Myokine Musclin Is Critical for Exercise-Induced Cardiac Conditioning.

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.

Elucidating the role of Rgs2 expression in the PVN for metabolic homeostasis in mice.

OBJECTIVE: RGS2 is a GTPase activating protein that modulates GPCR-Gα 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.

Pharmacological FGF21 signals to glutamatergic neurons to enhance leptin action and lower body weight during obesity.

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, ß-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.

Atrial-paced, exercise-similar heart rate envelope induces myocardial protection from ischaemic injury.

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.

Neuroanatomical organization and functional roles of PVN MC4R pathways in physiological and behavioral regulations.

OBJECTIVE: The paraventricular nucleus of hypothalamus (PVN), an integrative center in the brain, orchestrates a wide range of physiological and behavioral responses. While the PVN melanocortin 4 receptor (MC4R) signaling (PVNMC4R+) is involved in feeding regulation, the neuroanatomical organization of PVNMC4R+ connectivity and its role in other physiological regulations are incompletely understood. Here we aimed to better characterize the input-output organization of PVNMC4R+ neurons and test their physiological functions beyond feeding. METHODS: Using a combination of viral tools, we mapped PVNMC4R+ circuits and tested the effects of chemogenetic activation of PVNMC4R+ neurons on thermoregulation, cardiovascular control, and other behavioral responses beyond feeding. RESULTS: We found that PVNMC4R+ neurons innervate many different brain regions that are known to be important not only for feeding but also for neuroendocrine and autonomic control of thermoregulation and cardiovascular function, including but not limited to the preoptic area, median eminence, parabrachial nucleus, pre-locus coeruleus, nucleus of solitary tract, ventrolateral medulla, and thoracic spinal cord. Contrary to these broad efferent projections, PVNMC4R+ neurons receive monosynaptic inputs mainly from other hypothalamic nuclei (preoptic area, arcuate and dorsomedial hypothalamic nuclei, supraoptic nucleus, and premammillary nucleus), the circumventricular organs (subfornical organ and vascular organ of lamina terminalis), the bed nucleus of stria terminalis, and the parabrachial nucleus. Consistent with their broad efferent projections, chemogenetic activation of PVNMC4R+ neurons not only suppressed feeding but also led to an apparent increase in heart rate, blood pressure, and brown adipose tissue temperature. These physiological changes accompanied acute transient hyperactivity followed by hypoactivity and resting-like behavior. CONCLUSIONS: Our results elucidate the neuroanatomical organization of PVNMC4R+ circuits and shed new light on the roles of PVNMC4R+ pathways in autonomic control of thermoregulation, cardiovascular function, and biphasic behavioral activation.

ADH5-mediated NO bioactivity maintains metabolic homeostasis in brown adipose tissue.

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.

Endocannabinoid Receptor-1 and Sympathetic Nervous System Mediate the Beneficial Metabolic Effects of Gastric Bypass.

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.

Pdgfrα-Cre mediated knockout of the aryl hydrocarbon receptor protects mice from high-fat diet induced obesity and hepatic steatosis.

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.

Musclin, A Myokine Induced by Aerobic Exercise, Retards Muscle Atrophy During Cancer Cachexia in Mice.

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.

Impaired skeletal muscle mitochondrial pyruvate uptake rewires glucose metabolism to drive whole-body leanness.

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.

Liver Derived FGF21 Maintains Core Body Temperature During Acute Cold Exposure.

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 ß-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.

Loss of ATP-Sensitive Potassium Channel Surface Expression in Heart Failure Underlies Dysregulation of Action Potential Duration and Myocardial Vulnerability to Injury.

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.

Disruption of ATP-sensitive potassium channel function in skeletal muscles promotes production and secretion of musclin.

Sarcolemmal ATP-sensitive potassium (KATP) channels control skeletal muscle energy use through their ability to adjust membrane excitability and related cell functions in accordance with cellular metabolic status. Mice with disrupted skeletal muscle KATP channels exhibit reduced adipocyte size and increased fatty acid release into the circulation. As yet, the molecular mechanisms underlying this link between skeletal muscle KATP channel function and adipose mobilization have not been established. Here, we demonstrate that skeletal muscle-specific disruption of KATP channel function in transgenic (TG) mice promotes production and secretion of musclin. Musclin is a myokine with high homology to atrial natriuretic peptide (ANP) that enhances ANP signaling by competing for elimination. Augmented musclin production in TG mice is driven by a molecular cascade resulting in enhanced acetylation and nuclear exclusion of the transcription factor forkhead box O1 (FOXO1) - an inhibitor of transcription of the musclin encoding gene. Musclin production/secretion in TG is paired with increased mobilization of fatty acids and a clear trend toward increased circulating ANP, an activator of lipolysis. These data establish KATP channel-dependent musclin production as a potential mechanistic link coupling "local" skeletal muscle energy consumption with mobilization of bodily resources from fat. Understanding such mechanisms is an important step toward designing interventions to manage metabolic disorders including those related to excess body fat and associated co-morbidities.

Musclin is an activity-stimulated myokine that enhances physical endurance.

Exercise remains the most effective way to promote physical and metabolic wellbeing, but molecular mechanisms underlying exercise tolerance and its plasticity are only partially understood. In this study we identify musclin-a peptide with high homology to natriuretic peptides (NP)-as an exercise-responsive myokine that acts to enhance exercise capacity in mice. We use human primary myoblast culture and in vivo murine models to establish that the activity-related production of musclin is driven by Ca(2+)-dependent activation of Akt1 and the release of musclin-encoding gene (Ostn) transcription from forkhead box O1 transcription factor inhibition. Disruption of Ostn and elimination of musclin secretion in mice results in reduced exercise tolerance that can be rescued by treatment with recombinant musclin. Reduced exercise capacity in mice with disrupted musclin signaling is associated with a trend toward lower levels of plasma atrial NP (ANP) and significantly smaller levels of cyclic guanosine monophosphate (cGMP) and peroxisome proliferator-activated receptor gamma coactivator 1-α in skeletal muscles after exposure to exercise. Furthermore, in agreement with the established musclin ability to interact with NP clearance receptors, but not with NP guanyl cyclase-coupled signaling receptors, we demonstrate that musclin enhances cGMP production in cultured myoblasts only when applied together with ANP. Elimination of the activity-related musclin-dependent boost of ANP/cGMP signaling results in significantly lower maximum aerobic capacity, mitochondrial protein content, respiratory complex protein expression, and succinate dehydrogenase activity in skeletal muscles. Together, these data indicate that musclin enhances physical endurance by promoting mitochondrial biogenesis.

Inhibition of MCU forces extramitochondrial adaptations governing physiological and pathological stress responses in heart.

Myocardial mitochondrial Ca(2+) entry enables physiological stress responses but in excess promotes injury and death. However, tissue-specific in vivo systems for testing the role of mitochondrial Ca(2+) are lacking. We developed a mouse model with myocardial delimited transgenic expression of a dominant negative (DN) form of the mitochondrial Ca(2+) uniporter (MCU). DN-MCU mice lack MCU-mediated mitochondrial Ca(2+) entry in myocardium, but, surprisingly, isolated perfused hearts exhibited higher O2 consumption rates (OCR) and impaired pacing induced mechanical performance compared with wild-type (WT) littermate controls. In contrast, OCR in DN-MCU-permeabilized myocardial fibers or isolated mitochondria in low Ca(2+) were not increased compared with WT, suggesting that DN-MCU expression increased OCR by enhanced energetic demands related to extramitochondrial Ca(2+) homeostasis. Consistent with this, we found that DN-MCU ventricular cardiomyocytes exhibited elevated cytoplasmic [Ca(2+)] that was partially reversed by ATP dialysis, suggesting that metabolic defects arising from loss of MCU function impaired physiological intracellular Ca(2+) homeostasis. Mitochondrial Ca(2+) overload is thought to dissipate the inner mitochondrial membrane potential (ΔΨm) and enhance formation of reactive oxygen species (ROS) as a consequence of ischemia-reperfusion injury. Our data show that DN-MCU hearts had preserved ΔΨm and reduced ROS during ischemia reperfusion but were not protected from myocardial death compared with WT. Taken together, our findings show that chronic myocardial MCU inhibition leads to previously unanticipated compensatory changes that affect cytoplasmic Ca(2+) homeostasis, reprogram transcription, increase OCR, reduce performance, and prevent anticipated therapeutic responses to ischemia-reperfusion injury.

Sarcolemmal ATP-sensitive potassium channels modulate skeletal muscle function under low-intensity workloads.

ATP-sensitive potassium (KATP) channels have the unique ability to adjust membrane excitability and functions in accordance with the metabolic status of the cell. Skeletal muscles are primary sites of activity-related energy consumption and have KATP channels expressed in very high density. Previously, we demonstrated that transgenic mice with skeletal muscle-specific disruption of KATP channel function consume more energy than wild-type littermates. However, how KATP channel activation modulates skeletal muscle resting and action potentials under physiological conditions, particularly low-intensity workloads, and how this can be translated to muscle energy expenditure are yet to be determined. Here, we developed a technique that allows evaluation of skeletal muscle excitability in situ, with minimal disruption of the physiological environment. Isometric twitching of the tibialis anterior muscle at 1 Hz was used as a model of low-intensity physical activity in mice with normal and genetically disrupted KATP channel function. This workload was sufficient to induce KATP channel opening, resulting in membrane hyperpolarization as well as reduction in action potential overshoot and duration. Loss of KATP channel function resulted in increased calcium release and aggravated activity-induced heat production. Thus, this study identifies low-intensity workload as a trigger for opening skeletal muscle KATP channels and establishes that this coupling is important for regulation of myocyte function and thermogenesis. These mechanisms may provide a foundation for novel strategies to combat metabolic derangements when energy conservation or dissipation is required.

Regulation of cardiac ATP-sensitive potassium channel surface expression by calcium/calmodulin-dependent protein kinase II.

Cardiac ATP-sensitive potassium (K(ATP)) channels are key sensors and effectors of the metabolic status of cardiomyocytes. Alteration in their expression impacts their effectiveness in maintaining cellular energy homeostasis and resistance to injury. We sought to determine how activation of calcium/calmodulin-dependent protein kinase II (CaMKII), a central regulator of calcium signaling, translates into reduced membrane expression and current capacity of cardiac K(ATP) channels. We used real-time monitoring of K(ATP) channel current density, immunohistochemistry, and biotinylation studies in isolated hearts and cardiomyocytes from wild-type and transgenic mice as well as HEK cells expressing wild-type and mutant K(ATP) channel subunits to track the dynamics of K(ATP) channel surface expression. Results showed that activation of CaMKII triggered dynamin-dependent internalization of K(ATP) channels. This process required phosphorylation of threonine at 180 and 224 and an intact (330)YSKF(333) endocytosis motif of the K(ATP) channel Kir6.2 pore-forming subunit. A molecular model of the µ2 subunit of the endocytosis adaptor protein, AP2, complexed with Kir6.2 predicted that µ2 docks by interaction with (330)YSKF(333) and Thr-180 on one and Thr-224 on the adjacent Kir6.2 subunit. Phosphorylation of Thr-180 and Thr-224 would favor interactions with the corresponding arginine- and lysine-rich loops on µ2. We concluded that calcium-dependent activation of CaMKII results in phosphorylation of Kir6.2, which promotes endocytosis of cardiac K(ATP) channel subunits. This mechanism couples the surface expression of cardiac K(ATP) channels with calcium signaling and reveals new targets to improve cardiac energy efficiency and stress resistance.

Reduction in number of sarcolemmal KATP channels slows cardiac action potential duration shortening under hypoxia.

The cardiovascular system operates under demands ranging from conditions of rest to extreme stress. One mechanism of cardiac stress tolerance is action potential duration shortening driven by ATP-sensitive potassium (K(ATP)) channels. K(ATP) channel expression has a significant physiologic impact on action potential duration shortening and myocardial energy consumption in response to physiologic heart rate acceleration. However, the effect of reduced channel expression on action potential duration shortening in response to severe metabolic stress is yet to be established. Here, transgenic mice with myocardium-specific expression of a dominant negative K(ATP) channel subunit were compared with littermate controls. Evaluation of K(ATP) channel whole cell current and channel number/patch was assessed by patch clamp in isolated ventricular cardiomyocytes. Monophasic action potentials were monitored in retrogradely perfused, isolated hearts during the transition to hypoxic perfusate. An 80-85% reduction in cardiac K(ATP) channel current density results in a similar magnitude, but significantly slower rate, of shortening of the ventricular action potential duration in response to severe hypoxia, despite no significant difference in coronary flow. Therefore, the number of functional cardiac sarcolemmal K(ATP) channels is a critical determinant of the rate of adaptation of myocardial membrane excitability, with implications for optimization of cardiac energy consumption and consequent cardioprotection under conditions of severe metabolic stress.

Exercise-induced expression of cardiac ATP-sensitive potassium channels promotes action potential shortening and energy conservation.

Physical activity is one of the most important determinants of cardiac function. The ability of the heart to increase delivery of oxygen and metabolic fuels relies on an array of adaptive responses necessary to match bodily demand while avoiding exhaustion of cardiac resources. The ATP-sensitive potassium (K(ATP)) channel has the unique ability to adjust cardiac membrane excitability in accordance with ATP and ADP levels, and up-regulation of its expression that occurs in response to exercise could represent a critical element of this adaption. However, the mechanism by which K(ATP) channel expression changes result in a beneficial effect on cardiac excitability and function remains to be established. Here, we demonstrate that an exercise-induced rise in K(ATP) channel expression enhanced the rate and magnitude of action potential shortening in response to heart rate acceleration. This adaptation in membrane excitability promoted significant reduction in cardiac energy consumption under escalating workloads. Genetic disruption of normal K(ATP) channel pore function abolished the exercise-related changes in action potential duration adjustment and caused increased cardiac energy consumption. Thus, an expression-driven enhancement in the K(ATP) channel-dependent membrane response to alterations in cardiac workload represents a previously unrecognized mechanism for adaptation to physical activity and a potential target for cardioprotection.

Sarcolemmal ATP-sensitive K(+) channels control energy expenditure determining body weight.

Metabolic processes that regulate muscle energy use are major determinants of bodily energy balance. Here, we find that sarcolemmal ATP-sensitive K(+) (K(ATP)) channels, which couple membrane excitability with cellular metabolic pathways, set muscle energy expenditure under physiological stimuli. Disruption of K(ATP) channel function provoked, under conditions of unaltered locomotor activity and blood substrate availability, an extra energy cost of cardiac and skeletal muscle performance. Inefficient fuel metabolism in K(ATP) channel-deficient striated muscles reduced glycogen and fat body depots, promoting a lean phenotype. The propensity to lesser body weight imposed by K(ATP) channel deficit persisted under a high-fat diet, yet obesity restriction was achieved at the cost of compromised physical endurance. Thus, sarcolemmal K(ATP) channels govern muscle energy economy, and their downregulation in a tissue-specific manner could present an antiobesity strategy by rendering muscle increasingly thermogenic at rest and less fuel efficient during exercise.