Dysfunctional adipocyte calcium handling is implicated in obesity and thermogenesis. Junctophilins (JPs) stabilize calcium microdomain junctions between the plasma membrane and endoplasmic reticulum, but whether JPs are required for adipocyte function is not known. We show that JP2 is enriched in thermogenic brown adipose tissue (BAT) relative to other fat depots and is downregulated under conditions of nutrient overload. Conditional knockdown of JP2 in adipocytes, and more selectively in BAT, exacerbates cold intolerance and susceptibility to diet induced obesity. Mechanistically, JP2-depleted brown adipocytes exhibit calcium handling dysfunction with elevated cytosolic calcium levels at baseline but diminished norepinephrine-induced calcium transients, reduced store-operated calcium entry. Basal cytosolic calcium overload accounts for an increase in calpain activation and ensuing downregulation of STIM1 and hormone-sensitive lipase in JP2-depleted cells. Furthermore, JP2 silencing in brown adipocytes reduced oxygen consumption rates and compromised mitochondrial structure and quality. Together, these findings demonstrate that JP2 is essential for normal calcium homeostasis in brown adipocytes and reveal a critical role for JP2 in thermogenesis and resistance to diet-induced metabolic dysregulation.

This study investigates the role and mechanisms by which the myokine musclin promotes exercise-induced cardiac conditioning. Exercise is one of the most powerful triggers of cardiac conditioning with proven benefits for healthy and diseased hearts. There is an emerging understanding that muscles produce and secrete myokines, which mediate local and systemic “crosstalk” to promote exercise tolerance and overall health, including cardiac conditioning. The myokine musclin, highly conserved across animal species, has been shown to be upregulated in response to physical activity. However, musclin effects on exercise-induced cardiac conditioning are not established. Following completion of a treadmill exercise protocol, wild type (WT) mice and mice with disruption of the musclin-encoding gene, Ostn, had their hearts extracted and exposed to an ex vivo ischemia-reperfusion protocol or biochemical studies. Disruption of musclin signaling abolished the ability of exercise to mitigate cardiac ischemic injury. This impaired cardioprotection was associated with reduced mitochondrial content and function linked to blunted cyclic guanosine monophosphate (cGMP) signaling. Genetic deletion of musclin reduced the nuclear abundance of protein kinase G (PKGI) and cyclic adenosine monophosphate (cAMP) response element binding (CREB), resulting in suppression of the master regulator of mitochondrial biogenesis, peroxisome proliferator-activated receptor γ coactivator 1α (PGC1α), and its downstream targets in response to physical activity. Synthetic musclin peptide pharmacokinetic parameters were defined and used to calculate the infusion rate necessary to maintain its plasma level comparable to that observed after exercise. This infusion was found to reproduce the cardioprotective benefits of exercise in sedentary WT and Ostn-KO mice. Musclin is essential for exercise-induced cardiac protection. Boosting musclin signaling might serve as a novel therapeutic strategy for cardioprotection.

Downregulation of endothelial Sirtuin1 (Sirt1) in insulin resistant states contributes to vascular dysfunction. Furthermore, Sirt1 deficiency in skeletal myocytes promotes insulin resistance. Here, we show that deletion of endothelial Sirt1, while impairing endothelial function, paradoxically improves skeletal muscle insulin sensitivity. Compared to wild-type mice, male mice lacking endothelial Sirt1 (E-Sirt1-KO) preferentially utilize glucose over fat, and have higher insulin sensitivity, glucose uptake, and Akt signaling in fast-twitch skeletal muscle. Enhanced insulin sensitivity of E-Sirt1-KO mice is transferrable to wild-type mice via the systemic circulation. Endothelial Sirt1 deficiency, by inhibiting autophagy and activating nuclear factor-kappa B signaling, augments expression and secretion of thymosin beta-4 (Tβ4) that promotes insulin signaling in skeletal myotubes. Thus, unlike in skeletal myocytes, Sirt1 deficiency in the endothelium promotes glucose homeostasis by stimulating skeletal muscle insulin sensitivity through a blood-borne mechanism, and augmented secretion of Tβ4 by Sirt1-deficient endothelial cells boosts insulin signaling in skeletal muscle cells.

AIMS The study investigates the role and mechanisms of clinically translatable exercise heart rate (HR) envelope effects, without dyssynchrony, on myocardial ischaemia tolerance compared to standard preconditioning methods. Since the magnitude and duration of exercise HR acceleration are tightly correlated with beneficial cardiac outcomes, it is hypothesized that a paced exercise-similar HR envelope, delivered in a maximally physiologic way that avoids the toxic effects of chamber dyssynchrony, may be more than simply a readout, but rather also a significant trigger of myocardial conditioning and stress resistance. METHODS AND RESULTS For 8 days over 2 weeks, sedated mice were atrial-paced once daily via an oesophageal electrode to deliver an exercise-similar HR pattern with preserved atrioventricular and interventricular synchrony. Effects on cardiac calcium handling, protein expression/modification, and tolerance to ischaemia-reperfusion (IR) injury were assessed and compared to those in sham-paced mice and to the effects of exercise and ischaemic preconditioning (IPC). The paced cohort displayed improved myocardial IR injury tolerance vs. sham controls with an effect size similar to that afforded by treadmill exercise or IPC. Hearts from paced mice displayed changes in Ca2+ handling, coupled with changes in phosphorylation of calcium/calmodulin protein kinase II, phospholamban and ryanodine receptor channel, and transcriptional remodelling associated with a cardioprotective paradigm. CONCLUSIONS The HR pattern of exercise, delivered by atrial pacing that preserves intracardiac synchrony, induces cardiac conditioning and enhances ischaemic stress resistance. This identifies the HR pattern as a signal for conditioning and suggests the potential to repurpose atrial pacing for cardioprotection.

ABSTRACT Objective The paraventricular nucleus of hypothalamus (PVN) is an integrative center in the brain orchestrating a wide range of physiological and behavioral responses. While the PVN melanocortin 4 receptor (MC4R) signaling (PVN MC4R+ ) is undoubtedly involved in feeding regulation, the neuroanatomical organization of PVN MC4R+ pathway and its role in diverse physiological and behavioral regulations have not been fully understood. Here we aimed to better characterize the input-output organization of PVN MC4R+ neurons and further test their potential functional roles beyond feeding. Methods Using a combination of viral tools, we performed a comprehensive mapping of PVN MC4R+ circuits and tested the effects of chemogenetic activation of PVN MC4R+ neurons on thermogenesis, cardiovascular control and other behavioral regulations beyond feeding. Results We found that PVN MC4R+ neurons broadly innervate many different brain regions known to be important not only for feeding but also for neuroendocrine and autonomic control of thermogenesis and cardiovascular function, including but not limited to preoptic area, median eminence, parabrachial nucleus, locus coeruleus, nucleus of solitary tract, ventrolateral medulla and thoracic spinal cord. Contrary to broad efferent projections, PVN MC4R+ neurons receive monosynaptic inputs from limited brain regions, including medial preoptic nucleus, arcuate and dorsomedial hypothalamic nuclei, and supraoptic nucleus. Consistent with broad efferent projections, chemogenetic activation of PVN MC4R+ neurons not only suppressed feeding but also led to an apparent increase in heart rate, blood pressure and brown adipose tissue thermogenesis. Strikingly, these physiological changes accompanied an unexpected repetitive bedding-removing behavior followed by hypoactivity and resting-like behavior. Conclusions Our results clarify the neuroanatomical organization of PVN MC4R+ circuits and shed new light on the roles of PVN MC4R+ pathways in autonomic control of thermogenesis, cardiovascular function and other behavioral regulations.

Objective: RGS2 is a GTPase activating protein that modulates GPCR-Ga signaling and mice lacking RGS2 globally exhibit metabolic alterations. While RGS2 is known to be broadly expressed throughout the body including the brain, the relative contribution of brain RGS2 to metabolic homeostasis remains unknown. The purpose of this study was to characterize RGS2 expression in the paraventricular nucleus of hypothalamus (PVN) and test its role in metabolic homeostasis.Methods: We used a combination of RNAscope in situ hybridization (ISH), immunohistochemistry, and bioinformatic analyses to characterize the pattern of Rgs2 expression in the PVN. We then created mice lacking Rgs2 either prenatally or postnatally in the PVN and evaluated their metabolic consequences.Results: RNAscope ISH analysis revealed a broad but regionally enriched Rgs2 mRNA expression throughout the mouse brain, with the highest expression being observed in the PVN along with several other brain regions, such as the arcuate nucleus of hypothalamus and the dorsal raphe nucleus. Within the PVN, we found that Rgs2 is specifically enriched in CRH+ endocrine neurons and is further increased by calorie restriction. Functionally, although Sim1-Cre-mediated prenatal deletion of Rgs2 in PVN neurons had no major effects on metabolic homeostasis, AAV-mediated adult deletion of Rgs2 in the PVN led to significantly increased food intake, body weight (both fat and fat-free masses), body length, and blood glucose levels in both male and female mice. Strikingly, we found that prolonged postnatal loss of Rgs2 leads to neuronal cell death in the PVN, while rapid body weight gain in the early phase of viral-mediated PVN Rgs2 deletion is independent of PVN neuronal loss. Conclusions: Our results provide the first evidence to show that PVN Rgs2 expression is not only sensitive to metabolic challenge but also critically required for PVN endocrine neurons to function and maintain metabolic homeostasis.(c) 2022 The Author(s). Published by Elsevier GmbH. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

Objective: Fibroblast growth factor 21 (FGF21) is a peripherally-derived endocrine hormone that acts on the central nervous system (CNS) to regulate whole body energy homeostasis. Pharmacological administration of FGF21 promotes weight loss in obese animal models and human subjects with obesity. However, the central targets mediating these effects are incompletely defined. Methods: To explore the mechanism for FGF21's effects to lower body weight, we pharmacologically administer FGF21 to genetic animal models lacking the obligate FGF21 co-receptor, β-klotho (KLB), in either glutamatergic (Vglut2-Cre) or GABAergic (Vgat-Cre) neurons. In addition, we abolish FGF21 signaling to leptin receptor (LepR-Cre) positive cells. Finally, we examine the synergistic effects of FGF21 and leptin to lower body weight and explore the importance of physiological leptin levels in FGF21-mediated regulation of body weight. Results: Here we show that FGF21 signaling to glutamatergic neurons is required for FGF21 to modulate energy expenditure and promote weight loss. In addition, we demonstrate that FGF21 signals to leptin receptor-expressing cells to regulate body weight, and that central leptin signaling is required for FGF21 to fully stimulate body weight loss during obesity. Interestingly, co-administration of FGF21 and leptin synergistically leads to robust weight loss. Conclusions: These data reveal an important endocrine crosstalk between liver- and adipose-derived signals which integrate in the CNS to modulate energy homeostasis and body weight regulation.

Objectives: The hypothalamic ventromedial nucleus (VMH) plays a major role in metabolic control, but the molecular mechanisms involved remain poorly defined. We analyzed the relevance of the BBSome, a protein complex composed of 8 Bardet-Biedl syndrome (BBS) proteins including BBS1, in VMH steroidogenic factor 1 (SF1) neurons for the control of energy homeostasis and related physiological processes. Methods: We generated mice bearing selective BBSome disruption, through Bbs1 gene deletion, in SF1 neurons (SF1(Cre)/Bbs1(fl/fl)). We analyzed the consequence on body weight, glucose homeostasis, and cardiovascular autonomic function of BBSome loss in SF1 neurons. Results: SF1(Cre)/Bbs1(fl/fl) mice had increased body weight and adiposity under normal chow conditions. Food intake, energy absorption, and digestive efficiency were not altered by Bbs1 gene deletion in SF1 neurons. SF1(Cre)/Bbs1(fl/fl) mice exhibited lower energy expenditure, particularly during the dark cycle. Consistent with this finding, SF1(Cre)/Bbs1(fl/fl) mice displayed reduced sympathetic nerve traffic and expression of markers of thermogenesis in brown adipose tissue. SF1(Cre)/Bbs1(fl/fl) mice also had lower sympathetic nerve activity to subcutaneous white adipose tissue that was associated with a protein expression profile that promotes lipid accumulation. Notably, despite obesity and hyperinsulinemia, SF1(Cre)/Bbs1(fl/fl) mice did not exhibit significant changes in glucose metabolism, insulin sensitivity, blood pressure, and baroreflex sensitivity. Conclusions: Our findings demonstrate that the SF1 neuron BBSome is necessary for the regulation of energy homeostasis through modulation of the activity of the sympathetic nervous system and that the SF1 neuron BBSome is required for the development of obesity-related comorbidities. (C) 2021 The Authors. Published by Elsevier GmbH. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

Brown adipose tissue (BAT) thermogenic activity is tightly regulated by cellular redox status, but the underlying molecular mechanisms are incompletely understood. Protein S-nitrosylation, the nitric-oxide-mediated cysteine thiol protein modification, plays important roles in cellular redox regulation. Here we show that diet induced obesity (DIO) and acute cold exposure elevate BAT protein S-nitrosylation, including UCP1. This thermogenic-induced nitric oxide bioactivity is regulated by S-nitrosoglutathione reductase (GSNOR; alcohol dehydrogenase 5 [ADH5]), a denitrosylase that balances the intracellular nitroso-redox status. Loss of ADH5 in BAT impairs cold-induced UCP1-dependent thermogenesis and worsens obesity-associated metabolic dysfunction. Mechanistically, we demonstrate that Adh5 expression is induced by the transcription factor heat shock factor 1 (HSF1), and administration of an HSF1 activator to BAT of DIO mice increases Adh5 expression and significantly improves UCP1-mediated respiration. Together, these data indicate that ADH5 controls BAT nitroso-redox homeostasis to regulate adipose thermogenesis, which may be therapeutically targeted to improve metabolic health.

Central melanocortin pathways, mainly via acting on melanocortin-4 receptor (MC4R), play an important role in both energy homeostasis and sympathetic control of cardiovascular function. The paraventricular nucleus of hypothalamus (PVN) is one of hypothalamic nuclei with highest MC4R expression and is well known for its role in metabolic and autonomic regulations; however, the role of PVN-MC4R neural pathways in this regard remains incompletely understood. In this study, we first tested whether chemogenetic activation of PVN-MC4R neurons affects cardiovascular function. Adeno-associated virus (AAV) expressing excitatory (hM3Dq) Designer Receptors Exclusively Activated by Designer Drugs (DREADD) receptors were directly injected into the PVN of MC4R-Cre knock-in mice that are implanted with radio-telemetry for chronic monitoring of blood pressure. DREADD activation of PVN-MC4R neurons significantly elevated mean arterial pressure and heart rate. Next, we prepared a separate group of mice and subjected them to a battery of tests to gain insight into the role of these neurons in other behavioral and physiological regulations. Whole-body infrared thermal imaging upon DREADD activation of PVN-MC4R neurons revealed significant increase of temperature in near neck region, lower back, and tail. Behavioral monitoring in PhenoTyper cage further showed that DREADD activation of PVN-MC4R neurons leads to hypoactivity, suppressed feeding, and trend toward increased drinking behavior. In order to gain further insight into the neuroanatomical organization of PVN-MC4R neurons, we comprehensively mapped afferent projections of PVN-MC4R neurons by stereotaxically delivering Cre-dependent AAV driving expression of eYFP into the PVN of MC4R-Cre mice, which revealed a broad innervation of PVN-MC4R neurons to the brain regions that are known to be important for metabolic homeostasis and autonomic-cardiovascular control, including but not limited to parabrachial nucleus, nucleus of solitary tract, ventrolateral medulla, and spinal cord. Additionally, using a combination of sophisticated viral tools, we also mapped the brain regions where neurons provide monosynaptic inputs to PVN-MC4R neurons. In addition to the brain regions in which neurons are known to send monosynaptic inputs to PVN-MC4R neurons, such as arcuate nucleus, few discreate presynaptic neurons were also observed in organum vasculosum of the lamina terminalis, bed nucleus of the stria terminalis, supraoptic nucleus (SON) and ventral subiculum (vSub). These results provide an important insight into the neuroanatomical organization and multifaceted role of PVN-MC4R neural pathways in behavioral and physiological regulations.

The protein kinases Unc‐51 like autophagy activating kinase 1 and 2 (ULK1 and ULK2) have evolved from a single yeast gene, autophagy related 1 (Atg1). Although ULK1 and ULK2 are thought to redundantly stimulate autophagy, other functions are emerging. Interestingly, skeletal muscle expresses both ULK1 and ULK2 at high levels (i.e., >2‐fold the median expression of 91 mouse tissues and cell lines) suggesting an important, yet unknown role for their combined elevated expression in muscle. Therefore, to investigate the joint role of ULK1 and ULK2 on muscle homeostasis and whole‐body metabolism we generated mice with skeletal muscle‐specific knockout of these genes (ULK2/1 mKO). Here, we demonstrate that ULK2/1 mKO mice have larger muscles (~12%) that are actually weaker (~21% decrease in relative force) in comparison with wild type littermates. Loss of ULK2/1 in skeletal muscle also affects whole‐body metabolic homeostasis as indicated by decreased body fat mass (~20% at 7wk of age), attenuated fat mass gains with aging (~40% at 21mo. of age), and life‐threatening inability to maintain euglycemia under fasting conditions (~32% reduction of blood glucose compared to WT). Mechanistically, loss of ULK 2/1 not only impairs skeletal muscle autophagy but also AMPK function as indicated by accumulation of LC3, p62, NBR1, and ubiquitinated proteins, as well as reduced phosphorylation of ACC (a surrogate of AMPK activity), respectively. These results identify a previously unknown role for ULK2 and ULK1 in jointly coupling skeletal muscle size with force. They also reveal skeletal muscle ULK2 and ULK1 as fundamental proteins for survival under conditions of reduced nutrient availability.Support or Funding InformationThis work is funded by:AHA 16SDG30360001 (V.A.L.). and NASA Iowa Space Grant Graduate Fellowship Program

The exact mechanisms underlying the metabolic effects of bariatric surgery remain unclear. Here, we demonstrate, using a combination of direct and indirect calorimetry, an increase in total resting metabolic rate (RMR) and specifically anaerobic RMR after Roux-en-Y gastric bypass (RYGB), but not sleeve gastrectomy (SG). We also show an RYGB-specific increase in splanchnic sympathetic nerve activity and "browning" of visceral mesenteric fat. Consequently, selective splanchnic denervation abolishes all beneficial metabolic outcomes of gastric bypass that involve changes in the endocannabinoid signaling within the small intestine. Furthermore, we demonstrate that administration of rimonabant, an endocannabinoid receptor-1 (CB1) inverse agonist, to obese mice mimics RYGB-specific effects on energy balance and splanchnic nerve activity. On the other hand, arachidonoylethanolamide (AEA), a CB1 agonist, attenuates the weight loss and metabolic signature of this procedure. These findings identify CB1 as a key player in energy regulation post-RYGB via a pathway involving the sympathetic nervous system.

Aryl hydrocarbon receptor (AHR) agonists such as dioxin have been associated with obesity and the development of diabetes. Whole-body Ahr knockout mice on high-fat diet (HFD) have been shown to resist obesity and hepatic steatosis. Tissue-specific knockout of Ahr in mature adipocytes via adiponectin-Cre exacerbates obesity while knockout in liver increases steatosis without having significant effects on obesity. Our previous studies demonstrated that treatment of subcutaneous preadipocytes with exogenous or endogenous AHR agonists disrupts maturation into functional adipocytes in vitro. Here, we used platelet-derived growth factor receptor alpha (Pdgfrα)-Cre mice, a Cre model previously established to knock out genes in preadipocyte lineages and other cell types, but not liver cells, to further define AHR's role in obesity. We demonstrate that Pdgfrα-Cre Ahr-floxed (Ahrfl/fl) knockout mice are protected from HFD-induced obesity compared to non-knockout Ahrfl/fl mice (control mice). The Pdgfrα-Cre Ahrfl/fl knockout mice were also protected from increased adiposity, enlargement of adipocyte size, and liver steatosis while on the HFD compared to control mice. On a regular control diet, knockout and non-knockout mice showed no differences in weight gain, indicating the protective phenotype arises only when animals are challenged by a HFD. At the cellular level, cultured cells from brown adipose tissue (BAT) of Pdgfrα-Cre Ahrfl/fl mice were more responsive than cells from controls to transcriptional activation of the thermogenic uncoupling protein 1 (Ucp1) gene by norepinephrine, suggesting an ability to burn more energy under certain conditions. Collectively, our results show that knockout of Ahr mediated by Pdgfrα-Cre is protective against diet-induced obesity and suggest a mechanism by which enhanced UCP1 activity within BAT might confer these effects.

Metabolic cycles are a fundamental element of cellular and organismal function. Among the most critical in higher organisms is the Cori Cycle, the systemic cycling between lactate and glucose. Here, skeletal muscle-specific Mitochondrial Pyruvate Carrier (MPC) deletion in mice diverted pyruvate into circulating lactate. This switch disinhibited muscle fatty acid oxidation and drove Cori Cycling that contributed to increased energy expenditure. Loss of muscle MPC activity led to strikingly decreased adiposity with complete muscle mass and strength retention. Notably, despite decreasing muscle glucose oxidation, muscle MPC disruption increased muscle glucose uptake and whole-body insulin sensitivity. Furthermore, chronic and acute muscle MPC deletion accelerated fat mass loss on a normal diet after high fat diet-induced obesity. Our results illuminate the role of the skeletal muscle MPC as a whole-body carbon flux control point. They highlight the potential utility of modulating muscle pyruvate utilization to ameliorate obesity and type 2 diabetes.

Fibroblast Growth Factor 21 (FGF21) elicits an array of metabolic effects. However, the physiological role of FGF21 during thermal challenges is not clear. In this study, we assessed the tissue source of FGF21 and its site of action to regulate core body temperature in response to cold. Using mice lacking FGF21 specifically in the liver (FGF21 LivKO) or adipose tissues (FGF21 AdipoKO), we performed a series of cold exposure studies to examine the tissue specific induction of FGF21 in response to cold. We also examined the physiological site of FGF21 action during cold exposure by impairing FGF21 signaling to adipose tissues or the central nervous system (CNS) using genetic ablation of the FGF21 co-receptor β-klotho in adipose tissues (KLB AdipoKO) or pharmacological blockage of FGF21 signaling. We found that only liver-derived FGF21 enters circulation during acute cold exposure and is critical for thermoregulation. While FGF21 signaling directly to adipose tissues during cold is dispensable for thermoregulation, central FGF21 signaling is necessary for maximal sympathetic drive to brown adipose tissue to maintain thermoregulation during cold. These data demonstrate a previously unrecognized role for FGF21 in the maintenance of body temperature in response to cold.

Physical activity improves the prognosis of cancer patients, partly by contrasting the associated muscle wasting (cachexia), through still unknown mechanisms. We asked whether aerobic exercise causes secretion by skeletal muscles of proteins (myokines) that may contrast cachexia. Media conditioned by peroxisome proliferator-activated receptor γ coactivator 1α (PGC1α)-expressing myotubes, reproducing some metabolic adaptations of aerobic exercise, as increased mitochondrial biogenesis and oxidative phosphorylation, restrained constitutively active Forkhead box-containing subfamily O3 (caFoxO3)-induced proteolysis. Microarray analysis identified amphiregulin (AREG), natriuretic peptide precursor B (NppB), musclin and fibroblast growth factor 18 (FGF18) as myokines highly induced by PGC1α. Notably, only musclin tended to be low in muscle of mice with a rare human renal carcinoma; it was reduced in plasma and in muscles of C26-bearing mice and in atrophying myotubes, where PGC1α expression is impaired. Therefore, we electroporated the Tibialis Anterior (TA) of C26-bearing mice with musclin or (its receptor) natriuretic peptide receptor 3 (Npr3)-encoding plasmids and found a preserved fiber area, as a result of restrained proteolysis. Musclin knockout (KO) mice lose more muscle tissue during growth of two distinct cachexia-causing tumors. Running protected C26-bearing mice from cachexia, not changing tumor growth, and rescued the C26-induced downregulation of musclin in muscles and plasma. Musclin expression did not change in overloaded plantaris of mice, recapitulating partially muscle adaptations to anaerobic exercise. Musclin might, therefore, be beneficial to cancer patients who cannot exercise and are at risk of cachexia and may help to explain how aerobic exercise alleviates cancer-induced muscle wasting.

Exercise remains the most effective way to promote physical and metabolic wellbeing, but molecular mechanisms underlying exercise tolerance are only partially understood.This study is focused on the recently discovered myokine musclin. Musclin expression has been linked to insulin‐induced activation of protein kinase B (Akt) that phosphorylates the forkhead box O1 transcription factor (FOXO1), thus releasing musclin encoding gene inhibition. Musclin contains two putative serine protease cleavage sites and a region homologous to members of the natriuretic peptide (NP) family. Musclin does not have two cysteine residues needed to form the W‐like structure characteristic for NPs. In line with these characteristics musclin binds to the NP clearance receptor, NPRC, with affinity comparable to NPs, but exhibits only weak binding to NPRA and NPRB without activating the linked guanylyl cyclase. The physiological role of musclin production in skeletal muscles has remained elusive.Here we show that musclin production and secretion into the systemic circulation are stimulated by exercise. Mice subjected to daily treadmill exercise demonstrate an ~100% increase in skeletal muscle musclin protein and mRNA. Plasma levels of circulating musclin are increased from 27.71±5.54 pg/ml in sedentary control mice, to 46.24±4.69 pg/ml in the post‐exercise group.Furthermore, we demonstrate that in our exercise model that Akt is phosphorylated and FOXO1 exported from nuclei in response to exercise. We also show that musclin production is stimulated by Ca2+‐ionophore in human and murine myoblast cell culture in the presence of Ca2+ and is nonresponsive to ionophore in a Ca2+‐free environment. Application of an Akt inhibitor eliminates the stimulatory effect of Ca2+‐ionophore in cell culture establishing that musclin production is driven by Ca2+‐dependent activation of Akt.To establish the physiological role of systemically secreted musclin we created an Ostn (musclin‐encoding gene)‐KO mouse model. Disruption of Ostn and elimination of musclin secretion in mice results in reduced exercise tolerance that can be rescued by treatment with synthetic musclin. Specifically, Ostn‐KO mice demonstrate a significant deficit in exertional tolerance with respect to duration (71±6 vs. 91±6 min) and distance (769±102 vs. 1147±121 meters) on the treadmill. Similarly, when mice are offered the opportunity for voluntary exercise on running wheels, Ostn‐KO mice demonstrate significantly lower mean velocity (35±4 vs. 52±4 rotations/5 min), duration (303±38 vs. 383±64 min) and distance (1505±231 vs.2218±253 meters). Musclin delivered by osmotic pump equalizes running wheel activity between Ostn‐KO and WT mice.In agreement with the previously established musclin ability to interact with NP clearance receptors, Ostn‐KO mice show a trend toward higher plasma ANP levels compared to WT (140.4±19.9 pg/ml vs. 98.2±4.5) and significantly lower cGMP concentrations in muscle (23.13±.88 fmol/mg vs. 20.62±.60 skeletal muscle tissue). Experiments in primary myoblast culture show that induction of cGMP by the combination of musclin and ANP is associated with a significant increase in intracellular cGMP.In summary, this study defines musclin as an exercise‐responsive factor promoting exercise endurance.Support or Funding InformationThis work was supported by the National Institutes of Health [HL113089 to D.H‐Z., HL093368 and DK092412 to L.Z.]; the VA Merit Review Program [1I0BX000718 to L.Z.]; and the Fraternal Order of Eagles Diabetes Research Center.

The search for new approaches to treatment and prevention of heart failure is a major challenge in medicine. The adenosine triphosphate-sensitive potassium (KATP) channel has been long associated with the ability to preserve myocardial function and viability under stress. High surface expression of membrane KATP channels ensures a rapid energy-sparing reduction in action potential duration (APD) in response to metabolic challenges, while cellular signaling that reduces surface KATP channel expression blunts APD shortening, thus sacrificing energetic efficiency in exchange for greater cellular calcium entry and increased contractile force. In healthy hearts, calcium/calmodulin-dependent protein kinase II (CaMKII) phosphorylates the Kir6.2 KATP channel subunit initiating a cascade responsible for KATP channel endocytosis. Here, activation of CaMKII in a transaortic banding (TAB) model of heart failure is coupled with a 35-40% reduction in surface expression of KATP channels compared to hearts from sham-operated mice. Linkage between KATP channel expression and CaMKII is verified in isolated cardiomyocytes in which activation of CaMKII results in downregulation of KATP channel current. Accordingly, shortening of monophasic APD is slowed in response to hypoxia or heart rate acceleration in failing compared to non-failing hearts, a phenomenon previously shown to result in significant increases in oxygen consumption. Even in the absence of coronary artery disease, failing myocardium can be further injured by ischemia due to a mismatch between metabolic supply and demand. Ischemia-reperfusion injury, following ischemic preconditioning, is diminished in hearts with CaMKII inhibition compared to wild-type hearts and this advantage is largely eliminated when myocardial KATP channel expression is absent, supporting that the myocardial protective benefit of CaMKII inhibition in heart failure may be substantially mediated by KATP channels. Recognition of CaMKII-dependent downregulation of KATP channel expression as a mechanism for vulnerability to injury in failing hearts points to strategies targeting this interaction for potential preventives or treatments.