János Pálóczi

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.

PARP inhibition protects against alcoholic and non-alcoholic steatohepatitis

BACKGROUND & AIMS Mitochondrial dysfunction, oxidative stress, inflammation, and metabolic reprograming are crucial contributors to hepatic injury and subsequent liver fibrosis. Poly(ADP-ribose) polymerases (PARP) and their interactions with sirtuins play an important role in regulating intermediary metabolism in this process. However, there is little research into whether PARP inhibition affects alcoholic and non-alcoholic steatohepatitis (ASH/NASH). METHODS We investigated the effects of genetic deletion of PARP1 and pharmacological inhibition of PARP in models of early alcoholic steatohepatitis, as well as on Kupffer cell activation in vitro using biochemical assays, real-time PCR, and histological analyses. The effects of PARP inhibition were also evaluated in high fat or methionine and choline deficient diet-induced steatohepatitis models in mice. RESULTS PARP activity was increased in livers due to excessive alcohol intake, which was associated with decreased NAD+ content and SIRT1 activity. Pharmacological inhibition of PARP restored the hepatic NAD+ content, attenuated the decrease in SIRT1 activation and beneficially affected the metabolic-, inflammatory-, and oxidative stress-related alterations due to alcohol feeding in the liver. PARP1-/- animals were protected against alcoholic steatohepatitis and pharmacological inhibition of PARP or genetic deletion of PARP1 also attenuated Kupffer cell activation in vitro. Furthermore, PARP inhibition decreased hepatic triglyceride accumulation, metabolic dysregulation, or inflammation and/or fibrosis in models of NASH. CONCLUSION Our results suggests that PARP inhibition is a promising therapeutic strategy in steatohepatitis with high translational potential, considering the availability of PARP inhibitors for clinical treatment of cancer. LAY SUMMARY Poly(ADP-ribose) polymerases (PARP) are the most abundant nuclear enzymes. The PARP inhibitor olaparib (Lynparza) is a recently FDA-approved therapy for cancer. This study shows that PARP is overactivated in livers of subjects with alcoholic liver disease and that pharmacological inhibition of this enzyme with 3 different PARP inhibitors, including olaparib, attenuates high fat or alcohol induced liver injury, abnormal metabolic alteration, fat accumulation, inflammation and/or fibrosis in preclinical models of liver disease. These results suggest that PARP inhibition is a promising therapeutic strategy in the treatment of alcoholic and non-alcoholic liver diseases.

Chronic plus binge ethanol feeding induces myocardial oxidative stress, mitochondrial and cardiovascular dysfunction, and steatosis

Alcoholic cardiomyopathy in humans develops in response to chronic excessive alcohol consumption; however, good models of alcohol-induced cardiomyopathy in mice are lacking. Herein we describe mouse models of alcoholic cardiomyopathies induced by chronic and binge ethanol (EtOH) feeding and characterize detailed hemodynamic alterations, mitochondrial function, and redox signaling in these models. Mice were fed a liquid diet containing 5% EtOH for 10, 20, and 40 days (d) combined with single or multiple EtOH binges (5 g/kg body wt). Isocalorically pair-fed mice served as controls. Left ventricular (LV) function and morphology were assessed by invasive pressure-volume conductance approach and by echocardiography. Mitochondrial complex (I, II, IV) activities, 3-nitrotyrosine (3-NT) levels, gene expression of markers of oxidative stress (gp91phox, p47phox), mitochondrial biogenesis (PGC1α, peroxisome proliferator-activated receptor α), and fibrosis were examined. Cardiac steatosis and fibrosis were investigated by histological/immunohistochemical methods. Chronic and binge EtOH feeding (already in 10 days EtOH plus single binge group) was characterized by contractile dysfunction (decreased slope of end-systolic pressure-volume relationship and preload recruitable stroke work), impaired relaxation (decreased time constant of LV pressure decay and maximal slope of systolic pressure decrement), and vascular dysfunction (impaired arterial elastance and lower total peripheral resistance). This was accompanied by enhanced myocardial oxidative/nitrative stress (3-NT; gp91phox; p47phox; angiotensin II receptor, type 1a) and deterioration of mitochondrial complex I, II, IV activities and mitochondrial biogenesis, excessive cardiac steatosis, and higher mortality. Collectively, chronic plus binge EtOH feeding in mice leads to alcohol-induced cardiomyopathies (National Institute on Alcohol Abuse and Alcoholism models) characterized by increased myocardial oxidative/nitrative stress, impaired mitochondrial function and biogenesis, and enhanced cardiac steatosis.

Moderate inhibition of myocardial matrix metalloproteinase-2 by ilomastat is cardioprotective.

Pharmacological inhibition of matrix metalloproteinase-2 (MMP-2) is a promising target for acute cardioprotection against ischemia/reperfusion injury. Therefore, here we investigated if the MMP inhibitor ilomastat administered either before ischemia or before reperfusion is able to reduce infarct size via inhibition of MMP-2, the most abundant MMP in the rat heart. Infarct-size limiting effect of ilomastat (0.3-6.0μmol/kg) was tested in an in vivo rat model of myocardial infarction induced by 30min coronary occlusion/120min reperfusion. Ilomastat at 0.75 and 1.5μmol/kg decreased infarct size significantly as compared to the vehicle-treated (dimethyl sulfoxide) group (from 66.1±4.6% to 45.3±7.0% and 46.7±5.5% of area at risk, p<0.0.5, respectively), when administered 5min before the onset of ischemia. Ilomastat at 6.0μmol/kg significantly reduced infarct size from its control value of 65.4±2.5% to 52.8±3.7% of area at risk (p<0.05), when administered 5min before the onset of reperfusion. Area at risk was not significantly affected by ilomastat treatments. To further assess the cytoprotective effect of ilomastat, primary cardiomyocytes isolated from neonatal rats were subjected to 240min simulated ischemia followed by 120min simulated reperfusion in the presence of ilomastat (5nM-5μM). Ilomastat at 500nM and 5μM significantly increased cell viability when compared to vehicle treated group. To assess the in situ MMP-2 inhibitory effect of ilomastat, in separate experiments in situ zymography was performed in cardiomyocytes. The cytoprotective concentration of ilomastat (500nM) showed a moderate (approximately 25%) inhibition of intracellular MMP-2 in ischemic/reperfused cardiomyocytes. In these cells, MMP-2 immunostaining showed a 90% colocalization with the in situ gelatinolytic activity. We conclude that the MMP inhibitor ilomastat reduces infarct size when administered either before the onset of ischemia or before the onset of reperfusion in vivo. Furthermore, this is the first demonstration that a moderate inhibition of intracellular MMP-2 is sufficient to confer cardiocytoprotection.

Activation of PPARβ/δ protects cardiac myocytes from oxidative stress-induced apoptosis by suppressing generation of reactive oxygen/nitrogen species and expression of matrix metalloproteinases

Heart failure still remains one of the leading causes of morbidity and mortality worldwide. A major contributing factor is reactive oxygen/nitrogen species (RONS) overproduction which is associated with cardiac remodeling partly through cardiomyocyte apoptosis. Peroxisome proliferator-activated receptors (PPARs) are ligand-activated transcription factors that belong to the nuclear receptor superfamily and have been implicated in cardioprotection. However, the molecular mechanisms are largely unexplored. In this study we sought to investigate the potential beneficial effects evoked by activation of PPARβ/δ under the setting of oxidative stress induced by H2O2 in adult rat cardiac myocytes. The selective PPARβ/δ agonist GW0742 inhibited the H2O2-induced apoptosis and increased cell viability. In addition, generation of RONS was attenuated in cardiac myocytes in the presence of PPARβ/δ agonist. These effects were abolished in the presence of the PPARβ/δ antagonist indicating that the effect was through PPARβ/δ receptor activation. Treatment with PPARβ/δ agonist was also associated with attenuation of caspase-3 and PARP cleavage, upregulation of anti-apoptotic Bcl-2 and concomitant downregulation of pro-apoptotic Bax. In addition, activation of PPARβ/δ inhibited the oxidative-stress-induced MMP-2 and MMP-9 mRNA upregulation. It is concluded that PPARβ/δ activation exerts a cytoprotective effect in adult rat cardiac myocytes subjected to oxidative stress via inhibition of oxidative stress, MMP expression, and apoptosis. Our data suggest that the novel connection between PPAR signaling and MMP down-regulation in cardiac myocytes might represent a new target for the management of oxidative stress-induced cardiac dysfunction.

Cholesterol-enriched diet inhibits cardioprotection by ATP-sensitive K+ channel activators cromakalim and diazoxide

It has been previously shown that hyperlipidemia interferes with cardioprotective mechanisms. Here, we investigated the interaction of hyperlipidemia with cardioprotection induced by pharmacological activators of ATP-sensitive K(+) (KATP) channels. Hearts isolated from rats fed a 2% cholesterol-enriched diet or normal diet for 8 wk were subjected to 30 min of global ischemia and 120 min of reperfusion in the presence or absence of KATP modulators. In normal diet-fed rats, either the nonselective KATP activator cromakalim at 10(-5) M or the selective mitochondrial (mito)KATP opener diazoxide at 3 × 10(-5) M significantly decreased infarct size compared with vehicle-treated control rats. Their cardioprotective effect was abolished by coadministration of the nonselective KATP blocker glibenclamide or the selective mitoKATP blocker 5-hydroxydecanoate, respectively. However, in cholesterol-fed rats, the cardioprotective effect of cromakalim or diazoxide was not observed. Therefore, we further investigated how cholesterol-enriched diet influences cardiac KATP channels. Cardiac expression of a KATP subunit gene (Kir6.1) was significantly downregulated in cholesterol-fed rats; however, protein levels of Kir6.1 and Kir6.2 were not changed. The cholesterol diet significantly decreased cardiac ATP, increased lactate content, and enhanced myocardial oxidative stress, as shown by increased cardiac superoxide and dityrosine formation. This is the first demonstration that cardioprotection by KATP channel activators is impaired in cholesterol-enriched diet-induced hyperlipidemia. The background mechanism may include hyperlipidemia-induced attenuation of mitoKATP function by altered energy metabolism and increased oxidative stress in the heart.

β‐Caryophyllene protects against alcoholic steatohepatitis by attenuating inflammation and metabolic dysregulation in mice

β‐Caryophyllene (BCP) is a plant‐derived FDA approved food additive with anti‐inflammatory properties. Some of its beneficial effects in vivo are reported to involve activation of cannabinoid CB2 receptors that are predominantly expressed in immune cells. Here, we evaluated the translational potential of BCP using a well‐established model of chronic and binge alcohol‐induced liver injury.

Pepcan-12 (RVD-hemopressin) is a CB2 receptor positive allosteric modulator constitutively secreted by adrenals and in liver upon tissue damage

Pepcan-12 (RVD-hemopressin; RVDPVNFKLLSH) is the major peptide of a family of endogenous peptide endocannabinoids (pepcans) shown to act as negative allosteric modulators (NAM) of cannabinoid CB1 receptors. Noradrenergic neurons have been identified to be a specific site of pepcan production. However, it remains unknown whether pepcans occur in the periphery and interact with peripheral CB2 cannabinoid receptors. Here, it is shown that pepcan-12 acts as a potent (Ki value ~50 nM) hCB2 receptor positive allosteric modulator (PAM). It significantly potentiated the effects of CB2 receptor agonists, including the endocannabinoid 2-arachidonoyl glycerol (2-AG), for [35S]GTPγS binding and cAMP inhibition (5–10 fold). In mice, the putative precursor pepcan-23 (SALSDLHAHKLRVDPVNFKLLSH) was identified with pepcan-12 in brain, liver and kidney. Pepcan-12 was increased upon endotoxemia and ischemia reperfusion damage where CB2 receptors play a protective role. The adrenals are a major endocrine site of production/secretion of constitutive pepcan-12, as shown by its marked loss after adrenalectomy. However, upon I/R damage pepcan-12 was strongly increased in the liver (from ~100 pmol/g to ~500 pmol/g) independent of adrenals. The wide occurrence of this endogenous hormone-like CB2 receptor PAM, with unforeseen opposite allosteric effects on cannabinoid receptors, suggests its potential role in peripheral pathophysiological processes.

Cytoprotection by the NO-donor SNAP against ischemia/reoxygenation injury in mouse embryonic stem cell-derived cardiomyocytes.

Embryonic stem cell (ESC)-derived cardiomyocytes are a promising cell source for the screening for potential cytoprotective molecules against ischemia/reperfusion injury, however, little is known on their behavior in hypoxia/reoxygenation conditions. Here we tested the cytoprotective effect of the NO-donor SNAP and its downstream cellular pathway. Mouse ESC-derived cardiomyocytes were subjected to 150-min simulated ischemia (SI) followed by 120-min reoxygenation or corresponding non-ischemic conditions. The following treatments were applied during SI or normoxia: the NO-donor S-Nitroso-N-acetyl-d,l-penicillamine (SNAP), the protein kinase G (PKG) inhibitor, the KATP channel blocker glibenclamide, the particulate guanylate cyclase activator brain type natriuretic peptide (BNP), and a non-specific NO synthase inhibitor (N-Nitro-l-arginine, l-NNA) alone or in different combinations. Viability of cells was assayed by propidium iodide staining. SNAP attenuated SI-induced cell death in a concentration-dependent manner, and this protection was attenuated by inhibition of either PKG or KATP channels. However, SI-induced cell death was not affected by BNP or by l-NNA. We conclude that SNAP protects mESC-derived cardiomyocytes against SI/R injury and that soluble guanylate-cyclase, PKG, and KATP channels play a role in the downstream pathway of SNAP-induced cytoprotection. The present mESC-derived cardiomyocyte-based screening platform is a useful tool for discovery of cytoprotective molecules.

Novel, selective EPO receptor ligands lacking erythropoietic activity reduce infarct size in acute myocardial infarction in rats.

Erythropoietin (EPO) has been shown to protect the heart against acute myocardial infarction in pre-clinical studies, however, EPO failed to reduce infarct size in clinical trials and showed significant safety problems. Here, we investigated cardioprotective effects of two selective non-erythropoietic EPO receptor ligand dimeric peptides (AF41676 and AF43136) lacking erythropoietic activity, EPO, and the prolonged half-life EPO analogue, darbepoetin in acute myocardial infarction (AMI) in rats. In a pilot study, EPO at 100U/mL significantly decreased cell death compared to vehicle (33.8±2.3% vs. 40.3±1.5%, p<0.05) in rat neonatal cardiomyocytes subjected to simulated ischemia/reperfusion. In further studies (studies 1-4), in vivo AMI was induced by 30min coronary occlusion and 120min reperfusion in male Wistar rats. Test compounds and positive controls for model validation (B-type natriuretic peptide, BNP or cyclosporine A, CsA) were administered iv. before the onset of reperfusion. Infarct size (IS) was measured by standard TTC staining. In study 1, 5000U/kg EPO reduced infarct size significantly compared to vehicle (45.3±4.8% vs. 59.8±4.5%, p<0.05). In study 2, darbepoetin showed a U-shaped dose-response curve with maximal infarct size-reducing effect at 5μg/kg compared to the vehicle (44.4±5.7% vs. 65.9±2.7%, p<0.01). In study 3, AF41676 showed a U-shaped dose-response curve, where 3mg/kg was the most effective dose compared to the vehicle (24.1±3.9% vs. 44.3±2.5%, p<0.001). The positive control BNP significantly decreased infarct size in studies 1-3 by approximately 35%. In study 4, AF43136 at 10mg/kg decreased infarct size, similarly to the positive control CsA compared to the appropriate vehicle (39.4±5.9% vs. 58.1±5.4% and 45.9±2.4% vs. 63.8±4.1%, p<0.05, respectively). This is the first demonstration that selective, non-erythropoietic EPO receptor ligand dimeric peptides AF41676 and AF43136 administered before reperfusion are able to reduce infarct size in a rat model of AMI. Therefore, non-erythropoietic EPO receptor peptide ligands may be promising cardioprotective agents.