Kynurenic acid protects against ischemia/reperfusion injury by modulating apoptosis in cardiomyocytes.

Acute myocardial infarction, often associated with ischemia/reperfusion injury (I/R), is a leading cause of death worldwide. Although the endogenous tryptophan metabolite kynurenic acid (KYNA) has been shown to exert protection against I/R injury, its mechanism of action at the cellular and molecular level is not well understood yet. Therefore, we examined the potential involvement of antiapoptotic mechanisms, as well as N-methyl-D-aspartate (NMDA) receptor modulation in the protective effect of KYNA in cardiac cells exposed to simulated I/R (SI/R). KYNA was shown to attenuate cell death induced by SI/R dose-dependently in H9c2 cells or primary rat cardiomyocytes. Analysis of morphological and molecular markers of apoptosis (i.e., membrane blebbing, apoptotic nuclear morphology, DNA double-strand breaks, activation of caspases) revealed considerably increased apoptotic activity in cardiac cells undergoing SI/R. The investigated apoptotic markers were substantially improved by treatment with the cytoprotective dose of KYNA. Although cardiac cells were shown to express NMDA receptors, another NMDA antagonist structurally different from KYNA was unable to protect against SI/R-induced cell death. Our findings provide evidence that the protective effect of KYNA against SI/R-induced cardiac cell injury involves antiapoptotic mechanisms, that seem to evoke independently of NMDA receptor signaling.

Diet-Induced Hypercholesterolemia Leads to Cardiac Dysfunction and Alterations in the Myocardial Proteome.

Elevated blood cholesterol is a major risk factor for coronary heart disease. Moreover, direct effects on the myocardium also contribute to the adverse effects of hypercholesterolemia. Here, we investigated the effect of hypercholesterolemia on the cardiac proteome. Male Wistar rats were fed with a laboratory rodent chow supplemented with 2% cholesterol for 8 weeks to induce hypercholesterolemia. The protein expression data obtained from the proteomic characterization of left ventricular samples from normo- and hypercholesterolemic animals were subjected to gene ontology (GO) and protein interaction analyses. Elevated circulating cholesterol levels were accompanied by diastolic dysfunction in cholesterol-fed rats. The proteomic characterization of left ventricular samples revealed altered expression of 45 proteins due to hypercholesterolemia. Based on the Gene Ontology analysis, hypercholesterolemia was associated with disturbed expression of cytoskeletal and contractile proteins. Beta-actin was downregulated in the hypercholesterolemic myocardium, and established a prominent hub of the protein interaction network. Analysis of the unfiltered dataset revealed concordant downregulated expression patterns in proteins associated with the arrangement of the contractile system (e.g., cardiac-specific troponins and myosin complex), and in subunits of the mitochondrial respiratory chain. We conclude that the observed changes in the cardiac proteome may contribute to the development of diastolic dysfunction in hypercholesterolemia.

Modulatory Effect of Myokines on Reactive Oxygen Species in Ischemia/Reperfusion.

There is a growing body of evidence showing the importance of physical activity against acute ischemic events in various organs. Ischemia/reperfusion injury (I/R) is characterized by tissue damage as a result of restriction and subsequent restoration of blood supply to an organ. Oxidative stress due to increased reactive oxygen species formation and/or insufficient antioxidant defense is considered to play an important role in I/R. Physical activity not only decreases the general risk factors for ischemia but also confers direct anti-ischemic protection via myokine production. Myokines are skeletal muscle-derived cytokines, representing multifunctional communication channels between the contracting skeletal muscle and other organs through an endocrine manner. In this review, we discuss the most prominent members of the myokines (i.e., brain-derived neurotrophic factor (BDNF), cathepsin B, decorin, fibroblast growth factors-2 and -21, follistatin, follistatin-like, insulin-like growth factor-1; interleukin-6, interleukin-7, interleukin-15, irisin, leukemia inhibitory factor, meteorin-like, myonectin, musclin, myostatin, and osteoglycin) with a particular interest in their potential influence on reactive oxygen and nitrogen species formation or antioxidant capacity. A better understanding of the mechanism of action of myokines and particularly their participation in the regulation of oxidative stress may widen their possible therapeutic use and, thereby, may support the fight against I/R.

Hypercholesterolemia Interferes with Induction of miR-125b-1-3p in Preconditioned Hearts.

Ischemic preconditioning (IPre) reduces ischemia/reperfusion (I/R) injury in the heart. The non-coding microRNA miR-125b-1-3p has been demonstrated to play a role in the mechanism of IPre. Hypercholesterolemia is known to attenuate the cardioprotective effect of preconditioning; nevertheless, the exact underlying mechanisms are not clear. Here we investigated, whether hypercholesterolemia influences the induction of miR-125b-1-3p by IPre. Male Wistar rats were fed with a rodent chow supplemented with 2% cholesterol and 0.25% sodium-cholate hydrate for 8 weeks to induce high blood cholesterol levels. The hearts of normo- and hypercholesterolemic animals were then isolated and perfused according to Langendorff, and were subjected to 35 min global ischemia and 120 min reperfusion with or without IPre (3 × 5 min I/R cycles applied before index ischemia). IPre significantly reduced infarct size in the hearts of normocholesterolemic rats; however, IPre was ineffective in the hearts of hypercholesterolemic animals. Similarly, miR-125b-1-3p was upregulated by IPre in hearts of normocholesterolemic rats, while in the hearts of hypercholesterolemic animals IPre failed to increase miR-125b-1-3p significantly. Phosphorylation of cardiac Akt, ERK, and STAT3 was not significantly different in any of the groups at the end of reperfusion. Based on these results we propose here that hypercholesterolemia attenuates the upregulation of miR-125b-1-3p by IPre, which seems to be associated with the loss of cardioprotection.

Effects of Cardiovascular Risk Factors on Cardiac STAT3.

Nuclear, mitochondrial and cytoplasmic signal transducer and activator of transcription 3 (STAT3) regulates many cellular processes, e.g., the transcription or opening of mitochondrial permeability transition pore, and its activity depends on the phosphorylation of Tyr705 and/or Ser727 sites. In the heterogeneous network of cardiac cells, STAT3 promotes cardiac muscle differentiation, vascular element formation and extracellular matrix homeostasis. Overwhelming evidence suggests that STAT3 is beneficial for the heart, plays a role in the prevention of age-related and postpartum heart failure, protects the heart against cardiotoxic doxorubicin or ischaemia/reperfusion injury, and is involved in many cardioprotective strategies (e.g., ischaemic preconditioning, perconditioning, postconditioning, remote or pharmacological conditioning). Ischaemic heart disease is still the leading cause of death worldwide, and many cardiovascular risk factors contribute to the development of the disease. This review focuses on the effects of various cardiovascular risk factors (diabetes, aging, obesity, smoking, alcohol, depression, gender, comedications) on cardiac STAT3 under non-ischaemic baseline conditions, and in settings of ischaemia/reperfusion injury with or without cardioprotective strategies.

Low-Dose Endotoxin Induces Late Preconditioning, Increases Peroxynitrite Formation, and Activates STAT3 in the Rat Heart.

Administration of low-dose endotoxin (lipopolysaccharide, LPS) 24 h before a lethal ischemia induces pharmacological late preconditioning. The exact mechanism of this phenomenon is not clear. Here we aimed to investigate whether low-dose LPS exerts late effects on peroxynitrite formation and activation of Akt, Erk, and STAT3 in the heart. Male Wistar rats were injected with LPS (S. typhimurium; 0.5 mg/kg i.p.) or saline. Twenty-four hours later, hearts were isolated, perfused for 10 min, and then used for biochemical analyses. LPS pretreatment enhanced cardiac formation of the peroxynitrite marker 3-nitrotyrosine. LPS pretreatment also increased cardiac levels of the peroxynitrite precursor nitric oxide (NO) and superoxide. The activities of Ca2+-independent NO synthase and xanthine oxidoreductase increased in LPS-pretreated hearts. LPS pretreatment resulted in significantly enhanced phosphorylation of STAT3 and non-significantly increased phosphorylation of Akt without affecting the activation of Erk. In separate experiments, isolated working hearts were subjected to 30 min global ischemia and 20 min reperfusion. LPS pretreatment significantly improved ischemia-reperfusion-induced deterioration of cardiac function. We conclude that LPS pretreatment enhances cardiac peroxynitrite formation and activates STAT3 24 h later, which may contribute to LPS-induced late preconditioning.

The cytoprotective effect of biglycan core protein involves Toll-like receptor 4 signaling in cardiomyocytes.

AIMS: Exogenously administered biglycan (core protein with high-molecular weight glycosaminoglycan chains) has been shown to protect neonatal cardiomyocytes against simulated ischemia/reperfusion injury (SI/R), however, the mechanism of action is not clear. In this study we aimed to investigate, which structural component of biglycan is responsible for its cardiocytoprotective effect and to further explore the molecular mechanisms involved in the cytoprotection. METHODS AND RESULTS: A pilot study was conducted to demonstrate that both native (glycanated) and deglycanated biglycan can attenuate cell death induced by SI/R in a dose-dependent manner in primary neonatal cardiomyocytes isolated from Wistar rats. In separate experiments, we have shown that similarly to glycanated biglycan, recombinant human biglycan core protein (rhBGNc) protects cardiomyocytes against SI/R injury. In contrast, the glycosaminoglycan component dermatan sulfate had no significant effect on cell viability, while chondroitin sulfate further enhanced cell death induced by SI/R. Treatment of cardiomyocytes with rhBGNc reverses the effect of SI/R upon markers of necrosis, apoptosis, mitochondrial membrane potential, and autophagy. We have also shown that pharmacological blockade of Toll-like receptor 4 (TLR4) signaling or its downstream mediators (IRAK1/4, ERK, JNK and p38 MAP kinases) abolished the cytoprotective effect of rhBGNc against SI/R injury. Pretreatment of cardiomyocytes with rhBGNc for 20h resulted in increased Akt phosphorylation and NO production without having significant effect on phosphorylation of ERK1/2, STAT3, and on the production of superoxide. Treatment over 10min and 1h with rhBGNc increased ERK1 phosphorylation, while the SI/R-induced increase in superoxide production was attenuated by rhBGNc. Blockade of NO synthesis also prevented the cardiocytoprotective effect of rhBGNc. CONCLUSIONS: The core protein of exogenous biglycan protects myocardial cells from SI/R injury via TLR4-mediated mechanisms involving activation of ERK, JNK and p38 MAP kinases and increased NO production. The cytoprotective effect of rhBGNc is due to modulation of SI/R-induced changes in necrosis, apoptosis and autophagy.

Transcriptomic alterations in the heart of non-obese type 2 diabetic Goto-Kakizaki rats.

BACKGROUND: There is a spectacular rise in the global prevalence of type 2 diabetes mellitus (T2DM) due to the worldwide obesity epidemic. However, a significant proportion of T2DM patients are non-obese and they also have an increased risk of cardiovascular diseases. As the Goto-Kakizaki (GK) rat is a well-known model of non-obese T2DM, the goal of this study was to investigate the effect of non-obese T2DM on cardiac alterations of the transcriptome in GK rats. METHODS: Fasting blood glucose, serum insulin and cholesterol levels were measured at 7, 11, and 15 weeks of age in male GK and control rats. Oral glucose tolerance test and pancreatic insulin level measurements were performed at 11 weeks of age. At week 15, total RNA was isolated from the myocardium and assayed by rat oligonucleotide microarray for 41,012 genes, and then expression of selected genes was confirmed by qRT-PCR. Gene ontology and protein-protein network analyses were performed to demonstrate potentially characteristic gene alterations and key genes in non-obese T2DM. RESULTS: Fasting blood glucose, serum insulin and cholesterol levels were significantly increased, glucose tolerance and insulin sensitivity were significantly impaired in GK rats as compared to controls. In hearts of GK rats, 204 genes showed significant up-regulation and 303 genes showed down-regulation as compared to controls according to microarray analysis. Genes with significantly altered expression in the heart due to non-obese T2DM includes functional clusters of metabolism (e.g. Cyp2e1, Akr1b10), signal transduction (e.g. Dpp4, Stat3), receptors and ion channels (e.g. Sln, Chrng), membrane and structural proteins (e.g. Tnni1, Mylk2, Col8a1, Adam33), cell growth and differentiation (e.g. Gpc3, Jund), immune response (e.g. C3, C4a), and others (e.g. Lrp8, Msln, Klkc1, Epn3). Gene ontology analysis revealed several significantly enriched functional inter-relationships between genes influenced by non-obese T2DM. Protein-protein interaction analysis demonstrated that Stat is a potential key gene influenced by non-obese T2DM. CONCLUSIONS: Non-obese T2DM alters cardiac gene expression profile. The altered genes may be involved in the development of cardiac pathologies and could be potential therapeutic targets in non-obese T2DM.

The effect of a preparation of minerals, vitamins and trace elements on the cardiac gene expression pattern in male diabetic rats.

BACKGROUND: Diabetic patients have an increased risk of developing cardiovascular diseases, which are the leading cause of death in developed countries. Although multivitamin products are widely used as dietary supplements, the effects of these products have not been investigated in the diabetic heart yet. Therefore, here we investigated if a preparation of different minerals, vitamins, and trace elements (MVT) affects the cardiac gene expression pattern in experimental diabetes. METHODS: Two-day old male Wistar rats were injected with streptozotocin (i.p. 100 mg/kg) or citrate buffer to induce diabetes. From weeks 4 to 12, rats were fed with a vehicle or a MVT preparation. Fasting blood glucose measurement and oral glucose tolerance test were performed at week 12, and then total RNA was isolated from the myocardium and assayed by rat oligonucleotide microarray for 41012 oligonucleotides. RESULTS: Significantly elevated fasting blood glucose concentration and impaired glucose tolerance were markedly improved by MVT-treatment in diabetic rats at week 12. Genes with significantly altered expression due to diabetes include functional clusters related to cardiac hypertrophy (e.g. caspase recruitment domain family, member 9; cytochrome P450, family 26, subfamily B, polypeptide; FXYD domain containing ion transport regulator 3), stress response (e.g. metallothionein 1a; metallothionein 2a; interleukin-6 receptor; heme oxygenase (decycling) 1; and glutathione S-transferase, theta 3), and hormones associated with insulin resistance (e.g. resistin; FK506 binding protein 5; galanin/GMAP prepropeptide). Moreover the expression of some other genes with no definite cardiac function was also changed such as e.g. similar to apolipoprotein L2; brain expressed X-linked 1; prostaglandin b2 synthase (brain). MVT-treatment in diabetic rats showed opposite gene expression changes in the cases of 19 genes associated with diabetic cardiomyopathy. In healthy hearts, MVT-treatment resulted in cardiac gene expression changes mostly related to immune response (e.g. complement factor B; complement component 4a; interferon regulatory factor 7; hepcidin). CONCLUSIONS: MVT-treatment improved diagnostic markers of diabetes. This is the first demonstration that MVT-treatment significantly alters cardiac gene expression profile in both control and diabetic rats. Our results and further studies exploring the mechanistic role of individual genes may contribute to the prevention or diagnosis of cardiac complications in diabetes.

Tissue-specific Gene Expression in Rat Hearts and Aortas in a Model of Vascular Nitrate Tolerance.

Nitroglycerin exerts a direct myocardial anti-ischemic effect even in the state of vascular nitrate tolerance. To examine the potentially diverse molecular responses in vascular and cardiac tissues, we investigated the gene expression profile of the heart and the aorta by DNA microarray in male Wistar rats that were previously made tolerant to the vascular effects of nitroglycerin. The blood pressure-lowering effect of nitroglycerin (1-100 µg/kg) was markedly attenuated in rats pretreated for 3 days with 3 × 100 mg/kg nitroglycerin. Nitric oxide content was significantly elevated in the heart but not in the aorta of nitrate-tolerant animals, which indicated tissue-specific differences in nitroglycerin bioconversion. Of 7742 genes analyzed by DNA microarray, we found that although the expression of 25 genes changed significantly in the heart (increased: Tas2r119, Map6, Cd59, Kcnh2, Kcnh3, Senp6, Mcpt1, Tshb, Haus1, Vipr1, Lrn3, Lifr; decreased: Ihh, Fgfr1, Cryge, Krt9, Agrn, C4bpb, Fcer1a, Csf3, Hsd17b11, Hsd11b2, Ctnnbl1, Prpg1, Hsf1), only 14 genes were altered in the aorta (increased: Tas2r119, Ihh, Rrad, Npm1, Snai1; decreased: Tubb2b, Usp15, Sema6c, Wfdc2, Rps21, Ramp2, Galr1, Atxn1, Lhx1) in vascular nitrate tolerance. Quantitative reverse transcription polymerase chain reaction analysis of genes related to oxidative/nitrative/nitrosative stress also showed differential expression pattern in the heart and aorta. This is the first pharmacogenomic analysis showing that nitroglycerin treatment leading to vascular nitrate tolerance differentially impacts gene expression in vascular and cardiac tissues, which indicates different tissue-specific downstream signaling pathways.

MicroRNAs associated with ischemia-reperfusion injury and cardioprotection by ischemic pre- and postconditioning: protectomiRs.

We aimed to characterize early changes in microRNA expression in acute cardioprotection by ischemic pre- and postconditioning in rat hearts. Hearts isolated from male Wistar rats were subjected to 1) time-matched nonischemic perfusion, 2) ischemia-reperfusion (30 min of coronary occlusion and 120 min of reperfusion), 3) preconditioning (3 × 5 min of coronary occlusion) followed by ischemia-reperfusion, or 4) ischemia-reperfusion with postconditioning (6 × 10 s of global ischemia-reperfusion at the onset of reperfusion). Infarct size was significantly reduced by both interventions. Of 350 different microRNAs assessed by microarray analysis, 147-160 microRNAs showed detectable expression levels. Compared with microRNA alterations induced by ischemia-reperfusion versus time-matched nonischemic controls, five microRNAs were significantly affected by both pre- and postconditioning (microRNA-125b*, microRNA-139-3p, microRNA-320, microRNA-532-3p, and microRNA-188), four microRNAs were significantly affected by preconditioning (microRNA-487b, microRNA-139-5p, microRNA-192, and microRNA-212), and nine microRNAs were significantly affected by postconditioning (microRNA-1, microRNA let-7i, microRNA let-7e, microRNA let-7b, microRNA-181a, microRNA-208, microRNA-328, microRNA-335, and microRNA-503). Expression of randomly selected microRNAs was validated by quantitative real-time PCR. By a systematic comparison of the direction of microRNA expression changes in all groups, we identified microRNAs, specific mimics, or antagomiRs that may have pre- and postconditioning-like cardioprotective effects (protectomiRs). Transfection of selected protectomiRs (mimics of microRNA-139-5p, microRNA-125b*, microRNA let-7b, and inhibitor of microRNA-487b) into cardiac myocytes subjected to simulated ischemia-reperfusion showed a significant cytoprotective effect. This is the first demonstration that the ischemia-reperfusion-induced microRNA expression profile is significantly influenced by both pre- and postconditioning, which shows the involvement of microRNAs in cardioprotective signaling. Moreover, by analysis of microRNA expression patterns in cardioprotection by pre- and postconditioning, specific protectomiRs can be revealed as potential therapeutic tools for the treatment of ischemia-reperfusion injury.

Effect of Eccentric Exercise on Metabolic Health in Diabetes and Obesity.

There is a growing body of evidence showing the importance of physical activity against civilization-induced metabolic diseases, including type 2 diabetes (T2DM) and obesity. Eccentric contraction, when skeletal muscles generate force by lengthening, is a unique type of skeletal muscle activity. Eccentric contraction may lead to better power production characteristics of the muscle because eccentric contraction requires less energy and can result in higher tension. Therefore, it is an ideal tool in the rehabilitation program of patients. However, the complex metabolic effect (i.e., fat mass reduction, increased lipid oxidation, improvement in blood lipid profile, and increased insulin sensitivity) of the eccentric contraction alone has scarcely been investigated. This paper aims to review the current literature to provide information on whether eccentric contraction can influence metabolic health and body composition in T2DM or obesity. We also discussed the potential role of myokines in mediating the effects of eccentric exercise. A better understanding of the mechanism of eccentric training and particularly their participation in the regulation of metabolic diseases may widen their possible therapeutic use and, thereby, may support the fight against the leading global risks for mortality in the world.

Preconditioning protects the heart in a prolonged uremic condition.

Metabolic diseases such as hyperlipidemia and diabetes attenuate the cardioprotective effect of ischemic preconditioning. In the present study, we examined whether another metabolic disease, prolonged uremia, affects ischemia/reperfusion injury and cardioprotection by ischemic preconditioning. Uremia was induced by partial nephrectomy in male Wistar rats. The development of uremia was verified 29 wk after surgery. Transthoracic echocardiography was performed to monitor cardiac function. At week 30, hearts of nephrectomized and sham-operated rats were isolated and subjected to a 30-min coronary occlusion followed by 120 min reperfusion with or without preceding preconditioning induced by three intermittent cycles of brief ischemia and reperfusion. In nephrectomized rats, plasma uric acid, carbamide, and creatinine as well as urine protein levels were increased as compared with sham-operated controls. Systolic anterior and septal wall thicknesses were increased in nephrectomized rats, suggesting the development of a minimal cardiac hypertrophy. Ejection fraction was decreased and isovolumic relaxation time was shortened in nephrectomized rats demonstrating a mild systolic and diastolic dysfunction. Infarct size was not affected significantly by nephrectomy itself. Ischemic preconditioning significantly decreased infarct size from 24.8 ± 5.2% to 6.6 ± 1.3% in the sham-operated group and also in the uremic group from 35.4 ± 9.5% to 11.9 ± 3.1% of the area at risk. Plasma ANG II and nitrotyrosine were significantly increased in the uremic rats. We conclude that although prolonged experimental uremia leads to severe metabolic changes and the development of a mild myocardial dysfunction, the cardioprotective effect of ischemic preconditioning is still preserved.

Stellaria media tea protects against diabetes-induced cardiac dysfunction in rats without affecting glucose tolerance.

Background and aim: Common chickweed (Stellaria media) tea has traditionally been applied for treatment of various metabolic diseases including diabetes in folk medicine; however, experimental evidence to support this practice is lacking. Therefore, we aimed to assess the effect of Stellaria media tea on glucose homeostasis and cardiac performance in a rat model of diabetes. Experimental procedure: Hot water extract of Stellaria media herb were analyzed and used in this study, where diabetes was induced by fructose-enriched diet supplemented with a single injection of streptozotocin. Half of the animals received Stellaria media tea (100 mg/kg) by oral gavage. At the end of the 20-week experimental period, blood samples were collected and isolated working heart perfusions were performed. Results and conclusion: Compared to the animals receiving standard chow, serum fasting glucose level was increased and glucose tolerance was diminished in diabetic rats. Stellaria media tea did not affect significantly fasting hyperglycemia and glucose intolerance; however, it attenuated diabetes-induced deterioration of cardiac output and cardiac work. Analysis of the chemical composition of Stellaria media tea suggested the presence of rutin and various apigenin glycosides which have been reported to alleviate diabetic cardiomyopathy. Moreover, Stellaria media prevented diabetes-induced increase in cardiac STAT3 phosphorylation. We demonstrated for the first time that Stellaria media tea may beneficially affect cardiac dysfunction induced by diabetes without improvement of glucose homeostasis. Rutin and/or apigenin glycosides as well as modulation of STAT3 signaling may be implicated in the protection of Stellaria media tea against diabetic cardiomyopathy.

Kynurenine Pathway Metabolites as Potential Clinical Biomarkers in Coronary Artery Disease.

Coronary artery disease (CAD) is one of the leading cause of mortality worldwide. Several risk factors including unhealthy lifestyle, genetic background, obesity, diabetes, hypercholesterolemia, hypertension, smoking, age, etc. contribute to the development of coronary atherosclerosis and subsequent coronary artery disease. Inflammation plays an important role in coronary artery disease development and progression. Pro-inflammatory signals promote the degradation of tryptophan via the kynurenine pathway resulting in the formation of several immunomodulatory metabolites. An unbalanced kynurenic pathway has been implicated in the pathomechanisms of various diseases including CAD. Significant improvements in detection methods in the last decades may allow simultaneous measurement of multiple metabolites of the kynurenine pathway and such a thorough analysis of the kynurenine pathway may be a valuable tool for risk stratification and determination of CAD prognosis. Nevertheless, imbalance in the activities of different branches of the kynurenine pathway may require careful interpretation. In this review, we aim to summarize clinical evidence supporting a possible use of kynurenine pathway metabolites as clinical biomarkers in various manifestations of CAD.

Chronic kidney disease induces left ventricular overexpression of the pro-hypertrophic microRNA-212.

Chronic kidney disease (CKD) is a public health problem that increases the risk of cardiovascular morbidity and mortality. Heart failure with preserved ejection fraction (HFpEF) characterized by left ventricular hypertrophy (LVH) and diastolic dysfunction is a common cardiovascular complication of CKD. MicroRNA-212 (miR-212) has been demonstrated previously to be a crucial regulator of pathologic LVH in pressure-overload-induced heart failure via regulating the forkhead box O3 (FOXO3)/calcineurin/nuclear factor of activated T-cells (NFAT) pathway. Here we aimed to investigate whether miR-212 and its hypertrophy-associated targets including FOXO3, extracellular signal-regulated kinase 2 (ERK2), and AMP-activated protein kinase (AMPK) play a role in the development of HFpEF in CKD. CKD was induced by 5/6 nephrectomy in male Wistar rats. Echocardiography and histology revealed LVH, fibrosis, preserved systolic function, and diastolic dysfunction in the CKD group as compared to sham-operated animals eight and/or nine weeks later. Left ventricular miR-212 was significantly overexpressed in CKD. However, expressions of FOXO3, AMPK, and ERK2 failed to change significantly at the mRNA or protein level. The protein kinase B (AKT)/FOXO3 and AKT/mammalian target of rapamycin (mTOR) pathways are also proposed regulators of LVH induced by pressure-overload. Interestingly, phospho-AKT/total-AKT ratio was increased in CKD without significantly affecting phosphorylation of FOXO3 or mTOR. In summary, cardiac overexpression of miR-212 in CKD failed to affect its previously implicated hypertrophy-associated downstream targets. Thus, the molecular mechanism of the development of LVH in CKD seems to be independent of the FOXO3, ERK1/2, AMPK, and AKT/mTOR-mediated pathways indicating unique features in this form of LVH.

Lack of Contribution of p66shc and Its Mitochondrial Translocation to Ischemia-Reperfusion Injury and Cardioprotection by Ischemic Preconditioning.

Whereas high amounts of reactive oxygen species (ROS) contribute to cardiac damage following ischemia and reperfusion (IR), low amounts function as trigger molecules in the cardioprotection by ischemic preconditioning (IPC). The mitochondrial translocation and contribution of the hydrogen peroxide-generating protein p66shc in the cardioprotection by IPC is unclear yet. In the present study, we investigated the mitochondrial translocation of p66shc, addressed the impact of p66shc on ROS formation after IR, and characterized the role of p66shc in IR injury per se and in the cardioprotection by IPC. The amount of p66shc in subsarcolemmal (SSM) and interfibrillar mitochondria (IFM) isolated from wildtype mouse left ventricles (LV) was determined after 40 min normoxic perfusion and after 30 min ischemia and 10 min reperfusion without and with IPC. The p66shc content in SSM (in % of normoxic controls, n = 5) was 174 ± 16% (n = 6, p < 0.05) after IR, and was reduced to 128 ± 13% after IPC (n = 6, p = ns). In IFM, the amount of p66shc remained unchanged (IR: 81 ± 7%, n = 6; IPC: 110 ± 5%, n = 6, p = ns). IR induced an increase in ROS formation in SSM and IFM isolated from mouse wildtype LV, which was more pronounced in SSM than in IFM (1.18 ± 0.18 vs. 0.81 ± 0.16, n = 6, p < 0.05). In mitochondria from p66shc-knockout mice (p66shc-KO), the increase in ROS formation by IR was not different between SSM and IFM (0.90 ± 0.11 vs. 0.73 ± 0.08, n = 6, p = ns). Infarct size (in % of the left ventricle) was 51.7 ± 2.9% in wildtype and 59.7 ± 3.8% in p66shc-KO hearts in vitro and was significantly reduced to 35.8 ± 4.4% (wildtype) and 34.7 ± 5.6% (p66shc-KO) by IPC, respectively. In vivo, infarct size was 57.8 ± 2.9% following IR (n = 9) and was reduced to 40.3 ± 3.5% by IPC (n = 11, p < 0.05) in wildtype mice. In p66shc-knockout mice, infarct sizes were similar to those measured in wildtype animals (IR: 56.2 ± 4.3%, n = 11; IPC: 42.1 ± 3.9%, n = 13, p < 0.05). Taken together, the mitochondrial translocation of p66shc following IR and IPC differs between mitochondrial populations. However, similar infarct sizes after IR and preserved infarct size reductions by IPC in p66shc-KO mice suggest that p66shc-derived ROS are not involved in the cardioprotection by IPC nor do they contribute to IR injury per se.

Alternative Splicing of NOX4 in the Failing Human Heart.

Increased oxidative stress is a major contributor to the development and progression of heart failure, however, our knowledge on the role of the distinct NADPH oxidase (NOX) isoenzymes, especially on NOX4 is controversial. Therefore, we aimed to characterize NOX4 expression in human samples from healthy and failing hearts. Explanted human heart samples (left and right ventricular, and septal regions) were obtained from patients suffering from heart failure of ischemic or dilated origin. Control samples were obtained from donor hearts that were not used for transplantation. Deep RNA sequencing of the cardiac transcriptome indicated extensive alternative splicing of the NOX4 gene in heart failure as compared to samples from healthy donor hearts. Long distance PCR analysis with a universal 5'-3' end primer pair, allowing amplification of different splice variants, confirmed the presence of the splice variants. To assess translation of the alternatively spliced transcripts we determined protein expression of NOX4 by using a specific antibody recognizing a conserved region in all variants. Western blot analysis showed up-regulation of the full-length NOX4 in ischemic cardiomyopathy samples and confirmed presence of shorter isoforms both in control and failing samples with disease-associated expression pattern. We describe here for the first time that NOX4 undergoes extensive alternative splicing in human hearts which gives rise to the expression of different enzyme isoforms. The full length NOX4 is significantly upregulated in ischemic cardiomyopathy suggesting a role for NOX4 in ROS production during heart failure.

Modulation of Hypercholesterolemia-Induced Oxidative/Nitrative Stress in the Heart.

Hypercholesterolemia is a frequent metabolic disorder associated with increased risk for cardiovascular morbidity and mortality. In addition to its well-known proatherogenic effect, hypercholesterolemia may exert direct effects on the myocardium resulting in contractile dysfunction, aggravated ischemia/reperfusion injury, and diminished stress adaptation. Both preclinical and clinical studies suggested that elevated oxidative and/or nitrative stress plays a key role in cardiac complications induced by hypercholesterolemia. Therefore, modulation of hypercholesterolemia-induced myocardial oxidative/nitrative stress is a feasible approach to prevent or treat deleterious cardiac consequences. In this review, we discuss the effects of various pharmaceuticals, nutraceuticals, some novel potential pharmacological approaches, and physical exercise on hypercholesterolemia-induced oxidative/nitrative stress and subsequent cardiac dysfunction as well as impaired ischemic stress adaptation of the heart in hypercholesterolemia.

Rapid ventricular pacing-induced postconditioning attenuates reperfusion injury: effects on peroxynitrite, RISK and SAFE pathways.

BACKGROUND AND PURPOSE: Rapid ventricular pacing (RVP) applied before an index ischaemia has anti-ischaemic effects. Here, we investigated whether RVP applied after index ischaemia attenuates reperfusion injury and whether peroxynitrite, reperfusion injury salvage kinase (RISK) and survival activating factor enhancement (SAFE) pathways as well as haem oxygenase 1 (HO1) are involved in the mechanism of RVP-induced postconditioning. EXPERIMENTAL APPROACH: Langendorff perfused rat hearts were subjected to 30 min regional ischaemia and 120 min reperfusion with or without ischaemic postconditioning (6 × 10/10 s reperfusion/ischaemia; IPost) or RVP (6 × 10/10 s non-pacing/rapid pacing at 600 bpm) applied at the onset of reperfusion. KEY RESULTS: Meta-analysis of our previous studies revealed an association between longer reperfusion-induced ventricular tachycardia/fibrillation with decreased infarct size. In the present experiments, we tested whether RVP is cardioprotective and found that both IPost and RVP significantly decreased infarct size; however, only RVP attenuated the incidence of reperfusion-induced ventricular tachycardia. Both postconditioning methods increased the formation of cardiac 3-nitrotyrosine and superoxide, and non-significantly enhanced Akt phosphorylation at the beginning of reperfusion without affecting ERK1/2 and STAT3, while IPost alone induced HO1. Application of brief ischaemia/reperfusion cycles or RVP without preceding index ischaemia also facilitated peroxynitrite formation; nevertheless, only brief RVP increased STAT3 phosphorylation. CONCLUSIONS AND IMPLICATIONS: Short periods of RVP at the onset of reperfusion are cardioprotective and increase peroxynitrite formation similarly to IPost and thus may serve as an alternative postconditioning method. However, downstream mechanisms of the protection elicited by IPost and RVP seem to be partially different. LINKED ARTICLES: This article is part of a themed section on Conditioning the Heart - Pathways to Translation. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2015.172.issue-8.