities were greater in COPD. Muscle glycogen utilization (P = 0.022) and lactate accumulation (P = 0.025) per unit of work were greater in COPD. We conclude that cycling exercise induced changes in quadriceps metabolism in patients with COPD that were of similar magnitude to those of healthy controls. These intramuscular events required a much lower exercise work load and time to occur in COPD. Our data suggest a greater reliance on glycolysis during exercise in COPD, which may contribute to exercise intolerance in COPD.