Search

SMYD1, a striated muscle-specific lysine methyltransferase, was originally shown to play a key role in embryonic cardiac development but more recently we demonstrated that loss of Smyd1 in the murine adult heart leads to cardiac hypertrophy and failure. However, the effects of SMYD1 overexpression in the heart and its molecular function in the cardiomyocyte in response to ischemic stress are unknown. In this study, we show that inducible, cardiomyocyte-specific overexpression of SMYD1a in mice protects the heart from ischemic injury as seen by a > 50% reduction in infarct size and decreased myocyte cell death. We also demonstrate that attenuated pathological remodeling is a result of enhanced mitochondrial respiration efficiency, which is driven by increased mitochondrial cristae formation and stabilization of respiratory chain supercomplexes within the cristae. These morphological changes occur concomitant with increased OPA1 expression, a known driver of cristae morphology and supercomplex formation. Together, these analyses identify OPA1 as a novel downstream target of SMYD1a whereby cardiomyocytes upregulate energy efficiency to dynamically adapt to the energy demands of the cell. In addition, these findings highlight a new epigenetic mechanism by which SMYD1a regulates mitochondrial energetics and functions to protect the heart from ischemic injury.

Weight loss from an overweight state is associated with a disproportionate decrease in whole-body energy expenditure that may contribute to the heightened risk for weight regain. Evidence suggests that this energetic mismatch originates from lean tissue. Although this phenomenon is well documented, the mechanisms have remained elusive. We hypothesized that increased mitochondrial energy efficiency in skeletal muscle is associated with reduced expenditure under weight loss. Wildtype (WT) male C57BL6/N mice were fed with high-fat diet for 10 weeks, followed by a subset of mice that were maintained on the obesogenic diet (OB) or switched to standard chow to promote weight loss (WL) for additional 6 weeks. Mitochondrial energy efficiency was evaluated using high-resolution respirometry and fluorometry. Mass spectrometric analyses were employed to describe the mitochondrial proteome and lipidome. Weight loss promoted ~50% increase in the efficiency of oxidative phosphorylation (ATP produced per O2 consumed, or P/O) in skeletal muscle. However, Weight loss did not appear to induce significant changes in mitochondrial proteome, nor any changes in respiratory supercomplex formation. Instead, it accelerated the remodeling of mitochondrial cardiolipin (CL) acyl-chains to increase tetralinoleoyl CL (TLCL) content, a species of lipids thought to be functionally critical for the respiratory enzymes. We further show that lowering TLCL by deleting the CL transacylase tafazzin was sufficient to reduce skeletal muscle P/O and protect mice from diet-induced weight gain. These findings implicate skeletal muscle mitochondrial efficiency as a novel mechanism by which weight loss reduces energy expenditure in obesity.

SummaryReactive oxygen species (ROS) accumulation is a cardinal feature of skeletal muscle atrophy. ROS refers to a collection of radical molecules whose cellular signals are vast, and it is unclear which downstream consequences of ROS are responsible for the loss of muscle mass and strength. Here we show that lipid hydroperoxides (LOOH) are increased with age and disuse, and the accumulation of LOOH by deletion of glutathione peroxidase 4 (GPx4) is sufficient to augment muscle atrophy. LOOH promoted atrophy in a lysosomal-dependent, proteasomal-independent manner. In young and old mice, genetic and pharmacologic neutralization of LOOH or their secondary reactive lipid aldehydes robustly prevented muscle atrophy and weakness, indicating that LOOH-derived carbonyl stress mediate age- and disuse-induced muscle dysfunction. Our findings provide novel insights for the role of LOOH in sarcopenia including a therapeutic implication by pharmacologic suppression.

Thermogenesis by uncoupling protein 1 (UCP1) is one of the primary mechanisms by which brown adipose tissue (BAT) increases energy expenditure. UCP1 resides in the inner mitochondrial membrane (IMM), where it dissipates membrane potential independent of adenosine triphosphate (ATP) synthase. Here, we provide evidence that phosphatidylethanolamine (PE) modulates UCP1-dependent proton conductance across the IMM to modulate thermogenesis. Mitochondrial lipidomic analyses revealed PE as a signature molecule whose abundance bidirectionally responds to changes in thermogenic burden. Reduction in mitochondrial PE by deletion of phosphatidylserine decarboxylase (PSD) made mice cold intolerant and insensitive to β3 adrenergic receptor agonist–induced increase in whole-body oxygen consumption. High-resolution respirometry and fluorometry of BAT mitochondria showed that loss of mitochondrial PE specifically lowers UCP1-dependent respiration without compromising electron transfer efficiency or ATP synthesis. These findings were confirmed by a reduction in UCP1 proton current in PE-deficient mitoplasts. Thus, PE performs a previously unknown role as a temperature-responsive rheostat that regulates UCP1-dependent thermogenesis.

Weight loss is associated with a disproportionate decrease in whole-body energy expenditure that may contribute to the heightened risk for weight-regain. Evidence suggests that this energetic mismatch originates from lean tissue. Although this phenomenon is well documented, the mechanisms have remained elusive. We hypothesized that increased mitochondrial energy efficiency in skeletal muscle is associated with reduced expenditure under weight loss. Wildtype male C57BL6/N mice were fed with high-fat diet for 10 wks, followed by a subset of mice that were maintained on the obesogenic diet (OB) or switched to standard chow to promote weight loss (WL) for additional 6 wks. Mitochondrial energy efficiency was evaluated using high-resolution respirometry and fluorometry. Mass spectrometric analyses were employed to describe the mitochondrial proteome and lipidome. Weight loss promoted ~50% increase in the efficiency of oxidative phosphorylation (ATP produced per O2consumed, or P/O) in skeletal muscle. However, weight loss did not appear to induce significant changes in mitochondrial proteome, nor any changes in respiratory supercomplex formation. Instead, it accelerated the remodeling of mitochondrial cardiolipin (CL) acyl-chains to increase tetralinoleoyl CL (TLCL) content, a species of lipids thought to be functionally critical for the respiratory enzymes. We further show that lowering TLCL by deleting the CL transacylase tafazzin was sufficient to reduce skeletal muscle P/O and protect mice from diet-induced weight gain. These findings implicate skeletal muscle mitochondrial efficiency as a novel mechanism by which weight loss reduces energy expenditure in obesity.

Carbohydrates and lipids provide the majority of substrates to fuel mitochondrial oxidative phosphorylation (OXPHOS). Metabolic inflexibility, defined as an impaired ability to switch between these fuels, is implicated in a number of metabolic diseases. Here we explore the mechanism by which physical inactivity promotes metabolic inflexibility in skeletal muscle. We developed a mouse model of sedentariness by small mouse cage (SMC) that, unlike other classic models of disuse in mice, faithfully recapitulates metabolic responses that occur in humans. Bioenergetic phenotyping of mitochondria displayed metabolic inflexibility induced by physical inactivity, demonstrated by a reduction in pyruvate-stimulated respiration (JO2) in absence of a change in palmitate-stimulatedJO2. Pyruvate resistance in these mitochondria was likely driven by a decrease in phosphatidylethanolamine (PE) abundance in the mitochondrial membrane. Reduction in mitochondrial PE by deletion of phosphatidylserine decarboxylase (PSD) was sufficient to induce metabolic inflexibility measured at the whole-body level, as well as at the level of skeletal muscle mitochondria. Low mitochondrial PE was sufficient to increase glucose flux towards lactate. We further implicate that resistance to pyruvate metabolism is due to attenuated mitochondrial entry via mitochondrial pyruvate carrier (MPC). These findings suggest a novel mechanism by which mitochondrial PE directly regulates MPC activity to modulate metabolic flexibility.

Accumulation of reactive oxygen species (ROS) is known to promote cellular damage in multiple cell-types. In skeletal muscle, ROS has been implicated in disuse-induced muscle atrophy. However, the molecular origin and mechanism of how disuse promotes ROS and muscle dysfunction remains unclear.Recently, we implicated membrane lipids of mitochondria to be a potential source of ROS to promote muscle atrophy.In this review, we discuss evidence that changes in mitochondrial lipids represent a physiologically-relevant process by which disuse promotes mitochondrial electron leak and oxidative stress.We further discuss lipid hydroperoxides (LOOH) as a potential downstream mediator of ROS to induce muscle atrophy.

SUMMARYThermogenesis by uncoupling protein 1 (UCP1) is one of the primary mechanisms by which brown adipose tissue (BAT) increases energy expenditure. UCP1 resides in the inner mitochondrial membrane (IMM), where it dissipates membrane potential independent of ATP synthase. Here we provide evidence that mitochondrial phosphatidylethanolamine (PE) directly regulates UCP1-dependent proton conductance across IMM to modulate thermogenesis. Mitochondrial lipidomic analyses revealed PE as a signature molecule whose abundance bidirectionally responds to changes in thermogenic burden. Reduction in mitochondrial PE by deletion of phosphatidylserine decarboxylase (PSD) made mice cold intolerant and insensitive to β3 adrenergic receptor agonist-induced increase in whole-body oxygen consumption. High-resolution respirometry and fluorometry of BAT mitochondria showed that loss of mitochondrial PE specifically lowers UCP1-dependent respiration without compromising electron transfer efficiency or ATP synthesis. These findings were confirmed by a reduction in UCP1 proton current in PSD-deficient mitoplasts. Thus, PE performs a previously unknown role as a temperature-responsive rheostat that regulates UCP1-dependent thermogenesis.

The histone lysine methyltransferase SMYD1 has been shown to be critical for embryonic cardiac development and in maintaining cardiomyocyte homeostasis in adult mice. Subsequently, we reported that loss of Smyd1 in the adult mouse myocardium leads to progressive cardiac hypertrophy and heart failure, which is accompanied with downregulation of mitochondrial proteins involved in oxidative phosphorylation, including Ppargc1a , and reduction of mitochondrial respiration capacity. To build upon these results and evaluate if SMYD1a can attenuate disease-induced remodeling in an animal model, we generated transgenic mice which inducibly express SMYD1a (the human ortholog) in cardiomyocytes and subjected them to permanent occlusion (PO) of the LAD. This lead to >50% reduction in infarct size and preserved cardiac function, as compared to littermate controls. Additionally, we demonstrated that under physiological conditions SMYD1a maintains metabolic homeostasis by regulating expression of Ppargc1a and its downstream targets, including components of the electron transport chain. Our molecular analysis shows that observed protection from ischemic injury results from enhanced mitochondrial respiration through Complex I and II as well as increased ATP production. This is associated with increased mitochondria cristae, and formation and stabilization of respiratory chain supercomplexes within the cristae. These changes in cristae structure occur concomitant with enhanced OPA1 expression, a major regulator of mitochondrial fusion and cristae morphology. Through this work we have established that OPA1 is a novel, functionally important downstream target of SMYD1a by which cardiomyocytes upregulate energy efficiency, protecting them from ischemic injury. These results also highlight SMYD1a as the only known epigenetic regulator of cristae morphology and provide broad implications for understanding the epigenetic mechanisms driving cardiac metabolism. Ultimately this work has identified a novel signaling pathway by which cardiomyocytes regulate energy efficiency, protecting them from ischemic injury.

Mitochondrial dysfunction due to calcium overload is common in heart disease and results in heart failure. In a mouse model of mitochondrial cardiomyopathies we noted a substantial downregulation of the gene encoding for the EF hand containing domain 1 protein (EFHD1). EFHD1 is a mitochondrial protein which binds calcium and has been shown to stimulate both apoptosis and mitoflashes. These processes involve mitochondrial calcium overload. We hypothesized that EFHD1 down regulation could represent a natural response by failing hearts to prevent mitochondrial calcium overload by increasing the calcium handling of mitochondria. We therefore studied the calcium handling properties of mice where EFHD1 had been knocked out using CRISPR Cas9. These mice are viable and we have found no adverse cardiac or metabolic phenotypes. We find that EFHD1 is expressed in the intermembrane space of the mitochondria and associates strongly with endoplasmic reticulum (ER)-associated mitochondrial membranes. Mitochondria isolated from Efhd1 knockout mice also exhibited a

There are no current standard-of-care treatments for sarcopenia, an age-associated decline in muscle mass and strength. A new study shows that genetically or pharmacologically countering the age-associated accumulation of sphingolipids in skeletal muscle can ameliorate sarcopenia in mice. The authors also identify genetic variants linked to sphingolipid biosynthesis that associate with muscle function in aged humans.

Obesity and aging reduce skeletal muscle contractile function. However, it remains unclear whether obesity additively promotes muscle contractile dysfunction in the setting of aging. In this study, we investigated skeletal muscle contractile function ex vivo and intracellular Ca(2+) release in male C57BL/6J mice fed a low-fat diet (LFD) or a high-fat diet (HFD) for 4 or 20 mo. Tetanic force production in the extensor digitorum longus muscle was decreased by aging or HFD feeding, and the further reduction was observed in aged HFD mice. The 20-mo HFD-fed mice, not the 20-mo LFD-fed mice or 4-mo HFD-fed mice, showed reduced intracellular Ca(2+) peak levels by high concentration of caffeine (25 mM) compared with 4-mo LFD mice. Aging and HFD feeding additively increased intramyocellular lipid (IMCL) levels and were associated with the degree of impaired muscle contractile force and peak Ca(2+) level. These data suggest that impairment in the contractile force in aged muscle is aggravated by HFD, which may be due, at least in part, to dysfunction in intracellular Ca(2+) release. The IMCL level may be a marker for impaired muscle contractile force caused by aging and HFD. NEW & NOTEWORTHY The aim of this study was to examine the effect of high-fat diet (HFD)-induced obesity on contractile function and Ca(2+) release capacity in aged skeletal muscle. Not only were the force production and peak Ca(2+) levels decreased by aging and HFD feeding, respectively, but also, these interventions had an additive effect in aged HFD-fed mice. These data suggest that the impairment in the contractile force in aged muscle is aggravated by a HFD, which may be due to synergistic dysfunction in intracellular Ca(2+) release.

Objective Inactivity and inflammation are linked to obesity and insulin resistance. It was hypothesized that MyD88 (mediates inflammation) knockout from muscle (MusMyD88-/- ) would prevent, whereas miR146a-/- (MyD88 inhibitor) would exacerbate, inactivity-induced metabolic disturbances. Methods Cre-control, MusMyD88-/- , and miR146a-/- mice were given running wheels for 5 weeks to model an active phenotype. Afterward, half were placed into a small mouse cage (SMC) to restrict movement for 8 days. Body composition, muscle (3 H)2-deoxyglucose uptake, visceral fat histology, and tissue weight (hind limb muscles, visceral fat, and liver) were assessed. In skeletal muscle and visceral fat, RNA sequencing and mitochondrial function were performed on female MusMyD88-/- and Cre-control SMC mice. Results The SMC induced adiposity, hyperinsulinemia, and muscle insulin-stimulated glucose uptake, which was worsened in miR146a-/- mice. In females, MusMyD88-/- mice were protected. Female MusMyD88-/- mice during the SMC period (vs. Cre-control) exhibited higher Igf1 and decreased Ip6k3 and Trim63 muscle expression. Visceral fat transcript changes corresponded to improved lipid metabolism, decreased adipose expansion (Gulp1↑, Anxa2↓, Ehd1↓) and meta-inflammation (Hmox1↓), and increased beiging (Fgf10↑). Ralgapa2, negative regulator of GLUT4 translocation, and inflammation-related gene 993011J21Rik2 were decreased in both muscle and fat. Conclusions Whole-body miR146a-/- exacerbated inactivity-induced fat gain and muscle insulin resistance, whereas MusMyD88-/- prevented insulin resistance in female mice.

BACKGROUND AND AIMS. Metformin is the most commonly prescribed medication to treat diabetes. Emerging evidence suggests that metformin could have off target effects that might help promote healthy muscle aging, but these effects have not been thoroughly studied in glucose tolerant older individuals. The purpose of this study was to investigate the short-term effects of metformin consumption on skeletal muscle mitochondrial bioenergetics in healthy older adults. METHODS. We obtained muscle biopsy samples from 16 healthy older adults previously naïve to metformin and treated with metformin (METF; 3F, 5M), or placebo (CON; 3F, 5M), for two weeks using a randomized and blinded study design. Samples were analyzed using high-resolution respirometry, immunofluorescence, and immunoblotting to assess muscle mitochondrial bioenergetics, satellite cell (SC) content, and associated protein markers. RESULTS. We found that metformin treatment did not alter maximal mitochondrial respiration rates in muscle compared to CON. In contrast, mitochondrial H(2)O(2) emission and production were elevated in muscle samples from METF versus CON (METF emission: 2.59 ± 0.72 SE Fold, P=0.04; METF production: 2.29 ± 0.53 SE Fold, P=0.02). Furthermore, the change in H(2)O(2) emission was positively correlated with the change in type 1 myofiber SC content and this was biased in METF participants (Pooled: R(2)=0.5816, P=0.0006; METF: R(2)=0.674, P=0.0125). CONCLUSIONS. These findings suggest that acute exposure to metformin does not impact mitochondrial respiration in aged, glucose-tolerant muscle, but rather, influences mitochondrial-free radical and SC dynamics. CLINICAL TRIAL REGISTRATION. NCT03107884, clinicaltrials.gov

Introduction: Diabetes is a major cause of heart failure with preserved ejection fraction (HFpEF), which has limited diagnostic tools and therapeutic options. Abnormal remodeling in the t-tubule calcium handling cardiac bridging integrator 1 (cBIN1)-microdomains has emerged as key to the pathophysiology of failing cardiomyocytes, which can be targeted for cardiac functional improvement. It remains unclear whether cBIN1 replacement therapy is effective in restoring cardiac function in diabetic HFpEF. Hypothesis: We hypothesized that cBIN1 gene therapy rescues diastolic failure in mice with diabetic cardiomyopathy. Methods: 9-wks old male and female db/db mice that received retro-orbital injection of 1x10 11 vg of AAV9 transducing GFP or cBIN1 were compared to non-diabetic control db/m littermates injected with AAV9-GFP (N = 16 mice per group). Mice were terminated 8-wks later for tissue acquisition, imaging, as well as biochemical analysis. Echocardiography, exercise tolerance, and plasma cBIN1 score (CS, an inverse index of plasma cBIN1 concentration) were obtained before and after gene therapy. Results: db/db mice developed diastolic failure with reduced E/A, increased E/e’, exercise intolerance, and cardiac hypertrophy with reduced tissue cBIN1 levels and corresponding elevations in plasma CS. Supporting our hypothesis, AAV9-cBIN1 normalized E/A and E/e’ and improved exercise tolerance in db/db mice, which can be distinguished by plasma CS as an inverse marker of cBIN1-microfolds and cardiomyocyte remodeling (Figure 1). Preservation of cBIN1-microdomains in db/db cardiomyocytes reorganized functional SERCA2a and rescued mitochondrial respiration with diminished oxidative stress. Conclusions: Exogenous cBIN1 normalizes lusitropic function of diabetic hearts through microdomain-organized effective calcium handling and mitochondrial respiration. cBIN1 gene therapy is a new and effective method for guided treatment of diabetic HFpEF.

Altered levels of intracellular calcium (Ca2+) is a highly prevalent feature in different forms of cardiac injury, producing changes in contractility, arrhythmias, and mitochondrial dysfunction. In cardiac ischemia-reperfusion injury, mitochondrial Ca2+ overload leads to pathological production of reactive oxygen species (ROS), activates the permeability transition, and cardiomyocyte death. Here we investigated the cardiac phenotype caused by deletion of EF-hand domain-containing protein D1 (Efhd1-/-), a Ca2+-binding mitochondrial protein whose function is poorly understood. Efhd1-/- mice are viable and have no adverse cardiac phenotypes. They feature reductions in basal ROS levels and mitoflash events, both important precursors for mitochondrial injury, though cardiac mitochondria have normal susceptibility to Ca2+ overload. Notably, we also find that Efhd1-/- mice and their cardiomyocytes are resistant to hypoxic injury.

Altered levels of intracellular calcium (Ca(2+)) are a highly prevalent feature in different forms of cardiac injury, producing changes in contractility, arrhythmias, and mitochondrial dysfunction. In cardiac ischemia-reperfusion injury, mitochondrial Ca(2+) overload leads to pathological production of reactive oxygen species (ROS), activates the permeability transition, and cardiomyocyte death. Here we investigated the cardiac phenotype caused by deletion of EF-hand domain-containing protein D1 (Efhd1(−/−)), a Ca(2+)-binding mitochondrial protein whose function is poorly understood. Efhd1(−/−) mice are viable and have no adverse cardiac phenotypes. They feature reductions in basal ROS levels and mitoflash events, both important precursors for mitochondrial injury, though cardiac mitochondria have normal susceptibility to Ca(2+) overload. Notably, we also find that Efhd1(−/−) mice and their cardiomyocytes are resistant to hypoxic injury.

Obesity alters skeletal muscle lipidome and promotes myopathy, but it is unknown whether aberrant muscle lipidome contributes to the reduction in skeletal muscle contractile force-generating capacity. Comprehensive lipidomic analyses of mouse skeletal muscle revealed a very strong positive correlation between the abundance of lysophosphatidylcholine (lyso-PC), a class of lipids that is known to be downregulated with obesity, with maximal tetanic force production. The level of lyso-PC is regulated primarily by lyso-PC acyltransferase 3 (LPCAT3), which acylates lyso-PC to form phosphatidylcholine. Tamoxifen-inducible skeletal muscle-specific overexpression of LPCAT3 (LPCAT3-MKI) was sufficient to reduce muscle lyso-PC content in both standard chow diet- and high-fat diet (HFD)-fed conditions. Strikingly, the assessment of skeletal muscle force-generating capacity ex vivo revealed that muscles from LPCAT3-MKI mice were weaker regardless of diet. Defects in force production were more apparent in HFD-fed condition, where tetanic force production was 40% lower in muscles from LPCAT3-MKI compared to that of control mice. These observations were partly explained by reductions in the cross-sectional area in type IIa and IIx fibers, and signs of muscle edema in the absence of fibrosis. Future studies will pursue the mechanism by which LPCAT3 may alter protein turnover to promote myopathy.

SMYD1a, a myosin-specific histone lysine methyltransferase, plays a major role in regulating disease-induced remodeling in the adult heart. Previously, we demonstrated that the inducible loss of this chromatin-bound enzyme is sufficient to induce cardiac hypertrophy and failure in vivo , which is preceded by downregulation of mitochondrial proteins involved in oxidative phosphorylation (OXPHOS), and reduction of mitochondrial respiration capacity. However, our most recent data in transgenic mice (TG) displaying inducible, cardiomyocyte-specific overexpression of SMYD1a show that these mice are protected from ischemic injury after permanent occlusion (PO) of the LAD manifested by reduced infarct size and cardiac dysfunction compared to littermate controls (WT), suggesting that SMYD1 plays a protective role in the heart and mitigates disease-induced remodeling. Additionally, global proteomic evaluation of cardiac tissue from TG mice showed unique expression of metabolic enzymes, including proteins from the electron transport chain, and our high-resolution mitochondrial respirometry analysis showed that overexpression of SMYD1a leads to increased oxygen consumption rates through Complex I and II. To further asses OXPHOS efficiency in TG mice we subjected them to permanent occlusion of the LAD and evaluated ATP production rates in isolated mitochondria from TG and WT mice, by measuring the molar amount of ATP produced per mole of atomic oxygen consumed (known as ATP:O ratio). Interestingly, we observed a significant increase in ATP:O ratio in TG mice 24h after PO suggesting that they are much more efficient at producing ATP. Finally, we show that the global regulation of mitochondrial respiration in TG mice occurs through transcriptional control of Ppargc1α . Our results confirm that cardiac expression of Ppargc1α was significantly reduced in WT mice (48h after PO) but maintained at basal levels in TG mice, which also corroborated with our ChIP-qPCR data showing SMYD1a binding to the Ppargc1α promoter and regulating its expression. Overall, these results show that SMYD1a can mitigate ischemic injury and adverse remodeling in the adult myocardium, which occurs through Ppargc1α expression and regulation of cardiac energetics and metabolism.

Mitochondrial dysfunction due to calcium overload is common in heart disease and results in heart failure. In a mouse model of mitochondrial cardiomyopathies we noted a substantial downregulation of the gene encoding for the EF hand containing domain 1 protein (EFHD1). EFHD1 is a mitochondrial protein which binds calcium and has been shown to stimulate both apoptosis and mitoflashes. These processes involve mitochondrial calcium overload. We therefore hypothesized that EFHD1 down regulation could represent a natural response by failing hearts to prevent mitochondrial calcium overload. We therefore studied the calcium handling properties of mice where Efhd1 had been knocked out (Efhd1-KO). These mice are viable and have no obvious adverse cardiac or metabolic phenotypes. We find that EFHD1 is expressed in the cytoplasm and mitochondrial outer membrane and intermembrane space. It associates strongly with endoplasmic reticulum (ER)-associated mitochondrial membranes. Moreover, mitochondria isolated from Efhd1-KO mice also exhibited 250±10 % improvement in calcium retention in liver but not heart, while neonatal mouse hearts were 3 times more resistant to hypoxia. These findings suggest that EFHD1 may modulate mitochondrial calcium uptake.

Loss of muscle mass and strength after disuse followed by impaired muscle recovery commonly occurs with aging. Metformin and leucine individually have shown positive effects in skeletal muscle during atrophy conditions but have not been evaluated in combination nor tested as a remedy to enhance muscle recovery following disuse atrophy in aging. The purpose of this study was to determine if a dual treatment of metformin and leucine (MET+LEU) would prevent disuse-induced atrophy and/or promote muscle recovery in aged mice and if these muscle responses correspond to changes in satellite cells and collagen remodeling. Aged mice (22–24 mo) underwent 14 days of hindlimb unloading (HU) followed by 7 or 14 days of reloading (7 or 14d RL). Metformin (MET), leucine (LEU), or MET+LEU was administered via drinking water and were compared to Vehicle (standard drinking water) and Ambulatory baseline (AMB). We observed that during HU, MET+LEU resolved whole body grip strength and soleus muscle specific force decrements caused by HU. Gastrocnemius satellite cell abundance was increased with MET+LEU treatment but did not alter muscle size during disuse or recovery conditions. Moreover, MET+LEU treatment alleviated gastrocnemius collagen accumulation caused by HU and increased collagen turnover during 7 and 14d RL driven by a decrease in collagen IV content. Transcriptional pathway analysis revealed that MET+LEU altered muscle hallmark pathways related to inflammation and myogenesis during HU. Together, the dual treatment of metformin and leucine was able to increase muscle function, satellite cell content, and reduce collagen accumulation, thus improving muscle quality during disuse and recovery in aging.

Parent-of-origin effects are unexpectedly common in complex traits, including metabolic and neurological diseases. Parent-of-origin effects can be modified by the environment, but the architecture of these gene-by-environmental effects on phenotypes remains to be unraveled. Previously, quantitative trait loci (QTL) showing context-specific parent-of-origin effects on metabolic traits were mapped in the F16generation of an advanced intercross between LG/J and SM/J inbred mice. However, these QTL were not enriched for known imprinted genes, suggesting another mechanism is needed to explain these parent-of-origin effects phenomena. We propose that non-imprinted genes can generate complex parent-of-origin effects on metabolic traits through interactions with imprinted genes. Here, we employ data from mouse populations at different levels of intercrossing (F0, F1, F2, F16) of the LG/J and SM/J inbred mouse lines to test this hypothesis. Using multiple populations and incorporating genetic, genomic, and physiological data, we leverage orthogonal evidence to identify networks of genes through which parent-of-origin effects propagate. We identify a network comprised of 3 imprinted and 6 non-imprinted genes that show parent-of-origin effects. This epistatic network forms a nutritional responsive pathway and the genes comprising it jointly serve cellular functions associated with growth. We focus on 2 genes,NnatandF2r, whose interaction associates with serum glucose levels across generations in high fat-fed females. Single-cell RNAseq reveals thatNnatandF2rare negatively correlated in pre-adipocytes along an adipogenic trajectory, a result that is consistent with our observations in bulk white adipose tissue.

Objective Estrogen receptor-α (ERα) is a nuclear receptor family member thought to substantially contribute to the metabolic regulation of skeletal muscle. However, previous mouse models utilized to assess the necessity of ERα signaling in skeletal muscle were confounded by altered developmental programming and/or influenced by secondary effects, making it difficult to assign a causal role for ERα. The objective of this study was to determine the role of skeletal muscle ERα in regulating metabolism in the absence of confounding factors of development. Methods A novel mouse model was developed allowing for induced deletion of ERα in adult female skeletal muscle (ERαKOism). ERαshRNA was also used to knockdown ERα (ERαKD) in human myotubes cultured from primary human skeletal muscle cells isolated from muscle biopsies from healthy and obese insulin-resistant women. Results Twelve weeks of HFD exposure had no differential effects on body composition, VO2, VCO2, RER, energy expenditure, and activity counts across genotypes. Although ERαKOism mice exhibited greater glucose intolerance than wild-type (WT) mice after chronic HFD, ex vivo skeletal muscle glucose uptake was not impaired in the ERαKOism mice. Expression of pro-inflammatory genes was altered in the skeletal muscle of the ERαKOism, but the concentrations of these inflammatory markers in the systemic circulation were either lower or remained similar to the WT mice. Finally, skeletal muscle mitochondrial respiratory capacity, oxidative phosphorylation efficiency, and H2O2 emission potential was not affected in the ERαKOism mice. ERαKD in human skeletal muscle cells neither altered differentiation capacity nor caused severe deficits in mitochondrial respiratory capacity. Conclusions Collectively, these results suggest that ERα function is superfluous in protecting against HFD-induced skeletal muscle metabolic derangements after postnatal development is complete., Highlights • Induced skeletal muscle specific ERαKO (ERαKOism) examines the role of ERα without confounding factors of development. • Skeletal muscle glucose uptake is not impaired in ERαKOism. • Skeletal muscle mitochondrial function is not impaired in ERαKOism. • ERαKD in human myotubes does not severely affect mitochondrial respiratory capacity.

The biophysical environment of membrane phospholipids affects structure, function, and stability of membrane-bound proteins.1,2 Obesity can disrupt membrane lipids, and in particular, alter the activity of sarco/endoplasmic reticulum (ER/SR) Ca2+-ATPase (SERCA) to affect cellular metabolism.3-5 Recent evidence suggests that transport efficiency (Ca2+ uptake / ATP hydrolysis) of skeletal muscle SERCA can be uncoupled to increase energy expenditure and protect mice from diet-induced obesity.6,7 In isolated SR vesicles, membrane phospholipid composition is known to modulate SERCA efficiency.8-11 Here we show that skeletal muscle SR phospholipids can be altered to decrease SERCA efficiency and increase whole-body metabolic rate. The absence of skeletal muscle phosphatidylethanolamine (PE) methyltransferase (PEMT) promotes an increase in skeletal muscle and whole-body metabolic rate to protect mice from diet-induced obesity. The elevation in metabolic rate is caused by a decrease in SERCA Ca2+-transport efficiency, whereas mitochondrial uncoupling is unaffected. Our findings support the hypothesis that skeletal muscle energy efficiency can be reduced to promote protection from obesity.

Muscle progenitor cells (MPCs) in aged muscle exhibit impaired activation into proliferating myoblasts, thereby impairing fusion and changes in secreted factors. The antihyperglycemic drug metformin, currently studied as a candidate antiaging therapy, may have potential to promote function of aged MPCs. We evaluated the impact of 2 wk of metformin ingestion on primary myoblast function measured in vitro after being extracted from muscle biopsies of older adult participants. MPCs were isolated from muscle biopsies of community-dwelling older (4 male/4 female, ∼69 yr) adult participants before (pre) and after (post) the metformin ingestion period and studied in vitro. Cells were extracted from Young participants (4 male/4 female, ∼27 yr) to serve as a “youthful” comparator. MPCs from Old subjects had lower fusion index and myoblast-endothelial cell homing compared with Young, while Old MPCs, extracted after short-term metformin ingestion, performed better at both tasks. Transcriptomic analyses of Old MPCs (vs. Young) revealed decreased histone expression and increased myogenic pathway activity, yet this phenotype was partially restored by metformin. However, metformin ingestion exacerbated pathways related to inflammation signaling. Together, this study demonstrated that 2 wk of metformin ingestion induced persistent effects on Old MPCs that improved function in vitro and altered their transcriptional signature including histone and chromatin remodeling.

Physical inactivity influences the development of muscle insulin resistance yet is far less understood than diet-induced muscle insulin resistance. Progress in understanding the mechanisms of physical inactivity-induced insulin resistance is limited by a lack of an appropriate preclinical model of muscle insulin resistance. Here, we discuss differences between diet and physical inactivity-induced insulin resistance, the advantages and disadvantages of the available rodent inactivity models to study insulin resistance, and our current understanding of the mechanisms of muscle insulin resistance derived from such preclinical inactivity designs. The burgeoning rise of health complications emanating from metabolic disease presents an alarming issue with mounting costs for health care and a reduced quality of life. There exists a pressing need for more complete understanding of mechanisms behind the development and progression of metabolic dysfunction. Since lifestyle modifications such as poor diet and lack of physical activity are primary catalysts of metabolic dysfunction, rodent models have been formed to explore mechanisms behind these issues. Particularly, the use of a high-fat diet has been pervasive and has been an instrumental model to gain insight into mechanisms underlying diet-induced insulin resistance (IR). However, physical inactivity (and to some extent muscle disuse) is an often overlooked and much less frequently studied lifestyle modification, which some have contended is the primary contributor in the initial development of muscle IR. In this mini-review we highlight some of the key differences between diet- and physical inactivity-induced development of muscle IR and propose reasons for the sparse volume of academic research into physical inactivity-induced IR including infrequent use of clearly translatable rodent physical inactivity models.

Cells harbor two systems for fatty acid synthesis, one in the cytoplasm (catalyzed by fatty acid synthase, FASN) and one in the mitochondria (mtFAS). In contrast to FASN, mtFAS is poorly characterized, especially in higher eukaryotes, with the major product(s), metabolic roles, and cellular function(s) being essentially unknown. Here we show that hypomorphic mtFAS mutant mouse skeletal myoblast cell lines display a severe loss of electron transport chain (ETC) complexes and exhibit compensatory metabolic activities including reductive carboxylation. This effect on ETC complexes appears to be independent of protein lipoylation, the best characterized function of mtFAS, as mutants lacking lipoylation have an intact ETC. Finally, mtFAS impairment blocks the differentiation of skeletal myoblasts in vitro. Together, these data suggest that ETC activity in mammals is profoundly controlled by mtFAS function, thereby connecting anabolic fatty acid synthesis with the oxidation of carbon fuels., eLife digest In human, plant and other eukaryotic cells, fats are an important source of energy and also play many other roles including waterproofing, thermal insulation and energy storage. Eukaryotic cells have two systems that make the building blocks of fats (known as fatty acids) and one of these systems, called the mtFAS pathway, operates in small compartments known as mitochondria. This pathway only has one known product, a small fat molecule called lipoic acid, which mitochondria attach to several enzymes to allow them to work properly. The main role of mitochondria is to break down fats and other molecules to release chemical energy that powers many processes in cells. They achieve this using large groups of proteins known as ETC complexes. To build these complexes, families of proteins known as ETC assembly factors carefully coordinate the assembly of many proteins and small molecules into specific structures. However, it remains unclear precisely how this process works. Here, Nowinski et al. used a gene editing technique to mutate the genes encoding three enzymes in the mtFAS pathway in mammalian cells. The experiments found that the mutant cells had fewer ETC complexes and seemed to be less able to break down fats and other molecules than ‘normal’ cells. Furthermore, a family of ETC assembly factors were less stable in the mutant cells. These findings suggest that the mtFAS pathway controls how mitochondria assemble ETC complexes. Further experiments indicated that lipoic acid is not involved in the assembly of ETC complexes and that the mtFAS pathway produces another, as yet unidentified, product that regulates this process, instead. MEPAN syndrome is a rare neurological disorder that leads to progressive loss of control of movement, slurred speech and impaired vision in children. Patients with this syndrome have genetic mutations affecting components of the mtFAS pathway, therefore, a better understanding of how the pathway works may help researchers develop new treatments in the future. More broadly, these findings will have important ramifications for many other situations in which the activity of ETC complexes in mitochondria is modified.

Excess reactive oxygen species (ROS) induced by physical inactivity is associated with muscle atrophy and muscle weakness. However, the role of mitochondrial ROS on disuse-induced muscle atrophy is not fully understood. The purpose of this study was to utilize a genetic strategy to examine the effect of neutralizing mitochondrial ROS on disuse-induced skeletal muscle atrophy. This was accomplished by placing wild-type (WT) and mitochondrial-targeted catalase-expressing (MCAT) littermate mice on 7 days of hindlimb unloading. After assessment of body weight and composition, muscles were analyzed for individual muscle mass, force-generating capacity, fiber type, cross-sectional area, and mitochondrial function, including H(2)O(2) production. Despite a successful attenuation of mitochondrial ROS, MCAT mice were not protected from muscle atrophy. No differences were observed in body composition, lean mass, individual muscle masses, force-generating capacity, or muscle fiber cross-sectional area. These data suggest that neutralizing mitochondrial ROS is insufficient to suppress disuse-induced loss of skeletal muscle mass and contractile function. NEW & NOTEWORTHY The premise of this study was to examine the efficacy of genetic suppression of mitochondrial reactive oxygen species (ROS) to attenuate disuse-induced muscle atrophy and muscle weakness. Neutralization of mitochondrial ROS by MCAT expression was insufficient to rescue muscle atrophy and muscle weakness.

The biophysical environment of membrane phospholipids affects structure, function, and stability of membrane-bound proteins.1,2 Obesity can disrupt membrane lipids, and in particular, alter the activity of sarco/endoplasmic reticulum (ER/SR) Ca2+-ATPase (SERCA) to affect cellular metabolism.3–5 Recent evidence suggests that transport efficiency (Ca2+ uptake / ATP hydrolysis) of skeletal muscle SERCA can be uncoupled to increase energy expenditure and protect mice from diet-induced obesity.6,7 In isolated SR vesicles, membrane phospholipid composition is known to modulate SERCA efficiency.8–11 Here we show that skeletal muscle SR phospholipids can be altered to decrease SERCA efficiency and increase whole-body metabolic rate. The absence of skeletal muscle phosphatidylethanolamine (PE) methyltransferase (PEMT) promotes an increase in skeletal muscle and whole-body metabolic rate to protect mice from diet-induced obesity. The elevation in metabolic rate is caused by a decrease in SERCA Ca2+-transport efficiency, whereas mitochondrial uncoupling is unaffected. Our findings support the hypothesis that skeletal muscle energy efficiency can be reduced to promote protection from obesity.

Cells harbor two systems for fatty acid synthesis, one in the cytoplasm (catalyzed by fatty acid synthase, FASN) and one in the mitochondria (mtFAS). In contrast to FASN, mtFAS is poorly characterized, especially in higher eukaryotes, with the major product(s), metabolic roles, and cellular function(s) being essentially unknown. Here we show that hypomorphic mtFAS mutants display a severe loss of electron transport chain (ETC) complexes and exhibit compensatory metabolic activities including reductive carboxylation. This effect on ETC complexes appears to be independent of protein lipoylation, the best characterized function of mtFAS, as mutants lacking lipoylation have an intact ETC. Finally, mtFAS impairment blocks the differentiation of skeletal myoblasts in vitro. Together, these data suggest that ETC activity in mammals is profoundly controlled by mtFAS function, thereby connecting anabolic fatty acid synthesis with the oxidation of carbon fuels.

Peroxisomes perform essential functions in lipid metabolism, including fatty acid oxidation and plasmalogen synthesis. Here, we describe a role for peroxisomal lipid metabolism in mitochondrial dynamics in brown and beige adipocytes. Adipose tissue peroxisomal biogenesis was induced in response to cold exposure through activation of the thermogenic coregulator PRDM16. Adipose-specific knockout of the peroxisomal biogenesis factor Pex16 (Pex16-AKO) in mice impaired cold tolerance, decreased energy expenditure, and increased diet-induced obesity. Pex16 deficiency blocked cold-induced mitochondrial fission, decreased mitochondrial copy number, and caused mitochondrial dysfunction. Adipose-specific knockout of the peroxisomal β-oxidation enzyme acyl-CoA oxidase 1 (Acox1-AKO) was not sufficient to affect adiposity, thermogenesis, or mitochondrial copy number, but knockdown of the plasmalogen synthetic enzyme glyceronephosphate O-acyltransferase (GNPAT) recapitulated the effects of Pex16 inactivation on mitochondrial morphology and function. Plasmalogens are present in mitochondria and decreased with Pex16 inactivation. Dietary supplementation with plasmalogens increased mitochondrial copy number, improved mitochondrial function, and rescued thermogenesis in Pex16-AKO mice. These findings support a surprising interaction between peroxisomes and mitochondria regulating mitochondrial dynamics and thermogenesis.

Sugar- and lipid-derived aldehydes are reactive carbonyl species (RCS) frequently used as surrogate markers of oxidative stress in obesity. A pathogenic role for RCS in metabolic diseases of obesity remains controversial, however, partly because of their highly diffuse and broad reactivity and the lack of specific RCS-scavenging therapies. Naturally occurring histidine dipeptides (e.g., anserine and carnosine) show RCS reactivity, but their therapeutic potential in humans is limited by serum carnosinases. Here, we present the rational design, characterization, and pharmacological evaluation of carnosinol, i.e., (2S)-2-(3-amino propanoylamino)-3-(1H-imidazol-5-yl)propanol, a derivative of carnosine with high oral bioavailability that is resistant to carnosinases. Carnosinol displayed a suitable ADMET (absorption, distribution, metabolism, excretion, and toxicity) profile and was determined to have the greatest potency and selectivity toward α,β-unsaturated aldehydes (e.g., 4-hydroxynonenal, HNE, ACR) among all others reported thus far. In rodent models of diet-induced obesity and metabolic syndrome, carnosinol dose-dependently attenuated HNE adduct formation in liver and skeletal muscle, while simultaneously mitigating inflammation, dyslipidemia, insulin resistance, and steatohepatitis. These improvements in metabolic parameters with carnosinol were not due to changes in energy expenditure, physical activity, adiposity, or body weight. Collectively, our findings illustrate a pathogenic role for RCS in obesity-related metabolic disorders and provide validation for a promising new class of carbonyl-scavenging therapeutic compounds rationally derived from carnosine.

INTRODUCTION: Evidence suggests that the energy efficiency of key ATPases involved in skeletal muscle contractile activity are improved in a hypothermic condition. However, it is unclear how a decrease in temperature affects skeletal muscle O(2) consumption (mVO(2)) induced by muscle contraction. METHODS: Isolated mouse extensor digitorum longus (EDL) muscles were incubated in a temperature-controlled (37°C or 25°C) bath that included an O(2) probe. EDL muscles from one limb were subjected to the measurement of resting mVO(2), and the contralateral EDL muscles were used for the measurement of mVO(2) with electrically-stimulated contraction. For the resting protocol, muscles were suspended at resting tension for 15 mins with continuous O(2) recordings. For the contraction protocol, EDL muscles underwent ten electrically-stimulated isometric contractions with continuous O(2) recordings for 15 mins. The rate of O(2) disappearance was quantified as μmole O(2) per minute and normalized to the wet weight of the muscle. RESULTS: Resting mVO(2) was greater at 37°C than at 25°C, consistent with the idea that lower temperature reduces basal metabolic rate. Electrically-stimulated contraction robustly increased mVO(2) at both 37°C and 25°C, which was sustained for ~3 min post-contraction. During that period, mVO(2) was elevated ~5-fold at both 37°C and 25°C. Greater contraction-induced mVO(2) at 37°C compared to 25°C occurred despite lower force generated at 37°C than at 25°C. CONCLUSION: Together, O(2) cost for muscle contraction (force-time integral per O(2) consumed) was greater at 37°C than at 25°C. Levels of high-energy phosphates were consistent with greater energy demand at 37°C compared to 25°C. In conclusion, these results indicate that muscle contraction that occurs at subnormal temperature requires less O(2) than at 37°C.

Barth Syndrome (BTHS) is an X-linked recessive disorder characterized by cardiomyopathy and muscle weakness. The underlying cause of BTHS is a mutation in the tafazzin (TAZ) gene, a key enzyme of cardiolipin biosynthesis. The lack of CL arising from loss of TAZ function results in destabilization of the electron transport system, promoting oxidative stress that is thought to contribute to development of cardioskeletal myopathy. Indeed, in vitro studies demonstrate that mitochondria-targeted antioxidants improve contractile capacity in TAZ-deficient cardiomyocytes. The purpose of the present study was to determine if resolving mitochondrial oxidative stress would be sufficient to prevent cardiomyopathy and skeletal myopathy in vivo using a mouse model of BTHS. To this end we crossed mice that overexpress catalase in the mitochondria (MCAT mice) with TAZ-deficient mice (TAZKD) to produce TAZKD mice that selectively overexpress catalase in the mitochondria (TAZKD+MCAT mice). TAZKD+MCAT mice exhibited decreased mitochondrial H(2)O(2) emission and lipid peroxidation compared to TAZKD littermates, indicating decreased oxidative stress. Despite the improvements in oxidative stress, TAZKD+MCAT mice developed cardiomyopathy and mild muscle weakness similar to TAZKD littermates. These findings indicate that resolving oxidative stress is not sufficient to suppress cardioskeletal myopathy associated with BTHS.