Exclusion of sulfide:quinone oxidoreductase from mitochondria causes Leigh-like disease in mice by impairing sulfide metabolism.

Leigh syndrome is the most common inherited mitochondrial disease in children and is often fatal within the first few years of life. In 2020, mutations in the gene encoding sulfide:quinone oxidoreductase (SQOR), a mitochondrial protein, were identified as a cause of Leigh syndrome. Here, we report that mice with a mutation in the gene encoding SQOR (SqorΔN/ΔN mice), which prevented SQOR from entering mitochondria, had clinical and pathological manifestations of Leigh syndrome. SqorΔN/ΔN mice had increased blood lactate levels that were associated with markedly decreased complex IV activity and increased hydrogen sulfide (H2S) levels. Because H2S is produced by both gut microbiota and host tissue, we tested whether metronidazole (a broad-spectrum antibiotic) or a sulfur-restricted diet rescues SqorΔN/ΔN mice from developing Leigh syndrome. Daily treatment with metronidazole alleviated increased H2S levels, normalized complex IV activity and blood lactate levels, and prolonged the survival of SqorΔN/ΔN mice. Similarly, a sulfur-restricted diet normalized blood lactate levels and inhibited the development of Leigh syndrome. Taken together, these observations suggest that mitochondrial SQOR is essential to prevent systemic accumulation of H2S. Metronidazole administration and a sulfur-restricted diet may be therapeutic approaches to treatment of patients with Leigh syndrome caused by mutations in SQOR.

Supersulfide catalysis for nitric oxide and aldehyde metabolism.

Abundant formation of endogenous supersulfides, which include reactive persulfide species and sulfur catenated residues in thiols and proteins (supersulfidation), has been observed. We found here that supersulfides catalyze S-nitrosoglutathione (GSNO) metabolism via glutathione-dependent electron transfer from aldehydes by exploiting alcohol dehydrogenase 5 (ADH5). ADH5 is a highly conserved bifunctional enzyme serving as GSNO reductase (GSNOR) that down-regulates NO signaling and formaldehyde dehydrogenase (FDH) that detoxifies formaldehyde in the form of glutathione hemithioacetal. C174S mutation significantly reduced the supersulfidation of ADH5 and almost abolished GSNOR activity but spared FDH activity. Notably, Adh5C174S/C174S mice manifested improved cardiac functions possibly because of GSNOR elimination and consequent increased NO bioavailability. Therefore, we successfully separated dual functions (GSNOR and FDH) of ADH5 (mediated by the supersulfide catalysis) through the biochemical analysis for supersulfides in vitro and characterizing in vivo phenotypes of the GSNOR-deficient organisms that we established herein. Supersulfides in ADH5 thus constitute a substantial catalytic center for GSNO metabolism mediating electron transfer from aldehydes.

Redox-dependent internalization of the purinergic P2Y6 receptor limits colitis progression.

After ligand stimulation, many G protein­coupled receptors (GPCRs) undergo ß-arrestin­dependent desensitization, during which they are internalized and either degraded or recycled to the plasma membrane. Some GPCRs are not subject to this type of desensitization because they lack the residues required to interact with ß-arrestins. We identified a mechanism of redox-dependent alternative internalization (REDAI) that promotes the internalization and degradation of the purinergic P2Y6 receptor (P2Y6R). Synthetic and natural compounds containing electrophilic isothiocyanate groups covalently modified P2Y6R at Cys220, which promoted the ubiquitylation of Lys137 and receptor internalization and degradation in various mouse and human cultured cell lines. Endogenous electrophiles also promoted ligand-dependent P2Y6R internalization and degradation. P2Y6R is highly abundant in inflammatory cells and promotes the pathogenesis of colitis. Deficiency in P2Y6R protected mice against experimentally induced colitis, and mice expressing a form of P2Y6R in which Cys220 was mutated to nonmodifiable serine were more sensitive to the induction of colitis. Several other GPCRs, including A2BAR, contain cysteine and lysine residues at the appropriate positions to mediate REDAI, and isothiocyanate stimulated the internalization of A2BAR and of a form of P2Y2R with insertions of the appropriate residues. Thus, endogenous and exogenous electrophiles may limit colitis progression through cysteine modification of P2Y6R and may also mediate internalization of other GPCRs.

Modulation of P2Y6R expression exacerbates pressure overload-induced cardiac remodeling in mice.

Cardiac tissue remodeling caused by hemodynamic overload is a major clinical outcome of heart failure. Uridine-responsive purinergic P2Y6 receptor (P2Y6R) contributes to the progression of cardiovascular remodeling in rodents, but it is not known whether inhibition of P2Y6R prevents or promotes heart failure. We demonstrate that inhibition of P2Y6R promotes pressure overload-induced sudden death and heart failure in mice. In neonatal cardiomyocytes, knockdown of P2Y6R significantly attenuated hypertrophic growth and cell death caused by hypotonic stimulation, indicating the involvement of P2Y6R in mechanical stress-induced myocardial dysfunction. Unexpectedly, compared with wild-type mice, deletion of P2Y6R promoted pressure overload-induced sudden death, as well as cardiac remodeling and dysfunction. Mice with cardiomyocyte-specific overexpression of P2Y6R also exhibited cardiac dysfunction and severe fibrosis. In contrast, P2Y6R deletion had little impact on oxidative stress-mediated cardiac dysfunction induced by doxorubicin treatment. These findings provide overwhelming evidence that systemic inhibition of P2Y6R exacerbates pressure overload-induced heart failure in mice, although P2Y6R in cardiomyocytes contributes to the progression of cardiac fibrosis.

Depolysulfidation of Drp1 induced by low-dose methylmercury exposure increases cardiac vulnerability to hemodynamic overload.

Chronic exposure to methylmercury (MeHg), an environmental electrophilic pollutant, reportedly increases the risk of human cardiac events. We report that exposure to a low, non-neurotoxic dose of MeHg precipitated heart failure induced by pressure overload in mice. Exposure to MeHg at 10 ppm did not induce weight loss typical of higher doses but caused mitochondrial hyperfission in myocardium through the activation of Drp1 by its guanine nucleotide exchange factor filamin-A. Treatment of neonatal rat cardiomyocytes with cilnidipine, an inhibitor of the interaction between Drp1 and filamin-A, suppressed mitochondrial hyperfission caused by low-dose MeHg exposure. Modification of cysteine residues in proteins with polysulfides is important for redox signaling and mitochondrial homeostasis in mammalian cells. We found that MeHg targeted rat Drp1 at Cys624, a redox-sensitive residue whose SH side chain forms a bulky and nucleophilic polysulfide (Cys624-S(n)H). MeHg exposure induced the depolysulfidation of Cys624-S(n)H in Drp1, which led to filamin-dependent activation of Drp1 and mitochondrial hyperfission. Treatment with NaHS, which acts as a donor for reactive polysulfides, reversed MeHg-evoked Drp1 depolysulfidation and vulnerability to mechanical load in rodent and human cardiomyocytes and mouse hearts. These results suggest that depolysulfidation of Drp1 at Cys624-S(n)H by low-dose MeHg increases cardiac fragility to mechanical load through filamin-dependent mitochondrial hyperfission.

Hypoxia-induced interaction of filamin with Drp1 causes mitochondrial hyperfission-associated myocardial senescence.

Defective mitochondrial dynamics through aberrant interactions between mitochondria and actin cytoskeleton is increasingly recognized as a key determinant of cardiac fragility after myocardial infarction (MI). Dynamin-related protein 1 (Drp1), a mitochondrial fission-accelerating factor, is activated locally at the fission site through interactions with actin. Here, we report that the actin-binding protein filamin A acted as a guanine nucleotide exchange factor for Drp1 and mediated mitochondrial fission-associated myocardial senescence in mice after MI. In peri-infarct regions characterized by mitochondrial hyperfission and associated with myocardial senescence, filamin A colocalized with Drp1 around mitochondria. Hypoxic stress induced the interaction of filamin A with the GTPase domain of Drp1 and increased Drp1 activity in an actin-binding-dependent manner in rat cardiomyocytes. Expression of the A1545T filamin mutant, which potentiates actin aggregation, promoted mitochondrial hyperfission under normoxia. Furthermore, pharmacological perturbation of the Drp1-filamin A interaction by cilnidipine suppressed mitochondrial hyperfission-associated myocardial senescence and heart failure after MI. Together, these data demonstrate that Drp1 association with filamin and the actin cytoskeleton contributes to cardiac fragility after MI and suggests a potential repurposing of cilnidipine, as well as provides a starting point for innovative Drp1 inhibitor development.

TRPC5-eNOS Axis Negatively Regulates ATP-Induced Cardiomyocyte Hypertrophy.

Cardiac hypertrophy, induced by neurohumoral factors, including angiotensin II and endothelin-1, is a major predisposing factor for heart failure. These ligands can induce hypertrophic growth of neonatal rat cardiomyocytes (NRCMs) mainly through Ca2+-dependent calcineurin/nuclear factor of activated T cell (NFAT) signaling pathways activated by diacylglycerol-activated transient receptor potential canonical 3 and 6 (TRPC3/6) heteromultimer channels. Although extracellular nucleotide, adenosine 5'-triphosphate (ATP), is also known as most potent Ca2+-mobilizing ligand that acts on purinergic receptors, ATP never induces cardiomyocyte hypertrophy. Here we show that ATP-induced production of nitric oxide (NO) negatively regulates hypertrophic signaling mediated by TRPC3/6 channels in NRCMs. Pharmacological inhibition of NO synthase (NOS) potentiated ATP-induced increases in NFAT activity, protein synthesis, and transcriptional activity of brain natriuretic peptide. ATP significantly increased NO production and protein kinase G (PKG) activity compared to angiotensin II and endothelin-1. We found that ATP-induced Ca2+ signaling requires inositol 1,4,5-trisphosphate (IP3) receptor activation. Interestingly, inhibition of TRPC5, but not TRPC6 attenuated ATP-induced activation of Ca2+/NFAT-dependent signaling. As inhibition of TRPC5 attenuates ATP-stimulated NOS activation, these results suggest that NO-cGMP-PKG axis activated by IP3-mediated TRPC5 channels underlies negative regulation of TRPC3/6-dependent hypertrophic signaling induced by ATP stimulation.

Purinergic P2Y6 receptors: A new therapeutic target of age-dependent hypertension.

Aging has a remarkable effect on cardiovascular homeostasis and it is known as the major non-modifiable risk factor in the development of hypertension. Medications targeting sympathetic nerve system and/or renin-angiotensin-aldosterone system are widely accepted as a powerful therapeutic strategy to improve hypertension, although the control rates remain unsatisfactory especially in the elder patients with hypertension. Purinergic receptors, activated by adenine, uridine nucleotides and nucleotide sugars, play pivotal roles in many biological processes, including platelet aggregation, neurotransmission and hormone release, and regulation of cardiovascular contractility. Since clopidogrel, a selective inhibitor of G protein-coupled purinergic P2Y12 receptor (P2Y12R), achieved clinical success as an anti-platelet drug, P2YRs has been attracted more attention as new therapeutic targets of cardiovascular diseases. We have revealed that UDP-responsive P2Y6R promoted angiotensin type 1 receptor (AT1R)-stimulated vascular remodeling in mice, in an age-dependent manner. Moreover, the age-related formation of heterodimer between AT1R and P2Y6R was disrupted by MRS2578, a P2Y6R-selective inhibitor. These findings suggest that P2Y6R is a therapeutic target to prevent age-related hypertension.