Exercise and Ischemia-Activated Pathways in Limb Muscle Angiogenesis and Vascular Regeneration.

Exercise has a profound effect on cardiovascular disease, particularly through vascular remodeling and regeneration. Peripheral artery disease (PAD) is one such cardiovascular condition that benefits from regular exercise or rehabilitative physical therapy in terms of slowing the progression of disease and delaying amputations. Various rodent pre-clinical studies using models of PAD and exercise have shed light on molecular pathways of vascular regeneration. Here, I review key exercise-activated signaling pathways (nuclear receptors, kinases, and hypoxia inducible factors) in the skeletal muscle that drive paracrine regenerative angiogenesis. The rationale for highlighting the skeletal muscle is that it is the largest organ recruited during exercise. During exercise, skeletal muscle releases several myokines, including angiogenic factors and cytokines that drive tissue vascular regeneration via activation of endothelial cells, as well as by recruiting immune and endothelial progenitor cells. Some of these core exercise-activated pathways can be extrapolated to vascular regeneration in other organs. I also highlight future areas of exercise research (including metabolomics, single cell transcriptomics, and extracellular vesicle biology) to advance our understanding of how exercise induces vascular regeneration at the molecular level, and propose the idea of "exercise-mimicking" therapeutics for vascular recovery.

Estrogen-Related Receptor Gamma Gene Therapy Promotes Therapeutic Angiogenesis and Muscle Recovery in Preclinical Model of PAD.

Background Peripheral arterial disease and critical limb ischemia are cardiovascular complications associated with vascular insufficiency, oxidative metabolic dysfunction, and myopathy in the limbs. Estrogen-related receptor gamma (ERRγ) has emerged as a dual regulator of paracrine angiogenesis and oxidative metabolism through transgenic mouse studies. Here our objective was to investigate whether postischemic intramuscular targeting of ERRγ via gene therapy promotes ischemic recovery in a preclinical model of peripheral arterial disease/critical limb ischemia. Methods and Results Adeno-associated virus 9 (AAV9) Esrrg gene delivery vector was developed and first tested via intramuscular injection in murine skeletal muscle. AAV9-Esrrg robustly increased ERRγ protein expression, induced angiogenic and oxidative genes, and boosted capillary density and succinate dehydrogenase oxidative metabolic activity in skeletal muscles of C57Bl/6J mice. Next, hindlimb ischemia was induced via unilateral femoral vessel ligation in mice, followed by intramuscular AAV9-Esrrg (or AAV9-green fluorescent protein) gene delivery 24 hours after injury. ERRγ overexpression increased ischemic neoangiogenesis and markers of endothelial activation, and significantly improved ischemic revascularization measured using laser Doppler flowmetry. Moreover, ERRγ overexpression restored succinate dehydrogenase oxidative metabolic capacity in ischemic muscle, which correlated with increased mitochondrial respiratory complex protein expression. Most importantly, myofiber size to number quantification revealed that AAV9-Esrrg restores myofibrillar size and mitigates ischemia-induced myopathy. Conclusions These results demonstrate that intramuscular AAV9-Esrrg delivery rescues ischemic pathology after hindlimb ischemia, underscoring that Esrrg gene therapy or pharmacological activation could be a promising strategy for the management of peripheral arterial disease/critical limb ischemia.

Estrogen-related Receptor Signaling in Skeletal Muscle Fitness.

Skeletal muscle is a highly plastic tissue that can alter its metabolic and contractile features, as well as regenerative potential in response to exercise and other conditions. Multiple signaling factors including metabolites, kinases, receptors, and transcriptional factors have been studied in the regulation of skeletal muscle plasticity. Recently, estrogen-related receptors (ERRs) have emerged as a critical transcriptional hub in control of skeletal muscle homeostasis. ERRα and ERRγ - the two highly expressed ERR sub-types in the muscle respond to various extracellular cues such as exercise, hypoxia, fasting and dietary factors, in turn regulating gene expression in the skeletal muscle. On the other hand, conditions such as diabetes and muscular dystrophy suppress expression of ERRs in the skeletal muscle, likely contributing to disease progression. We highlight key functions of ERRs in the skeletal muscle including the regulation of fiber type, mitochondrial metabolism, vascularization, and regeneration. We also describe how ERRs are regulated in the skeletal muscle, and their interaction with important muscle regulators (e. g. AMPK and PGCs). Finally, we identify critical gaps in our understanding of ERR signaling in the skeletal muscle, and suggest future areas of investigation to advance ERRs as potential targets for function promoting therapeutics in muscle diseases.

Innately expressed estrogen-related receptors in the skeletal muscle are indispensable for exercise fitness.

Transcriptional determinants in the skeletal muscle that govern exercise capacity, while poorly defined, could provide molecular insights into how exercise improves fitness. Here, we have elucidated the role of nuclear receptors, estrogen-related receptor alpha and gamma (ERRα/γ) in regulating myofibrillar composition, contractility, and exercise capacity in skeletal muscle. We used muscle-specific single or double (DKO) ERRα/γ knockout mice to investigate the effect of ERRα/γ deletion on muscle and exercise parameters. Individual knockout of ERRα/γ did not have a significant impact on the skeletal muscle. On the other hand, DKO mice exhibit pale muscles compared to wild-type (WT) littermates. RNA-seq analysis revealed a predominant decrease in expression of genes linked to mitochondrial and oxidative metabolism in DKO versus WT muscles. DKO muscles exhibit marked repression of oxidative enzymatic capacity, as well as mitochondrial number and size compared to WT muscles. Mitochondrial function is also impaired in single myofibers isolated from DKO versus WT muscles. In addition, mutant muscles exhibit reduced angiogenic gene expression and decreased capillarity. Consequently, DKO mice have a significantly reduced exercise capacity, further reflected in poor fatigue resistance of DKO mice in in vivo contraction assays. These results show that ERRα and ERRγ together are a critical link between muscle aerobic capacity and exercise tolerance. The ERRα/γ mutant mice could be valuable for understanding the long-term impact of impaired mitochondria and vascular supply on the pathogenesis of muscle-linked disorders.

Targeted ablation of Fn14 receptor improves exercise capacity and inhibits neurogenic muscle atrophy.

Skeletal muscle atrophy is a prevalent complication in multiple chronic diseases and disuse conditions. Fibroblast growth factor-inducible 14 (Fn14) is a member of the TNF receptor superfamily and a bona fide receptor of the TWEAK cytokine. Accumulating evidence suggests that Fn14 levels are increased in catabolic conditions as well as during exercise. However, the role of Fn14 in the regulation of skeletal muscle mass and function remains poorly understood. In this study, through the generation of novel skeletal muscle-specific Fn14-knockout mice, we have investigated the muscle role of Fn14 in the regulation of exercise capacity and denervation-induced muscle atrophy. Our results demonstrate that there was no difference in skeletal muscle mass between control and muscle-specific Fn14-knockout mice. Nevertheless, the deletion of Fn14 in skeletal muscle significantly improved exercise capacity and resistance to fatigue. This effect of Fn14 deletion is associated with an increased proportion of oxidative myofibers and higher capillaries number per myofiber in skeletal muscle. Furthermore, our results demonstrate that targeted deletion of Fn14 inhibits denervation-induced muscle atrophy in adult mice. Deletion of Fn14 reduced the expression of components of the ubiquitin-proteasome system and non-canonical NF-kappa B signaling in denervated skeletal muscle, as well as increased the phosphorylation of Akt kinase and FoxO3a transcription factor. Collectively, our results demonstrate that targeted inhibition of Fn14 improves exercise tolerance and inhibits denervation-induced muscle atrophy in adult mice.

Estrogen-related receptor alpha is an AMPK-regulated factor that promotes ischemic muscle revascularization and recovery in diet-induced obese mice.

Obesity and type II diabetes are leading causes of peripheral arterial disease (PAD), which is characterized by vascular insufficiency and ischemic damage in the limb skeletal muscle. Glycemic control is not sufficient to prevent progression of PAD, and molecular targets that can promote muscle neo-angiogenesis in obesity and diabetes remain poorly defined. Here, we have investigated whether nuclear receptor estrogen-related receptor alpha (ERRα) can promote ischemic revascularization in the skeletal muscles of diet-induced obese (DIO) mice. Using muscle-specific ERRα transgenic mice, we found that ERRα overexpression promotes revascularization, marked by increased capillary staining and muscle perfusion in DIO mice after hindlimb ischemic injury. Furthermore, ERRα facilitates repair and restoration of skeletal muscle myofiber size after limb ischemia in DIO mice. The ameliorative effects of ERRα overexpression did not involve the prevention of weight gain, hyperglycemia or glucose/insulin intolerance, suggesting a direct role for ERRα in promoting angiogenesis. Interestingly, levels of endogenous ERRα protein are suppressed in the skeletal muscles of DIO mice compared to lean controls, coinciding with the suppression of angiogenic gene expression, and reduced AMPK signaling in the DIO skeletal muscles. Upon further investigating the link between AMPK and ERRα, we found that AMPK activation increases the expression and recruitment of ERRα protein to specific angiogenic gene promoters in muscle cells. Further, the induction of angiogenic factors by AMPK activators in muscle cells is blocked by repressing ERRα. In summary, our results identify an AMPK/ERRα-dependent angiogenic gene program in the skeletal muscle, which is repressed by DIO, and demonstrate that forced ERRα activation can promote ischemic revascularization and muscle recovery in obesity.

Estrogen-related receptor α is involved in angiogenesis and skeletal muscle revascularization in hindlimb ischemia.

Skeletal muscle ischemia is a major consequence of peripheral arterial disease (PAD) or critical limb ischemia (CLI). Although therapeutic options for resolving muscle ischemia in PAD/CLI are limited, the issue is compounded by poor understanding of the mechanisms driving muscle vascularization. We found that nuclear receptor estrogen-related receptor alpha (ERRα) expression is induced in murine skeletal muscle by hindlimb ischemia (HLI), and in cultured myotubes by hypoxia, suggesting a potential role for ERRα in ischemic response. To test this, we generated skeletal muscle-specific ERRα transgenic (TG) mice. In these mice, ERRα drives myofiber type switch from glycolytic type IIB to oxidative type IIA/IIX myofibers, which are typically associated with more vascular supply in muscle. Indeed, RNA sequencing and functional enrichment analysis of TG muscle revealed that "paracrine angiogenesis" is the top-ranked transcriptional program activated by ERRα in the skeletal muscle. Immunohistochemistry and angiography showed that ERRα overexpression increases baseline capillarity, arterioles and non-leaky blood vessel formation in the skeletal muscles. Moreover, ERRα overexpression facilitates ischemic neo-angiogenesis and perfusion recovery in hindlimb musculature of mice subjected to HLI. Therefore, ERRα is a hypoxia inducible nuclear receptor that is involved in skeletal muscle angiogenesis and could be potentially targeted for treating PAD/CLI.

TAK1 preserves skeletal muscle mass and mitochondrial function through redox homeostasis.

Skeletal muscle atrophy is debilitating consequence of a large number of chronic disease states, aging, and disuse conditions. Skeletal muscle mass is regulated through coordinated activation of a number of signaling cascades. Transforming growth factor-ß activated kinase 1 (TAK1) is a central kinase that mediates the activation of multiple signaling pathways in response to various growth factors, cytokines, and microbial products. Accumulating evidence suggests that TAK1 promotes skeletal muscle growth and essential for the maintenance of muscle mass in adults. Targeted inactivation of TAK1 leads to severe muscle wasting and kyphosis in mice. However, the mechanisms by which TAK1 prevents loss of muscle mass remain poorly understood. Through generation of inducible skeletal muscle-specific Tak1-knockout mice, we demonstrate that targeted ablation of TAK1 disrupts redox signaling leading to the accumulation of reactive oxygen species and loss of skeletal muscle mass and contractile function. Suppression of oxidative stress using Trolox improves muscle contractile function and inhibits the activation of catabolic signaling pathways in Tak1-deficient muscle. Moreover, Trolox inhibits the activation of ubiquitin-proteasome system and autophagy markers in skeletal muscle of Tak1-deficient mice. Furthermore, inhibition of oxidative stress using Trolox prevents the slow-to-fast type fiber transition and improves mitochondrial respiration in skeletal muscle of Tak1-deficient mice. Overall, our results demonstrate that TAK1 maintains skeletal muscle mass and health through redox homeostasis.

Loss of Estrogen-Related Receptor Alpha Facilitates Angiogenesis in Endothelial Cells.

Estrogen-related receptors (ERRs) have emerged as major metabolic regulators in various tissues. However, their expression and function in the vasculature remains unknown. Here, we report the transcriptional program and cellular function of ERRα in endothelial cells (ECs), a cell type with a multifaceted role in vasculature. Of the three ERR subtypes, ECs exclusively express ERRα. Gene expression profiling of ECs lacking ERRα revealed that ERRα predominantly acts as a transcriptional repressor, targeting genes linked with angiogenesis, cell migration, and cell adhesion. ERRα-deficient ECs exhibit decreased proliferation but increased migration and tube formation. ERRα depletion increased basal as well as vascular endothelial growth factor A (VEGFA)- and ANG1/2-stimulated angiogenic sprouting in endothelial spheroids. Moreover, retinal angiogenesis is enhanced in ERRα knockout mice compared to that in wild-type mice. Surprisingly, ERRα is dispensable for the regulation of its classic targets, such as metabolism, mitochondrial biogenesis, and cellular respiration in the ECs. ERRα is enriched at the promoters of angiogenic, migratory, and cell adhesion genes. Further, VEGFA increased ERRα recruitment to angiogenesis-associated genes and simultaneously decreased their expression. Despite increasing its gene occupancy, proangiogenic stimuli decrease ERRα expression in ECs. Our work shows that endothelial ERRα plays a repressive role in angiogenesis and potentially fine-tunes growth factor-mediated angiogenesis.

GPCRs profiling and identification of GPR110 as a potential new target in HER2+ breast cancer.

PURPOSE: G protein-coupled receptors (GPCRs) represent the largest family of druggable targets in human genome. Although several GPCRs can cross-talk with the human epidermal growth factor receptors (HERs), the expression and function of most GPCRs remain unknown in HER2+ breast cancer (BC). In this study, we aimed to evaluate gene expression of GPCRs in tumorigenic or anti-HER2 drug-resistant cells and to understand the potential role of candidate GPCRs in HER2+ BC. METHODS: Gene expression of 352 GPCRs was profiled in Aldeflur+ tumorigenic versus Aldeflur- population and anti-HER2 therapy-resistant derivatives versus parental cells of HER2+ BT474 cells. The GPCR candidates were confirmed in 7 additional HER2+ BC cell line models and publicly available patient dataset. Anchorage-dependent and anchorage-independent cell growth, mammosphere formation, and migration/invasion were evaluated upon GPR110 knockdown by siRNA in BT474 and SKBR3 parental and lapatinib+ trastuzumab-resistant (LTR) cells. RESULTS: Adhesion and class A GPCRs were overexpressed in Aldeflur+ and anti-HER2 therapy-resistant population of BT474 cells, respectively. GPR110 was the only GPCR overexpressed in Aldeflur+ and anti-HER2 therapy-resistant population in BT474, SKBR3, HCC1569, MDA-MB-361, AU565, and/or HCC202 cells and in HER2+ BC subtype in patient tumors. Using BT474 and SKBR3 parental and LTR cells, we found that GPR110 knockdown significantly reduced anchorage-dependent/independent cell growth as well as migration/invasion of parental and LTR cells and mammosphere formation in LTR derivatives and not in parental cells. CONCLUSION: Our data suggest a potential role of GPR110 in tumorigenicity and in tumor cell dissemination in HER2+ BC.

Muscle Arnt/Hif1ß Is Dispensable in Myofiber Type Determination, Vascularization and Insulin Sensitivity.

Aryl Hydrocarbon Receptor Nuclear Translocator/ hypoxia-inducible factor 1 beta (ARNT/ HIF1ß), a member of bHLH-PAS family of transcriptional factors, plays a critical role in metabolic homeostasis, insulin resistance and glucose intolerance. The contributions of ARNT in pancreas, liver and adipose tissue to energy balance through gene regulation have been described. Surprisingly, the impact of ARNT signaling in the skeletal muscles, one of the major organs involved in glucose disposal, has not been investigated, especially in type II diabetes. Here we report that ARNT is expressed in the skeletal muscles, particularly in the energy-efficient oxidative slow-twitch myofibers, which are characterized by increased oxidative capacity, mitochondrial content, vascular supply and insulin sensitivity. However, muscle-specific deletion of ARNT did not change myofiber type distribution, oxidative capacity, mitochondrial content, capillarity, or the expression of genes associated with these features. Consequently, the lack of ARNT in the skeletal muscle did not affect weight gain, lean/fat mass, insulin sensitivity and glucose tolerance in lean mice, nor did it impact insulin resistance and glucose intolerance in high fat diet-induced obesity. Therefore, skeletal muscle ARNT is dispensable for controlling muscle fiber type and metabolic regulation, as well as diet-induced weight control, insulin sensitivity and glucose tolerance.

Enhanced exercise and regenerative capacity in a mouse model that violates size constraints of oxidative muscle fibres.

A central tenet of skeletal muscle biology is the existence of an inverse relationship between the oxidative fibre capacity and its size. However, robustness of this relationship is unknown. We show that superimposition of Estrogen-related receptor gamma (Errγ) on the myostatin (Mtn) mouse null background (Mtn(-/-)/Errγ(Tg/+)) results in hypertrophic muscle with a high oxidative capacity thus violating the inverse relationship between fibre size and oxidative capacity. We also examined the canonical view that oxidative muscle phenotype positively correlate with Satellite cell number, the resident stem cells of skeletal muscle. Surprisingly, hypertrophic fibres from Mtn(-/-)/Errγ(Tg/+) mouse showed satellite cell deficit which unexpectedly did not affect muscle regeneration. These observations 1) challenge the concept of a constraint between fibre size and oxidative capacity and 2) indicate the important role of the microcirculation in the regenerative capacity of a muscle even when satellite cell numbers are reduced.

Nuclear Perilipin 5 integrates lipid droplet lipolysis with PGC-1α/SIRT1-dependent transcriptional regulation of mitochondrial function.

Dysfunctional cellular lipid metabolism contributes to common chronic human diseases, including type 2 diabetes, obesity, fatty liver disease and diabetic cardiomyopathy. How cells balance lipid storage and mitochondrial oxidative capacity is poorly understood. Here we identify the lipid droplet protein Perilipin 5 as a catecholamine-triggered interaction partner of PGC-1α. We report that during catecholamine-stimulated lipolysis, Perilipin 5 is phosphorylated by protein kinase A and forms transcriptional complexes with PGC-1α and SIRT1 in the nucleus. Perilipin 5 promotes PGC-1α co-activator function by disinhibiting SIRT1 deacetylase activity. We show by gain-and-loss of function studies in cells that nuclear Perilipin 5 promotes transcription of genes that mediate mitochondrial biogenesis and oxidative function. We propose that Perilipin 5 is an important molecular link that couples the coordinated catecholamine activation of the PKA pathway and of lipid droplet lipolysis with transcriptional regulation to promote efficient fatty acid catabolism and prevent mitochondrial dysfunction.

Exercise-like effects by Estrogen-related receptor-gamma in muscle do not prevent insulin resistance in db/db mice.

Dissecting exercise-mimicking pathways that can replicate the benefits of exercise in obesity and diabetes may lead to promising treatments for metabolic disorders. Muscle estrogen-related receptor gamma (ERRγ) is induced by exercise, and when over-expressed in the skeletal muscle mimics exercise by stimulating glycolytic-to-oxidative myofiber switch, mitochondrial biogenesis and angiogenesis in lean mice. The objective of this study was to test whether muscle ERRγ in obese mice mitigates weight gain and insulin resistance. To do so, ERRγ was selectively over-expressed in the skeletal muscle of obese and diabetic db/db mice. Muscle ERRγ over-expression successfully triggered glycolytic-to-oxidative myofiber switch, increased functional mitochondrial content and boosted vascular supply in the db/db mice. Despite aerobic remodeling, ERRγ surprisingly failed to improve whole-body energy expenditure, block muscle accumulation of triglycerides, toxic diacylglycerols (DAG) and ceramides or suppress muscle PKCε sarcolemmal translocation in db/db mice. Consequently, muscle ERRγ did not mitigate impaired muscle insulin signaling or insulin resistance in these mice. In conclusion, obesity and diabetes in db/db mice are not amenable to selective ERRγ-directed programming of classic exercise-like effects in the skeletal muscle. Other biochemical pathways or integrated whole-body effects of exercise may be critical for resisting diabetes and obesity.

Muscle ERRγ mitigates Duchenne muscular dystrophy via metabolic and angiogenic reprogramming.

Treatment of Duchenne muscular dystrophy (DMD) by replacing mutant dystrophin or restoring dystrophin-associated glycoprotein complex (DAG) has been clinically challenging. Instead, identifying and targeting muscle pathways deregulated in DMD will provide new therapeutic avenues. We report that the expression of nuclear receptor estrogen-related receptor-γ (ERRγ), and its metabolic and angiogenic targets are down-regulated (50-85%) in skeletal muscles of mdx mice (DMD model) vs. wild-type mice. Corelatively, oxidative myofibers, muscle vasculature, and exercise tolerance (33%) are decreased in mdx vs. wild-type mice. Overexpressing ERRγ selectively in the dystrophic muscles of the mdx mice restored metabolic and angiogenic gene expression compared with control mdx mice. Further, ERRγ enhanced muscle oxidative myofibers, vasculature, and blood flow (by 33-66%) and improved exercise tolerance (by 75%) in the dystrophic mice. Restoring muscle ERRγ pathway ameliorated muscle damage and also prevented DMD hallmarks of postexercise muscle damage, hypoxia, and fatigue in mdx mice. Notably, ERRγ did not restore sarcolemmal DAG complex, which is thus dispensable for antidystrophic effects of ERRγ. In summary, ERRγ-dependent metabolic and angiogenic gene program is defective in DMD, and we demonstrate that its restoration is a potential strategy for treating muscular dystrophy.

Revascularization of ischemic skeletal muscle by estrogen-related receptor-γ.

RATIONALE: Oxidative myofibers in the skeletal muscles express high levels of angiogenic factors, have dense vasculature, and promptly revascularize during ischemia. Estrogen-related receptor-gamma (ERRγ) activates genes that govern metabolic and vascular features typical to oxidative myofibers. Therefore, ERRγ-dependent remodeling of the myofibers may promote neoangiogenesis and restoration of blood perfusion in skeletal muscle ischemia. OBJECTIVE: To investigate the muscle fiber type remodeling by ERRγ and its role in the vascular recovery of ischemic muscle. METHODS AND RESULTS: Using immunohistology, we show that skeletal muscle-specific transgenic overexpression of ERRγ increases the proportions of oxidative and densely vascularized type IIA and IIX myofibers and decreases glycolytic and less vascularized type IIB myofibers. This myofiber remodeling results in a higher basal blood flow in the transgenic skeletal muscle. By applying unilateral hind limb ischemia to transgenic and wild-type mice, we found accelerated revascularization (fluorescent microangiography), restoration of blood perfusion (laser Doppler flowmetry), and muscle repair (Evans blue dye exclusion) in transgenic compared to wild-type ischemic muscles. This ameliorative effect is linked to enhanced neoangiogenesis (CD31 staining and microfil perfusion) by ERRγ. Using cultured muscle cells in which ERRγ is inactivated, we show that the receptor is dispensable for the classical hypoxic response of transcriptional upregulation and secretion of vascular endothelial growth factor A. Rather, the ameliorative effect of ERRγ is linked to the receptor-mediated increase in oxidative myofibers that inherently express and secrete high levels of angiogenic factors. CONCLUSIONS: The ERRγ is a hypoxia-independent inducer of neoangiogenesis that can promote reparative revascularization.

Exercise training attenuates the hypermuscular phenotype and restores skeletal muscle function in the myostatin null mouse.

Myostatin regulates both muscle mass and muscle metabolism. The myostatin null (MSTN(-/-)) mouse has a hypermuscular phenotype owing to both hypertrophy and hyperplasia of the myofibres. The enlarged muscles display a reliance on glycolysis for energy production; however, enlarged muscles that develop in the absence of myostatin have compromised force-generating capacity. Recent evidence has suggested that endurance exercise training increases the oxidative properties of muscle. Here, we aimed to identify key changes in the muscle phenotype of MSTN(-/-) mice that can be induced by training. To this end, we subjected MSTN(-/-) mice to two different forms of training, namely voluntary wheel running and swimming, and compared the response at the morphological, myocellular and molecular levels. We found that both regimes normalized changes of myostatin deficiency and restored muscle function. We showed that both exercise training regimes increased muscle capillary density and the expression of Ucp3, Cpt1α, Pdk4 and Errγ, key markers for oxidative metabolism. Cross-sectional area of hypertrophic myofibres from MSTN(-/-) mice decreased towards wild-type values in response to exercise and, in this context, Bnip3, a key autophagy-related gene, was upregulated. This reduction in myofibre size caused an increase of the nuclear-to-cytoplasmic ratio towards wild-type values. Importantly, both training regimes increased muscle force in MSTN(-/-) mice. We conclude that impaired skeletal muscle function in myostatin-deficient mice can be improved through endurance exercise-mediated remodelling of muscle fibre size and metabolic profile.

Exercise and PGC-1α-independent synchronization of type I muscle metabolism and vasculature by ERRγ.

How type I skeletal muscle inherently maintains high oxidative and vascular capacity in the absence of exercise is unclear. We show that nuclear receptor ERRγ is highly expressed in type I muscle and, when transgenically expressed in anaerobic type II muscles (ERRGO mice), dually induces metabolic and vascular transformation in the absence of exercise. ERRGO mice show increased expression of genes promoting fat metabolism, mitochondrial respiration, and type I fiber specification. Muscles in ERRGO mice also display an activated angiogenic program marked by myofibrillar induction and secretion of proangiogenic factors, neovascularization, and a 100% increase in running endurance. Surprisingly, the induction of type I muscle properties by ERRγ does not involve PGC-1α. Instead, ERRγ genetically activates the energy sensor AMPK in mediating the metabovascular changes in ERRGO mice. Therefore, ERRγ represents a previously unrecognized determinant that specifies intrinsic vascular and oxidative metabolic features that distinguish type I from type II muscle.

Endurance exercise mimetics in skeletal muscle.

Regular exercise promotes favorable structural and metabolic adaptations, especially in the skeletal muscle, to boost endurance and cardiovascular health. These changes are driven by a network of incompletely understood molecular pathways that trigger transcriptional remodeling of the skeletal muscle. In this article, we describe recent advances in the understanding of the key components of this circuitry [namely peroxisome proliferator activator receptor delta (PPARdelta), adenosine monophosphate (AMP)-activated protein kinase (AMPK), silent information regulator two protein 1 (SIRT1), and peroxisome proliferator-activated receptor gamma coactivator-1alpha (PGC-1alpha)] that govern aerobic transformation of the skeletal muscles. We also discuss recent discoveries that raise the possibility of synthetically mimicking exercise with pathway-specific drugs to improve aerobic capacity and, in turn, health.

Dopamine recruits D1A receptors to Na-K-ATPase-rich caveolar plasma membranes in rat renal proximal tubules.

Activation of dopamine D(1A) receptors in renal proximal tubules causes inhibition of sodium transporters (Na-K-ATPase and Na/H exchanger), leading to a decrease in sodium reabsorption. In addition to being localized on the plasma membrane, D(1A) receptors are mainly present in intracellular compartments under basal conditions. We observed, using [(3)H]SCH-23390 binding and immunoblotting, that dopamine recruits D(1A) receptors to the plasma membrane in rat renal proximal tubules. Furthermore, radioligand binding and/or immunoblotting experiments using pharmacological modulators showed that dopamine-induced D(1A) receptor recruitment requires activation of cell surface D(1)-like receptors, activation of adenylyl cyclase, and intact endocytic vesicles with internal acidic pH. A key finding of this study was that these recruited D(1A) receptors were functional because they potentiated dopamine-induced [(35)S]GTPgammaS binding, cAMP accumulation, and Na-K-ATPase inhibition. Interestingly, dopamine increased immunoreactivity of D(1A) receptors specifically in caveolin-rich plasma membranes isolated by a sucrose density gradient. In support of this observation, coimmunoprecipitation studies showed that D(1A) receptors interacted with caveolin-2 in an agonist-dependent fashion. The caveolin-rich plasma membranes had a high content of the alpha(1)-subunit of Na-K-ATPase, which is a downstream target of D(1A) receptor signaling in proximal tubules. These results show that dopamine, via the D(1)-like receptor-adenylyl cyclase pathway, recruits D(1A) receptors to the plasma membrane. These newly recruited receptors couple to G proteins, increase cAMP, and participate in dopamine-mediated inhibition of Na-K-ATPase in proximal tubules. Moreover, dopamine-induced recruitment of D(1A) receptors to the caveolin-rich plasma membranes brings them in close proximity to targets such as Na-K-ATPase in proximal tubules of Sprague-Dawley rats.