A key role for peroxynitrite-mediated inhibition of cardiac ERG (Kv11.1) K+ channels in carbon monoxide-induced proarrhythmic early afterdepolarizations.

Exposure to CO causes early afterdepolarization arrhythmias. Previous studies in rats have indicated that arrhythmias arose as a result of augmentation of the late Na+ current. The purpose of the present study was to examine the basis for CO-induced arrhythmias in guinea pig myocytes in which action potentials (APs) more closely resemble those of human myocytes. Whole-cell current- and voltage-clamp recordings were made from isolated guinea pig myocytes as well as from human embryonic kidney 293 (HEK293) cells that express wild-type or a C723S mutant form of ether-a-go-go-related gene (ERG; Kv11.1). We also monitored the formation of peroxynitrite (ONOO-) in HEK293 cells fluorimetrically. CO-applied as the CO-releasing molecule, CORM-2-prolonged the APs and induced early afterdepolarizations in guinea pig myocytes. In HEK293 cells, CO inhibited wild-type, but not C723S mutant, Kv11.1 K+ currents. Inhibition was prevented by an antioxidant, mitochondrial inhibitors, or inhibition of NO formation. CO also raised ONOO- levels, an effect that was reversed by the ONOO- scavenger, FeTPPS [5,10,15,20-tetrakis-(4-sulfonatophenyl)-porphyrinato-iron(III)], which also prevented the CO inhibition of Kv11.1 currents and abolished the effects of CO on Kv11.1 tail currents and APs in guinea pig myocytes. Our data suggest that CO induces arrhythmias in guinea pig cardiac myocytes via the ONOO--mediated inhibition of Kv11.1 K+ channels.-Al-Owais, M. M., Hettiarachchi, N. T., Kirton, H. M., Hardy, M. E., Boyle, J. P., Scragg, J. L., Steele, D. S., Peers, C. A key role for peroxynitrite-mediated inhibition of cardiac ERG (Kv11.1) K+ channels in carbon monoxide-induced proarrhythmic early afterdepolarizations.

Oxygen-dependent hydroxylation by FIH regulates the TRPV3 ion channel.

Factor inhibiting HIF (FIH, also known as HIF1AN) is an oxygen-dependent asparaginyl hydroxylase that regulates the hypoxia-inducible factors (HIFs). Several proteins containing ankyrin repeat domains (ARDs) have been characterised as substrates of FIH, although there is little evidence for a functional consequence of hydroxylation on these substrates. This study demonstrates that the transient receptor potential vanilloid 3 (TRPV3) channel is hydroxylated by FIH on asparagine 242 within the cytoplasmic ARD. Hypoxia, FIH inhibitors and mutation of asparagine 242 all potentiated TRPV3-mediated current, without altering TRPV3 protein levels, indicating that oxygen-dependent hydroxylation inhibits TRPV3 activity. This novel mechanism of channel regulation by oxygen-dependent asparaginyl hydroxylation is likely to extend to other ion channels.

Regulation of the T-type Ca(2+) channel Cav3.2 by hydrogen sulfide: emerging controversies concerning the role of H2 S in nociception.

Ion channels represent a large and growing family of target proteins regulated by gasotransmitters such as nitric oxide, carbon monoxide and, as described more recently, hydrogen sulfide. Indeed, many of the biological actions of these gases can be accounted for by their ability to modulate ion channel activity. Here, we report recent evidence that H2 S is a modulator of low voltage-activated T-type Ca(2+) channels, and discriminates between the different subtypes of T-type Ca(2+) channel in that it selectively modulates Cav3.2, whilst Cav3.1 and Cav3.3 are unaffected. At high concentrations, H2 S augments Cav3.2 currents, an observation which has led to the suggestion that H2 S exerts its pro-nociceptive effects via this channel, since Cav3.2 plays a central role in sensory nerve excitability. However, at more physiological concentrations, H2 S is seen to inhibit Cav3.2. This inhibitory action requires the presence of the redox-sensitive, extracellular region of the channel which is responsible for tonic metal ion binding and which particularly distinguishes this channel isoform from Cav3.1 and 3.3. Further studies indicate that H2 S may act in a novel manner to alter channel activity by potentiating the zinc sensitivity/affinity of this binding site. This review discusses the different reports of H2 S modulation of T-type Ca(2+) channels, and how such varying effects may impact on nociception given the role of this channel in sensory activity. This subject remains controversial, and future studies are required before the impact of T-type Ca(2+) channel modulation by H2 S might be exploited as a novel approach to pain management.

Inhibition of the cardiac Na⁺ channel Nav1.5 by carbon monoxide.

Sublethal carbon monoxide (CO) exposure is frequently associated with myocardial arrhythmias, and our recent studies have demonstrated that these may be attributable to modulation of cardiac Na(+) channels, causing an increase in the late current and an inhibition of the peak current. Using a recombinant expression system, we demonstrate that CO inhibits peak human Nav1.5 current amplitude without activation of the late Na(+) current observed in native tissue. Inhibition was associated with a hyperpolarizing shift in the steady-state inactivation properties of the channels and was unaffected by modification of channel gating induced by anemone toxin (rATX-II). Systematic pharmacological assessment indicated that no recognized CO-sensitive intracellular signaling pathways appeared to mediate CO inhibition of Nav1.5. Inhibition was, however, markedly suppressed by inhibition of NO formation, but NO donors did not mimic or occlude channel inhibition by CO, indicating that NO alone did not account for the actions of CO. Exposure of cells to DTT immediately before CO exposure also dramatically reduced the magnitude of current inhibition. Similarly, l-cysteine and N-ethylmaleimide significantly attenuated the inhibition caused by CO. In the presence of DTT and the NO inhibitor N(ω)-nitro-L-arginine methyl ester hydrochloride, the ability of CO to inhibit Nav1.5 was almost fully prevented. Our data indicate that inhibition of peak Na(+) current (which can lead to Brugada syndrome-like arrhythmias) occurs via a mechanism distinct from induction of the late current, requires NO formation, and is dependent on channel redox state.

Heme oxygenase-1 regulates cell proliferation via carbon monoxide-mediated inhibition of T-type Ca2+ channels.

Induction of the antioxidant enzyme heme oxygenase-1 (HO-1) affords cellular protection and suppresses proliferation of vascular smooth muscle cells (VSMCs) associated with a variety of pathological cardiovascular conditions including myocardial infarction and vascular injury. However, the underlying mechanisms are not fully understood. Over-expression of Cav3.2 T-type Ca(2+) channels in HEK293 cells raised basal [Ca(2+)]i and increased proliferation as compared with non-transfected cells. Proliferation and [Ca(2+)]i levels were reduced to levels seen in non-transfected cells either by induction of HO-1 or exposure of cells to the HO-1 product, carbon monoxide (CO) (applied as the CO releasing molecule, CORM-3). In the aortic VSMC line A7r5, proliferation was also inhibited by induction of HO-1 or by exposure of cells to CO, and patch-clamp recordings indicated that CO inhibited T-type (as well as L-type) Ca(2+) currents in these cells. Finally, in human saphenous vein smooth muscle cells, proliferation was reduced by T-type channel inhibition or by HO-1 induction or CO exposure. The effects of T-type channel blockade and HO-1 induction were non-additive. Collectively, these data indicate that HO-1 regulates proliferation via CO-mediated inhibition of T-type Ca(2+) channels. This signalling pathway provides a novel means by which proliferation of VSMCs (and other cells) may be regulated therapeutically.

H2S does not regulate proliferation via T-type Ca2+ channels.

T-type Ca(2+) channels (Cav3.1, 3.2 and 3.3) strongly influence proliferation of various cell types, including vascular smooth muscle cells (VSMCs) and certain cancers. We have recently shown that the gasotransmitter carbon monoxide (CO) inhibits T-type Ca(2+) channels and, in so doing, attenuates proliferation of VSMC. We have also shown that the T-type Ca(2+) channel Cav3.2 is selectively inhibited by hydrogen sulfide (H2S) whilst the other channel isoforms (Cav3.1 and Cav3.3) are unaffected. Here, we explored whether inhibition of Cav3.2 by H2S could account for the anti-proliferative effects of this gasotransmitter. H2S suppressed proliferation in HEK293 cells expressing Cav3.2, as predicted by our previous observations. However, H2S was similarly effective in suppressing proliferation in wild type (non-transfected) HEK293 cells and those expressing the H2S insensitive channel, Cav3.1. Further studies demonstrated that T-type Ca(2+) channels in the smooth muscle cell line A7r5 and in human coronary VSMCs strongly influenced proliferation. In both cell types, H2S caused a concentration-dependent inhibition of proliferation, yet by far the dominant T-type Ca(2+) channel isoform was the H2S-insensitive channel, Cav3.1. Our data indicate that inhibition of T-type Ca(2+) channel-mediated proliferation by H2S is independent of the channels' sensitivity to H2S.

Hydrogen sulfide inhibits Cav3.2 T-type Ca2+ channels.

The importance of H2S as a physiological signaling molecule continues to develop, and ion channels are emerging as a major family of target proteins through which H2S exerts many actions. The purpose of the present study was to investigate its effects on T-type Ca(2+) channels. Using patch-clamp electrophysiology, we demonstrate that the H2S donor, NaHS (10 µM-1 mM) selectively inhibits Cav3.2 T-type channels heterologously expressed in HEK293 cells, whereas Cav3.1 and Cav3.3 channels were unaffected. The sensitivity of Cav3.2 channels to H2S required the presence of the redox-sensitive extracellular residue H191, which is also required for tonic binding of Zn(2+) to this channel. Chelation of Zn(2+) with N,N,N',N'-tetra-2-picolylethylenediamine prevented channel inhibition by H2S and also reversed H2S inhibition when applied after H2S exposure, suggesting that H2S may act via increasing the affinity of the channel for extracellular Zn(2+) binding. Inhibition of native T-type channels in 3 cell lines correlated with expression of Cav3.2 and not Cav3.1 channels. Notably, H2S also inhibited native T-type (primarily Cav3.2) channels in sensory dorsal root ganglion neurons. Our data demonstrate a novel target for H2S regulation, the T-type Ca(2+) channel Cav3.2, and suggest that such modulation cannot account for the pronociceptive effects of this gasotransmitter.

Heme Oxygenase-1 Influences Apoptosis via CO-mediated Inhibition of K+ Channels.

Hypoxic/ischemic episodes can trigger oxidative stress-mediated loss of central neurons via apoptosis, and low pO2 is also a feature of the tumor microenvironment, where cancer cells are particularly resistant to apoptosis. In the CNS, ischemic insult increases expression of the CO-generating enzyme heme oxygenase-1 (HO-1), which is commonly constitutively active in cancer cells. It has been proposed that apoptosis can be regulated by the trafficking and activity of K(+) channels, particularly Kv2.1. We have explored the idea that HO-1 may influence apoptosis via regulation of Kv2.1. Overexpression of Kv2.1 in HEK293 cells increased their vulnerability to oxidant-induced apoptosis. CO (applied as the donor CORM-2) protected cells against apoptosis and inhibited Kv2.1 channels. Similarly in hippocampal neurones, CO selectively inhibited Kv2.1 and protected neurones against oxidant-induced apoptosis. In medulloblastoma sections we identified constitutive expression of HO-1 and Kv2.1, and in the medulloblastoma-derived cell line DAOY, hypoxic HO-1 induction or exposure to CO protected cells against apoptosis, and also selectively inhibited Kv2.1 channels expressed in these cells. These studies are consistent with a central role for Kv2.1 in apoptosis in both central neurones and cancer cells. They also suggest that HO-1 expression can strongly influence apoptosis via CO-mediated regulation of Kv2.1 activity.

Inhibition of T-type Ca2+ Channels by Hydrogen Sulfide.

T-type Ca(2+) channels are a distinct family of low voltage-activated Ca(2+) channels which serve many roles in different tissues. Several studies have implicated them, for example, in the adaptive responses to chronic hypoxia in the cardiovascular and endocrine systems. Hydrogen sulfide (H(2)S) was more recently discovered as an important signalling molecule involved in many functions, including O(2) sensing. Since ion channels are emerging as an important family of target proteins for modulation by H(2)S, and both T-type Ca(2+) channels and H(2)S are involved in cellular responses to hypoxia, we have investigated whether recombinant and native T-type Ca(2+) channels are a target for modulation by H(2)S. Using patch-clamp electrophysiology, we demonstrate that the H(2)S donor, NaHS, selectively inhibits Cav3.2 T-type Ca(2+) channels heterologously expressed in HEK293 cells, whilst Cav3.1 and Cav3.3 channels were unaffected. Sensitivity of Cav3.2 channels to H2S required the presence of the redox-sensitive extracellular residue H191, which is also required for tonic binding of Zn(2+) to this channel. Chelation of Zn(2+) using TPEN prevented channel inhibition by H(2)S. H2S also selectively inhibited native T-type channels (primarily Cav3.2) in sensory dorsal root ganglion neurons. Our data demonstrate a novel target for H(2)S regulation, the T-type Ca(2+) channel Cav3.2. Results have important implications for the proposed pro-nociceptive effects of this gasotransmitter. Implications for the control of cellular responses to hypoxia await further study.

T-Type Ca2+ Channel Regulation by CO: A Mechanism for Control of Cell Proliferation.

T-type Ca(2+) channels regulate proliferation in a number of tissue types, including vascular smooth muscle and various cancers. In such tissues, up-regulation of the inducible enzyme heme oxygenase-1 (HO-1) is often observed, and hypoxia is a key factor in its induction. HO-1 degrades heme to generate carbon monoxide (CO) along with Fe(2+) and biliverdin. Since CO is increasingly recognized as a regulator of ion channels (Peers et al. 2015), we have explored the possibility that it may regulate proliferation via modulation of T-type Ca(2+) channels.Whole-cell patch-clamp recordings revealed that CO (applied as the dissolved gas or via CORM donors) inhibited all 3 isoforms of T-type Ca(2+) channels (Cav3.1-3.3) when expressed in HEK293 cells with similar IC(50) values, and induction of HO-1 expression also suppressed T-type currents (Boycott et al. 2013). CO/HO-1 induction also suppressed the elevated basal [Ca(2+) ](i) in cells expressing these channels and reduced their proliferative rate to levels seen in non-transfected control cells (Duckles et al. 2015).Proliferation of vascular smooth muscle cells (both A7r5 and human saphenous vein cells) was also suppressed either by T-type Ca(2+) channel inhibitors (mibefradil and NNC 55-0396), HO-1 induction or application of CO. Effects of these blockers and CO were non additive. Although L-type Ca(2+) channels were also sensitive to CO (Scragg et al. 2008), they did not influence proliferation. Our data suggest that HO-1 acts to control proliferation via CO modulation of T-type Ca(2+) channels.

Carbon monoxide inhibition of Cav3.2 T-type Ca2+ channels reveals tonic modulation by thioredoxin.

T-type Ca(2+) channels play diverse roles in tissues such as sensory neurons, vascular smooth muscle, and cancers, where increased expression of the cytoprotective enzyme, heme oxygenase-1 (HO-1) is often found. Here, we report regulation of T-type Ca(2+) channels by carbon monoxide (CO) a HO-1 by-product. CO (applied as CORM-2) caused a concentration-dependent, poorly reversible inhibition of all T-type channel isoforms (Cav3.1-3.3, IC50 ∼3 µM) expressed in HEK293 cells, and native T-type channels in NG108-15 cells and primary rat sensory neurons. No recognized CO-sensitive signaling pathway could account for the CO inhibition of Cav3.2. Instead, CO sensitivity was mediated by an extracellular redox-sensitive site, which was also highly sensitive to thioredoxin (Trx). Trx depletion (using auranofin, 2-5 µM) reduced Cav3.2 currents and their CO sensitivity by >50% but increased sensitivity to dithiothreitol ∼3-fold. By contrast, Cav3.1 and Cav3.3 channels, and their sensitivity to CO, were unaffected in identical experiments. Our data propose a novel signaling pathway in which Trx acts as a tonic, endogenous regulator of Cav3.2 channels, while HO-1-derived CO disrupts this regulation, causing channel inhibition. CO modulation of T-type channels has widespread implications for diverse physiological and pathophysiological mechanisms, such as excitability, contractility, and proliferation.

Carbon monoxide mediates the anti-apoptotic effects of heme oxygenase-1 in medulloblastoma DAOY cells via K+ channel inhibition.

Tumor cell survival and proliferation is attributable in part to suppression of apoptotic pathways, yet the mechanisms by which cancer cells resist apoptosis are not fully understood. Many cancer cells constitutively express heme oxygenase-1 (HO-1), which catabolizes heme to generate biliverdin, Fe(2+), and carbon monoxide (CO). These breakdown products may play a role in the ability of cancer cells to suppress apoptotic signals. K(+) channels also play a crucial role in apoptosis, permitting K(+) efflux which is required to initiate caspase activation. Here, we demonstrate that HO-1 is constitutively expressed in human medulloblastoma tissue, and can be induced in the medulloblastoma cell line DAOY either chemically or by hypoxia. Induction of HO-1 markedly increases the resistance of DAOY cells to oxidant-induced apoptosis. This effect was mimicked by exogenous application of the heme degradation product CO. Furthermore we demonstrate the presence of the pro-apoptotic K(+) channel, Kv2.1, in both human medulloblastoma tissue and DAOY cells. CO inhibited the voltage-gated K(+) currents in DAOY cells, and largely reversed the oxidant-induced increase in K(+) channel activity. p38 MAPK inhibition prevented the oxidant-induced increase of K(+) channel activity in DAOY cells, and enhanced their resistance to apoptosis. Our findings suggest that CO-mediated inhibition of K(+) channels represents an important mechanism by which HO-1 can increase the resistance to apoptosis of medulloblastoma cells, and support the idea that HO-1 inhibition may enhance the effectiveness of current chemo- and radiotherapies.

Carbon monoxide induces cardiac arrhythmia via induction of the late Na+ current.

RATIONALE: Clinical reports describe life-threatening cardiac arrhythmias after environmental exposure to carbon monoxide (CO) or accidental CO poisoning. Numerous case studies describe disruption of repolarization and prolongation of the QT interval, yet the mechanisms underlying CO-induced arrhythmias are unknown. OBJECTIVES: To understand the cellular basis of CO-induced arrhythmias and to identify an effective therapeutic approach. METHODS: Patch-clamp electrophysiology and confocal Ca(2+) and nitric oxide (NO) imaging in isolated ventricular myocytes was performed together with protein S-nitrosylation to investigate the effects of CO at the cellular and molecular levels, whereas telemetry was used to investigate effects of CO on electrocardiogram recordings in vivo. MEASUREMENTS AND MAIN RESULTS: CO increased the sustained (late) component of the inward Na(+) current, resulting in prolongation of the action potential and the associated intracellular Ca(2+) transient. In more than 50% of myocytes these changes progressed to early after-depolarization-like arrhythmias. CO elevated NO levels in myocytes and caused S-nitrosylation of the Na(+) channel, Na(v)1.5. All proarrhythmic effects of CO were abolished by the NO synthase inhibitor l-NAME, and reversed by ranolazine, an inhibitor of the late Na(+) current. Ranolazine also corrected QT variability and arrhythmias induced by CO in vivo, as monitored by telemetry. CONCLUSIONS: Our data indicate that the proarrhythmic effects of CO arise from activation of NO synthase, leading to NO-mediated nitrosylation of Na(V)1.5 and to induction of the late Na(+) current. We also show that the antianginal drug ranolazine can abolish CO-induced early after-depolarizations, highlighting a novel approach to the treatment of CO-induced arrhythmias.

Inhibition of the voltage-gated potassium channel Kv1.5 by hydrogen sulfide attenuates remodeling through S-nitrosylation-mediated signaling.

The voltage-gated K+ channel plays a key role in atrial excitability, conducting the ultra-rapid rectifier K+ current (IKur) and contributing to the repolarization of the atrial action potential. In this study, we examine its regulation by hydrogen sulfide (H2S) in HL-1 cardiomyocytes and in HEK293 cells expressing human Kv1.5. Pacing induced remodeling resulted in shorting action potential duration, enhanced both Kv1.5 channel and H2S producing enzymes protein expression in HL-1 cardiomyocytes. H2S supplementation reduced these remodeling changes and restored action potential duration through inhibition of Kv1.5 channel. H2S also inhibited recombinant hKv1.5, lead to nitric oxide (NO) mediated S-nitrosylation and activated endothelial nitric oxide synthase (eNOS) by increased phosphorylation of Ser1177, prevention of NO formation precluded these effects. Regulation of Ikur by H2S has important cardiovascular implications and represents a novel and potential therapeutic target.

Carbon monoxide protects against oxidant-induced apoptosis via inhibition of Kv2.1.

Oxidative stress induces neuronal apoptosis and is implicated in cerebral ischemia, head trauma, and age-related neurodegenerative diseases. An early step in this process is the loss of intracellular K(+) via K(+) channels, and evidence indicates that K(v)2.1 is of particular importance in this regard, being rapidly inserted into the plasma membrane in response to apoptotic stimuli. An additional feature of neuronal oxidative stress is the up-regulation of the inducible enzyme heme oxygenase-1 (HO-1), which catabolizes heme to generate biliverdin, Fe(2+), and carbon monoxide (CO). CO provides neuronal protection against stresses such as stroke and excitotoxicity, although the underlying mechanisms are not yet elucidated. Here, we demonstrate that CO reversibly inhibits K(v)2.1. Channel inhibition by CO involves reactive oxygen species and protein kinase G activity. Overexpression of K(v)2.1 in HEK293 cells increases their vulnerability to oxidant-induced apoptosis, and this is reversed by CO. In hippocampal neurons, CO selectively inhibits K(v)2.1, reverses the dramatic oxidant-induced increase in K(+) current density, and provides marked protection against oxidant-induced apoptosis. Our results provide a novel mechanism to account for the neuroprotective effects of CO against oxidative apoptosis, which has potential for therapeutic exploitation to provide neuronal protection in situations of oxidative stress.

Hydrogen sulfide regulates hippocampal neuron excitability via S-sulfhydration of Kv2.1.

Hydrogen sulfide (H2S) is gaining interest as a mammalian signalling molecule with wide ranging effects. S-sulfhydration is one mechanism that is emerging as a key post translational modification through which H2S acts. Ion channels and neuronal receptors are key target proteins for S-sulfhydration and this can influence a range of neuronal functions. Voltage-gated K+ channels, including Kv2.1, are fundamental components of neuronal excitability. Here, we show that both recombinant and native rat Kv2.1 channels are inhibited by the H2S donors, NaHS and GYY4137. Biochemical investigations revealed that NaHS treatment leads to S-sulfhydration of the full length wild type Kv2.1 protein which was absent (as was functional regulation by H2S) in the C73A mutant form of the channel. Functional experiments utilising primary rat hippocampal neurons indicated that NaHS augments action potential firing and thereby increases neuronal excitability. These studies highlight an important role for H2S in shaping cellular excitability through S-sulfhydration of Kv2.1 at C73 within the central nervous system.

Kv1.3 voltage-gated potassium channels link cellular respiration to proliferation through a non-conducting mechanism.

Cellular energy metabolism is fundamental for all biological functions. Cellular proliferation requires extensive metabolic reprogramming and has a high energy demand. The Kv1.3 voltage-gated potassium channel drives cellular proliferation. Kv1.3 channels localise to mitochondria. Using high-resolution respirometry, we show Kv1.3 channels increase oxidative phosphorylation, independently of redox balance, mitochondrial membrane potential or calcium signalling. Kv1.3-induced respiration increased reactive oxygen species production. Reducing reactive oxygen concentrations inhibited Kv1.3-induced proliferation. Selective Kv1.3 mutation identified that channel-induced respiration required an intact voltage sensor and C-terminal ERK1/2 phosphorylation site, but is channel pore independent. We show Kv1.3 channels regulate respiration through a non-conducting mechanism to generate reactive oxygen species which drive proliferation. This study identifies a Kv1.3-mediated mechanism underlying the metabolic regulation of proliferation, which may provide a therapeutic target for diseases characterised by dysfunctional proliferation and cell growth.

Brown and beige adipose tissue regulate systemic metabolism through a metabolite interorgan signaling axis.

Brown and beige adipose tissue are emerging as distinct endocrine organs. These tissues are functionally associated with skeletal muscle, adipose tissue metabolism and systemic energy expenditure, suggesting an interorgan signaling network. Using metabolomics, we identify 3-methyl-2-oxovaleric acid, 5-oxoproline, and ß-hydroxyisobutyric acid as small molecule metabokines synthesized in browning adipocytes and secreted via monocarboxylate transporters. 3-methyl-2-oxovaleric acid, 5-oxoproline and ß-hydroxyisobutyric acid induce a brown adipocyte-specific phenotype in white adipocytes and mitochondrial oxidative energy metabolism in skeletal myocytes both in vitro and in vivo. 3-methyl-2-oxovaleric acid and 5-oxoproline signal through cAMP-PKA-p38 MAPK and ß-hydroxyisobutyric acid via mTOR. In humans, plasma and adipose tissue 3-methyl-2-oxovaleric acid, 5-oxoproline and ß-hydroxyisobutyric acid concentrations correlate with markers of adipose browning and inversely associate with body mass index. These metabolites reduce adiposity, increase energy expenditure and improve glucose and insulin homeostasis in mouse models of obesity and diabetes. Our findings identify beige adipose-brown adipose-muscle physiological metabokine crosstalk.

Unique Transcriptome Signature Distinguishes Patients With Heart Failure With Myopathy.

Background People with chronic heart failure (CHF) experience severe skeletal muscle dysfunction, characterized by mitochondrial abnormalities, which exacerbates the primary symptom of exercise intolerance. However, the molecular triggers and characteristics underlying mitochondrial abnormalities caused by CHF remain poorly understood. Methods and Results We recruited 28 patients with CHF caused by reduced ejection fraction and 9 controls. We simultaneously biopsied skeletal muscle from the pectoralis major in the upper limb and from the vastus lateralis in the lower limb. We phenotyped mitochondrial function in permeabilized myofibers from both sites and followed this by complete RNA sequencing to identify novel molecular abnormalities in CHF skeletal muscle. Patients with CHF presented with upper and lower limb skeletal muscle impairments to mitochondrial function that were of a similar deficit and indicative of a myopathy. Mitochondrial abnormalities were strongly correlated to symptoms. Further RNA sequencing revealed a unique transcriptome signature in CHF skeletal muscle characterized by a novel triad of differentially expressed genes related to deficits in energy metabolism including adenosine monophosphate deaminase 3, pyridine nucleotide-disulphide oxidoreductase domain 2, and lactate dehydrogenase C. Conclusions Our data suggest an upper and lower limb metabolic myopathy that is characterized by a unique transcriptome signature in skeletal muscle of humans with CHF.

Piezo1 channel activation mimics high glucose as a stimulator of insulin release.

Glucose and hypotonicity induced cell swelling stimulate insulin release from pancreatic ß-cells but the mechanisms are poorly understood. Recently, Piezo1 was identified as a mechanically-activated nonselective Ca2+ permeable cationic channel in a range of mammalian cells. As cell swelling induced insulin release could be through stimulation of Ca2+ permeable stretch activated channels, we hypothesised a role for Piezo1 in cell swelling induced insulin release. Two rat ß-cell lines (INS-1 and BRIN-BD11) and freshly-isolated mouse pancreatic islets were studied. Intracellular Ca2+ measurements were performed using the fura-2 Ca2+ indicator dye and ionic current was recorded by whole cell patch-clamp. Piezo1 agonist Yoda1, a competitive antagonist of Yoda1 (Dooku1) and an inactive analogue of Yoda1 (2e) were used as chemical probes. Piezo1 mRNA and insulin secretion were measured by RT-PCR and ELISA respectively. Piezo1 mRNA was detected in both ß-cell lines and mouse islets. Yoda1 evoked Ca2+ entry was inhibited by Yoda1 antagonist Dooku1 as well as other Piezo1 inhibitors gadolinium and ruthenium red, and not mimicked by 2e. Yoda1, but not 2e, stimulated Dooku1-sensitive insulin release from ß-cells and pancreatic islets. Hypotonicity and high glucose increased intracellular Ca2+ and enhanced Yoda1 Ca2+ influx responses. Yoda1 and hypotonicity induced insulin release were significantly inhibited by Piezo1 specific siRNA. Pancreatic islets from mice with haploinsufficiency of Piezo1 released less insulin upon exposure to Yoda1. The data show that Piezo1 channel agonist induces insulin release from ß-cell lines and mouse pancreatic islets suggesting a role for Piezo1 in cell swelling induced insulin release. Hence Piezo1 agonists have the potential to be used as enhancers of insulin release.