Majonoside-R2 Postconditioning Protects Cardiomyocytes Against Hypoxia/Reoxygenation Injury by Attenuating the Expression of HIF1α and Activating RISK Pathway.

Reoxygenation of hypoxic cardiac myocytes can paradoxically induce myocardial injury and affect the recovery processes. Pharmacological postconditioning is an efficient strategy used in clinical practice that protects cardiomyocytes from hypoxia/reoxygenation (HR) injury. Natural products or foods have been known to possess effective cardioprotective properties. Majonoside-R2 (MR2) is a dominant saponin component of Vietnamese ginseng that has several biological effects. In this study, we evaluated the protective effect of MR2 on HR-stimulated cardiomyocytes and investigated the related molecular mechanisms. H9C2 cardiomyocytes were exposed to HR conditions with or without MR2 supplementation. Samples from experimental groups were used to analyze the expression of apoptosis- and activating reperfusion injury salvage kinase (RISK)-related factors in response to HR injury by using enzyme-linked immunosorbent assay, real-time polymerase chain reaction, and Western blotting. Post-treatment, MR2 enhanced cell viability under HR conditions. We found that MR2 suppressed the expression of hypoxia-inducible factor 1-alpha (HIF1α) and transforming growth factor beta 1 (TGFß1), modulated Akt/GSK3ß/cAMP response element-binding signaling, and regulated gene expression related to apoptosis (B cell lymphoma-extra-large [Bcl-xl], Bcl-2 homologous killer [Bak], Bcl-2 associated X [Bax], and connexin 43 [Cnx43]). Thus, the present findings demonstrate that MR2 protects cardiomyocytes against HR injury by suppressing the expression of HIF1α and activating the RISK pathway.

Majonoside-R2 extracted from Vietnamese ginseng protects H9C2 cells against hypoxia/reoxygenation injury via modulating mitochondrial function and biogenesis.

Vietnamese ginseng has a therapeutic effect on various diseases; however its bioactivity against cardiac hypoxia/reoxygenation (HR) injury remains unclear. In this study, we evaluated the protective roles of total saponin extract (TSE) and majonoside-R2 (MR2) targeting mitochondria in HR-induced rat cardiomyocyte H9C2 cells. The results showed that both TSE and MR2 effectively protected the cells from HR damage. Particularly, 9 µM of MR2 significantly increased the viability of HR-induced cells (p < 0.05). Interestingly, MR2 treatment markedly prevented the loss of mitochondrial membrane potential and cardiolipin content, and an increase in reactive oxygen species production in HR-treated H9C2 cells. Moreover, MR2 treatment altered the mRNA expression of genes involved in mitochondrial biogenesis under HR conditions. The present study documented for the first time the cardioprotective effects of MR2 against HR injury by maintaining mitochondrial function and modulating mitochondrial biogenesis.

BH4 activates CaMKK2 and rescues the cardiomyopathic phenotype in rodent models of diabetes.

Diabetic cardiomyopathy (DCM) is a major cause of mortality/morbidity in diabetes mellitus patients. Although tetrahydrobiopterin (BH4) shows therapeutic potential as an endogenous cardiovascular target, its effect on myocardial cells and mitochondria in DCM and the underlying mechanisms remain unknown. Here, we determined the involvement of BH4 deficiency in DCM and the therapeutic potential of BH4 supplementation in a rodent DCM model. We observed a decreased BH4:total biopterin ratio in heart and mitochondria accompanied by cardiac remodeling, lower cardiac contractility, and mitochondrial dysfunction. Prolonged BH4 supplementation improved cardiac function, corrected morphological abnormalities in cardiac muscle, and increased mitochondrial activity. Proteomics analysis revealed oxidative phosphorylation (OXPHOS) as the BH4-targeted biological pathway in diabetic hearts as well as BH4-mediated rescue of down-regulated peroxisome proliferator-activated receptor-γ coactivator 1-α (PGC-1α) signaling as a key modulator of OXPHOS and mitochondrial biogenesis. Mechanistically, BH4 bound to calcium/calmodulin-dependent protein kinase kinase 2 (CaMKK2) and activated downstream AMP-activated protein kinase/cAMP response element binding protein/PGC-1α signaling to rescue mitochondrial and cardiac dysfunction in DCM. These results suggest BH4 as a novel endogenous activator of CaMKK2.

Cardiovascular Protective Effects and Clinical Applications of Resveratrol.

Resveratrol is a naturally occurring phenol that is generated by plant species following injury or attack by bacterial and fungal pathogens. This compound was first described as the French Paradox in 1992. Later in 2003, resveratrol was reported to activate sirtuins in yeast cells. Recent experimental studies have found that resveratrol offers a variety of benefits that include both anticarcinogenic and anti-inflammatory effects in addition to the ability to reverse obesity, attenuate hyperglycemia and hyperinsulinemia, protect heart and endothelial function, and increase the life span. Multiple molecular targets are associated with the cardioprotective capabilities of resveratrol, and therefore, resveratrol has potential for a wide range of new therapeutic strategies for atherosclerosis, ischemia/reperfusion, metabolic syndrome, cardiac failure, and inflammatory alterations during aging. Expectations for application in human patients, however, suffer from a lack of sufficient clinical evidence in support of these beneficial effects. This article reviews recently reported basic research results that describe the beneficial effects of resveratrol in an attempt to condense the evidence observed in clinical trials and provide support for the future development of novel clinical therapeutics in patients with cardiovascular diseases.

Low abundance of mitochondrial DNA changes mitochondrial status and renders cells resistant to serum starvation and sodium nitroprusside insult.

Mutation or depletion of mitochondrial DNA (mtDNA) can cause severe mitochondrial malfunction, originating from the mitochondrion itself, or from the crosstalk between nuclei and mitochondria. However, the changes that would occur if the amount of mtDNA is diminished are less known. Thus, we generated rat myoblast H9c2 cells containing lower amounts of mtDNA via ethidium bromide and uridine supplementation. After confirming the depletion of mtDNA by quantitative PCR and gel electrophoresis analysis, we investigated the changes in mitochondrial physical parameters by using flow cytometry. We also evaluated the resistance of these cells to serum starvation and sodium nitroprusside. H9c2 cells with diminished mtDNA contents showed decreased mitochondrial membrane potential, mass, free calcium, and zinc ion contents as compared to naïve H9c2 cells. Furthermore, cytosolic and mitochondrial reactive oxygen species levels were significantly higher in mtDNA-lowered H9c2 cells than in the naïve cells. Although the oxygen consumption rate and cell proliferation were decreased, mtDNA-lowered H9c2 cells were more resistant to serum deprivation and nitroprusside insults than the naïve H9c2 cells. Taken together, we conclude that the low abundance of mtDNA cause changes in cellular status, such as changes in reactive oxygen species, calcium, and zinc ion levels inducing resistance to stress.

Yoga training improves metabolic parameters in obese boys.

Yoga has been known to have stimulatory or inhibitory effects on the metabolic parameters and to be uncomplicated therapy for obesity. The purpose of the present study was to test the effect of an 8-week of yoga-asana training on body composition, lipid profile, and insulin resistance (IR) in obese adolescent boys. Twenty volunteers with body mass index (BMI) greater than the 95th percentile were randomly assigned to yoga (age 14.7±0.5 years, n=10) and control groups (age 14.6±1.0 years, n=10). The yoga group performed exercises three times per week at 40~60% of heart-rate reserve (HRR) for 8 weeks. IR was determined with the homeostasis model assessment of insulin resistance (HOMA-IR). After yoga training, body weight, BMI, fat mass (FM), and body fat % (BF %) were significantly decreased, and fat-free mass and basal metabolic rate were significantly increased than baseline values. FM and BF % were significantly improved in the yoga group compared with the control group (p<0.05). Total cholesterol (TC) was significantly decreased in the yoga group (p<0.01). HDL-cholesterol was decreased in both groups (p<0.05). No significant changes were observed between or within groups for triglycerides, LDL-cholesterol, glucose, insulin, and HOMA-IR. Our findings show that an 8-week of yoga training improves body composition and TC levels in obese adolescent boys, suggesting that yoga training may be effective in controlling some metabolic syndrome factors in obese adolescent boys.

Effect of eplerenone, enalapril and their combination treatment on diabetic nephropathy in type II diabetic rats.

BACKGROUND: Recent data suggest that aldosterone antagonists have beneficial effects on diabetic nephropathy. In this study, we investigated the dose-dependent effect of eplerenone and a combined treatment with eplerenone and enalapril compared with each drug alone on renal function in type II diabetic rats. To further explore the molecular mechanism of action of combination therapy, we also performed in vitro study. METHODS: The animals were divided into six groups as follows: normal control Long-Evans Tokushima Otsuka (LETO) rats, Otsuka Long-Evans Tokushima Fatty (OLETF) rats, OLETF rats treated with low dose of eplerenone (50 mg/kg/day), OLETF rats treated with high dose of eplerenone (200 mg/kg/day), OLETF rats treated with enalapril (10 mg/kg/day) and OLETF rats treated with a combination of both drugs (eplerenone 200 mg/kg/day and enalapril 10 mg/kg/day) for 6 months. RESULTS: Treatment of OLETF rats had no significant effect on body weight, kidney weight and blood glucose levels. However, urinary albumin excretion, glomerular filtration rate and glomerulosclerosis were significantly improved in the enalapril group and improvement was observed in a dose-dependent manner in the eplerenone groups; the most dramatic decreases were observed in the combination group. In accordance with these findings, renal expressions of TGF-beta1, type IV collagen and PAI-1 were also markedly decreased in the treatment groups, with the combined treatment providing the most significant level of improvement. In cultured mesangial cells, combined treatment resulted in an additive decrease in TGF-beta1, PAI-1 and collagen gene expressions and protein production induced by high glucose and aldosterone stimulation. CONCLUSIONS: Aldosterone receptor antagonism provided additional benefits beyond blockade of the renin-angiotensin system in type II diabetic nephropathy.

Inflammatory cytokines and lipopolysaccharide induce Fas-mediated apoptosis in renal tubular cells.

BACKGROUND/AIMS: Increased susceptibility of the kidney to acute renal failure (ARF) in the setting of sepsis even in the absence of systemic hypotension is well known. In the hypothesis that the proinflammatory cytokines and lipopolysaccharide (LPS) in gram-negative sepsis can directly cause renal tubular cell apoptosis via Fas- and caspase-mediated pathways, we examined apoptosis and Fas, Fas ligand, FADD expression, as well as PARP cleavage in cultured human proximal tubular cells under the cytokine and LPS-stimulated conditions. METHODS: HK-2 cell, immortalized human proximal tubular cell lines, were treated with 5 and 30 ng/ml of tumor necrosis factor-alpha (TNF-alpha), 5 and 20 ng/ml of interleukin-1beta (IL-1beta) and 30 ng/ml LPS for 24 h. Fas expression was examined by RT-PCR and Fas ligand, Fas-associated protein with death domain (FADD) and poly ADP ribose polymerase (PARP) cleavage were examined by Western blot analysis. Apoptosis was assessed by flow cytometer using Annexin V-FITC and propidium iodide (PI) staining and also by terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling (TUNEL) methods. RESULTS: Fas mRNA expression (ratio of Fas/L-19) increased in the TNF-alpha 5, 30 ng/ml and LPS treated group (p < 0.01, p < 0.01, p = 0.02), but there was no difference between the low- and high-dose TNF-alpha groups. Fas ligand protein expression did not increase in the low-dose TNF-alpha treated group, but it increased significantly in the high-dose TNF-alpha treated group (p < 0.01), IL-1beta- and LPS-treated groups (p < 0.01, p = 0.01, p < 0.01, p = 0.02). The intracellular adaptor protein, FADD expression also increased significantly in the high-dose TNF-alpha- and IL-beta-treated groups (p = 0.04, p = 0.04), but in the low-dose TNF-alpha and IL-beta treated group, it did not show statistically significant differences. In the LPS group, FADD expression also showed an increased tendency, but it was not statistically significant (p = 0.09). Western blot for PARP, a DNA repair enzyme mainly cleaved by caspase 3, showed increased 89- and 24-kD PARP cleavage products in TNF-alpha, IL-1beta and LPS treated cells. The degree of apoptosis examined by DNA fragmentation and translocation of membrane phosphatidyl serine significantly increased in cytokines and LPS treated groups. CONCLUSION: These results suggest that Fas- and caspase-mediated apoptosis of tubular cells by inflammatory cytokines and LPS can be one of the possible mechanisms of renal dysfunction in endotoxemia.