Sudhakar Veeranki

Defective homocysteine metabolism: potential implications for skeletal muscle malfunction.

Hyperhomocysteinemia (HHcy) is a systemic medical condition and has been attributed to multi-organ pathologies. Genetic, nutritional, hormonal, age and gender differences are involved in abnormal homocysteine (Hcy) metabolism that produces HHcy. Homocysteine is an intermediate for many key processes such as cellular methylation and cellular antioxidant potential and imbalances in Hcy production and/or catabolism impacts gene expression and cell signaling including GPCR signaling. Furthermore, HHcy might damage the vagus nerve and superior cervical ganglion and affects various GPCR functions; therefore it can impair both the parasympathetic and sympathetic regulation in the blood vessels of skeletal muscle and affect long-term muscle function. Understanding cellular targets of Hcy during HHcy in different contexts and its role either as a primary risk factor or as an aggravator of certain disease conditions would provide better interventions. In this review we have provided recent Hcy mediated mechanistic insights into different diseases and presented potential implications in the context of reduced muscle function and integrity. Overall, the impact of HHcy in various skeletal muscle malfunctions is underappreciated; future studies in this area will provide deeper insights and improve our understanding of the association between HHcy and diminished physical function.

Dysregulation of Mfn2 and Drp-1 proteins in heart failure

Therapeutic approaches for cardiac regenerative mechanisms have been explored over the past decade to target various cardiovascular diseases (CVD). Structural and functional aberrations of mitochondria have been observed in CVD. The significance of mitochondrial maturation and function in cardiomyocytes is distinguished by their attribution to embryonic stem cell differentiation into adult cardiomyocytes. An abnormal fission process has been implicated in heart failure, and treatment with mitochondrial division inhibitor 1 (Mdivi-1), a specific inhibitor of dynamin related protein-1 (Drp-1), has been shown to improve cardiac function. We recently observed that the ratio of mitofusin 2 (Mfn2; a fusion protein) and Drp-1 (a fission protein) was decreased during heart failure, suggesting increased mitophagy. Treatment with Mdivi-1 improved cardiac function by normalizing this ratio. Aberrant mitophagy and enhanced oxidative stress in the mitochondria contribute to abnormal activation of MMP-9, leading to degradation of the important gap junction protein connexin-43 (Cx-43) in the ventricular myocardium. Reduced Cx-43 levels were associated with increased fibrosis and ventricular dysfunction in heart failure. Treatment with Mdivi-1 restored MMP-9 and Cx-43 expression towards normal. In this review, we discuss mitochondrial dynamics, its relation to MMP-9 and Cx-43, and the therapeutic role of fission inhibition in heart failure.

Hyperhomocysteinemia associated skeletal muscle weakness involves mitochondrial dysfunction and epigenetic modifications

HHcy has been implicated in elderly frailty, but the underlying mechanisms are poorly understood. Using C57 and CBS+/- mice and C2C12 cell line, we investigated mechanisms behind HHcy induced skeletal muscle weakness and fatigability. Possible alterations in metabolic capacity (levels of LDH, CS, MM-CK and COX-IV), in structural proteins (levels of dystrophin) and in mitochondrial function (ATP production) were examined. An exercise regimen was employed to reverse HHcy induced changes. CBS+/- mice exhibited more fatigability, and generated less contraction force. No significant changes in muscle morphology were observed. However, there is a corresponding reduction in large muscle fiber number in CBS+/- mice. Excess fatigability was not due to changes in key enzymes involved in metabolism, but was due to reduced ATP levels. A marginal reduction in dystrophin levels along with a decrease in mitochondrial transcription factor A (mtTFA) were observed. There was also an increase in the mir-31, and mir-494 quantities that were implicated in dystrophin and mtTFA regulation respectively. The molecular changes elevated during HHcy, with the exception of dystrophin levels, were reversed after exercise. In addition, the amount of NRF-1, one of the transcriptional regulators of mtTFA, was significantly decreased. Furthermore, there was enhancement in mir-494 levels and a concomitant decline in mtTFA protein quantity in homocysteine treated cells. These changes in C2C12 cells were also accompanied by an increase in DNMT3a and DNMT3b proteins and global DNA methylation levels. Together, these results suggest that HHcy plays a causal role in enhanced fatigability through mitochondrial dysfunction which involves epigenetic changes.

Moderate intensity exercise prevents diabetic cardiomyopathy associated contractile dysfunction through restoration of mitochondrial function and connexin 43 levels in db/db mice

AIMS Although the cardiovascular benefits of exercise are well known, exercise induced effects and mechanisms in prevention of cardiomyopathy are less clear during obesity associated type-2 diabetes. The current study assessed the impact of moderate intensity exercise on diabetic cardiomyopathy by examining cardiac function and structure and mitochondrial function. METHODS Obese-diabetic (db/db), and lean control (db/+) mice, were subjected to a 5 week, 300 m run on a tread-mill for 5 days/week at the speeds of 10-11 m/min. Various physiological parameters were recorded and the heart function was evaluated with M-mode echocardiography. Contraction parameters and calcium transits were examined on isolated cardiomyocytes. At the molecular level: connexin 43 and 37 (Cx43 and 37) levels, mitochondrial biogenesis regulators: Mfn2 and Drp-1 levels, mitochondrial trans-membrane potential and cytochrome c leakage were assessed through western blotting immunohistochemistry and flow cytometry. Ability of exercise to reverse oxygen consumption rate (OCR), tissue ATP levels, and cardiac fibrosis were also determined. RESULTS The exercise regimen was able to prevent diabetic cardiac functional deficiencies: ejection fraction (EF) and fractional shortening (FS). Improvements in contraction velocity and contraction maximum were noted with the isolated cardiomyocytes. Restoration of interstitial and micro-vessels associated Cx43 levels and improved gap junction intercellular communication (GJIC) were observed. The decline in the Mfn2/Drp-1 ratio in the db/db mice hearts was prevented after exercise. The exercise regimen further attenuated transmembrane potential decline and cytochrome c leakage. These corrections further led to improvements in OCR and tissue ATP levels and reduction in cardiac fibrosis. CONCLUSIONS Moderate intensity exercise produced significant cardiovascular benefits by improving mitochondrial function through restoration of Cx43 networks and mitochondrial trans-membrane potential and prevention of excessive mitochondrial fission.

Role of mitochondrial fission and fusion in cardiomyocyte contractility

BACKGROUND Mitochondria constitute 30% of cell volume and are engaged in two dynamic processes called fission and fusion, regulated by Drp-1 (dynamin related protein) and mitofusin 2 (Mfn2). Previously, we showed that Drp-1 inhibition attenuates cardiovascular dysfunction following pressure overload in aortic banding model and myocardial infarction. As dynamic organelles, mitochondria are capable of changing their morphology in response to stress. However, whether such changes can alter their function and in turn cellular function is unknown. Further, a direct role of fission and fusion in cardiomyocyte contractility has not yet been studied. In this study, we hypothesize that disrupted fission and fusion balance by increased Drp-1 and decreased Mfn2 expression in cardiomyocytes affects their contractility through alterations in the calcium and potassium concentrations. METHODS To verify this, we used freshly isolated ventricular myocytes from wild type mouse and transfected them with either siRNA to Drp-1 or Mfn2. Myocyte contractility studies were performed by IonOptix using a myopacer. Intracellular calcium and potassium measurements were done using flow cytometry. Immunocytochemistry (ICC) was done to evaluate live cell mitochondria and its membrane potential. Protein expression was done by western blot and immunocytochemistry. RESULTS We found that silencing mitochondrial fission increased the myocyte contractility, while fusion inhibition decreased contractility with simultaneous changes in calcium and potassium. Also, we observed that increase in fission prompted decrease in Serca-2a and increase in cytochrome c leakage leading to mitophagy. CONCLUSION Our results suggested that regulating mitochondrial fission and fusion have direct effects on overall cardiomyocyte contractility and thus function.

Role of hydrogen sulfide in skeletal muscle biology and metabolism

Hydrogen sulfide (H2S) is a novel endogenous gaseous signal transducer (gasotransmitter). Its emerging role in multiple facets of inter- and intra-cellular signaling as a metabolic, inflammatory, neuro and vascular modulator has been increasingly realized. Although H2S is known for its effects as an anti-hypertensive, anti-inflammatory and anti-oxidant molecule, the relevance of these effects in skeletal muscle biology during health and during metabolic syndromes is unclear. H2S has been implicated in vascular relaxation and vessel tone enhancement, which might lead to mitigation of vascular complications caused by the metabolic syndromes. Metabolic complications may also lead to mitochondrial remodeling by interfering with fusion and fission, therefore, leading to mitochondrial mitophagy and skeletal muscle myopathy. Mitochondrial protection by H2S enhancing treatments may mitigate deterioration of muscle function during metabolic syndromes. In addition, H2S might upregulate uncoupling proteins and might also cause browning of white fat, resulting in suppression of imbalanced cytokine signaling caused by abnormal fat accumulation. Likewise, as a source for H(+) ions, it has the potential to augment anaerobic ATP synthesis. However, there is a need for studies to test these putative H2S benefits in different patho-physiological scenarios before its full-fledged usage as a therapeutic molecule. The present review highlights current knowledge with regard to exogenous and endogenous H2S roles in skeletal muscle biology, metabolism, exercise physiology and related metabolic disorders, such as diabetes and obesity, and also provides future directions.

Atherogenesis: hyperhomocysteinemia interactions with LDL, macrophage function, paraoxonase 1, and exercise

Despite great strides in understanding the atherogenesis process, the mechanisms are not entirely known. In addition to diet, cigarette smoking, genetic predisposition, and hypertension, hyperhomocysteinemia (HHcy), an accumulation of the noncoding sulfur‐containing amino acid homocysteine (Hcy), is a significant contributor to atherogenesis. Although exercise decreases HHcy and increases longevity, the complete mechanism is unclear. In light of recent evidence, in this review, we focus on the effects of HHcy on macrophage function, differentiation, and polarization. Though there is need for further evidence, it is most likely that HHcy‐mediated alterations in macrophage function are important contributors to atherogenesis, and HHcy‐countering strategies, such as nutrition and exercise, should be included in the combinatorial regimens for effective prevention and regression of atherosclerotic plaques. Therefore, we also included a discussion on the effects of exercise on the HHcy‐mediated atherogenic process.

Hyperhomocysteinemia attenuates angiogenesis through reduction of HIF-1α and PGC-1α levels in muscle fibers during hindlimb ischemia

Hyperhomocysteinemia (HHcy) is associated with elderly frailty, skeletal muscle injury and malfunction, reduced vascular integrity and function, and mortality. Although HHcy has been implicated in the impairment of angiogenesis after hindlimb ischemia in murine models, the underlying mechanisms are still unclear. We hypothesized that HHcy compromises skeletal muscle perfusion, collateral formation, and arteriogenesis by diminishing postischemic vasculogenic responses in muscle fibers. To test this hypothesis, we created femoral artery ligation in wild-type and heterozygous cystathionine β-synthase (CBS(+/-)) mice (a model for HHcy) and assessed tissue perfusion, collateral vessel formation, and skeletal muscle function using laser-Doppler perfusion imaging, barium angiography, and fatigue tests. In addition, we assessed postischemic levels of VEGF and levels of its muscle-specific regulators: hypoxia-inducible factor (HIF)-1α and peroxisome proliferator-activated receptor-γ coactivator (PGC)-1α. The observations indicated dysregulation of VEGF, HIF-1α, and PGC-1α levels in ischemic skeletal muscles of CBS(+/-) mice. Concomitant with the reduced ischemic angiogenic responses, we also observed diminished leptin expression and attenuated Akt signaling in ischemic muscle fibers of CBS(+/-) mice. Moreover, there was enhanced atrogene, ubiquitin ligases that conjugate proteins for degradation during muscle atrophy, transcription, and reduced muscle function after ischemia in CBS(+/-) mice. These results suggest that HHcy adversely affects muscle-specific ischemic responses and contributes to muscle frailty.

Hyperhomocysteinemia inhibits satellite cell regenerative capacity through p38 alpha/beta MAPK signaling

Chronic failure in maintenance and regeneration of skeletal muscles leads to lower muscle mass (sarcopenia), muscle weakness, and poor response to injury. Evidence suggests that aberrant p38 MAPK signaling undermines the repair process after injury in aged mice. Previous studies have shown that hyperhomocysteinemia (HHcy) has been associated with muscle weakness and lower than normal body weights. However, whether or not HHcy condition also compromises skeletal muscle regenerative capabilities is not clear. In the current study, we show that CBS-/+ mice, a model for HHcy condition, exhibited compromised regenerative function and cell proliferation upon injury. However, there was no significant difference in Pax7 expression levels in the satellite cells from CBS-/+ mouse skeletal muscles. Interestingly, the satellite cells from CBS-/+ mice not only exhibited diminished in vitro proliferative capabilities, but also there was heightened oxidative stress. In addition, there was enhanced p38 MAPK activation as well as p16 and p21 expression in the CBS-/+ mouse satellite cells. Moreover, the C2C12 myoblasts also exhibited higher p38 MAPK activation and p16 expression upon treatment with homocysteine in addition to enhanced ROS presence. Tissue engraftment potential and regeneration after injury were restored to some extent upon treatment with the p38-MAPK inhibitor, SB203580, in the CBS-/+ mice. These results together suggest that HHcy-induced diminished satellite cell proliferation involves excessive oxidative stress and p38 MAPK signaling. Our study further proposes that HHcy is a potential risk factor for elderly frailty, and need to be considered as a therapeutic target while designing the alleviation interventions/postinjury rehabilitation measures for adults with HHcy.

Exercise mitigates the adverse effects of hyperhomocysteinemia on macrophages, MMP-9, skeletal muscle, and white adipocytes.

Regular exercise is a great medicine with its benefits encompassing everything from prevention of cardiovascular risk to alleviation of different muscular myopathies. Interestingly, elevated levels of homocysteine (Hcy), also known as hyperhomocysteinemia (HHcy), antagonizes beta-2 adrenergic receptors (β2AR), gamma amino butyric acid (GABA), and peroxisome proliferator-activated receptor-gamma (PPARγ) receptors. HHcy also stimulates an elevation of the M1/M2 macrophage ratio, resulting in a more inflammatory profile. In this review we discuss several potential targets altered by HHcy that result in myopathy and excessive fat accumulation. Several of these HHcy mediated changes can be countered by exercise and culminate into mitigation of HHcy induced myopathy and metabolic syndrome. We suggest that exercise directly impacts levels of Hcy, matrix metalloproteinase 9 (MMP-9), macrophages, and G-protein coupled receptors (GPCRs, especially Gs). While HHcy promotes the M1 macrophage phenotype, it appears that exercise may diminish the M1/M2 ratio, resulting in a less inflammatory phenotype. HHcy through its influence on GPCRs, specifically β₂AR, PPARγ and GABA receptors, promotes accumulation of white fat, whereas exercise enhances the browning of white fat and counters HHcy-mediated effects on GPCRs. Alleviation of HHcy-associated pathologies with exercise also includes reversal of excessive MMP-9 activation. Moreover, exercise, by reducing plasma Hcy levels, may prevent skeletal muscle myopathy, improve exercise capacity and rescue the obese phenotype. The purpose of this review is to summarize the pathological conditions surrounding HHcy and to clarify the importance of regular exercise as a method of disease prevention.