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• W2891145500 abstract "•Sarcolipin (SLN) regulates thermogenesis and energy metabolism in skeletal muscle•Loss of SLN leads to obesity, whereas overexpression of SLN resists against obesity•SLN promotes mitochondrial biogenesis and oxidative phenotype in glycolytic muscle•SLN activates the CamKII-PGC1α signaling pathway to promote mitochondrial biogenesis The major objective of this study was to understand the molecular basis of how sarcolipin uncoupling of SERCA regulates muscle oxidative metabolism. Using genetically engineered sarcolipin (SLN) mouse models and primary muscle cells, we demonstrate that SLN plays a crucial role in mitochondrial biogenesis and oxidative metabolism in muscle. Loss of SLN severely compromised muscle oxidative capacity without affecting fiber-type composition. Mice overexpressing SLN in fast-twitch glycolytic muscle reprogrammed mitochondrial phenotype, increasing fat utilization and protecting against high-fat diet-induced lipotoxicity. We show that SLN affects cytosolic Ca2+ transients and activates the Ca2+/calmodulin-dependent protein kinase II (CamKII) and PGC1α axis to increase mitochondrial biogenesis and oxidative metabolism. These studies provide a fundamental framework for understanding the role of sarcoplasmic reticulum (SR)-Ca2+ cycling as an important factor in mitochondrial health and muscle metabolism. We propose that SLN can be targeted to enhance energy expenditure in muscle and prevent metabolic disease. The major objective of this study was to understand the molecular basis of how sarcolipin uncoupling of SERCA regulates muscle oxidative metabolism. Using genetically engineered sarcolipin (SLN) mouse models and primary muscle cells, we demonstrate that SLN plays a crucial role in mitochondrial biogenesis and oxidative metabolism in muscle. Loss of SLN severely compromised muscle oxidative capacity without affecting fiber-type composition. Mice overexpressing SLN in fast-twitch glycolytic muscle reprogrammed mitochondrial phenotype, increasing fat utilization and protecting against high-fat diet-induced lipotoxicity. We show that SLN affects cytosolic Ca2+ transients and activates the Ca2+/calmodulin-dependent protein kinase II (CamKII) and PGC1α axis to increase mitochondrial biogenesis and oxidative metabolism. These studies provide a fundamental framework for understanding the role of sarcoplasmic reticulum (SR)-Ca2+ cycling as an important factor in mitochondrial health and muscle metabolism. We propose that SLN can be targeted to enhance energy expenditure in muscle and prevent metabolic disease. Skeletal muscle constitutes 40%–50% of body mass in the human adult and is a key determinant of basal metabolic rate and whole-body energy metabolism. Muscle is the major consumer of glucose (nearly 80% of insulin-mediated glucose uptake) and fatty acids (DeFronzo et al., 1981DeFronzo R.A. Jacot E. Jequier E. Maeder E. Wahren J. Felber J.P. The effect of insulin on the disposal of intravenous glucose. Results from indirect calorimetry and hepatic and femoral venous catheterization.Diabetes. 1981; 30: 1000-1007Crossref PubMed Scopus (1392) Google Scholar, Thiebaud et al., 1982Thiebaud D. Jacot E. DeFronzo R.A. Maeder E. Jequier E. Felber J.P. The effect of graded doses of insulin on total glucose uptake, glucose oxidation, and glucose storage in man.Diabetes. 1982; 31: 957-963Crossref PubMed Scopus (328) Google Scholar). It has the ability to increase its energy expenditure 20- to 30-fold during intense exercise by stimulating insulin-independent glucose uptake and by switching to higher fatty acid uptake and oxidation (Lowell and Spiegelman, 2000Lowell B.B. Spiegelman B.M. Towards a molecular understanding of adaptive thermogenesis.Nature. 2000; 404: 652-660Crossref PubMed Scopus (1308) Google Scholar, Zurlo et al., 1990Zurlo F. Larson K. Bogardus C. Ravussin E. Skeletal muscle metabolism is a major determinant of resting energy expenditure.J. Clin. Invest. 1990; 86: 1423-1427Crossref PubMed Scopus (644) Google Scholar). Many studies suggest that enhancing energy expenditure in muscle through physical activity could be the most effective strategy for controlling obesity and diabetes, second only to caloric restriction (Gabriel and Zierath, 2017Gabriel B.M. Zierath J.R. The limits of exercise physiology: from performance to health.Cell Metab. 2017; 25: 1000-1011Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar). Apart from physical activity, muscle expends a significant amount of energy to maintain muscle mass through protein synthesis, repair, and regeneration. In addition to contractile function, muscle is the primary organ of heat production in most mammals through shivering and nonshivering thermogenesis, demanding a considerable amount of energy (Arruda et al., 2007Arruda A.P. Nigro M. Oliveira G.M. de Meis L. Thermogenic activity of Ca2+-ATPase from skeletal muscle heavy sarcoplasmic reticulum: the role of ryanodine Ca2+ channel.Biochim. Biophys. Acta. 2007; 1768: 1498-1505Crossref PubMed Scopus (33) Google Scholar, Bal et al., 2012Bal N.C. Maurya S.K. Sopariwala D.H. Sahoo S.K. Gupta S.C. Shaikh S.A. Pant M. Rowland L.A. Bombardier E. Goonasekera S.A. et al.Sarcolipin is a newly identified regulator of muscle-based thermogenesis in mammals.Nat. Med. 2012; 18: 1575-1579Crossref PubMed Scopus (367) Google Scholar, Block, 1994Block B.A. Thermogenesis in muscle.Annu. Rev. Physiol. 1994; 56: 535-577Crossref PubMed Scopus (217) Google Scholar, Lowell and Spiegelman, 2000Lowell B.B. Spiegelman B.M. Towards a molecular understanding of adaptive thermogenesis.Nature. 2000; 404: 652-660Crossref PubMed Scopus (1308) Google Scholar, van Marken Lichtenbelt and Daanen, 2003van Marken Lichtenbelt W.D. Daanen H.A. Cold-induced metabolism.Curr. Opin. Clin. Nutr. Metab. Care. 2003; 6: 469-475Crossref PubMed Google Scholar). Data also suggest that muscle can expend energy in the form of heat during diet-induced thermogenesis; however, the mechanisms are not fully understood (Bachman et al., 2002Bachman E.S. Dhillon H. Zhang C.Y. Cinti S. Bianco A.C. Kobilka B.K. Lowell B.B. BetaAR signaling required for diet-induced thermogenesis and obesity resistance.Science. 2002; 297: 843-845Crossref PubMed Scopus (637) Google Scholar, Bal et al., 2012Bal N.C. Maurya S.K. Sopariwala D.H. Sahoo S.K. Gupta S.C. Shaikh S.A. Pant M. Rowland L.A. Bombardier E. Goonasekera S.A. et al.Sarcolipin is a newly identified regulator of muscle-based thermogenesis in mammals.Nat. Med. 2012; 18: 1575-1579Crossref PubMed Scopus (367) Google Scholar, Bombardier et al., 2013Bombardier 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. 2013; 27: 3871-3878Crossref PubMed Scopus (43) Google Scholar). Our laboratory has been exploring the role of sarcoplasmic reticulum (SR)-Ca2+ cycling, especially the role of sarcolipin (SLN) and the sarcoendoplasmic reticulum Ca2+ ATPase (SERCA) pump in nonshivering thermogenesis (NST). Although SERCA-mediated ATP hydrolysis is generally coupled to Ca2+ transport, we and others have shown that binding of SLN within the SERCA-transmembrane groove promotes uncoupling of SERCA Ca2+ transport from ATP hydrolysis; by this mechanism, SLN increases futile SERCA activity, ATP hydrolysis, and thus heat production (Mall et al., 2006Mall S. Broadbridge R. Harrison S.L. Gore M.G. Lee A.G. East J.M. The presence of sarcolipin results in increased heat production by Ca(2+)-ATPase.J. Biol. Chem. 2006; 281: 36597-36602Crossref PubMed Scopus (82) Google Scholar, Sahoo et al., 2013Sahoo S.K. Shaikh S.A. Sopariwala D.H. Bal N.C. Periasamy M. Sarcolipin protein interaction with sarco(endo)plasmic reticulum Ca2+ ATPase (SERCA) is distinct from phospholamban protein, and only sarcolipin can promote uncoupling of the SERCA pump.J. Biol. Chem. 2013; 288: 6881-6889Crossref PubMed Scopus (77) Google Scholar, Sahoo et al., 2015Sahoo S.K. Shaikh S.A. Sopariwala D.H. Bal N.C. Bruhn D.S. Kopec W. Khandelia H. Periasamy M. The N terminus of sarcolipin plays an important role in uncoupling sarco-endoplasmic reticulum Ca2+-ATPase (SERCA) ATP hydrolysis from Ca2+ transport.J. Biol. Chem. 2015; 290: 14057-14067Crossref PubMed Scopus (42) Google Scholar, Smith et al., 2002Smith W.S. Broadbridge R. East J.M. Lee A.G. Sarcolipin uncouples hydrolysis of ATP from accumulation of Ca2+ by the Ca2+-ATPase of skeletal-muscle sarcoplasmic reticulum.Biochem. J. 2002; 361: 277-286Crossref PubMed Scopus (105) Google Scholar, Toyoshima et al., 2013Toyoshima C. Iwasawa S. Ogawa H. Hirata A. Tsueda J. Inesi G. Crystal structures of the calcium pump and sarcolipin in the Mg2+-bound E1 state.Nature. 2013; 495: 260-264Crossref PubMed Scopus (163) Google Scholar, Winther et al., 2013Winther A.M. Bublitz M. Karlsen J.L. Møller J.V. Hansen J.B. Nissen P. Buch-Pedersen M.J. The sarcolipin-bound calcium pump stabilizes calcium sites exposed to the cytoplasm.Nature. 2013; 495: 265-269Crossref PubMed Scopus (143) Google Scholar). Using genetically engineered SLN mouse models, we previously reported that SLN plays an important role in muscle thermogenesis and energy metabolism (Bal et al., 2012Bal N.C. Maurya S.K. Sopariwala D.H. Sahoo S.K. Gupta S.C. Shaikh S.A. Pant M. Rowland L.A. Bombardier E. Goonasekera S.A. et al.Sarcolipin is a newly identified regulator of muscle-based thermogenesis in mammals.Nat. Med. 2012; 18: 1575-1579Crossref PubMed Scopus (367) Google Scholar, Bombardier et al., 2013Bombardier 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. 2013; 27: 3871-3878Crossref PubMed Scopus (43) Google Scholar). Loss of SLN in muscle predisposes mice to develop hypothermia and failure to maintain whole-body temperature during cold exposure. Our studies also revealed that mice lacking SLN were prone to diet-induced obesity, whereas overexpression of SLN in fast and slow twitch fibers led to increased energy expenditure and resistance against high-fat diet-induced obesity (Maurya et al., 2015Maurya S.K. Bal N.C. Sopariwala D.H. Pant M. Rowland L.A. Shaikh S.A. Periasamy M. Sarcolipin is a key determinant of the basal metabolic rate, and its overexpression enhances energy expenditure and resistance against diet-induced obesity.J. Biol. Chem. 2015; 290: 10840-10849Crossref PubMed Scopus (97) Google Scholar, Sopariwala et al., 2015Sopariwala D.H. Pant M. Shaikh S.A. Goonasekera S.A. Molkentin J.D. Weisleder N. Ma J. Pan Z. Periasamy M. Sarcolipin overexpression improves muscle energetics and reduces fatigue.J. Appl. Physiol. 2015; 118: 1050-1058Crossref PubMed Scopus (42) Google Scholar). Although these studies implicate SLN as an important regulator of muscle thermogenesis and energy expenditure, the detailed mechanism of how SLN programs and orchestrates oxidative metabolism is not understood. Therefore, in this study, we set out to address the mechanistic basis of SLN signaling both in vivo and in primary muscle cells derived for Sln knockout (KO) and wild-type (WT) mice. A major objective of this study was to determine the role of SLN in programming mitochondrial phenotype and enhancing fatty acid oxidation in skeletal muscle. SLN expression is induced severalfold during early neonatal stages of muscle development (in both fast and slow twitch muscle), but its role is not well understood. The neonatal stage is precarious in a newborn’s life, with a significant demand for energy to maintain thermogenesis and survive cold. Furthermore, the muscle is undergoing significant growth, differentiation, and maturation due to increased physical activity. To understand the role of SLN in neonatal physiology, we first investigated the relevance of SLN to mitochondrial biogenesis and oxidative metabolism in neonatal muscle of WT and Sln-KO mice. SLN expression peaks around birth and continues to be expressed at high levels in all skeletal muscles during the first 10 days of neonatal development (Babu et al., 2007aBabu G.J. Bhupathy P. Carnes C.A. Billman G.E. Periasamy M. Differential expression of sarcolipin protein during muscle development and cardiac pathophysiology.J. Mol. Cell. Cardiol. 2007; 43: 215-222Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar, Pant et al., 2015Pant M. Bal N.C. Periasamy M. Cold adaptation overrides developmental regulation of sarcolipin expression in mice skeletal muscle: SOS for muscle-based thermogenesis?.J Exp Biol. 2015; 218: 2321-2325Crossref PubMed Scopus (26) Google Scholar, Pant et al., 2016Pant M. Bal N.C. Periasamy M. Sarcolipin: a key thermogenic and metabolic regulator in skeletal muscle.Trends Endocrinol Metab. 2016; 27: 881-892Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar) (Figure 1A). By day 15 postnatally, SLN expression is downregulated in mature glycolytic muscles, including quadriceps, extensor digitorum longus (EDL), and tibialis anterior (TA), but continues to be expressed in oxidative and/or slow twitch muscle, including soleus, red gastrocnemius, and diaphragm (Pant et al., 2015Pant M. Bal N.C. Periasamy M. Cold adaptation overrides developmental regulation of sarcolipin expression in mice skeletal muscle: SOS for muscle-based thermogenesis?.J Exp Biol. 2015; 218: 2321-2325Crossref PubMed Scopus (26) Google Scholar, Pant et al., 2016Pant M. Bal N.C. Periasamy M. Sarcolipin: a key thermogenic and metabolic regulator in skeletal muscle.Trends Endocrinol Metab. 2016; 27: 881-892Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar). This raised the possibility that SLN is important for maintaining the oxidative metabolic phenotype in muscle. Loss of SLN had no effect on muscle fiber size (Figure S1A) and or the expression level of major contractile and SR proteins, including SERCA, calsequestrin 1 and 2 (CASQ1 and CASQ2), muscle α-actin, and α-actinin (Figure 1B). We next studied how loss of SLN expression affected muscle oxidative capacity in isolated quadriceps muscle from WT and Sln-KO mice (10 days old) using high-resolution respirometry. In Sln-KO muscle compared to WT, there was a significant decrease in fatty acid (palmitate)-supported oxygen consumption and a significant reduction in succinate-induced maximal respiration, a sign of reduced fatty acid oxidation (Figure 1C). This is supported by a significant reduction in mtDNA content (Figure 1D) and a drastic reduction in succinate dehydrogenase (SDH) activity in the Sln-KO muscle (Figure 1E), indicative of decreased oxidative capacity. Expression of mitochondrial oxidative phosphorylation (OXPHOS) proteins (Figure 1F) and enzymes involved in lipid metabolism, including lipoprotein lipase (LPL), carnitine palmitoyltransferase-1 mitochondrial (CPT1-M), long-chain acyl-coenzyme A (CoA) dehydrogenase (LCAD), hydroxyacyl-CoA dehydrogenase trifunctional multienzyme complex subunit beta (HADHB), citrate synthase, and adenine nucleotide translocator (ANT1/2), in Sln-KO muscle compared to WT (Figure 1G). In addition, electron microscopy of Sln-KO muscle revealed alteration in mitochondrial morphology, with fewer cristae of smaller length and width (Figures S1B and S1C). However, the expression of genes involved in mitochondrial dynamics, including fusion and fission, was not significantly altered (Figures S1D and S1E). Proteins involved in mitochondrial fusion and fission were higher when normalized to mitochondrial protein VDAC (Figures S1F and S1G). These data suggest that loss of SLN leads to decreased mitochondrial mass but increased mitochondrial dynamics in muscle. We next investigated the autophagy by performing western blotting of LC3 (Figures S1H and S1I) and immunostaining with LC3 and COX4-I1 antibodies (Figures S1J and S1K). The LC3-II/LC3-I ratio, which is an indicator of increased autophagy, is greater in KO muscle (Figures S1H and S1I). Similarly, KO muscle showed higher LC3 staining when normalized with mitochondrial COX4-I1 staining (Figures S1J and S1K). To our surprise, the 10-day-old Sln-KO quadriceps (quads) muscle expressed higher levels of glycolytic enzymes, including phosphofructokinase 1 (PFK1), hexokinase II (HKII), pyruvate kinase muscle (PKM2), and pyruvate dehydrogenase (PDH), which suggest that Sln-KO muscle primarily relies on glycolytic metabolism to compensate for decreased fatty acid utilization (Figure 1H). There was an activation of 5′ adenosine monophosphate-activated protein (AMP) kinase, alpha (AMPKα) (increased phospho-AMPKα level) in Sln-KO quads, indicating a compensatory mechanism to increase energy production (Narkar et al., 2008Narkar V.A. Downes M. Yu R.T. Embler E. Wang Y.X. Banayo E. Mihaylova M.M. Nelson M.C. Zou Y. Juguilon H. et al.AMPK and PPARdelta agonists are exercise mimetics.Cell. 2008; 134: 405-415Abstract Full Text Full Text PDF PubMed Scopus (941) Google Scholar, Ojuka, 2004Ojuka E.O. Role of calcium and AMP kinase in the regulation of mitochondrial biogenesis and GLUT4 levels in muscle.Proc. Nutr. Soc. 2004; 63: 275-278Crossref PubMed Scopus (192) Google Scholar) (Figure 1H). These data suggest that SLN is essential for the metabolic switch toward increased fatty acid oxidation in developing muscle. However, quads and gastrocnemius muscle of 10-day-old neonatal Sln-KO mice did not show alterations in the expression of myosin isoforms and fiber-type composition (Figures S1L–S1N). We investigated whether loss of SLN affected oxidative metabolism in soleus muscle of adult mice, which expresses SLN throughout life. In rodents, soleus muscle contains a mixture of both fast oxidative fibers, expressing myosin heavy-chain type IIA (MHC-IIA), and slow twitch fibers, containing the myosin heavy-chain type I (MHC-I) isoform (Rowe et al., 2013Rowe G.C. Patten I.S. Zsengeller Z.K. El-Khoury R. Okutsu M. Bampoh S. Koulisis N. Farrell C. Hirshman M.F. Yan Z. et al.Disconnecting mitochondrial content from respiratory chain capacity in PGC-1-deficient skeletal muscle.Cell Rep. 2013; 3: 1449-1456Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar), with higher mitochondrial content. Our data show that loss of SLN does not alter mtDNA content in soleus muscle (Figure 1I). Western blot analyses show that the expression level of mitochondrial OXPHOS proteins is not altered in Sln-KO soleus compared to WT (Figure 1J); however, there is a reduction in enzymes involved in lipid metabolism (LPL, CPT1-M, LCAD, and HADHB) (Figure 1K) and the ability to oxidize fatty acid (Figure 1L). Enzymes involved in glucose metabolism were not affected (Figure 1M). SDH activity staining confirmed that loss of SLN in soleus muscle decreases its oxidative capacity (Figure 1N). Surprisingly, loss of SLN does not affect the composition of MHC-IIA and MHC-I fibers in soleus muscle (Figure 1O). These data suggest that SLN is critical for maintaining muscle oxidative metabolism, but not fiber-type switching. Because SLN expression is downregulated in adult glycolytic muscle, we asked whether transgenic expression of SLN in glycolytic muscle promotes oxidative metabolism using the skeletal muscle-specific SLN overexpression (SlnOE) mouse model (Sopariwala et al., 2015Sopariwala D.H. Pant M. Shaikh S.A. Goonasekera S.A. Molkentin J.D. Weisleder N. Ma J. Pan Z. Periasamy M. Sarcolipin overexpression improves muscle energetics and reduces fatigue.J. Appl. Physiol. 2015; 118: 1050-1058Crossref PubMed Scopus (42) Google Scholar). Overexpression of SLN in glycolytic muscle (TA, quads, and gastrocnemius) did not result in fiber-type switching, and the numbers of oxidative and glycolytic muscle fibers were similar to those of WT muscle (Figures S2A and S2B). In addition, SLN overexpression did not affect mtDNA content (Figure 2A) or the expression level of OXPHOS proteins in TA muscle (Figure 2B). However, SLN overexpression caused an increase in fatty acid transport proteins, especially CD36, CPT1-M, LPL, and mitochondrial enzymes involved in fatty acid oxidation, like LCAD and HADHB (Figure 2C). Furthermore, glycolytic muscle fibers overexpressing SLN showed higher fatty acid oxidation as measured by oxidation of fatty acid substrates (palmitoylCoA + carnitine) in comparison to Sln-KO muscle (Figure 2D). Expression of fusion and fission genes was not significantly different (Figure S1C). However, when expression of fusion and fission proteins was normalized with VDAC protein levels, SlnOE muscle showed a lower abundance of mitochondrial dynamic proteins (Figures S2D and S2E). We next investigated whether higher SLN expression and activity can prevent high-fat diet (HFD)-induced metabolic abnormalities in muscle. HFD feeding induced a significant increase in mtDNA copy number (Figure 2E), the expression of OXPHOS protein complexes (Figure 2F), and enzymes involved in fatty acid metabolism in glycolytic muscle (TA) of SlnOE compared to WT muscle (Figure 2G). This increase in mitochondrial metabolic enzymes was supported by a significant increase in fatty acid oxidation (palmitatoylcarnitine) in SlnOE muscle (Figure 2H). TA muscle of SlnOE mice did not show lipid accumulation compared to WT muscle (very low SLN expression) (Figure 2I) and Sln-KO muscle (Figure S2F). Conversely, Sln-KO soleus showed significant accumulation of lipid droplets compared to WT soleus (high SLN expression) (Figure 2J), a sign of decreased fat utilization, whereas muscle overexpressing SLN did not show signs of lipid accumulation. Furthermore, we observed a lower intramuscular concentration of ceramide, diacylglycerides (DAGs), and acylcarnitines in SlnOE muscle compared to WT (Figure 2K). Studies have shown that excess accumulation of these lipid intermediates contributes to lipotoxicity and insulin resistance in skeletal muscle (Samuel and Shulman, 2012Samuel V.T. Shulman G.I. Mechanisms for insulin resistance: common threads and missing links.Cell. 2012; 148: 852-871Abstract Full Text Full Text PDF PubMed Scopus (1480) Google Scholar, Shulman, 2014Shulman G.I. Ectopic fat in insulin resistance, dyslipidemia, and cardiometabolic disease.N. Engl. J. Med. 2014; 371: 1131-1141Crossref PubMed Scopus (427) Google Scholar, Szendroedi et al., 2014Szendroedi J. Yoshimura T. Phielix E. Koliaki C. Marcucci M. Zhang D. Jelenik T. Müller J. Herder C. Nowotny P. et al.Role of diacylglycerol activation of PKCθ in lipid-induced muscle insulin resistance in humans.Proc. Natl. Acad. Sci. USA. 2014; 111: 9597-9602Crossref PubMed Scopus (271) Google Scholar); therefore, we next investigated the muscle glucose uptake and glucose clearance in live mice using hyperinsulinemic-euglycemic clamp (Figure 2L). As expected, SlnOE mice displayed a higher rate of muscle glucose uptake and clearance compared to WT mice, suggesting enhanced muscle insulin sensitivity. Conversely, Sln-KO mice showed a lower rate of muscle glucose uptake and clearance compared to WT mice, suggesting insulin resistance in Sln-KO muscle (Figure S2G). Altogether, these findings strongly suggest that increased SLN expression in muscle reprograms mitochondria, enhances fatty acid oxidation, and protects against lipotoxicity, thus improving insulin sensitivity. To explore the mechanistic details of how SLN regulates mitochondrial phenotype, we took advantage of primary muscle cell cultures derived from Sln-KO and WT mice (Figure 3A). SLN is not expressed in myoblast, but its expression is relatively high in differentiating WT myotubes (Figure 3B). Both WT and Sln-KO myotubes expressed normal levels of contractile (α-actinin and α-actin) and SR-Ca2+ transporter SERCA2 (Figure 3C). However, Sln-KO myotubes showed a significant decrease in mitochondrial content, as observed by lower mtDNA copy number (Figure 3D) and decreased expression of OXPHOS protein complexes (Figure 3E). However, the expression of genes involved in mitochondrial dynamics, including fusion and fission, was not significantly altered (Figures S3A and S3B). Sln-KO myotubes showed a lower rate of fatty acid oxidation and a decreased oxygen consumption rate (OCR), supporting the lower mitochondrial content (Figures 3F and 3G), whereas the rate of glycolysis and glycolytic capacity (as measured by the extracellular acidification rate [ECAR]) of Sln-KO myotubes was higher than that of WT myotubes (Figures S3C and S3D). We next tested whether re-expression of SLN through adenoviral gene transfer could rescue the mitochondrial phenotype in Sln-KO myotubes (Figure 3H). Re-expression of SLN in Sln-KO myotubes restored mtDNA content (Figure 3I), respiratory capacity (OCR) (Figure 3J), and PGC1α expression to WT control levels (Figure 3K). In WT myotubes, PGC1α expression increased progressively during differentiation (Figure 3L), whereas loss of SLN caused a decrease in the expression level of PGC1α, PPARδ, and TFAM (Figures 3L and 3M). PGC1α is the master regulator of mitochondrial biogenesis, especially in muscle. Therefore, we next addressed whether SLN mediates its effect through upregulation of PGC1α by using small interfering RNA (siRNA)-mediated knockdown (KD) of PGC1α in WT and Sln-KO primary myotubes. As previously reported, PGC1α KD in WT myotubes decreased mtDNA copy number, OXPHOS protein expression, and OCR (Figures 3N–3P). We tested whether the rescue of Sln-KO myotubes by adenoviral SLN gene transfer is mediated through PGC1α. We show that knocking down PGC1α blunted the effect of adenoviral SLN (Ad-SLN) on mitochondrial biogenesis, including mtDNA copy number, OXPHOS protein expression, and OCR (Figures 3N–3P; Figure S3C). Conversely, overexpression of PGC1α in Sln-KO myotubes partially rescued mitochondrial biogenesis compared to WT myotubes (Figure 3Q). These experiments demonstrate that SLN recruits PGC1α to promote mitochondrial biogenesis. To determine whether SLN affected cytosolic Ca2+ transients, we performed Ca2+ imaging using green fluorescent dye (Fluo-4) in C2C12 myotubes expressing high levels of SLN through Ad-SLN gene transfer (Figures 4A–4C). Myotubes expressing SLN showed a higher percentage of cytosolic Ca2+ transients (>18%) compared to nontransfected (NT) myotubes in response to caffeine-induced Ca2+ release. The Ad-SLN-transfected myotubes show an increase of >2 s in the average time of fluorescence in the cytosol, measured at 50% of the peak; these myotubes also showed a slower decay of the fluorescence in the cytosol, as measured by the slope (0.088 interval [int]/ms) compared to NT (0.090 int/ms) (Figure 4C). These data indicate a slower Ca2+ removal by SERCA in the presence of SLN. However, fluorescence measured 60 s post-stimulation with caffeine showed higher levels of cytosolic Ca2+ in Ad-SLN-transfected myotubes (>28%) compared to NT myotubes (Figure 4C). Cytosolic Ca2+ remained higher over the course of the experiment in Ad-SLN-transfected myotubes compared to NT control myotubes. These data suggest that SLN shapes cytosolic Ca2+ dynamics by increasing the duration of Ca2+ transients in the cytosol, which activates Ca2+-dependent signaling mechanisms. To determine whether SR-Ca2+ cycling is a prerequisite for SLN signaling, we manipulated SR-Ca2+ release in myotubes using caffeine to sensitize (Darcy et al., 2016Darcy Y.L. Diaz-Sylvester P.L. Copello J.A. K201 (JTV519) is a Ca2+-dependent blocker of SERCA and a partial agonist of ryanodine receptors in striated muscle.Mol. Pharmacol. 2016; 90: 106-115Crossref PubMed Scopus (7) Google Scholar) and dantrolene to inhibit RYR1, the primary Ca2+ release channel in skeletal muscle SR (Cherednichenko et al., 2008Cherednichenko G. Ward C.W. Feng W. Cabrales E. Michaelson L. Samso M. López J.R. Allen P.D. Pessah I.N. Enhanced excitation-coupled calcium entry in myotubes expressing malignant hyperthermia mutation R163C is attenuated by dantrolene.Mol. Pharmacol. 2008; 73: 1203-1212Crossref PubMed Scopus (89) Google Scholar). Treatment with 3.5 mM caffeine resulted in an increase in phosphorylated Ca2+/calmodulin-dependent protein kinase II (pCamKII) activation and Mef2c expression in WT myotubes, but not in Sln-KO myotubes (Figure 4D). Caffeine treatment of WT myotubes increased mitochondrial biogenesis, as measured by mtDNA content (Figure 4E), PGC1α, (Figure 4F), and PPARδ, TFAM, and metabolic enzymes (Figure 4G), whereas Sln-KO myotubes failed to show an increase in mitochondrial biogenesis (Figures 4D–4G). Furthermore, caffeine-treated WT myotubes showed a significant increase in mitochondrial respiration, but Sln-KO myotubes showed a poor response to caffeine treatment (Figure 4H). In contrast, inhibition of SR-Ca2+ release by dantrolene treatment (10 μM) decreased CamKII activation, mitochondrial biogenesis (Figures 4I–4L), and respiratory capacity only in SLN-expressing myotubes (Figure 4M). We show that inhibition of CamKII activation by pretreatment with KN93 decreased mitochondrial biogenesis and respiratory capacity in WT myotubes. In contrast, SLN re-expression in Sln-KO myotubes rescued the phenotype, as evident by increased CamKII activity, mtDNA content, and respiration (Figures 4N–4P). Altogether, these data demonstrate that SLN is essential for the Ca2+-dependent increase in mitochondrial biogenesis and this directly depends on SR-Ca2+ cycling and recruitment of the CamKII-PGC1α axis (Figure 4Q). SLN is an important regulator of the SERCA pump, which is expressed exclusively in striated muscle of all mammals, including humans (Paran et al., 2015Paran C.W. Verkerke A.R. Heden T.D. Park S. Zou K. Lawson H.A. Song H. Turk J. Houmard J.A. Funai K. Reduced efficiency of sarcolipin-dependent respiration in myocytes from humans with severe obesity.Obesity (Silver Spring). 2015; 23: 1440-1449Crossr" @default.
• W2891145500 created "2018-09-27" @default.
• W2891145500 creator A5000014440 @default.
• W2891145500 creator A5007553562 @default.
• W2891145500 creator A5012889147 @default.
• W2891145500 creator A5028299159 @default.
• W2891145500 creator A5060333286 @default.
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• W2891145500 date "2018-09-01" @default.
• W2891145500 modified "2023-10-18" @default.
• W2891145500 title "Sarcolipin Signaling Promotes Mitochondrial Biogenesis and Oxidative Metabolism in Skeletal Muscle" @default.
• W2891145500 cites W1575263278 @default.
• W2891145500 cites W1976555898 @default.
• W2891145500 cites W1982216040 @default.
• W2891145500 cites W1983069524 @default.
• W2891145500 cites W1983846086 @default.
• W2891145500 cites W1987441094 @default.
• W2891145500 cites W1991431964 @default.
• W2891145500 cites W1998414295 @default.
• W2891145500 cites W1999248384 @default.
• W2891145500 cites W2009265893 @default.
• W2891145500 cites W2015454275 @default.
• W2891145500 cites W2021052502 @default.
• W2891145500 cites W2022419155 @default.
• W2891145500 cites W2026678165 @default.
• W2891145500 cites W2028080851 @default.
• W2891145500 cites W2032245659 @default.
• W2891145500 cites W2034766774 @default.
• W2891145500 cites W2036841744 @default.
• W2891145500 cites W2040091217 @default.
• W2891145500 cites W2046771956 @default.
• W2891145500 cites W2047323330 @default.
• W2891145500 cites W2053765200 @default.
• W2891145500 cites W2055608265 @default.
• W2891145500 cites W2058258141 @default.
• W2891145500 cites W2061075485 @default.
• W2891145500 cites W2063936188 @default.
• W2891145500 cites W2064754499 @default.
• W2891145500 cites W2065650877 @default.
• W2891145500 cites W2065656177 @default.
• W2891145500 cites W2086102771 @default.
• W2891145500 cites W2087040379 @default.
• W2891145500 cites W2090875155 @default.
• W2891145500 cites W2105491011 @default.
• W2891145500 cites W2106141724 @default.
• W2891145500 cites W2121198137 @default.
• W2891145500 cites W2129266853 @default.
• W2891145500 cites W2130246533 @default.
• W2891145500 cites W2141248283 @default.
• W2891145500 cites W2145148177 @default.
• W2891145500 cites W2145537234 @default.
• W2891145500 cites W2147819535 @default.
• W2891145500 cites W2148979483 @default.
• W2891145500 cites W2151563579 @default.
• W2891145500 cites W2154449937 @default.
• W2891145500 cites W2158324747 @default.
• W2891145500 cites W2159233773 @default.
• W2891145500 cites W2160105048 @default.
• W2891145500 cites W2161081876 @default.
• W2891145500 cites W2166246053 @default.
• W2891145500 cites W2166598559 @default.
• W2891145500 cites W2169401893 @default.
• W2891145500 cites W2179494850 @default.
• W2891145500 cites W2215249885 @default.
• W2891145500 cites W2315898212 @default.
• W2891145500 cites W2400980195 @default.
• W2891145500 cites W2432906270 @default.
• W2891145500 cites W2493597249 @default.
• W2891145500 cites W2498400869 @default.
• W2891145500 cites W2520543091 @default.
• W2891145500 cites W2531174801 @default.
• W2891145500 cites W2552288287 @default.
• W2891145500 cites W2565398910 @default.
• W2891145500 cites W2610469329 @default.
• W2891145500 cites W2743591448 @default.
• W2891145500 cites W2765290991 @default.
• W2891145500 cites W2766894047 @default.
• W2891145500 cites W2767518339 @default.
• W2891145500 cites W2767572094 @default.
• W2891145500 cites W2767989432 @default.
• W2891145500 cites W2769445739 @default.
• W2891145500 cites W4234334798 @default.
• W2891145500 cites W68036047 @default.
• W2891145500 doi "https://doi.org/10.1016/j.celrep.2018.08.036" @default.
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