Gábor Koncsos

Diastolic dysfunction in prediabetic male rats: Role of mitochondrial oxidative stress

Although incidence and prevalence of prediabetes are increasing, little is known about its cardiac effects. Therefore, our aim was to investigate the effect of prediabetes on cardiac function and to characterize parameters and pathways associated with deteriorated cardiac performance. Long-Evans rats were fed with either control or high-fat chow for 21 wk and treated with a single low dose (20 mg/kg) of streptozotocin at week 4 High-fat and streptozotocin treatment induced prediabetes as characterized by slightly elevated fasting blood glucose, impaired glucose and insulin tolerance, increased visceral adipose tissue and plasma leptin levels, as well as sensory neuropathy. In prediabetic animals, a mild diastolic dysfunction was observed, the number of myocardial lipid droplets increased, and left ventricular mass and wall thickness were elevated; however, no molecular sign of fibrosis or cardiac hypertrophy was shown. In prediabetes, production of reactive oxygen species was elevated in subsarcolemmal mitochondria. Expression of mitofusin-2 was increased, while the phosphorylation of phospholamban and expression of Bcl-2/adenovirus E1B 19-kDa protein-interacting protein 3 (BNIP3, a marker of mitophagy) decreased. However, expression of other markers of cardiac auto- and mitophagy, mitochondrial dynamics, inflammation, heat shock proteins, Ca2+/calmodulin-dependent protein kinase II, mammalian target of rapamycin, or apoptotic pathways were unchanged in prediabetes. This is the first comprehensive analysis of cardiac effects of prediabetes indicating that mild diastolic dysfunction and cardiac hypertrophy are multifactorial phenomena that are associated with early changes in mitophagy, cardiac lipid accumulation, and elevated oxidative stress and that prediabetes-induced oxidative stress originates from the subsarcolemmal mitochondria.

Nagarse treatment of cardiac subsarcolemmal and interfibrillar mitochondria leads to artefacts in mitochondrial protein quantification

INTRODUCTION In the heart, subsarcolemmal (SSM), interfibrillar (IFM) and perinuclear mitochondria represent three subtypes of mitochondria. The most commonly used protease during IFM isolation is the nagarse, however, its effect on the detection of mitochondrial proteins is still unclear. Therefore, we investigated whether nagarse treatment influences the quantification of mitochondrial proteins. METHODS SSM and IFM were isolated from hearts of mice and rats. During IFM isolation, nagarse activity was either stopped by centrifugation (common protocol, IFM+N) or inhibited by phenylmethylsulfonyl fluoride (PMSF, IFM+N+I). The amounts of proteins located in different mitochondrial compartments (outer membrane: mitofusin 1 (MFN1) and 2 (MFN2); intermembrane space: p66shc; inner membrane (connexin 43 (Cx43)), and of protein deglycase DJ-1 were determined by Western blot. RESULTS MFN2 and Cx43 were found predominantly in SSM isolated from mouse and rat hearts. MFN1 and p66shc were present in similar amounts in SSM and IFM+N, whereas the level of DJ-1 was higher in IFM+N compared to SSM. In IFM+N+I samples from mice, the amount of MFN2, but not that of Cx43 increased. Nagarse or nagarse inhibition by PMSF had no effect on oxygen consumption of SSM or IFM. DISCUSSION Whereas the use of the common protocol indicates the localization of MFN2 predominantly in SSM, the inhibition of nagarse by PMSF increases the signal of MFN2 in IFM to that of in SSM, indicating an underestimation of MFN2 in IFM. Therefore, protease sensitivity should be considered when assessing distribution of mitochondrial proteins using nagarse-based isolation.

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.

Versatility of microglial bioenergetic machinery under starving conditions

Microglia are highly dynamic cells in the brain. Their functional diversity and phenotypic versatility brought microglial energy metabolism into the focus of research. Although it is known that microenvironmental cues shape microglial phenotype, their bioenergetic response to local nutrient availability remains unclear. In the present study effects of energy substrates on the oxidative and glycolytic metabolism of primary - and BV-2 microglial cells were investigated. Cellular oxygen consumption, glycolytic activity, the levels of intracellular ATP/ADP, autophagy, mTOR phosphorylation, apoptosis and cell viability were measured in the absence of nutrients or in the presence of physiological energy substrates: glutamine, glucose, lactate, pyruvate or ketone bodies. All of the oxidative energy metabolites increased the rate of basal and maximal respiration. However, the addition of glucose decreased microglial oxidative metabolism and glycolytic activity was enhanced. Increased ATP/ADP ratio and cell viability, activation of the mTOR and reduction of autophagic activity were observed in glutamine-supplemented media. Moreover, moderate and transient oxidation of ketone bodies was highly enhanced by glutamine, suggesting that anaplerosis of the TCA-cycle could stimulate ketone body oxidation. It is concluded that microglia show high metabolic plasticity and utilize a wide range of substrates. Among them glutamine is the most efficient metabolite. To our knowledge these data provide the first account of microglial direct metabolic response to nutrients under short-term starvation and demonstrate that microglia exhibit versatile metabolic machinery. Our finding that microglia have a distinct bioenergetic profile provides a critical foundation for specifying microglial contributions to brain energy metabolism.

Autophagosome formation is required for cardioprotection by chloramphenicol

Aims: Chloramphenicol (CAP), a broad spectrum antibiotic, was shown to protect the heart against ischemia/reperfusion (I/R) injury. CAP also induces autophagy, however, it is not known whether CAP‐induced cardioprotection is mediated by autophagy. Therefore, here we aimed to assess whether activation of autophagy is required for the infarct size limiting effect of CAP and to identify which component of CAP‐induced autophagy contributes to cardioprotection against I/R injury. Main methods: Hearts of Sprague‐Dawley rats were perfused in Langendorff mode with Krebs‐Henseleit solution containing either vehicle (CON), 300 &mgr;M CAP (CAP), CAP and an inhibitor of autophagosome‐lysosome fusion chloroquine (CAP + CQ), or an inhibitor of autophagosome formation, the functional null mutant TAT‐HA‐Atg5K130R protein (CAP + K130R), and K130R or CQ alone, respectively. After 35 min of aerobic perfusion, hearts were subjected to 30 min global ischemia and 2 h reperfusion. Autophagy was determined by immunoblot against LC3 from left atrial tissue. Infarct size was measured by TTC staining, coronary flow was measured, and the release of creatine kinase (CK) was assessed from the coronary effluent. Key findings: CAP treatment induced autophagy, increased phosphorylation of Erk1/2 in the myocardium and significantly reduced infarct size and CK release. Autophagy inhibitor TAT‐HA‐Atg5K130R abolished cardioprotection by CAP, while in CAP + CQ hearts infarct size and CK release were reduced similarly to as seen in the CAP‐treated group. Conclusion: This is the first demonstration that autophagosome formation but not autophagosomal clearance is required for CAP‐induced cardioprotection. Significance: Inducing autophagy sequestration might yield novel therapeutic options against acute ischemia/reperfusion injury.

Selegiline reduces adiposity induced by high‐fat, high‐sucrose diet in male rats

Incidence and severity of obesity are increasing worldwide, however, efficient and safe pharmacological treatments are not yet available. Certain MAO inhibitors reduce body weight, although their effects on metabolic parameters have not been investigated. Here, we have assessed effects of a widely used, selective MAO‐B inhibitor, selegiline, on metabolic parameters in a rat model of diet‐induced obesity.

Erratum to: Hypercholesterolemia downregulates autophagy in the rat heart

Erratum Following publication of the original article [1], it came to the attention of authors that the definition of the term “isolated hypercholesterolemia” used frequently in the article is missing. In this paper, authors define isolated hypercholesterolemia as a hypercholesterolemia without an accompanying hypertriglyceridemia. Therefore, we would like to amend the Background paragraph of the Abstract with the following sentence: “However, it is unknown whether isolated hypercholesterolemia (i.e., hypercholesterolemia without hypertriglyceridemia) disturbs autophagy or the mammalian target of rapamycin (mTOR) pathways.” In addition, the second sentence of Background section was also incorrectly phrased; which we would like to correct as follows: “The role of atherosclerosis is well studied in these pathologies; however, myocardial effects of hypercholesterolemia is less well understood.”