Sulfur metabolism as a new therapeutic target of heart failure.

Sulfur-based redox signaling has long attracted attention as critical mechanisms underlying the development of cardiac diseases and resultant heart failure. Especially, post-translational modifications of cysteine (Cys) thiols in proteins mediate oxidative stress-dependent cardiac remodeling including myocardial hypertrophy, senescence, and interstitial fibrosis. However, we recently revealed the existence of Cys persulfides and Cys polysulfides in cells and tissues, which show higher redox activities than Cys and substantially contribute to redox signaling and energy metabolism. We have established simple evaluation methods that can detect polysulfides in proteins and inorganic polysulfides in cells and revealed that polysulfides abundantly expressed in normal hearts are dramatically catabolized by exposure to ischemic/hypoxic and environmental electrophilic stress, which causes vulnerability of the heart to mechanical load. Accumulation of hydrogen sulfide, a nucleophilic catabolite of persulfides/polysulfides, may lead to reductive stress in ischemic hearts, and perturbation of polysulfide catabolism can improve chronic heart failure after myocardial infarction in mice. This review focuses on the (patho)physiological role of sulfur metabolism in hearts, and proposes that sulfur catabolism during ischemic/hypoxic stress has great potential as a new therapeutic strategy for the treatment of ischemic heart failure.

Supersulfide catabolism participates in maladaptive remodeling of cardiac cells.

The atrophic myocardium resulting from mechanical unloading and nutritional deprivation is considered crucial as maladaptive remodeling directly associated with heart failure, as well as interstitial fibrosis. Conversely, myocardial hypertrophy resulting from hemodynamic loading is perceived as compensatory stress adaptation. We previously reported the abundant presence of highly redox-active polysulfide molecules, termed supersulfide, with two or more sulfur atoms catenated in normal hearts, and the supersulfide catabolism in pathologic hearts after myocardial infarction correlated with worsened prognosis of heart failure. However, the impact of supersulfide on myocardial remodeling remains unclear. Here, we investigated the involvement of supersulfide metabolism in cardiomyocyte remodeling, using a model of adenosine 5'-triphosphate (ATP) receptor-stimulated atrophy and endothelin-1 receptor-stimulated hypertrophy in neonatal rat cardiomyocytes. Results revealed contrasting changes in intracellular supersulfide and its catabolite, hydrogen sulfide (H2S), between cardiomyocyte atrophy and hypertrophy. Stimulation of cardiomyocytes with ATP decreased supersulfide activity, while H2S accumulation itself did not affect cardiomyocyte atrophy. This supersulfide catabolism was also involved in myofibroblast formation of neonatal rat cardiac fibroblasts. Thus, unraveling supersulfide metabolism during myocardial remodeling may lead to the development of novel therapeutic strategies to improve heart failure.

Supersulfide prevents cigarette smoke extract-induced mitochondria hyperfission and cardiomyocyte early senescence by inhibiting Drp1-filamin complex formation.

Smoking is one of the most serious risk factors for cardiovascular diseases. Although cigarette mainstream and sidestream smoke are significant contributors to increased cardiovascular mortality and morbidity, the underlying mechanism is still unclear. Here, we report that exposure of rat neonatal cardiomyocytes to cigarette smoke extract (CSE) induces mitochondrial hyperfission-mediated myocardial senescence. CSE leads to mitochondrial fission and reactive oxygen species (ROS) production through the complex formation between mitochondrial fission factor Drp1 and actin-binding protein, filamin A. Pharmacological perturbation of interaction between Drp1 and filamin A by cilnidipine and gene knockdown of Drp1 or filamin A inhibited CSE-induced mitochondrial hyperfission and ROS production as well as myocardial senescence. We previously reported that Drp1 activity is controlled by supersulfide-induced Cys644 polysulfidation. The redox-sensitive Cys644 was critical for CSE-mediated interaction with filamin A. The administration of supersulfide donor, Na2S3 also improved mitochondrial hyperfission-mediated myocardial senescence induced by CSE. Our results suggest the important role of Drp1-filamin A complex formation on cigarette smoke-mediated cardiac risk and the contribution of supersulfide to mitochondrial fission-associated myocardial senescence.

Inorganic sulfides prevent osimertinib-induced mitochondrial dysfunction in human iPS cell-derived cardiomyocytes.

Despite the widespread recognition of the global concern regarding the onset of cardiovascular diseases in a significant number of patients following cancer treatment, definitive strategies for prevention and treatment remain elusive. In this study, we established systems to evaluate the influence of anti-cancer drugs on the quality control of mitochondria, pivotal for energy metabolism, using human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). Osimertinib, an epidermal growth factor receptor tyrosine kinase inhibitor used for treatment in lung cancer, reportedly increases the risk of cardiovascular disease. However, its underlying mechanism is largely unknown. Here, we found that the treatment of hiPSC-CMs with osimertinib and doxorubicin, but not trastuzumab and cisplatin, revealed a concentration-dependent impairment of respiratory function accompanied by mitochondrial fission. We previously reported the significant role of sulfur metabolism in maintaining mitochondrial quality in the heart. Co-treatment with various inorganic sulfur donors (Na2S, Na2S2, Na2S3) alongside anti-cancer drugs demonstrated that Na2S attenuated the cardiotoxicity of osimertinib but not doxorubicin. Osimertinib decreased intracellular reduced sulfur levels, while Na2S treatment suppressed the sulfur leakage, suggesting its potential in mitigating osimertinib-induced cardiotoxicity. These results imply the prospect of inorganic sulfides, such as Na2S, as a seed for precision pharmacotherapy to alleviate osimertinib's cardiotoxic effects.

Pharmacological Activation of TRPC6 Channel Prevents Colitis Progression.

We recently reported that transient receptor potential canonical (TRPC) 6 channel activity contributes to intracellular Zn2+ homeostasis in the heart. Zn2+ has also been implicated in the regulation of intestinal redox and microbial homeostasis. This study aims to investigate the role of TRPC6-mediated Zn2+ influx in the stress resistance of the intestine. The expression profile of TRPC1-C7 mRNAs in the actively inflamed mucosa from inflammatory bowel disease (IBD) patients was analyzed using the GEO database. Systemic TRPC3 knockout (KO) and TRPC6 KO mice were treated with dextran sulfate sodium (DSS) to induce colitis. The Zn2+ concentration and the mRNA expression levels of oxidative/inflammatory markers in colon tissues were quantitatively analyzed, and gut microbiota profiles were compared. TRPC6 mRNA expression level was increased in IBD patients and DSS-treated mouse colon tissues. DSS-treated TRPC6 KO mice, but not TRPC3 KO mice, showed severe weight loss and increased disease activity index compared with DSS-treated WT mice. The mRNA abundances of antioxidant proteins were basically increased in the TRPC6 KO colon, with changes in gut microbiota profiles. Treatment with TRPC6 activator prevented the DSS-induced colitis progression accompanied by increasing Zn2+ concentration. We suggest that TRPC6-mediated Zn2+ influx activity plays a key role in stress resistance against IBD, providing a new strategy for treating colitis.

Inhibition of Drp1-Filamin Protein Complex Prevents Hepatic Lipid Droplet Accumulation by Increasing Mitochondria-Lipid Droplet Contact.

Lipid droplet (LD) accumulation in hepatocytes is one of the major symptoms associated with fatty liver disease. Mitochondria play a key role in catabolizing fatty acids for energy production through ß-oxidation. The interplay between mitochondria and LD assumes a crucial role in lipid metabolism, while it is obscure how mitochondrial morphology affects systemic lipid metabolism in the liver. We previously reported that cilnidipine, an already existing anti-hypertensive drug, can prevent pathological mitochondrial fission by inhibiting protein-protein interaction between dynamin-related protein 1 (Drp1) and filamin, an actin-binding protein. Here, we found that cilnidipine and its new dihydropyridine (DHP) derivative, 1,4-DHP, which lacks Ca2+ channel-blocking action of cilnidipine, prevent the palmitic acid-induced Drp1-filamin interaction, LD accumulation and cytotoxicity of human hepatic HepG2 cells. Cilnidipine and 1,4-DHP also suppressed the LD accumulation accompanied by reducing mitochondrial contact with LD in obese model and high-fat diet-fed mouse livers. These results propose that targeting the Drp1-filamin interaction become a new strategy for the prevention or treatment of fatty liver disease.

Echinochrome Prevents Sulfide Catabolism-Associated Chronic Heart Failure after Myocardial Infarction in Mice.

Abnormal sulfide catabolism, especially the accumulation of hydrogen sulfide (H2S) during hypoxic or inflammatory stresses, is a major cause of redox imbalance-associated cardiac dysfunction. Polyhydroxynaphtoquinone echinochrome A (Ech-A), a natural pigment of marine origin found in the shells and needles of many species of sea urchins, is a potent antioxidant and inhibits acute myocardial ferroptosis after ischemia/reperfusion, but the chronic effect of Ech-A on heart failure is unknown. Reactive sulfur species (RSS), which include catenated sulfur atoms, have been revealed as true biomolecules with high redox reactivity required for intracellular energy metabolism and signal transduction. Here, we report that continuous intraperitoneal administration of Ech-A (2.0 mg/kg/day) prevents RSS catabolism-associated chronic heart failure after myocardial infarction (MI) in mice. Ech-A prevented left ventricular (LV) systolic dysfunction and structural remodeling after MI. Fluorescence imaging revealed that intracellular RSS level was reduced after MI, while H2S/HS- level was increased in LV myocardium, which was attenuated by Ech-A. This result indicates that Ech-A suppresses RSS catabolism to H2S/HS- in LV myocardium after MI. In addition, Ech-A reduced oxidative stress formation by MI. Ech-A suppressed RSS catabolism caused by hypoxia in neonatal rat cardiomyocytes and human iPS cell-derived cardiomyocytes. Ech-A also suppressed RSS catabolism caused by lipopolysaccharide stimulation in macrophages. Thus, Ech-A has the potential to improve chronic heart failure after MI, in part by preventing sulfide catabolism.

Knockout of Purinergic P2Y6 Receptor Fails to Improve Liver Injury and Inflammation in Non-Alcoholic Steatohepatitis.

Nonalcoholic steatohepatitis (NASH) is a disease that progresses from nonalcoholic fatty liver (NAFL) and which is characterized by inflammation and fibrosis. The purinergic P2Y6 receptor (P2Y6R) is a pro-inflammatory Gq/G12 family protein-coupled receptor and reportedly contributes to intestinal inflammation and cardiovascular fibrosis, but its role in liver pathogenesis is unknown. Human genomics data analysis revealed that the liver P2Y6R mRNA expression level is increased during the progression from NAFL to NASH, which positively correlates with inductions of C-C motif chemokine 2 (CCL2) and collagen type I α1 chain (Col1a1) mRNAs. Therefore, we examined the impact of P2Y6R functional deficiency in mice crossed with a NASH model using a choline-deficient, L-amino acid-defined, high-fat diet (CDAHFD). Feeding CDAHFD for 6 weeks markedly increased P2Y6R expression level in mouse liver, which was positively correlated with CCL2 mRNA induction. Unexpectedly, the CDAHFD treatment for 6 weeks increased liver weights with severe steatosis in both wild-type (WT) and P2Y6R knockout (KO) mice, while the disease marker levels such as serum AST and liver CCL2 mRNA in CDAHFD-treated P2Y6R KO mice were rather aggravated compared with those of CDAHFD-treated WT mice. Thus, P2Y6R may not contribute to the progression of liver injury, despite increased expression in NASH liver.

Protective effect of d-alanine against acute kidney injury.

Recent studies have revealed the connection between amino acid chirality and diseases. We have previously reported that the gut microbiota produces various d-amino acids in a murine acute kidney injury (AKI) model. Here, we further explored the pathophysiological role of d-alanine (d-Ala) in AKI. Levels of d-Ala were evaluated in a murine AKI model. We analyzed transcripts of the N-methyl-d-aspartate (NMDA) receptor, a receptor for d-Ala, in tubular epithelial cells (TECs). The therapeutic effect of d-Ala was then assessed in vivo and in vitro. Finally, the plasma level of d-Ala was evaluated in patients with AKI. The Grin genes encoding NMDA receptor subtypes were expressed in TECs. Hypoxic conditions change the gene expression of Grin1, Grin2A, and Grin2B. d-Ala protected TECs from hypoxia-related cell injury and induced proliferation after hypoxia. These protective effects are associated with the chirality of d-Ala. d-Ala inhibits reactive oxygen species (ROS) production and improves mitochondrial membrane potential, through NMDA receptor signaling. The ratio of d-Ala to l-Ala was increased in feces, plasma, and urine after the induction of ischemia-reperfusion (I/R). Moreover, Enterobacteriaceae, such as Escherichia coli and Klebsiella oxytoca, produce d-Ala. Oral administration of d-Ala ameliorated kidney injury after the induction of I/R in mice. Deficiency of NMDA subunit NR1 in tubular cells worsened kidney damage in AKI. In addition, the plasma level of d-Ala was increased and reflected the level of renal function in patients with AKI. In conclusion, d-Ala has protective effects on I/R-induced kidney injury. Moreover, the plasma level of d-Ala reflects the estimated glomerular filtration rate in patients with AKI. d-Ala could be a promising therapeutic target and potential biomarker for AKI.NEW & NOTEWORTHY d-Alanine has protective effects on I/R-induced kidney injury. d-Ala inhibits ROS production and improves mitochondrial membrane potential, resulting in reduced TEC necrosis by hypoxic stimulation. The administration of d-Ala protects the tubules from I/R injury in mice. Moreover, the plasma level of d-Ala is conversely associated with eGFR in patients with AKI. Our data suggest that d-Ala is an appealing therapeutic target and a potential biomarker for AKI.

SARS-CoV-2 induces barrier damage and inflammatory responses in the human iPSC-derived intestinal epithelium.

Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has rapidly spread and led to global health crises. COVID-19 causes well-known respiratory failure and gastrointestinal symptoms, such as diarrhea, nausea, and vomiting. Thus, human gastrointestinal cell models are urgently needed for COVID-19 research; however, it is difficult to obtain primary human intestinal cells. In this study, we examined whether human induced pluripotent stem cell (iPSC)-derived small intestinal epithelial cells (iPSC-SIECs) could be used as a SARS-CoV-2 infection model. We observed that iPSC-SIECs, such as absorptive and Paneth cells, were infected with SARS-CoV-2, and remdesivir treatment decreased intracellular SARS-CoV-2 replication in iPSC-SIECs. SARS-CoV-2 infection decreased expression levels of tight junction markers, ZO-3 and CLDN1, and transepithelial electrical resistance (TEER), which evaluates the integrity of tight junction dynamics. In addition, SARS-CoV-2 infection increased expression levels of proinflammatory genes, which are elevated in patients with COVID-19. These findings suggest iPSC-SIECs as a useful in vitro model for elucidating COVID-19 pathology and drug development.

Drug repurposing for the treatment of COVID-19.

Coronavirus disease 2019 (COVID-19) remains prevalent worldwide since its onset was confirmed in Wuhan, China in 2019. Vaccines against the causative virus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), have shown a preventive effect against the onset and severity of COVID-19, and social and economic activities are gradually recovering. However, the presence of vaccine-resistant variants has been reported, and the development of therapeutic agents for patients with severe COVID-19 and related sequelae remains urgent. Drug repurposing, also called drug repositioning or eco-pharma, is the strategy of using previously approved and safe drugs for a therapeutic indication that is different from their original indication. The risk of severe COVID-19 and mortality increases with advancing age, cardiovascular disease, hypertension, diabetes, and cancer. We have reported three protein-protein interactions that are related to heart failure, and recently identified that one mechanism increases the risk of SARS-CoV-2 infection in mammalian cells. This review outlines the global efforts and outcomes of drug repurposing research for the treatment of severe COVID-19. It also discusses our recent finding of a new protein-protein interaction that is common to COVID-19 aggravation and heart failure.

Astrocytic STAT3 activation and chronic itch require IP3R1/TRPC-dependent Ca2+ signals in mice.

BACKGROUND: Chronic itch is a debilitating symptom of inflammatory skin diseases, but the underlying mechanism is poorly understood. We have recently demonstrated that astrocytes in the spinal dorsal horn become reactive in models of atopic and contact dermatitis via activation of the transcription factor signal transducer and activator of transcription 3 (STAT3) and critically contribute to chronic itch. In general, STAT3 is transiently activated; however, STAT3 activation in reactive astrocytes of chronic itch model mice persistently occurs via an unknown mechanism. OBJECTIVE: We aimed to determine the mechanisms of persistent activation of astrocytic STAT3 in chronic itch conditions. METHODS: To determine the factors that are required for persistent activation of astrocytic STAT3, Western blotting and calcium imaging with cultured astrocytes or spinal cord slices were performed. Thereafter, chronic itch model mice were used for genetic and behavioral experiments to confirm the role of the factors determined to mediate persistent STAT3 activation from in vitro and ex vivo experiments in chronic itch. RESULTS: IP3 receptor type 1 (IP3R1) knockdown in astrocytes suppressed IL-6-induced persistent STAT3 activation and expression of lipocalin-2 (LCN2), an astrocytic STAT3-dependent inflammatory factor that is required for chronic itch. IP3R1-dependent astrocytic Ca2+ responses involved Ca2+ influx through the cation channel transient receptor potential canonical (TRPC), which was required for persistent STAT3 activation evoked by IL-6. IL-6 expression was upregulated in dorsal root ganglion neurons in a mouse model of chronic itch. Dorsal root ganglion neuron-specific IL-6 knockdown, spinal astrocyte-specific IP3R1 knockdown, and pharmacologic spinal TRPC inhibition attenuated LCN2 expression and chronic itch. CONCLUSION: Our findings suggest that IP3R1/TRPC channel-mediated Ca2+ signals elicited by IL-6 in astrocytes are necessary for persistent STAT3 activation, LCN2 expression, and chronic itch, and they may also provide new targets for therapeutic intervention.

Lysophosphatidic Acid Promotes the Expansion of Cancer Stem Cells via TRPC3 Channels in Triple-Negative Breast Cancer.

Triple-negative breast cancer (TNBC) is a highly aggressive cancer for which targeted therapeutic agents are limited. Growing evidence suggests that TNBC originates from breast cancer stem cells (BCSCs), and elucidation of the molecular mechanisms controlling BCSC proliferation will be crucial for new drug development. We have previously reported that the lysosphingolipid sphingosine-1-phosphate mediates the CSC phenotype, which can be identified as the ALDH-positive cell population in several types of human cancer cell lines. In this study, we have investigated additional lipid receptors upregulated in BCSCs. We found that lysophosphatidic acid (LPA) receptor 3 was highly expressed in ALDH-positive TNBC cells. The LPAR3 antagonist inhibited the increase in ALDH-positive cells after LPA treatment. Mechanistically, the LPA-induced increase in ALDH-positive cells was dependent on intracellular calcium ion (Ca2+), and the increase in Ca2+ was suppressed by a selective inhibitor of transient receptor potential cation channel subfamily C member 3 (TRPC3). Moreover, IL-8 production was involved in the LPA response via the activation of the Ca2+-dependent transcriptional factor nuclear factor of activated T cells. Taken together, our findings provide new insights into the lipid-mediated regulation of BCSCs via the LPA-TRPC3 signaling axis and suggest several potential therapeutic targets for TNBC.

TRPC3-Nox2 Protein Complex Formation Increases the Risk of SARS-CoV-2 Spike Protein-Induced Cardiomyocyte Dysfunction through ACE2 Upregulation.

Myocardial damage caused by the newly emerged coronavirus (SARS-CoV-2) infection is one of the key determinants of COVID-19 severity and mortality. SARS-CoV-2 entry to host cells is initiated by binding with its receptor, angiotensin-converting enzyme (ACE) 2, and the ACE2 abundance is thought to reflect the susceptibility to infection. Here, we report that ibudilast, which we previously identified as a potent inhibitor of protein complex between transient receptor potential canonical (TRPC) 3 and NADPH oxidase (Nox) 2, attenuates the SARS-CoV-2 spike glycoprotein pseudovirus-evoked contractile and metabolic dysfunctions of neonatal rat cardiomyocytes (NRCMs). Epidemiologically reported risk factors of severe COVID-19, including cigarette sidestream smoke (CSS) and anti-cancer drug treatment, commonly upregulate ACE2 expression level, and these were suppressed by inhibiting TRPC3-Nox2 complex formation. Exposure of NRCMs to SARS-CoV-2 pseudovirus, as well as CSS and doxorubicin (Dox), induces ATP release through pannexin-1 hemi-channels, and this ATP release potentiates pseudovirus entry to NRCMs and human iPS cell-derived cardiomyocytes (hiPS-CMs). As the pseudovirus entry followed by production of reactive oxygen species was attenuated by inhibiting TRPC3-Nox2 complex in hiPS-CMs, we suggest that TRPC3-Nox2 complex formation triggered by panexin1-mediated ATP release participates in exacerbation of myocardial damage by amplifying ACE2-dependent SARS-CoV-2 entry.

Cardiac robustness regulated by reactive sulfur species.

The human myocardium contains robust cells that constantly beat from birth to death without being replaced, even when exposed to various environmental stresses. Myocardial robustness is thought to depend primarily on the strength of the reducing power to protect the heart from oxidative stress. Myocardial antioxidant systems are controlled by redox reactions, primarily via the redox reaction of Cys sulfhydryl groups, such as found in thioredoxin and glutathione. However, the specific molecular entities that regulate myocardial reducing power have long been debated. Recently, reactive sulfide species, with excellent electron transfer ability, consisting of a series of multiple sulfur atoms, i.e., Cys persulfide and Cys polysulfides, have been found to play an essential role in maintaining mitochondrial quality and function, as well as myocardial robustness. This review presents the latest findings on the molecular mechanisms underlying mitochondrial energy metabolism and the maintenance of quality control by reactive sulfide species and provides a new insight for the prevention of chronic heart failure.

Deletion of TRPC3 or TRPC6 Fails to Attenuate the Formation of Inflammation and Fibrosis in Non-alcoholic Steatohepatitis.

Non-alcoholic steatohepatitis (NASH) is a disease that has progressed from non-alcoholic fatty liver disease (NAFLD) and is characterized by inflammation and fibrosis. Two transient receptor potential canonical (TRPC) subfamily members, TRPC3 and TRPC6 (TRPC3/6), reportedly participate in the development of fibrosis in cardiovascular and renal systems. We hypothesized that TRPC3/6 may also participate in NASH fibrosis. We evaluated the effects of TRPC3 or TRPC6 functional deficiency in a NASH mouse model using choline-deficient, L-amino acid-defined, high-fat diet (CDAHFD). Wild-type (WT) and TRPC3 or TRPC6 gene-deficient (KO) mice were fed with CDAHFD or standard diet for 6 weeks. The CDAHFD-induced body weight loss in TRPC6 KO mice was significantly lower compared with WT mice with CDAHFD. CDAHFD treatment significantly increased TRPC3 mRNA expression level and tissue weight in WT liver, which were suppressed in TRPC3 KO mice. However, either systemic deletion of TRPC3 or TRPC6 failed to attenuate liver steatosis, inflammation and fibrosis. These results imply that TRPC3 and TRPC6 are unlikely to be involved in liver dysfunction and fibrosis of NASH model mice.

Cold Atmospheric Plasma Modification of Amyloid ß.

Cold atmospheric plasma (CAP) has attracted much attention in the fields of biotechnology and medicine owing to its potential utility in clinical applications. Recently accumulating evidence has demonstrated that CAP influences protein structures. However, there remain open questions regarding the molecular mechanisms behind the CAP-induced structural perturbations of biomacromolecules. Here, we investigated the potential effects of CAP irradiation of amyloid ß (Aß), an amyloidogenic protein associated with Alzheimer's disease. Using nuclear magnetic resonance spectroscopy, we observed gradual spectral changes in Aß after a 10 s CAP pretreatment, which also suppressed its fibril formation, as revealed by thioflavin T assay. As per mass spectrometric analyses, these effects were attributed to selective oxidation of the methionine residue (Met) at position 35. Interestingly, this modification occurred when Aß was dissolved into a pre-irradiated buffer, indicating that some reactive species oxidize the Met residue. Our results strongly suggest that the H2O2 generated in the solution by CAP irradiation is responsible for Met oxidation, which inhibits Aß amyloid formation. The findings of the present study provide fundamental insights into plasma biology, giving clues for developing novel applications of CAP.

2-Oxo-histidine-containing dipeptides are functional oxidation products.

Imidazole-containing dipeptides (IDPs), such as carnosine and anserine, are found exclusively in various animal tissues, especially in the skeletal muscles and nerves. IDPs have antioxidant activity because of their metal-chelating and free radical-scavenging properties. However, the underlying mechanisms that would fully explain IDP antioxidant effects remain obscure. Here, using HPLC-electrospray ionization-tandem MS analyses, we comprehensively investigated carnosine and its related small peptides in the soluble fractions of mouse tissue homogenates and ubiquitously detected 2-oxo-histidine-containing dipeptides (2-oxo-IDPs) in all examined tissues. We noted enhanced production of the 2-oxo-IDPs in the brain of a mouse model of sepsis-associated encephalopathy. Moreover, in SH-SY5Y human neuroblastoma cells stably expressing carnosine synthase, H2O2 exposure resulted in the intracellular production of 2-oxo-carnosine, which was associated with significant inhibition of the H2O2 cytotoxicity. Notably, 2-oxo-carnosine showed a better antioxidant activity than endogenous antioxidants such as GSH and ascorbate. Mechanistic studies indicated that carnosine monooxygenation is mediated through the formation of a histidyl-imidazole radical, followed by the addition of molecular oxygen. Our findings reveal that 2-oxo-IDPs are metal-catalyzed oxidation products present in vivo and provide a revised paradigm for understanding the antioxidant effects of the IDPs.

Mitochondrial dynamics in exercise physiology.

A growing body of evidence suggests that exercise shows pleiotropic effects on the maintenance of systemic homeostasis through mitochondria. Dysregulation of mitochondrial dynamism is associated with metabolic inflexibility, resulting in many of the metabolic diseases and aging. Studies have suggested that exercise prevents and delays the progression of mitochondrial dysfunction by improving mitochondrial metabolism, biogenesis, and quality control. Exercise modulates functions of mitochondrial dynamics-regulating proteins through post-translational modification mechanisms. In this review, we discuss the putative mechanisms underlying maintenance of mitochondrial homeostasis by exercise, especially focusing on the post-translational modifications of several signaling proteins contributing to mitochondrial biogenesis, autophagy or mitophagy flux, and fission/fusion cycle. We also introduce novel small molecules that can potentially mimic exercise therapy through preserving mitochondrial dynamism. These recent advancements in the field of mitochondrial biology may lead to a greater understanding of exercise signaling.

TRPC6 regulates phenotypic switching of vascular smooth muscle cells through plasma membrane potential-dependent coupling with PTEN.

Vascular smooth muscle cells (VSMCs) play critical roles in the stability and tonic regulation of vascular homeostasis. VSMCs can switch back and forth between highly proliferative synthetic and fully differentiated contractile phenotypes in response to changes in the vessel environment. Although abnormal phenotypic switching of VSMCs is a hallmark of vascular disorders such as atherosclerosis and restenosis after angioplasty, how control of VSMC phenotypic switching is dysregulated in pathologic conditions remains obscure. We found that inhibition of canonical transient receptor potential 6 (TRPC6) channels facilitated contractile differentiation of VSMCs through plasma membrane hyperpolarization. TRPC6-deficient VSMCs exhibited more polarized resting membrane potentials and higher protein kinase B (Akt) activity than wild-type VSMCs in response to TGF-ß1 stimulation. Ischemic stress elicited by oxygen-glucose deprivation suppressed TGF-ß1-induced hyperpolarization and VSMC differentiation, but this effect was abolished by TRPC6 deletion. TRPC6-mediated Ca2+ influx and depolarization coordinately promoted the interaction of TRPC6 with lipid phosphatase and tensin homolog deleted from chromosome 10 (PTEN), a negative regulator of Akt activation. Given the marked up-regulation of TRPC6 observed in vascular disorders, our findings suggest that attenuation of TRPC6 channel activity in pathologic VSMCs could be a rational strategy to maintain vascular quality control by fine-tuning of VSMC phenotypic switching.-Numaga-Tomita, T., Shimauchi, T., Oda, S., Tanaka, T., Nishiyama, K., Nishimura, A., Birnbaumer, L., Mori, Y., Nishida, M. TRPC6 regulates phenotypic switching of vascular smooth muscle cells through plasma membrane potential-dependent coupling with PTEN.