Role of circadian clock system in the mitochondrial trans-sulfuration pathway and tissue remodeling.

Previous studies from our laboratory revealed that the gaseous molecule hydrogen sulfide (H2S), a metabolic product of epigenetics, involves trans-sulfuration pathway for ensuring metabolism and clearance of homocysteine (Hcy) from body, thereby mitigating the skeletal muscle's pathological remodeling. Although the master circadian clock regulator that is known as brain and muscle aryl hydrocarbon receptor nuclear translocator like protein 1 (i.e., BMAL 1) is associated with S-adenosylhomocysteine hydrolase (SAHH) and Hcy metabolism but how trans-sulfuration pathway is influenced by the circadian clock remains unexplored. We hypothesize that alterations in the functioning of circadian clock during sleep and wake cycle affect skeletal muscle's biology. To test this hypothesis, we measured serum matrix metalloproteinase (MMP) activities using gelatin gels for analyzing the MMP-2 and MMP-9. Further, employing casein gels, we also studied MMP-13 that is known to be influenced by the growth arrest and DNA damage-45 (GADD45) protein during sleep and wake cycle. The wild type and cystathionine ß synthase-deficient (CBS-/+) mice strains were treated with H2S and subjected to measurement of trans-sulfuration factors from skeletal muscle tissues. The results suggested highly robust activation of MMPs in the wake mice versus sleep mice, which appears somewhat akin to the "1-carbon metabolic dysregulation", which takes place during remodeling of extracellular matrix during muscular dystrophy. Interestingly, the levels of trans-sulfuration factors such as CBS, cystathionine γ lyase (CSE), methyl tetrahydrofolate reductase (MTHFR), phosphatidylethanolamine N-methyltransferase (PEMT), and Hcy-protein bound paraoxonase 1 (PON1) were attenuated in CBS-/+ mice. However, treatment with H2S mitigated the attenuation of the trans-sulfuration pathway. In addition, levels of mitochondrial peroxisome proliferator-activated receptor-gamma coactivator 1-α (PGC 1-α) and mitofusin-2 (MFN-2) were significantly improved by H2S intervention. Our findings suggest participation of the circadian clock in trans-sulfuration pathway that affects skeletal muscle remodeling and mitochondrial regeneration.

The role of the mitochondrial trans-sulfuration in cerebro-cardio renal dysfunction during trisomy down syndrome.

One in 700 children is born with the down syndrome (DS). In DS, there is an extra copy of X chromosome 21 (trisomy). Interestingly, the chromosome 21 also contains an extra copy of the cystathionine beta synthase (CBS) gene. The CBS activity is known to contribute in mitochondrial sulfur metabolism via trans-sulfuration pathway. We hypothesize that due to an extra copy of the CBS gene there is hyper trans-sulfuration in DS. We believe that understanding the mechanism of hyper trans-sulfuration during DS will be important in improving the quality of DS patients and towards developing new treatment strategies. We know that folic acid "1-carbon" metabolism (FOCM) cycle transfers the "1-carbon" methyl group to DNA (H3K4) via conversion of s-adenosyl methionine (SAM) to s-adenosyl homocysteine (SAH) by DNMTs (the gene writers). The demethylation reaction is carried out by ten-eleven translocation methylcytosine dioxygenases (TETs; the gene erasers) through epigenetics thus turning the genes off/on and opening the chromatin by altering the acetylation/HDAC ratio. The S-adenosyl homocysteine hydrolase (SAHH) hydrolyzes SAH to homocysteine (Hcy) and adenosine. The Hcy is converted to cystathionine, cysteine and hydrogen sulfide (H2S) via CBS/cystathioneγ lyase (CSE)/3-mercaptopyruvate sulfurtransferase (3MST) pathways. Adenosine by deaminase is converted to inosine and then to uric acid. All these molecules remain high in DS patients. H2S is a potent inhibitor of mitochondrial complexes I-IV, and regulated by UCP1. Therefore, decreased UCP1 levels and ATP production can ensue in DS subjects. Interestingly, children born with DS show elevated levels of CBS/CSE/3MST/Superoxide dismutase (SOD)/cystathionine/cysteine/H2S. We opine that increased levels of epigenetic gene writers (DNMTs) and decreased in gene erasers (TETs) activity cause folic acid exhaustion, leading to an increase in trans-sulphuration by CBS/CSE/3MST/SOD pathways. Thus, it is important to determine whether SIRT3 (inhibitor of HDAC3) can decrease the trans-sulfuration activity in DS patients. Since there is an increase in H3K4 and HDAC3 via epigenetics in DS, we propose that sirtuin-3 (Sirt3) may decrease H3K4 and HDAC3 and hence may be able to decrease the trans-sulfuration in DS. It would be worth to determine whether the lactobacillus, a folic acid producing probiotic, mitigates hyper-trans-sulphuration pathway in DS subjects. Further, as we know that in DS patients the folic acid is exhausted due to increase in CBS, Hcy and re-methylation. In this context, we suggest that folic acid producing probiotics such as lactobacillus might be able to improve re-methylation process and hence may help decrease the trans-sulfuration pathway in the DS patients.

Porphyromonas gingivalis induces cardiovascular dysfunction.

Porphyromonas gingivalis (P. gingivalis) is one of the most responsible periodontopathogenic bacteria in the development of periodontal disease (PD); however, its role in the development of other diseases still needs to be understood, specially its implications in the causation of cardiovascular pathogenesis. The aim of this study is to determine whether there is a direct association between P. gingivalis-induced PD with that of the development of cardiovascular disease, and whether a long-term administration of probiotic(s) could help improve the cardiovascular disease outcome. To test this hypothesis, we employed four different experimental groups of mice, designated as: Group I: Wild-type (WT) mice (C57BL/6J); Group II: Lactobacillus rhamnosus GG (LGG) (WT mice treated with a probiotic; LGG), Group III: PD (WT mice treated with P. gingivalis), and Group IV: PD + LGG (WT mice treated with P. gingivalis and LGG). PD was created by injecting 2 µL (i.e., 20 µg) of P. gingivalis lipopolysaccharide (LPS) intragingivally between the 1st and 2nd mandibular molars, two times a week for a total period of 6 weeks. The PD (LGG) intervention was done orally employing 2.5 × 105 CFU/day for a continuous period of 12 weeks. Immediately before the mice were sacrificed, echocardiography of the heart was performed, and after sacrifice, we collected serum samples, hearts, and the periodontal tissue. Histological assessment, cytokine analysis, and zymography of the cardiac tissue were performed. Results revealed inflammation of the heart muscle in the PD group that was marked by infiltration of neutrophils and monocytes, followed by fibrosis. Cytokine analysis of the mice sera revealed significantly elevated levels of tumor necrosis factor-α, IL-1ß, IL-6, and IL-17A in the PD group along with LPS-binding protein, and CD14. Most importantly, we observed elevated levels of P. gingivalis mRNAs in the heart tissues of PD mice. Zymographic analysis demonstrated matrix remodeling as revealed by increasing content of MMP-9 in the heart tissues of PD mice. Interestingly, LGG treatment was able to mitigate most of the pathological effects. The findings suggest that P. gingivalis could lead to cardiovascular system disorder and that probiotic intervention could alleviate, and most likely prevent bacteremia and its harmful effect(s) on the cardiovascular function.

Exercise mitigates calpain induced Purkinje cell loss in diabetes.

Calpain-1 is a ubiquitous calcium dependent cysteine protease and found in cytoplasm as well as mitochondria. We have earlier reported that active calpain-1 is translocated from cytosol to mitochondria and activates MMP9. Calpain-1 activation is detrimental to the heart in several different ways, but there is little evidence that it can degrade Purkinje cell protein (PCP-4) and impair contractility in diabetes. Our hypothesis is that in diabetes, PCP-4 is degraded by calpain-1, causing contractile dysfunction that can be mitigated by exercise. To test this hypothesis, we recruited four groups of mice, 1) db/+ control, 2) db/+ with exercise, 3) db/db, 4) db/db with exercise. The mice were exercised on treadmill for 8 weeks as per American Veterinary Research Guidelines. Adding calcium to isolated cardiomyocytes caused them to lose shape and die. Compared with live myocytes, we observed high calpain-1 levels as well as significantly lower levels of PCP-4 and increased levels of calmodulin and calmodulin kinase II (CaMKII) in dead myocytes. We used the CRISPR/Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats) plasmid to knock down calpain-1 in HL-1 myocytes which restored the levels of PCP-4 along with calmodulin and CaMKII. In vivo, we found upregulated levels of calpain-1 in db/db mice (diabetic) as compared to db/+ which were mitigated in the exercised mice. Conclusively our data strongly suggests that in diabetes there is high induction of calpain-1 with degrades PCP-4, a protein important for contractility and exercise can mitigate this.

Hydrogen sulfide mitigates skeletal muscle mitophagy-led tissue remodeling via epigenetic regulation of the gene writer and eraser function.

Ketone bodies (KB) serve as the food for mitochondrial biogenetics. Interestingly, probiotics are known to promote KB formation in the gut (especially those that belong to the Lactobacillus genus). Furthermore, Lactobacillus helps produce folate that lowers the levels of homocysteine (Hcy); a hallmark non-proteinogenic amino acid that defines the importance of epigenetics, and its landscape. In this study, we decided to test whether hydrogen sulfide (H2 S), another Hcy lowering agent regulates the epigenetic gene writer DNA methyltransferase (DNMT), eraser FTO and TET2, and thus mitigates the skeletal muscle remodeling. We treated hyperhomocysteinemic (HHcy, cystathionine beta-synthase heterozygote knockout; CBS+/- ) mice with NaHS (the H2 S donor). The results suggested multi-organ damage by HHcy in the CBS+/- mouse strain compared with WT control mice (CBS+/+ ). H2 S treatment abrogated most of the HHcy-induced damage. The levels of gene writer (DNMT2) and H3K9 (methylation) were higher in the CBS+/- mice, and the H2 S treatment normalized their levels. More importantly, the levels of eraser FTO, TET, and associated GADD45, and MMP-13 were decreased in the CBS+/- mice; however, H2 S treatment mitigated their respective decrease. These events were associated with mitochondrial fission, i.e., an increase in DRP1, and mitophagy. Although the MMP-2 level was lower in CBS+/- compared to WT but H2 S could further lower it in the CBS+/- mice. The MMPs levels were associated with an increase in interstitial fibrosis in the CBS+/- skeletal muscle. Due to fibrosis, the femoral artery blood flow was reduced in the CBS+/- mice, and that was normalized by H2 S. The bone and muscle strengths were found to be decreased in the CBS+/- mice but the H2 S treatment normalized skeletal muscle strength in the CBS+/- mice. Our findings suggest that H2 S mitigates the mitophagy-led skeletal muscle remodeling via epigenetic regulation of the gene writer and eraser function.

COVID-19 Mimics Pulmonary Dysfunction in Muscular Dystrophy as a Post-Acute Syndrome in Patients.

Although progressive wasting and weakness of respiratory muscles are the prominent hallmarks of Duchenne muscular dystrophy (DMD) and long-COVID (also referred as the post-acute sequelae of COVID-19 syndrome); however, the underlying mechanism(s) leading to respiratory failure in both conditions remain unclear. We put together the latest relevant literature to further understand the plausible mechanism(s) behind diaphragm malfunctioning in COVID-19 and DMD conditions. Previously, we have shown the role of matrix metalloproteinase-9 (MMP9) in skeletal muscle fibrosis via a substantial increase in the levels of tumor necrosis factor-α (TNF-α) employing a DMD mouse model that was crossed-bred with MMP9-knockout (MMP9-KO or MMP9-/-) strain. Interestingly, recent observations from clinical studies show a robust increase in neopterin (NPT) levels during COVID-19 which is often observed in patients having DMD. What seems to be common in both (DMD and COVID-19) is the involvement of neopterin (NPT). We know that NPT is generated by activated white blood cells (WBCs) especially the M1 macrophages in response to inducible nitric oxide synthase (iNOS), tetrahydrobiopterin (BH4), and tetrahydrofolate (FH4) pathways, i.e., folate one-carbon metabolism (FOCM) in conjunction with epigenetics underpinning as an immune surveillance protection. Studies from our laboratory, and others researching DMD and the genetically engineered humanized (hACE2) mice that were administered with the spike protein (SP) of SARS-CoV-2 revealed an increase in the levels of NPT, TNF-α, HDAC, IL-1ß, CD147, and MMP9 in the lung tissue of the animals that were subsequently accompanied by fibrosis of the diaphragm depicting a decreased oscillation phenotype. Therefore, it is of interest to understand how regulatory processes such as epigenetics involvement affect DNMT, HDAC, MTHFS, and iNOS that help generate NPT in the long-COVID patients.

Hyperhomocysteinemia: an instigating factor for periodontal disease.

Hyperhomocysteinemia (HHcy) affects bone remodeling, since a destructive process in cortical alveolar bone has been linked to it; however, the mechanism remains at large. HHcy increases proinflammatory cytokines viz. TNF-α, IL-1b, IL-6, and IL-8 that leads to a cascade that negatively impacts methionine metabolism and homocysteine cycling. Further, chronic inflammation decreases vitamins B12, B6, and folic acid that are required for methionine homocysteine homeostasis. This study aims to investigate a HHcy mouse model (cystathionine ß-synthase deficient, CBS+/-) for studying the potential pathophysiological changes, if any, in the periodontium (gingiva, periodontal ligament, cement, and alveolar bone). We compared the periodontium side-by-side in the CBS+/- model with that of the wild-type (C57BL/6J) mice. Histology and histomorphometry of the mandibular bone along with gene expression analyses were carried out. Also, proangiogenic proteins and metalloproteinases were studied. To our knowledge, this research shows, for the first time, a direct connection between periodontal disease during CBS deficiency, thereby suggesting the existence of disease drivers during the hyperhomocysteinemic condition. Our findings offer opportunities to develop diagnostics/therapeutics for people who suffer from chronic metabolic disorders like HHcy.

Regulation of the parental gene GRM4 by circGrm4 RNA transcript and glutamate-mediated neurovascular toxicity in eyes.

Epigenetic memory plays crucial roles in gene regulation. It not only modulates the expression of specific genes but also has ripple effects on transcription as well as translation of other genes. Very often an alteration in expression occurs either via methylation or demethylation. In this context, "1-carbon metabolism" assumes a special significance since its dysregulation by higher levels of homocysteine; Hcy (known as hyperhomocysteinemia; HHcy), a byproduct of "1-Carbon Metabolism" during methionine biosynthesis leads to serious implications in cardiovascular, renal, cerebrovascular systems, and a host of other conditions. Currently, the circular RNAs (circRNAs) generated via non-canonical back-splicing events from the pre-mRNA molecules are at the center stage for their essential roles in diseases via their epigenetic manifestations. We recently identified a circular RNA transcript (circGRM4) that is significantly upregulated in the eye of cystathionine ß-synthase-deficient mice. We also discovered a concurrent over-expression of the mGLUR4 receptor in the eyes of these mice. In brief, circGRM4 is selectively transcribed from its parental mGLUR4 receptor gene (GRM4) functions as a "molecular-sponge" for the miRNAs and results into excessive turnover of the mGLUR4 receptor in the eye in response to extremely high circulating glutamate concentration. We opine that this epigenetic manifestation potentially predisposes HHcy people to retinovascular malfunctioning.

High-methionine diet in skeletal muscle remodeling: epigenetic mechanism of homocysteine-mediated growth retardation.

Epigenetic DNA methylation (1-carbon metabolism) is crucial for gene imprinting/off-printing that ensures epigenetic memory but also generates a copious amount of homocysteine (Hcy), unequivocally. That is why during pregnancy, expectant mothers are recommended "folic acid" preemptively to avoid birth defects in the young ones because of elevated Hcy levels (i.e., hyperhomocysteinemia (HHcy)). As we know, children born with HHcy have several musculoskeletal abnormalities, including growth retardation. Here, we focus on the gut-dysbiotic microbiome implication(s) that we believe instigates the "1-carbon metabolism" and HHcy causing growth retardation along with skeletal muscle abnormalities. We test our hypothesis whether high-methionine diet (HMD) (an amino acid that is high in red meat), a substrate for Hcy, can cause skeletal muscle and growth retardation, and treatment with probiotics (PB) to mitigate skeletal muscle dysfunction. To test this, we employed cystathionine ß-synthase, CBS deficient mouse (CBS+/-) fed with/without HMD and with/without a probiotic (Lactobacillus rhamnosus) in drinking water for 16 weeks. Matrix metalloproteinase (MMP) activity, a hallmark of remodeling, was measured by zymography. Muscle functions were scored via electric stimulation. Our results suggest that compared to the wild-type, CBS+/- mice exhibited reduced growth phenotype. MMP-2 activity was robust in CBS+/- and HMD effects were successfully attenuated by PB intervention. Electrical stimulation magnitude was decreased in CBS+/- and CBS+/- treated with HMD. Interestingly; PB mitigated skeletal muscle growth retardation and atrophy. Collectively, results imply that individuals with mild/moderate HHcy seem more prone to skeletal muscle injury and its dysfunction.

Hidradenitis Suppurativa and 1-Carbon Metabolism: Role of Gut Microbiome, Matrix Metalloproteinases, and Hyperhomocysteinemia.

Hidradenitis suppurativa (HS) is a chronic, inflammatory skin condition characterized by painful nodules which suppurate and later develop into scar tissues followed by the development of hypodermal tracts. Although the mechanisms behind HS are not fully understood, it is known that dietary factors play important roles in flare frequency and severity. We hypothesize that the high fat diet (HFD) causes dysbiosis, systemic inflammation, and hyperhomocysteinemia (HHcy) in susceptible individuals, which subsequently elevate inflammatory cytokines such as IL-1ß, IL-6, IL-17, and tumor necrosis factor alpha (TNF-α). This increase in dysbiosis-led inflammation coupled with a dysregulation of the 1-carbon metabolism results in an increase in matrix metalloproteinases MMP-2, MMP-8, and MMP-9 along with tissue matrix remodeling in the development and maintenance of the lesions and tracts. This manuscript weaves together the potential roles played by the gut microbiome, HHcy, MMPs, and the 1-carbon metabolism toward HS disease causation in susceptible individuals.

Dysbiotic 1-carbon metabolism in cardiac muscle remodeling.

Unless there is a genetic defect/mutation/deletion in a gene, the causation of a given disease is chronic dysregulation of gut metabolism. Most of the time, if not always, starts within the gut; that is what we eat. Recent research shows that the imbalance between good versus bad microbial population, especially in the gut, causes systemic diseases. Thus, an appropriate balance of the gut microbiota (eubiosis over dysbiosis) needs to be maintained for normal health (Veeranki and Tyagi, 2017, Journal of Cellular Physiology, 232, 2929-2930). However, during various diseases such as metabolic syndrome, inflammatory bowel disease, diabetes, obesity, and hypertension the dysbiotic gut environment tends to prevail. Our research focuses on homocysteine (Hcy) metabolism that occupies a center-stage in many biochemically relevant epigenetic mechanisms. For example, dysbiotic bacteria methylate promoters to inhibit gene activities. Interestingly, the product of the 1-carbon metabolism is Hcy, unequivocally. Emerging studies show that host resistance to various antibiotics occurs due to inverton promoter inhibition, presumably because of promoter methylation. This results from modification of host promoters by bacterial products leading to loss of host's ability to drug compatibility and system sensitivity. In this study, we focus on the role of high methionine diet (HMD), an ingredient rich in red meat and measure the effects of a probiotic on cardiac muscle remodeling and its functions. We employed wild type (WT) and cystathionine beta-synthase heterozygote knockout (CBS+/- ) mice with and without HMD and with and without a probiotic; PB (Lactobacillus) in drinking water for 16 weeks. Results indicate that matrix metalloproteinase-2 (MMP-2) activity was robust in CBS+/- fed with HMD and that it was successfully attenuated by the PB treatment. Cardiomyocyte contractility and ECHO data revealed mitigation of the cardiac dysfunction in CBS+/- + HMD mice treated with PB. In conclusion, our data suggest that probiotics can potentially reverse the Hcy-meditated cardiac dysfunction.

Genes and genetics in hyperhomocysteinemia and the "1-carbon metabolism": implications for retinal structure and eye functions.

Homocysteine (Hcy), a sulfur-containing nonproteinogenic amino acid, is generated as a metabolic intermediate. Hcy constitutes an important part of the "1-carbon metabolism" during methionine turnover. Elevated levels of Hcy known as hyperhomocysteinemia (HHcy) results from vitamin B deficiency, lack of exercise, smoking, excessive alcohol intake, high-fat and methionine-rich diet, and the underlying genetic defects. These factors directly affect the "1-carbon metabolism (methionine-Hcy-folate)" of a given cell. In fact, the Hcy levels are determined primarily by dietary intake, vitamin status, and the genetic blueprint of the susceptible individual. Although Hcy performs an important role in cellular functions, genetic alterations in any of the key enzymes responsible for the "1-carbon metabolism" could potentially upset the metabolic cycle, thus causing HHcy environment in susceptible people. As such, HHcy relates to several clinical conditions like atherosclerosis, myocardial infarction, stroke, cognitive impairment, dementia, Parkinson's disease, multiple sclerosis, epilepsy, and ocular disorders, among others. This article summarizes the findings from our laboratory and public database regarding genetics of HHcy and its effects on ocular disorders, their respective management during dysregulation of the 1-carbon metabolism.

The cardioprotective effects of diallyl trisulfide on diabetic rats with ex vivo induced ischemia/reperfusion injury.

Diallyl trisulfide (DATS) is distinguished as the most potent polysulfide isolated from garlic. The aim of our study was to investigate effects of oral administration of DATS on healthy and diabetic rats, with special attention on heart function. Rats were randomly divided into four groups: CTRL (healthy rats), DATS (healthy rats treated with DATS), DM (diabetic rats), DM + DATS (diabetic rats treated with DATS). DATS (40 mg/kg of body weight) was administered every other day for 3 weeks, at the end of which rats underwent echocardiography, glycemic measurement and redox status assessment. Isolated rat hearts were subjected to 30 min global ischemia and 60 min reperfusion, after which heart tissue was counterstain with hematoxylin and eosin and cardiac Troponin T staining (cTnT), while expression of Bax, B cell lymphoma 2 (Bcl-2), caspase-3, caspase-9 and superoxide dismutase-2 were examined in the left ventricle. DATS treatment significantly reduced blood glucose levels of diabetic rats, and improved cardiac function recovery, diminished oxidation status, attenuated cardiac remodeling and inhibited myocardial apoptosis in healthy and diabetic rats. DATS treatment causes promising cardioprotective effects on ex vivo-induced ischemia/reperfusion (I/R) injury in diabetic and healthy rat heart probably mediated by inhibited myocardial apoptosis. Moreover, appropriate DATS consumption may provide potential co-therapy or prevention of hyperglycemia and various cardiac complications in rats with DM.

Dysregulation of 1-carbon metabolism and muscle atrophy: potential roles of forkhead box O proteins and PPARγ co-activator-1α.

Homocysteine, a non-proteinogenic amino acid but an important metabolic intermediate is generated as an integral component for the "1-carbon metabolism" during normal physiology. It is catabolized to cysteine via the transulfuration pathway resulting in the generation of hydrogen sulfide, a naturally endogenous byproduct. Genetics or metabolic derangement can alter homocysteine concentration leading to hyperhomocysteinemia (HHcy), a physiologically unfavorable condition that causes serious medical conditions including muscle wasting. HHcy environment can derail physiological processes by targeting biomolecules such as Akt; however, not much is known regarding the effects of HHcy on regulation of transcription factors such as forkhead box O (FOXO) proteins. Recently, hydrogen sulfide has been shown to be highly effective in alleviating the effects of HHcy by serving as an antiapoptotic factor, but role of FOXO and its interaction with hydrogen sulfide are yet to be established. In this review, we discuss role(s) of HHcy in skeletal muscle atrophy and how HHcy interact with FOXO and peroxisome proliferator-activated receptor gamma coactivator 1-alpha expressions that are relevant in musculoskeletal atrophy. Further, therapeutic intervention with hydrogen sulfide for harnessing its beneficial effects might help mitigate the dysregulated 1-carbon metabolism that happens to be the hallmark of HHcy-induced pathologies such as muscle atrophy.

Hydrogen sulfide intervention in cystathionine-ß-synthase mutant mouse helps restore ocular homeostasis.

AIM: To investigate the applications of hydrogen sulfide (H2S) in eye-specific ailments in mice. METHODS: Heterozygous cystathionine-ß-synthase (CBS+/-) and wild-type C57BL/6J (WT) mice fed with or without high methionine diet (HMD) were administered either phosphate buffered saline (PBS) or the slow-release H2S donor: GYY4137. Several analyses were performed to study GYY4137 effects by examining retinal lysates for key protein expressions along with plasma glutamate and glutathione estimations. Intraocular pressure (IOP) was monitored during GYY4137 treatment; barium sulfate and bovine serum albumin conjugated fluorescein isothiocyanate (BSA-FITC) angiographies were performed for examining vasculature and its permeability post-treatment. Vision-guided behavior was also tested employing novel object recognition test (NORT) and light-dark box test (LDBT) recordings. RESULTS: CBS deficiency (CBS+/-) coupled with HMD led disruption of methionine/homocysteine (Hcy) metabolism leading to hyperhomocysteinemia (HHcy) in CBS+/- mice as reflected by increased Hcy, and s-adenosylhomocysteine hydrolase (SAHH) levels. Unlike CBS, cystathionine-γ lyase (CSE), methylenetetrahydrofolate reductase (MTHFR) levels which were reduced but compensated by GYY4137 intervention. Heightened oxidative and endoplasmic reticulum (ER) stress responses were mitigated by GYY4137 effects along with enhanced glutathione (GSH) levels. Increased glutamate levels in CBS+/- strain were prominent than WT mice and these mice also exhibited higher IOP that was lowered by GYY4137 treatment. CBS deficiency also resulted in vision-guided behavioral impairment as revealed by NORT and LDBT findings. Interestingly, GYY4137 was able to improve CBS+/- mice behavior together with lowering their glutamate levels. Blood-retinal barrier (BRB) appeared compromised in CBS+/- with vessels' leakage that was mitigated in GYY4137 treated group. This corroborated the results for occludin (an integral plasma membrane protein of the cellular tight junctions) stabilization. CONCLUSION: Findings reveal that HHcy-induced glutamate excitotoxicity, oxidative damage, ER-stress and vascular permeability alone or together can compromise ocular health and that GYY4137 could serve as a potential therapeutic agent for treating HHcy induced ocular disorders.

Role of hydrogen sulfide in the musculoskeletal system.

Hydrogen sulfide (H2S) has been known as a gasotransmitter, and it contributes to various physiological and pathological processes. Multiple enzymes such as cystathionine-ß-synthase (CBS), cystathionine-γ-lyase (CSE) and 3-Mercaptopyruvate sulfurtransferase (MST) produce endogenous H2S, and these are differentially expressed in the various tissue systems including the skeletal system. However, abnormal H2S production is associated with deregulation of the signaling cascade and imbalanced tissue homeostasis. Several studies have previously provided evidence showing the essential regulatory action of H2S in skeletal homeostasis. In this review, we have emphasized the novel function of H2S in both bone and skeletal muscle anabolism, in particular. Additionally, we also reviewed the molecular and epigenetic basis of H2S signaling in bone development and skeletal muscle function.

Hydrogen sulfide attenuates homocysteine-induced osteoblast dysfunction by inhibiting mitochondrial toxicity.

Homocysteine (Hcy) is detrimental to bone health in a mouse model of diet-induced hyperhomocysteinemia (HHcy). However, little is known about Hcy-mediated osteoblast dysfunction via mitochondrial oxidative damage. Hydrogen sulfide (H2 S) has potent antioxidant, anti-inflammatory, and antiapoptotic effects. In this study, we hypothesized that the H2 S mediated recovery of osteoblast dysfunction by maintaining mitochondrial biogenesis in Hcy-treated osteoblast cultures in vitro. MC3T3-E1 osteoblastic cells were exposed to Hcy treatment in the presence or absence of an H2 S donor (NaHS). Cell viability, osteogenic differentiation, reactive oxygen species (ROS) production were determined. Mitochondrial DNA copy number, adenosine triphosphate (ATP) production, and oxygen consumption were also measured. Our results demonstrated that administration of Hcy increases the intracellular Hcy level and decreases intracellular H2 S level and expression of the cystathionine ß-synthase/Cystathionine γ-lyase system, thereby inhibiting osteogenic differentiation. Pretreatment with NaHS attenuated Hcy-induced mitochondrial toxicity (production of total ROS and mito-ROS, ratio of mitochondrial fission (DRP-1)/fusion (Mfn-2)) and restored ATP production and mitochondrial DNA copy numbers as well as oxygen consumption in the osteoblast as compared with the control, indicating its protective effects against Hcy-induced mitochondrial toxicity. In addition, NaHS also decreased the release of cytochrome c from the mitochondria to the cytosol, which induces cell apoptosis. Finally, flow cytometry confirmed that NaHS can rescue cells from apoptosis induced by Hcy. Our studies strongly suggest that NaHS has beneficial effects on mitochondrial toxicity, and could be developed as a potential therapeutic agent against HHcy-induced mitochondrial dysfunction in cultured osteoblasts in vitro.

Hyperhomocysteinemia induced endothelial progenitor cells dysfunction through hyper-methylation of CBS promoter.

Bone marrow (BM)-derived endothelial progenitor cells (EPCs) are the key players in angiogenesis and vascular function. Cystathionine-ß-synthase (CBS), an H2S-generating enzyme in methionine metabolism, regulates the function of these EPCs. This study aims to examine whether CBS hyper-methylation contributes to the bone marrow endothelial progenitor cell (BM-EPCs) function and subsequent bone blood flow in mice fed with a high methionine diet (HMD). Bone marrow (BM) cells were collected from HMD and control mice, differentiated into BM-EPCs, and were characterized by acLDL-DiI labeling. CBS mRNA expression was analyzed by real-time PCR, and the global methylation status and methylation of the CBS promoter were detected by nuclear 5-mC assay and methylation-specific PCR (qMSP) respectively. The result reveals that CBS promoter in BM-EPCs from HMD mice was hyper-methylated and the methylation level was, indeed, negatively correlated with CBS mRNA and angiogenic function of BM-EPCs. In addition, global methylation (5-mC) and DNA methyltransferase-1 (DNMT1) expression were increased in HMD condition. In vitro study also shows that HMD induced hyperhomocysteinemia (HHcy) impaired both adhesion and angiogenesis properties of BM-EPCs, accompanied by higher methylation level of CBS promoter that compared to control. Furthermore, bone blood flow was found to be decreased in HMD mice as compared to wild-type mice. To dissect the epigenetic mechanism, we also administrated DNMT inhibitor, 5-azacytidine (5-Aza) to HMD mice. The administration of 5-Aza in HMD mice restored the CBS expression, EPC mediated angiogenesis and blood flow by reducing abnormal DNA hyper-methylation. In conclusion, HHcy dismantles BM-EPCs function and bone blood flow through the hyper-methylation of the CBS promoter in HMD fed mice.

Remote ischemic conditioning as a cytoprotective strategy in vasculopathies during hyperhomocysteinemia: An emerging research perspective.

Higher levels of nonprotein amino acid homocysteine (Hcy), that is, hyperhomocysteinemia (HHcy) (~5% of general population) has been associated with severe vasculopathies in different organs; however, precise molecular mechanism(s) as to how HHcy plays havoc with body's vascular networks are largely unknown. Interventional modalities have not proven beneficial to counter multifactorial HHcy's effects on the vascular system. An ancient Indian form of exercise called 'yoga' causes transient ischemia as a result of various body postures however the cellular mechanisms are not clear. We discuss a novel perspective wherein we argue that application of remote ischemic conditioning (RIC) could, in fact, deliver anticipated results to patients who are suffering from chronic vascular dysfunction due to HHcy. RIC is the mechanistic phenomenon whereby brief episodes of ischemia-reperfusion events are applied to distant tissues/organs; that could potentially offer a powerful tool in mitigating chronic lethal ischemia in target organs during HHcy condition via simultaneous reduction of inflammation, oxidative and endoplasmic reticulum stress, extracellular matrix remodeling, fibrosis, and angiogenesis. We opine that during ischemic conditioning our organs cross talk by releasing cellular messengers in the form of exosomes containing messenger RNAs, circular RNAs, anti-pyroptotic factors, protective cytokines like musclin, transcription factors, small molecules, anti-inflammatory, antiapoptotic factors, antioxidants, and vasoactive gases. All these could help mobilize the bone marrow-derived stem cells (having tissue healing properties) to target organs. In that context, we argue that RIC could certainly play a savior's role in an unfortunate ischemic or adverse event in people who have higher levels of the circulating Hcy in their systems.

Restoration of skeletal muscle homeostasis by hydrogen sulfide during hyperhomocysteinemia-mediated oxidative/ER stress condition 1.

Elevated homocysteine (Hcy), i.e., hyperhomocysteinemia (HHcy), causes skeletal muscle myopathy. Among many cellular and metabolic alterations caused by HHcy, oxidative and endoplasmic reticulum (ER) stress are considered the major ones; however, the precise molecular mechanism(s) in this process is unclear. Nevertheless, there is no treatment option available to treat HHcy-mediated muscle injury. Hydrogen sulfide (H2S) is increasingly recognized as a potent anti-oxidant, anti-apoptotic/necrotic/pyroptotic, and anti-inflammatory compound and also has been shown to improve angiogenesis during ischemic injury. Patients with CBS mutation produce less H2S, making them vulnerable to Hcy-mediated cellular damage. Many studies have reported bidirectional regulation of ER stress in apoptosis through JNK activation and concomitant attenuation of cell proliferation and protein synthesis via PI3K/AKT axis. Whether H2S mitigates these detrimental effects of HHcy on muscle remains unexplored. In this review, we discuss molecular mechanisms of HHcy-mediated oxidative/ER stress responses, apoptosis, angiogenesis, and atrophic changes in skeletal muscle and how H2S can restore skeletal muscle homeostasis during HHcy condition. This review also highlights the molecular mechanisms on how H2S could be developed as a clinically relevant therapeutic option for chronic conditions that are aggravated by HHcy.