Impaired Ischemia-Reperfusion Responses in the Hearts of Aged Male and Female Offspring of Obese Rats.

Maternal obesity predisposes offspring (F1) to cardiovascular disease. To evaluate basal heart function and ischemia-reperfusion (IR) responses in F1 males and females of obese mothers, female Wistar rats (F0) were fed chow or an obesogenic (MO) diet from weaning through pregnancy and lactation. Non-sibling F1 males and females were weaned to chow at postnatal day (PND) 21 and euthanized at PND 550. Offspring of MO mothers (MOF1) rarely survive beyond PND 650. Hearts were immediately isolated from euthanized F1s and subjected to 30 min ischemia with 20 min reperfusion. Retroperitoneal fat, serum triglycerides, glucose, insulin, and insulin resistance were measured. Baseline left ventricular developed pressure (LVDP) was lower in male and female MOF1 than in controls. After global ischemia, LVDP in control (C) male and female F1 recovered 78 and 83%, respectively, while recovery in MO male and female F1 was significantly lower at 28 and 52%, respectively. Following the IR challenge, MO hearts showed a higher functional susceptibility to reperfusion injury, resulting in lower cardiac reserve than controls in both sexes. Female hearts were more resistant to IR. Retroperitoneal fat was increased in male MOF1 vs. CF1. Circulating triglycerides and insulin resistance were increased in male and female MOF1 vs. CF1. These data show that MO programming reduces F1 cardiac reserve associated with age-related insulin resistance in a sex-specific manner.

Genetic Variations on Redox Control in Cardiometabolic Diseases: The Role of Nrf2.

The transcription factor Nrf2 is a master regulator of multiple cytoprotective genes that maintain redox homeostasis and exert anti-inflammatory functions. The Nrf2-Keap1 signaling pathway is a paramount target of many cardioprotective strategies, because redox homeostasis is essential in cardiovascular health. Nrf2 gene variations, including single nucleotide polymorphisms (SNPs), are correlated with cardiometabolic diseases and drug responses. SNPs of Nrf2, KEAP1, and other related genes can impair the transcriptional activation or the activity of the resulting protein, exerting differential susceptibility to cardiometabolic disease progression and prevalence. Further understanding of the implications of Nrf2 polymorphisms on basic cellular processes involved in cardiometabolic diseases progression and prevalence will be helpful to establish more accurate protective strategies. This review provides insight into the association between the polymorphisms of Nrf2-related genes with cardiometabolic diseases. We also briefly describe that SNPs of Nrf2-related genes are potential modifiers of the pharmacokinetics that contribute to the inter-individual variability.

Mitochondrial Quality Control in Cardiac-Conditioning Strategies against Ischemia-Reperfusion Injury.

Mitochondria are the central target of ischemic preconditioning and postconditioning cardioprotective strategies, which consist of either the application of brief intermittent ischemia/reperfusion (I/R) cycles or the administration of pharmacological agents. Such strategies reduce cardiac I/R injury by activating protective signaling pathways that prevent the exacerbated production of reactive oxygen/nitrogen species, inhibit opening of mitochondrial permeability transition pore and reduce apoptosis, maintaining normal mitochondrial function. Cardioprotection also involves the activation of mitochondrial quality control (MQC) processes, which replace defective mitochondria or eliminate mitochondrial debris, preserving the structure and function of the network of these organelles, and consequently ensuring homeostasis and survival of cardiomyocytes. Such processes include mitochondrial biogenesis, fission, fusion, mitophagy and mitochondrial-controlled cell death. This review updates recent advances in MQC mechanisms that are activated in the protection conferred by different cardiac conditioning interventions. Furthermore, the role of extracellular vesicles in mitochondrial protection and turnover of these organelles will be discussed. It is concluded that modulation of MQC mechanisms and recognition of mitochondrial targets could provide a potential and selective therapeutic approach for I/R-induced mitochondrial dysfunction.

Cold exposure aggravates pulmonary arterial hypertension through increased miR-146a-5p, miR-155-5p and cytokines TNF-α, IL-1ß, and IL-6.

BACKGROUND: Cold temperatures can aggravate pulmonary diseases and promote pulmonary arterial hypertension (PAH); however, the underlying mechanism has not been fully explored. AIM: To explore the effect of chronic cold exposure on the production of inflammatory cytokines and microRNAs (miRNAs) in a monocrotaline (MCT)-induced PAH model. METHODS: Male Sprague Dawley rats were divided into a Control (23.5 ± 2 °C), Cold (5.0 ± 1 °C for ten days), MCT (60 mg/kg body weight i.p.), and MCT + Cold (ten days of cold exposure after 3 weeks of MCT injection). Hemodynamic parameters, right ventricle (RV) hypertrophy, and pulmonary arterial medial wall thickness were determined. IL-1ß, IL-6, and TNF-α levels were determined using western blotting. miR-21-5p and -3p, miR-146a-5p and -3p, and miR-155-5p and -3p and plasma extracellular vesicles (EVs) and mRNA expression of Cd68, Cd163, Bmpr2, Smad5, Tgfbr2, and Smad3 were determined using RT-qPCR. RESULTS: The MCT + Cold group had aggravated RV hypertrophy hemodynamic parameters, and pulmonary arterial medial wall thickness. In lungs of the MCT + Cold, group the protein levels of TNF-α, IL-1ß, and IL-6 were higher than those in the MCT group. The mRNA expression of Cd68 and Cd163 were higher in the MCT + Cold group. miR-146a-5p and miR-155-5p levels were higher in the plasma EVs and lungs of the MCT + Cold group. Cold exposure promoted a greater decrease in miR-21-5p, Bmpr2, Smad5, Tgfbr2, and Smad3 mRNA expression in lungs of the MCT + Cold group. CONCLUSION: Cold exposure aggravates MCT-induced PAH with an increase in inflammatory marker and miRNA levels in the plasma EVs and lungs.

Early inflammatory changes and CC chemokine ligand-8 upregulation in the heart contribute to uremic cardiomyopathy.

Uremic cardiomyopathy is a common complication in chronic kidney disease (CKD) patients, accounting for a high mortality rate. Several mechanisms have been proposed to link CKD and cardiac alterations; however, the early cardiac modifications that occur in CKD that may trigger cardiac remodeling and dysfunction remain largely unexplored. Here, in a mouse model of CKD induced by 5/6 nephrectomy, we first analyzed the early transcriptional and inflammatory changes that occur in the heart. Five days after 5/6 nephrectomy, RNA-sequencing showed the upregulation of 54 genes in the cardiac tissue of CKD mice and the enrichment of biological processes related to immune system processes. Increased cardiac infiltration of T-CD4+ lymphocytes, myeloid cells, and macrophages during early CKD was observed. Next, since CC chemokine ligand-8 (CCL8) was one of the most upregulated genes in the heart of mice with early CKD, we investigated the effect of acute and transient CCL8 inhibition on uremic cardiomyopathy severity. An increase in CCL8 protein levels was confirmed in the heart of early CKD mice. CCL8 inhibition attenuated the early infiltration of T-CD4+ lymphocytes and macrophages to the cardiac tissue, leading to a protection against chronic cardiac fibrotic remodeling, inflammation and cardiac dysfunction induced by CKD. Altogether, our data show the occurrence of transcriptional and inflammatory changes in the heart during the early phases of CKD and identify CCL8 as a key contributor to the early cardiac inflammatory state that triggers further cardiac remodeling and dysfunction in uremic cardiomyopathy.

Implications of Oxidative and Nitrosative Post-Translational Modifications in Therapeutic Strategies against Reperfusion Damage.

Post-translational modifications based on redox reactions "switch on-off" the biological activity of different downstream targets, modifying a myriad of processes and providing an efficient mechanism for signaling regulation in physiological and pathological conditions. Such modifications depend on the generation of redox components, such as reactive oxygen species and nitric oxide. Therefore, as the oxidative or nitrosative milieu prevailing in the reperfused heart is determinant for protective signaling, in this review we defined the impact of redox-based post-translational modifications resulting from either oxidative/nitrosative signaling or oxidative/nitrosative stress that occurs during reperfusion damage. The role that cardioprotective conditioning strategies have had to establish that such changes occur at different subcellular levels, particularly in mitochondria, is also presented. Another section is devoted to the possible mechanism of signal delivering of modified proteins. Finally, we discuss the possible efficacy of redox-based therapeutic strategies against reperfusion damage.

The regulation of protein acetylation influences the redox homeostasis to protect the heart.

The cellular damage caused by redox imbalance is involved in the pathogenesis of many cardiovascular diseases. Besides, redox imbalance is related to the alteration of protein acetylation processes, causing not only chromatin remodeling but also disturbances in so many processes where protein acetylation is involved, such as metabolism and signal transduction. The modulation of acetylases and deacetylases enzymes aids in maintaining the redox homeostasis, avoiding the deleterious cellular effects associated with the dysregulation of protein acetylation. Of note, regulation of protein acetylation has shown protective effects to ameliorate cardiovascular diseases. For instance, HDAC inhibition has been related to inducing cardiac protective effects and it is an interesting approach to the management of cardiovascular diseases. On the other hand, the upregulation of SIRT protein activity has also been implicated in the relief of cardiovascular diseases. This review focuses on the major protein acetylation modulators described, involving pharmacological and bioactive compounds targeting deacetylase and acetylase enzymes contributing to heart protection through redox homeostasis.

Interaction of Agaric Acid with the Adenine Nucleotide Translocase Induces Mitochondrial Oxidative Stress.

Mitochondrial permeability transition is characterized by the opening of a transmembranal pore that switches membrane permeability from specific to nonspecific. This structure allows the free traffic of ions, metabolites, and water across the mitochondrial inner membrane. The opening of the permeability transition pore is triggered by oxidative stress along with calcium overload. In this work, we explored if oxidative stress is a consequence, rather than an effector of the pore opening, by evaluating the interaction of agaric acid with the adenine nucleotide translocase, a structural component of the permeability transition pore. We found that agaric acid induces transition pore opening, increases the generation of oxygen-derived reactive species, augments the oxidation of unsaturated fatty acids in the membrane, and promotes the detachment of cytochrome c from the inner membrane. The effect of agaric acid was inhibited by the antioxidant tamoxifen in association with decreased binding of the thiol reagent eosin-3 maleimide to the adenine nucleotide translocase. We conclude that agaric acid promotes the opening of the pore, increasing ROS production that exerts oxidative modification of critical thiols in the adenine nucleotide translocase.

Redox signaling in ischemic postconditioning protection involves PKCε and Erk1/2 pathways and converges indirectly in Nrf2 activation.

Ischemic-postconditioning (iPostC) exerts cardioprotection by preserving redox homeostasis in the reperfused heart. This protective effect has been associated with the activation of endogenous antioxidant response driven by transcription factor Nrf2 and with the activation of 'reperfusion injury salvage kinases' (RISK) as PI3K, PKC and Erk1/2. Redox homeostasis is essential for normal cell physiology since reactive oxygen species (ROS) are crucial for processes that involve protein signaling. Thus, it has become clear that not only the perturbation of redox balance to oxidative state is deleterious but also towards a reductive state contributing to pathogenesis of diseases. However, there is still a scarce knowledge about the role of ROS in the cardioprotective signals mediated by RISK in postconditioned hearts. Therefore, we studied the role of ROS as initiator of RISK signaling molecules in iPostC-conferred cardioprotection. With the aim to study the relationship between redox-dependent RISK activation and the downstream activation of the transcription factor Nrf2, we evaluated the effect of redox signaling disruption by the effect of ascorbic acid in iPostC hearts. Our results showed that PKCε and Erk1/2 activation is redox-dependent and that concurs downstream with Nrf2 deficient activation. Besides, using inhibitors we found that neither PI3K nor Erk1/2 are directly related with Nrf2 activation, indicating that these kinases have other targets. We conclude that redox signaling participates in cardioprotection triggered by iPostC through the action of kinase-dependent and -independent mechanisms and concurred with the downstream regulation of Nrf2-mediated antioxidant response to prolonged redox balance during long reperfusion.

Adipogenesis: A Necessary but Harmful Strategy.

Obesity is considered to significantly increase the risk of the development of a vast range of metabolic diseases. However, adipogenesis is a complex physiological process, necessary to sequester lipids effectively to avoid lipotoxicity in other tissues, like the liver, heart, muscle, essential for maintaining metabolic homeostasis and has a crucial role as a component of the innate immune system, far beyond than only being an inert mass of energy storage. In pathophysiological conditions, adipogenesis promotes a pro-inflammatory state, angiogenesis and the release of adipokines, which become dangerous to health. It results in a hypoxic state, causing oxidative stress and the synthesis and release of harmful free fatty acids. In this review, we try to explain the mechanisms occurring at the breaking point, at which adipogenesis leads to an uncontrolled lipotoxicity. This review highlights the types of adipose tissue and their functions, their way of storing lipids until a critical point, which is associated with hypoxia, inflammation, insulin resistance as well as lipodystrophy and adipogenesis modulation by Krüppel-like factors and miRNAs.

Calcium Induces Mitochondrial Oxidative Stress Because of its Binding to Adenine Nucleotide Translocase.

Several studies have demonstrated that the mitochondrial membrane switches from selective to non-selective permeability because of its improved matrix Ca2+ accumulation and oxidative stress. This process, known as permeability transition, evokes severe dysfunction in mitochondria through the opening of a non-specific pore, whose chemical nature is still under discussion. There are some proposals regarding the components of the pore structure, e.g., the adenine nucleotide translocase and dimers of the F1 Fo-ATP synthase. Our results reveal that Ca2+ induces oxidative stress, which not only increases lipid peroxidation and ROS generation but also brings about both the collapse of the transmembrane potential and the membrane release of cytochrome c. Additionally, it is shown that Ca2+ increases the binding of the probe eosin-5-maleimide to adenine nucleotide translocase. Interestingly, these effects are diminished after the addition of ADP. It is suggested that pore opening is caused by the binding of Ca2+ to the adenine nucleotide translocase.

Cardioprotection by curcumin post-treatment in rats with established chronic kidney disease.

PURPOSE: The pathogenic mechanisms leading to cardiovascular disorders in patients with chronic kidney disease have not been clearly established, although increased oxidative stress has been pointed out as a potential cause. Therefore, as cardiovascular events are still the first cause of death in patients with chronic kidney disease and traditional drugs or therapies rarely have effects on cardiac complications, we sought to determine the effect of curcumin in treating cardiac dysfunction in rats with established chronic renal disease. METHODS AND RESULTS: Treatment consisted in daily administration of curcumin (120 mg/kg/day) dissolved in 0.05% carboxymethylcellulose via oral gavages during 30 days, beginning from day 30 after 5/6 nephrectomy (5/6Nx). Cardiac function, markers of oxidative stress, activation of PI3K/Akt/GSK3ß and MEK1/2-ERK1/2 pathway, metalloproteinase-II (MMP-2) content, overall gelatinolytic activity, ROS production and mitochondrial integrity were evaluated after 1-month treatment. Curcumin restored systolic blood pressure, diminished interventricular and rear wall thickening, decreased left ventricle dimension at end-systole (LVSd) and restored ejection fraction in nephrectomized rats. Also, it diminished metalloproteinase-II levels and overall gelatinase activity, decreased oxidative stress and inhibited the mitochondrial permeability transition pore opening. CONCLUSION: Our findings suggest that curcumin might have therapeutic potential in treatment of heart disease in patients with established CKD by attenuating oxidative stress-related events as cardiac remodeling, mitochondrial dysfunction and cell death.

Nrf2-regulated antioxidant response is activated by protein kinase C in postconditioned rat hearts.

Postconditioning (PostC) activates endogenous protective mechanisms that contend against reperfusion injury. Nevertheless, although PostC efficiency in both experimental studies and clinical trials has been demonstrated, a complete picture of the interacting mechanisms, particularly the relationship between kinase signaling and redox maintenance, is still lacking. To unravel such association, in this work we focus on the participation of protein kinase C (PKC) and the transcription factor nuclear factor E2-related factor 2 (Nrf2) in the cardioprotective response elicited by PostC. PostC was performed in an in vivo rat model by applying three repetitive cycles of ischemia and reperfusion (10 s each), followed by evaluation of heart function and infarct size measurements. PKC activation and Nrf2 phosphorylation were evaluated after 10 min of reperfusion, whereas Nrf2 activity and the content and activities of Nrf2-regulated antioxidant proteins were evaluated after 60 min of reperfusion in PostC hearts. Maintenance of heart function and diminution in infarct size concurred with PKC activation and Nrf2 phosphorylation. PKC inhibition diminished Nrf2 phosphorylation and transcriptional activity in association with diminished levels and activities of Nrf2-regulated antioxidant proteins. In conclusion, this study proposes that the novel pathway PKC/Nrf2 participates in the long-term protective mechanisms induced by PostC application by maintaining the antioxidant defense system.

Antiarrhythmic effect of tamoxifen on the vulnerability induced by hyperthyroidism to heart ischemia/reperfusion damage.

Hyperthyroidism, known to have deleterious effects on heart function, and is associated with an enhanced metabolic state, implying an increased production of reactive oxygen species. Tamoxifen is a selective antagonist of estrogen receptors. These receptors make the hyperthyroid heart more susceptible to ischemia/reperfusion. Tamoxifen is also well-known as an antioxidant. The aim of the present study was to explore the possible protective effect of tamoxifen on heart function in hyperthyroid rats. Rats were injected daily with 3,5,3'-triiodothyronine at 2mg/kg body weight during 5 days to induce hyperthyroidism. One group was treated with 10mg/kg tamoxifen and another was not. The protective effect of the drug on heart rhythm was analyzed after 5 min of coronary occlusion followed by 5 min reperfusion. In hyperthyroid rats not treated with tamoxifen, ECG tracings showed post-reperfusion arrhythmias, and heart mitochondria isolated from the ventricular free wall lost the ability to accumulate and retain matrix Ca(2+) and to form a high electric gradient. Both of these adverse effects were avoided with tamoxifen treatment. Hyperthyroidism-induced oxidative stress caused inhibition of cis-aconitase and disruption of mitochondrial DNA, effects which were also avoided by tamoxifen treatment. The current results support the idea that tamoxifen inhibits the hypersensitivity of hyperthyroid rat myocardium to reperfusion damage, probably because its antioxidant activity inhibits the mitochondrial permeability transition.

Mitochondrial inactivation by Anopheles albimanus cecropin 3: molecular mechanisms.

Cecropin 3 (Ccrp3) is an antimicrobial peptide from Anopheles albimanus, which is expressed during Plasmodium berghei infection. Here, we report that synthetic Ccrp3, aside from antibacterial activity, also shows cardio regulatory functions. In rats, Ccrp3 significantly diminishes blood pressure as well as the heartbeat frequency at nanomolar concentration. Ccrp3 affect the rat cardiac muscle mitochondria, inducing uncoupling of oxidative phosphorylation, oxygen consumption and transport of Ca(2). Ccrp3 treatment of the mitochondria causes mitochondrial damage promoting oxidative stress, causing overproduction of reactive oxygen species (ROS) and inhibition of superoxide dismutase. At nM concentration, Ccrp3 inhibits superoxide dismutase activity through direct interaction, diminishing by its enzymatic activity. Ccrp3 induces the release of the pro-apoptotic marker Bax from the mitochondria. Altogether, these results suggest that Ccrp3 pro-oxidative activity on cardiac muscle mitochondria could be responsible for triggering the heartbeat frequency and blood pressure lowering observed the Ccrp3 injected rats.

Titration of lysine residues on adenine nucleotide translocase by fluorescamine induces permeability transition.

Chemical modification of primary amino groups of mitochondrial membrane proteins by the fluorescent probe fluorescamine induces non-specific membrane permeabilisation. Titration of the lysine ϵ-amino group promoted efflux of accumulated Ca(2+), collapse of transmembrane potential and mitochondrial swelling. Ca(2+) release was inhibited by cyclosporin A. Considering the latter, we assumed that fluorescamine induces permeability transition. Carboxyatractyloside also inhibited the reaction. Using a polyclonal antibody for adenine nucleotide translocase, Western blot analysis showed that the carrier appeared labelled with the fluorescent probe. The results point out the importance of the ϵ-amino group of lysine residues, located in the adenine nucleotide carrier, on the modulation of membrane permeability, since its blockage suffices to promote opening of the non-specific nanopore.

Redox activation of Nrf2 & NF-κB: a double end sword?

Moderate concentrations of reactive oxygen species (ROS) are produced by diverse sources under physiological conditions. At such low levels, these molecules may act as upstream mediators of relevant signaling pathways; however an increase in their concentration with respect to the antioxidant system activity, changes their redox signaling function into a deleterious role. Thus, cell health depends, at least in part, on redox balance. This review includes global aspects of oxygen chemistry, ROS generation, antioxidant system, and redox signaling. It is also focused on the description of two relevant redox-sensitive transcription factors: nuclear factor erythroid 2-related factor 2 (Nrf2), which may be a potential target to confer cell protection, and nuclear factor κB (NF-κB), which is involved in deleterious effects in the cell. Finally, recent findings on the interplay between both factors for the development of different pathologies are discussed.

Curcumin maintains cardiac and mitochondrial function in chronic kidney disease.

Curcumin, a natural pigment with antioxidant activity obtained from turmeric and largely used in traditional medicine, is currently being studied in the chemoprevention of several diseases for its pleiotropic effects and nontoxicity. In chronic renal failure, the pathogenic mechanisms leading to cardiovascular disorders have been associated with increased oxidative stress, a process inevitably linked with mitochondrial dysfunction. Thus, in this study we aimed at investigating if curcumin pretreatment exerts cardioprotective effects in a rat model of subtotal nephrectomy (5/6Nx) and its impact on mitochondrial homeostasis. Curcumin was orally administered (120mg/kg) to Wistar rats 7 days before nephrectomy and after surgery for 60 days (5/6Nx+curc). Renal dysfunction was detected a few days after nephrectomy, whereas changes in cardiac function were observed until the end of the protocol. Our results indicate that curcumin treatment protects against pathological remodeling, diminishes ischemic events, and preserves cardiac function in uremic rats. Cardioprotection was related to diminished reactive oxygen species production, decreased oxidative stress markers, increased antioxidant response, and diminution of active metalloproteinase-2. We also observed that curcumin's cardioprotective effects were related to maintaining mitochondrial function. Aconitase activity was significantly higher in the 5/6Nx + curc (408.5±68.7nmol/min/mg protein) than in the 5/6Nx group (104.4±52.3nmol/min/mg protein, P<0.05), and mitochondria from curcumin-treated rats showed enhanced oxidative phosphorylation capacities with both NADH-linked substrates and succinate plus rotenone (3.6±1 vs 1.1±0.9 and 3.1±0.7 vs 1.2±0.8, respectively, P<0.05). The mechanisms involved in cardioprotection included both direct antioxidant effects and indirect strategies that could be related to protein kinase C-activated downstream signaling.

Targeting mitochondria for cardiac protection.

The critical role of mitochondria in cardiomyocyte survival and death has become an exciting field of research in cardiac biology. Indeed, it is accepted that mitochondrial dysfunction plays a crucial role in the pathogenesis of multiple cardiac diseases. Besides the obvious relevance of mitochondria in energy production, calcium homeostasis, and reactive oxygen species (ROS) production, new processes like mitochondrial fusion/fission, phosphorylation and nitrosylation modifications in mitochondrial proteins have been suggested to form part of a cast of key players in cardiac disease. This review describes currently studied drugs and compounds that target mitochondria in the scenario of cardiovascular diseases.