Search

Over past few years, there has been a dramatic increase in studying physiological mechanisms of the activity of various signaling low-molecular molecules that directly or indirectly initiate adaptive changes in the cardiovascular system cells (CVSC) to hypoxia. These molecules include biologically active endogenous gases or gasotransmitters (H2S, NO and CO) that influence on many cellular processes, including mitochondrial biogenesis, oxidative phosphorylation, K+/Ca2+ exchange, contractility of cardiomyocytes (CM) and vascular smooth muscle cells (VSMC) under conditions of oxygen deficiency. The present review focuses on the mechanistic role of the gasotransmitters (NO, H2S, CO) in cardioprotection. The structural components of these mechanisms involve mitochondrial enzyme complexes and redox signal proteins, K+ and Ca2+ channels, and mitochondrial permeability transition pore (MPTP) that have been considered as the final molecular targets of mechanisms underlying antioxidant and mild mitochondrial uncoupling effects, preconditioning, vasodilatation and adaptation to hypoxia. In this article, we have reviewed recent findings on the gasotransmitters and proposed a unifying model of mitochondrial mechanisms of cardioprotection.

Background Age‐related heart diseases are significant contributors to increased morbidity and mortality. Emerging evidence indicates that mitochondria within cardiomyocytes contribute to age‐related increased reactive oxygen species (ROS) generation that plays an essential role in aging‐associated cardiac diseases. Methods and Results The present study investigated differences between ROS production in cardiomyocytes isolated from adult (6 months) and aged (24 months) Fischer 344 rats, and in cardiac tissue of adult (18–65 years) and elderly (>65 years) patients with preserved cardiac function. Superoxide dismutase inhibitable ferricytochrome c reduction assay (1.32±0.63 versus 0.76±0.31 nMol/mg per minute; P=0.001) superoxide and H2O2 production, measured as dichlorofluorescein diacetate fluorescence (1646±428 versus 699±329, P=0.04), were significantly higher in the aged versus adult cardiomyocytes. Similarity in age‐related alteration between rats and humans was identified in mitochondrial‐electron transport chain‐complex‐I‐associated increased oxidative‐stress by MitoSOX fluorescence (53.66±18.58 versus 22.81±12.60; P=0.03) and in 4‐HNE adduct levels (187.54±54.8 versus 47.83±16.7 ng/mg protein, P=0.0063), indicative of increased peroxidation in the elderly. These differences correlated with changes in functional enrichment of genes regulating ROS homeostasis pathways in aged human and rat hearts. Functional merged collective network and pathway enrichment analysis revealed common genes prioritized in human and rat aging‐associated networks that underlay enriched functional terms of mitochondrial complex I and common pathways in the aging human and rat heart. Conclusions Aging sensitizes mitochondrial and extramitochondrial mechanisms of ROS buildup within the heart. Network analysis of the transcriptome highlights the critical elements involved with aging‐related ROS homeostasis pathways common in rat and human hearts as targets.

Abstract Aims Fibroblast to myofibroblast trans‐differentiation with altered bioenergetics precedes cardiac fibrosis (CF). Either prevention of differentiation or promotion of de‐differentiation could mitigate CF‐related pathologies. We determined whether 3‐hydroxy‐3‐methyl‐glutaryl‐coenzyme A (HMG‐CoA) reductase inhibitors—statins, commonly prescribed to patients at risk of heart failure (HF)—can de‐differentiate myofibroblasts, alter cellular bioenergetics, and impact the human ventricular fibroblasts (hVFs) in HF patients. Methods and results Either in vitro statin treatment of differentiated myofibroblasts (n = 3–6) or hVFs, isolated from human HF patients under statin therapy (HF + statin) vs. without statins (HF) were randomly used (n = 4–12). In vitro, hVFs were differentiated by transforming growth factor‐β1 (TGF‐β1) for 72 h (TGF‐72 h). Differentiation status and cellular oxygen consumption rate (OCR) were determined by α‐smooth muscle actin (α‐SMA) expression and Seahorse assay, respectively. Data are mean ± SEM except Seahorse (mean ± SD); P

Excessive cardiac fibrosis, characterized by increased collagen-rich extracellular matrix (ECM) deposition, is a major predisposing factor for mechanical and electrical dysfunction in heart failure (HF). The human ventricular fibroblast (hVF) remodeling mechanisms that cause excessive collagen deposition in HF are unclear, although reports suggest a role for intracellular free Ca2+ in fibrosis. Therefore, we determined the association of differences in cellular Ca2+ dynamics and collagen secretion/deposition between hVFs from failing and normal (control) hearts. Histology of left ventricle sections (Masson trichrome) confirmed excessive fibrosis in HF versus normal. In vitro, hVFs from HF showed increased secretion/deposition of soluble collagen in 48 h of culture compared with control [85.9±7.4 µg/106 cells vs 58.5±8.8 µg/106 cells, P

PurposeHigh calorie intake leads to obesity, a global socio-economic and health problem, reaching epidemic proportion in children and adolescents. Saturated and monounsaturated fatty acids from animal (lard) fat are major components of the western-pattern diet and its regular consumption leads to obesity, a risk factor for cardiovascular disease. However, no clear evidence exists whether consumption of diet rich in saturated (SFAs) and monounsaturated (MUFAs) fatty acids has detrimental effects on cardiac structure and energetics primarily due to excessive calories. We, therefore, sought to determine the impact of high calories versus fat content in diet on cardiac structure and mitochondrial energetics.MethodsSix-week-old C57BL/6J mice were fed with high calorie, high lard fat-based diet (60% fat, HFD), high-calorie and low lard fat-based diet (10% fat, LFD), and lower-calorie and fat diet (standard chow, 12% fat, SCD) for 10 weeks.ResultsThe HFD- and LFD-fed mice had higher body weight, ventricular mass and thickness of posterior and septal wall with increased cardiomyocytes diameter compared to the SCD-fed mice. These changes were associated with a reduction in the mitochondrial oxidative phosphorylation (OXPHOS) complexes I and III activity compared to the SCD-fed mice without significant differences between the HFD- and LFD-fed animals. The HFD-fed animals had higher level of malondialdehyde (MDA) than LFD and SCD-fed mice.ConclusionsWe assume that changes in cardiac morphology and selective reduction of the OXPHOS complexes activity observed in the HFD- and LFD-fed mice might be related to excessive calories with additional effect of fat content on oxidative stress.

Purpose: Excessive fibrosis has been suggested to result from persistence of fibroblasts in injured tissue due to impaired apoptosis, but signaling pathways are not fully defined. Methods: Suppression of apoptotic cell death following transforming growth factor-β1 (TGF-β1) exposure was studied using the culture of NIH/3T3 mouse embryonic fibroblasts. Caspase-3 activity, propidium iodide staining and annexin V binding induced by Fas-ligand (FasL) in NIH/3T3 fibroblasts in the absence and presence of TGF-β1 was determined, and relative contribution of signaling through Smad2/3 and noncanonical Erk1/2 and Akt pathways was dissected by assessing phosphorylation status of these kinases and caspase activity in the absence and presence of specific inhibitors (SB431542, PD0325901 and LY294002), respectively. Results: TGF-β1 treatment suppressed FasL-mediated fibroblasts apoptosis with a greater than threefold reduction of caspase-3 activity (from 894 ± 186 to 195 ± 56 nmol AFC/min/106 cells at 250 ng/mL of FasL) and reductions in cleaved caspase-8 and caspase-3 by 3.2-fold and 4.3-fold, respectively. The reduction in caspase activation was accompanied by a decrease in annexin V-positive cells by ~80%. TGF-β1 treatment phosphorylated Smad2/3, Erk1/2 and Akt, which were reduced by their selective inhibitors. Inhibition of Smad2/3 and Erk1/2 alone partially reduced the protective effect of TGF-β1 on caspase-3 activation, whereas inhibition of the Akt pathway had no significant effect. Concomitant inhibition of Smad2/3 and Erk1/2 completely reversed the protection by TGF-β1. Conclusions: TGF-β1-mediated suppression of apoptosis in fibroblasts involves both Smad2/3 and Erk1/2 pathways, but not the Akt pathway. A combined approach inhibiting Smad2/3 and Erk1/2 pathways can completely reverse the protective effect of TGF-β1 on apoptosis. These findings are proof of concept to help define strategies to reduce progression of fibrosis and resultant morbidities associated with conditions causing excessive fibrosis, including but not limited to keloid formation, transplant fibrosis and aging-associated fibrosis of the heart.

Cytokine-dependent activation of fibroblasts to myofibroblasts, a key event in fibrosis, is accompanied by phenotypic changes with increased secretory and contractile properties dependent on increased energy utilization, yet changes in the energetic profile of these cells are not fully described. We hypothesize that the TGF-β1-mediated transformation of myofibroblasts is associated with an increase in mitochondrial content and function when compared to naive fibroblasts.Cultured NIH/3T3 mouse fibroblasts treated with TGF-β1, a profibrotic cytokine, or vehicle were assessed for transformation to myofibroblasts (appearance of α-smooth muscle actin [α-SMA] stress fibers) and associated changes in mitochondrial content and functions using laser confocal microscopy, Seahorse respirometry, multi-well plate reader and biochemical protocols. Expression of mitochondrial-specific proteins was determined using western blotting, and the mitochondrial DNA quantified using Mitochondrial DNA isolation kit.Treatment with TGF-β1 (5 ng/mL) induced transformation of naive fibroblasts into myofibroblasts with a threefold increase in the expression of α-SMA (6.85 ± 0.27 RU) compared to cells not treated with TGF-β1 (2.52 ± 0.11 RU). TGF-β1 exposure increased the number of mitochondria in the cells, as monitored by membrane potential sensitive dye tetramethylrhodamine, and expression of mitochondria-specific proteins; voltage-dependent anion channels (0.54 ± 0.05 vs. 0.23 ± 0.05 RU) and adenine nucleotide transporter (0.61 ± 0.11 vs. 0.22 ± 0.05 RU), as well as mitochondrial DNA content (530 ± 12 μg DNA/106 cells vs. 307 ± 9 μg DNA/106 cells in control). TGF-β1 treatment was associated with an increase in mitochondrial function with a twofold increase in baseline oxygen consumption rate (2.25 ± 0.03 vs. 1.13 ± 0.1 nmol O2/min/106 cells) and FCCP-induced mitochondrial respiration (2.87 ± 0.03 vs. 1.46 ± 0.15 nmol O2/min/106 cells).TGF-β1 induced differentiation of fibroblasts is accompanied by energetic remodeling of myofibroblasts with an increase in mitochondrial respiration and mitochondrial content.