Sex Difference in Cardioprotection against Acute Myocardial Infarction in MAO-B Knockout Mice In Vivo.

The cardiomyocyte-specific knockout (KO) of monoamine oxidase (MAO)-B, an enzyme involved in the formation of reactive oxygen species (ROS), reduced myocardial ischemia/reperfusion (I/R) injury in vitro. Because sex hormones have a strong impact on MAO metabolic pathways, we analyzed the myocardial infarct size (IS) following I/R in female and male MAO-B KO mice in vivo. METHOD AND RESULTS: To induce the deletion of MAO-B, MAO-B KO mice (Myh6 Cre+/MAO-Bfl/fl) and wild-type (WT, Cre-negative MAO-Bfl/fl littermates) were fed with tamoxifen for 2 weeks followed by 10 weeks of normal mice chow. Myocardial infarction (assessed by TTC staining and expressed as a percentage of the area at risk as determined by Evans blue staining)) was induced by 45 min coronary occlusion followed by 120 min of reperfusion. RESULTS: The mortality following I/R was higher in male compared to female mice, with the lowest mortality found in MAO-B KO female mice. IS was significantly higher in male WT mice compared to female WT mice. MAO-B KO reduced IS in male mice but had no further impact on IS in female MAO-B KO mice. Interestingly, there was no difference in the plasma estradiol levels among the groups. CONCLUSION: The cardiomyocyte-specific knockout of MAO-B protects male mice against acute myocardial infarction but had no effect on the infarct size in female mice.

Proprotein Convertase Subtilisin Kexin Type 9 (PCSK9) Deletion but Not Inhibition of Extracellular PCSK9 Reduces Infarct Sizes Ex Vivo but Not In Vivo.

Hypoxia upregulates PCSK9 expression in the heart, and PCSK9 affects the function of myocytes. This study aimed to investigate the impact of PCSK9 on reperfusion injury in rats and mice fed normal or high-fat diets. Either the genetic knockout of PCSK9 (mice) or the antagonism of circulating PCSK9 via Pep2-8 (mice and rats) was used. Isolated perfused hearts were exposed to 45 min of ischemia followed by 120 min of reperfusion. In vivo, mice were fed normal or high-fat diets (2% cholesterol) for eight weeks prior to coronary artery occlusion (45 min of ischemia) and reperfusion (120 min). Ischemia/reperfusion upregulates PCSK9 expression (rats and mice) and releases it into the perfusate. The inhibition of extracellular PCSK9 does not affect infarct sizes or functional recovery. However, genetic deletion largely reduces infarct size and improves post-ischemic recovery in mice ex vivo but not in vivo. A high-fat diet reduced the survival rate during ischemia and reperfusion, but in a PCSK9-independent manner that was associated with increased plasma matrix metalloproteinase (MMP)9 activity. PCSK9 deletion, but not the inhibition of extracellular PCSK9, reduces infarct sizes in ex vivo hearts, but this effect is overridden in vivo by factors such as MMP9.

Matrix Metalloproteinases Repress Hypertrophic Growth in Cardiac Myocytes.

PURPOSE: Matrix metalloproteinases (MMPs) are identified as modulators of the extracellular matrix in heart failure progression. However, evidence for intracellular effects of MMPs is emerging. Pro- and anti-hypertrophic cardiac effects are described. This may be due to the various sources of different MMPs in the heart tissue. Therefore, the aim of the present study was to determine the role of MMPs in hypertrophic growth of isolated rat ventricular cardiac myocytes. METHODS: Cardiomyocytes were isolated form ventricular tissues of the rat hearts by collagenase perfusion. RT-qPCR, western blots, and zymography were used for expression and MMP activity analysis. Cross-sectional area and the rate of protein synthesis were determined as parameters for hypertrophic growth. RESULTS: MMP-1, MMP-2, MMP-3, MMP-9 and MMP-14 mRNAs were detected in cardiomyocytes, and protein expression of MMP-2, MMP-9, and MMP-14 was identified. Hypertrophic stimulation of cardiomyocytes did not enhance, but interestingly decreased expression of MMPs, indicating that downregulation of MMPs may promote hypertrophic growth. Indeed, the nonselective MMP inhibitors TAPI-0 or TIMP2 and the MMP-2-selective ARP-100 enhanced hypertrophic growth. Furthermore, TAPI-0 increased phosphorylation and thus activation of extracellular signaling kinase (ERK) and Akt (protein kinase B), as well as inhibition of glycogen synthase 3ß (GSK3ß). Abrogation of MEK/ERK- or phosphatidylinositol-3-kinase(PI3K)/Akt/GSK3ß-signaling with PD98059 or LY290042, respectively, inhibited hypertrophic growth under TAPI-0. CONCLUSION: MMPs' inhibition promotes hypertrophic growth in cardiomyocytes in vitro. Therefore, MMPs in the healthy heart may be important players to repress cardiac hypertrophy.

Ischaemic post-conditioning in rats: Responder and non-responder differ in transcriptome of mitochondrial proteins.

Ischaemic post-conditioning (IPoC) is a clinical applicable procedure to reduce reperfusion injury. Non-responsiveness to IPoC possibly caused by co-morbidities limits its clinical attractiveness. We analysed differences in the expression of mitochondrial proteins between IPoC responder (IPoC-R) and non-responder (IPoC-NR). Eighty rats were randomly grouped to sham, ischaemia/reperfusion (I/R), IPoC or ischaemic pre-conditioning (IPC, as positive cardioprotective intervention) in vivo. Infarct sizes were quantified by plasma troponin I levels 60 minutes after reperfusion. After 7 days, rats were sacrificed and left ventricular tissue was taken for post hoc analysis. The transcriptome was analysed by qRT-PCR and small RNA sequencing. Key findings were verified by immunoblots. I/R increased plasma troponin I levels compared to Sham. IPC reduced troponin I compared to I/R, whereas IPoC produced either excellent protection (IPoC-R) or no protection (IPoC-NR). Twenty-one miRs were up-regulated by I/R and modified by IPoC. qRT-PCR analysis revealed that IPoC-R differed from other groups by reduced expression of arginase-2 and bax, whereas the mitochondrial uncoupling protein (UCP)-2 was induced in IPC and IPoC-R. IPoC-R and IPoC-NR synergistically increased the expression of non-mitochondrial proteins like VEGF and SERCA2a independent of the infarct size. Cardiac function was more closely linked to differences in mitochondrial proteins than on regulation of calcium-handling proteins. In conclusion, healthy rats could not always be protected by IPoC. IPoC-NR displayed an incomplete responsiveness which is reflected by different changes in the mitochondrial transcriptome compared to IPoC-R. This study underlines the importance of mitochondrial proteins for successful long-term outcome.

Definition of hidden drug cardiotoxicity: paradigm change in cardiac safety testing and its clinical implications.

Unexpected cardiac adverse effects are the leading causes of discontinuation of clinical trials and withdrawal of drugs from the market. Since the original observations in the mid-90s, it has been well established that cardiovascular risk factors and comorbidities (such as ageing, hyperlipidaemia, and diabetes) and their medications (e.g. nitrate tolerance, adenosine triphosphate-dependent potassium inhibitor antidiabetic drugs, statins, etc.) may interfere with cardiac ischaemic tolerance and endogenous cardioprotective signalling pathways. Indeed drugs may exert unwanted effects on the diseased and treated heart that is hidden in the healthy myocardium. Hidden cardiotoxic effects may be due to (i) drug-induced enhancement of deleterious signalling due to ischaemia/reperfusion injury and/or the presence of risk factors and/or (ii) inhibition of cardioprotective survival signalling pathways, both of which may lead to ischaemia-related cell death and/or pro-arrhythmic effects. This led to a novel concept of 'hidden cardiotoxicity', defined as cardiotoxity of a drug that manifests only in the diseased heart with e.g. ischaemia/reperfusion injury and/or in the presence of its major comorbidities. Little is known on the mechanism of hidden cardiotoxocity, moreover, hidden cardiotoxicity cannot be revealed by the routinely used non-clinical cardiac safety testing methods on healthy animals or tissues. Therefore, here, we emphasize the need for development of novel cardiac safety testing platform involving combined experimental models of cardiac diseases (especially myocardial ischaemia/reperfusion and ischaemic conditioning) in the presence and absence of major cardiovascular comorbidities and/or cotreatments.

Capsaicin-Sensitive Sensory Nerves and the TRPV1 Ion Channel in Cardiac Physiology and Pathologies.

Cardiovascular diseases, including coronary artery disease, ischemic heart diseases such as acute myocardial infarction and postischemic heart failure, heart failure of other etiologies, and cardiac arrhythmias, belong to the leading causes of death. Activation of capsaicin-sensitive sensory nerves by the transient receptor potential vanilloid 1 (TRPV1) capsaicin receptor and other receptors, as well as neuropeptide mediators released from them upon stimulation, play important physiological regulatory roles. Capsaicin-sensitive sensory nerves also contribute to the development and progression of some cardiac diseases, as well as to mechanisms of endogenous stress adaptation leading to cardioprotection. In this review, we summarize the role of capsaicin-sensitive afferents and the TRPV1 ion channel in physiological and pathophysiological functions of the heart based mainly on experimental results and show their diagnostic or therapeutic potentials. Although the actions of several other channels or receptors expressed on cardiac sensory afferents and the effects of TRPV1 channel activation on different non-neural cell types in the heart are not precisely known, most data suggest that stimulation of the TRPV1-expressing sensory nerves or stimulation/overexpression of TRPV1 channels have beneficial effects in cardiac diseases.

Cardioprotective Effect of Novel Matrix Metalloproteinase Inhibitors.

BACKGROUND: We recently developed novel matrix metalloproteinase-2 (MMP-2) inhibitor small molecules for cardioprotection against ischemia/reperfusion injury and validated their efficacy in ischemia/reperfusion injury in cardiac myocytes. The aim of the present study was to test our lead compounds for cardioprotection in vivo in a rat model of acute myocardial infarction (AMI) in the presence or absence of hypercholesterolemia, one of the major comorbidities affecting cardioprotection. METHODS: Normocholesterolemic adult male Wistar rats were subjected to 30 min of coronary occlusion followed by 120 min of reperfusion to induce AMI. MMP inhibitors (MMPI)-1154 and -1260 at 0.3, 1, and 3 µmol/kg, MMPI-1248 at 1, 3, and 10 µmol/kg were administered at the 25th min of ischemia intravenously. In separate groups, hypercholesterolemia was induced by a 12-week diet (2% cholesterol, 0.25% cholic acid), then the rats were subjected to the same AMI protocol and single doses of the MMPIs that showed the most efficacy in normocholesterolemic animals were tested in the hypercholesterolemic animals. Infarct size/area at risk was assessed at the end of reperfusion in all groups by standard Evans blue and 2,3,5-triphenyltetrazolium chloride (TTC) staining, and myocardial microvascular obstruction (MVO) was determined by thioflavine-S staining. RESULTS: MMPI-1154 at 1 µmol/kg, MMPI-1260 at 3 µmol/kg and ischemic preconditioning (IPC) as the positive control reduced infarct size significantly; however, this effect was not seen in hypercholesterolemic animals. MVO in hypercholesterolemic animals decreased by IPC only. CONCLUSIONS: This is the first demonstration that MMPI-1154 and MMPI-1260 showed a dose-dependent infarct size reduction in an in vivo rat AMI model; however, single doses that showed the most efficacy in normocholesterolemic animals were abolished by hypercholesterolemia. The further development of these promising cardioprotective MMPIs should be continued with different dose ranges in the study of hypercholesterolemia and other comorbidities.

Swiprosin-1/EFhD-2 Expression in Cardiac Remodeling and Post-Infarct Repair: Effect of Ischemic Conditioning.

Swiprosin-1 (EFhD2) is a molecule that triggers structural adaptation of isolated adult rat cardiomyocytes to cell culture conditions by initiating a process known as cell spreading. This process mimics central aspects of cardiac remodeling, as it occurs subsequent to myocardial infarction. However, expression of swiprosin-1 in cardiac tissue and its regulation in vivo has not yet been addressed. The expression of swiprosin-1 was analyzed in mice, rat, and pig hearts undergoing myocardial infarction or ischemia/reperfusion with or without cardiac protection by ischemic pre- and postconditioning. In mouse hearts, swiprosin-1 protein expression was increased after 4 and 7 days in myocardial infarct areas specifically in cardiomyocytes as verified by immunoblotting and histology. In rat hearts, swiprosin-1 mRNA expression was induced within 7 days after ischemia/reperfusion but this induction was abrogated by conditioning. As in cultured cardiomyocytes, the expression of swiprosin-1 was associated with a coinduction of arrestin-2, suggesting a common mechanism of regulation. Rno-miR-32-3p and rno-miR-34c-3p were associated with the regulation pattern of both molecules. Moreover, induction of swiprosin-1 and ssc-miR-34c was also confirmed in the infarct zone of pigs. In summary, our data show that up-regulation of swiprosin-1 appears in the postischemic heart during cardiac remodeling and repair in different species.

Sensory Neuropathy Affects Cardiac miRNA Expression Network Targeting IGF-1, SLC2a-12, EIF-4e, and ULK-2 mRNAs.

BACKGROUND: Here we examined myocardial microRNA (miRNA) expression profile in a sensory neuropathy model with cardiac diastolic dysfunction and aimed to identify key mRNA molecular targets of the differentially expressed miRNAs that may contribute to cardiac dysfunction. METHODS: Male Wistar rats were treated with vehicle or capsaicin for 3 days to induce systemic sensory neuropathy. Seven days later, diastolic dysfunction was detected by echocardiography, and miRNAs were isolated from the whole ventricles. RESULTS: Out of 711 known miRNAs measured by miRNA microarray, the expression of 257 miRNAs was detected in the heart. As compared to vehicle-treated hearts, miR-344b, miR-466b, miR-98, let-7a, miR-1, miR-206, and miR-34b were downregulated, while miR-181a was upregulated as validated also by quantitative real time polymerase chain reaction (qRT-PCR). By an in silico network analysis, we identified common mRNA targets (insulin-like growth factor 1 (IGF-1), solute carrier family 2 facilitated glucose transporter member 12 (SLC2a-12), eukaryotic translation initiation factor 4e (EIF-4e), and Unc-51 like autophagy activating kinase 2 (ULK-2)) targeted by at least three altered miRNAs. Predicted upregulation of these mRNA targets were validated by qRT-PCR. CONCLUSION: This is the first demonstration that sensory neuropathy affects cardiac miRNA expression network targeting IGF-1, SLC2a-12, EIF-4e, and ULK-2, which may contribute to cardiac diastolic dysfunction. These results further support the need for unbiased omics approach followed by in silico prediction and validation of molecular targets to reveal novel pathomechanisms.

Effect of nitric oxide deficiency on the pulmonary PTHrP system.

Nitric oxide (NO) deficiency is common in pulmonary diseases, but its effect on pulmonary remodelling is still controversial. As pulmonary parathyroid hormone-related protein (PTHrP) expression is a key regulator of pulmonary fibrosis and development, the effect of chronic NO deficiency on the pulmonary PTHrP system and its relationship with oxidative stress was addressed. NO bioavailability in adult rats was reduced by systemic administration of L-NAME via tap water. To clarify the role of NO synthase (NOS)-3-derived NO on pulmonary expression of PTHrP, NOS-3-deficient mice were used. Captopril and hydralazine were used to reduce the hypertensive effect of L-NAME treatment and to interfere with the pulmonary renin-angiotensin system (RAS). Quantitative RT-PCR and immunoblot techniques were used to characterize the expression of key proteins involved in pulmonary remodelling. L-NAME administration significantly reduced pulmonary NO concentration and caused oxidative stress as characterized by increased pulmonary nitrite concentration and increased expression of NOX2, p47phox and p67phox. Furthermore, L-NAME induced the pulmonary expression of PTHrP and of its corresponding receptor, PTH-1R. Expression of PTHrP and PTH-1R correlated with the expression of two well-established PTHrP downstream targets, ADRP and PPARγ, suggesting an activation of the pulmonary PTHrP system by NO deficiency. Captopril reduced the expression of PTHrP, profibrotic markers and ornithine decarboxylase, but neither that of PTH-1R nor that of ADRP and PPARγ. All transcriptional changes were confirmed in NOS-3-deficient mice. In conclusion, NOS-3-derived NO suppresses pulmonary PTHrP and PTH-1R expression, thereby modifying pulmonary remodelling.

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.

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.

Renin-Angiotensin-Aldosterone Signaling Inhibitors-Losartan, Enalapril, and Cardosten-Prevent Infarction-induced Heart Failure Development in Rats.

CONTEXT: The activation of the renin-angiotensin-aldosterone system (RAAS) plays an important role in the pathophysiology of congestive heart failure, which is the reason that angiotensin-converting enzyme inhibitors (ACEIs) and angiotensin 2 receptor blockers (ARBs) have become established therapies for heart failure. However, it is still not known whether preventive treatment with losartan or enalapril can reduce symptoms of infarction-induced heart failure. Ultra-low dose (ULD) drug therapy is thought to exert specific activity, with a lower chance of side effects. OBJECTIVES • The research team had hypothesized that preventive treatment with inhibitors of RAAS signaling-losartan, enalapril, and a preparation of a ULD antibody (ie, cardosten), which target the angiotensin type 1 (AT1) receptor-might alleviate pathological hypertrophy and/or functional decline in infarction-induced heart failure. METHODS: The research team treated male Wistar rats orally for 30 d with 20 mg/kg of losartan, 10 mg/kg enalapril, 5 or 7.5 mL/kg of cardosten, or a control solution, started 1 d prior to permanent coronary occlusion. A sham-operated group functioned as a second control group. SETTINGS: The study was conducted at the Department of Biochemistry of the Faculty of Medicine at the University of Szeged in Szeged, Hungary, in cooperation with the Pharmahungary Group, also in Szeged, Hungary, and with OOO "NPF" Materia Medica Holding Ltd in Moscow, Russia. OUTCOME MEASURES: To determine cardiac functional parameters in vivo, the research team inserted a catheter into the left ventricle of the rats and measured the parameters of ventricular pressure, and cardiac output was determined by thermodilution. Morphological parameters were measured after heart isolation in transverse sections by a digital caliper. RESULTS: A total of 30 d after permanent coronary ligation, both losartan and enalapril, significantly decreased mean arterial blood pressure (MABP), attenuated the development of the left-ventricular anterior-wall and septum hypertrophy, and reduced scar thickness compared with the vehicle control group. The deterioration of cardiac output and the increase in total peripheral resistance (TPR) due to coronary ligation were significantly inhibited by both losartan and enalapril. The effects of cardosten were comparable with those of losartan and enalapril on cardiac morphology, left ventricular function, and TPR; however, it did not influence MABP. Moreover, in contrast to losartan and enalapril, cardosten did not decrease the rate of survival. CONCLUSIONS: The study was the first to have demonstrated that preventive treatment with losartan, enalapril, or cardosten can attenuate pathological hypertrophy in infarction-induced heart failure in rats.

Serum lipids and cardiac function correlate with nitrotyrosine and MMP activity in coronary artery disease patients.

AIMS: Peroxynitrite-matrix metalloproteinase (MMP) signalling has been shown to contribute to myocardial ischaemia/reperfusion injury and heart failure and to be influenced by hyperlipidaemia in preclinical models. Therefore, here we investigated the correlation between the markers of peroxynitrite-MMP signalling and hyperlipidaemia in patients with significant coronary stenosis. METHODS: Five minutes before percutaneous coronary intervention (PCI), arterial blood samples were collected from 36 consecutive patients with coronary artery disease (CAD) selected for elective PCI. RESULTS: Serum nitrotyrosine positively correlated with MMP-9 activity (r = 0·54, P = 0·01), but not with MMP-2 activity. Nitrotyrosine positively correlated with total (r = 0·58; P < 0·01) and LDL cholesterol (r = 0·55; P < 0·01), serum triglyceride (r = 0·47; P < 0·05), and creatinine (r = 0·42; P < 0·05) and negatively correlated with HDL cholesterol (r = -0·46; P < 0·05) and with left ventricular ejection fraction (LVEF; r = -0·55; P < 0·05), respectively. MMP-2 activity correlated positively with total (r = 0·55; P < 0·05) and LDL cholesterol (r = 0·45; P < 0·05). In statin-treated patients, a significantly reduced serum nitrotyrosine was found as compared to statin naives; however, MMP activities and serum cholesterol levels were not different. MMP-9 activity correlated with urea nitrogen (r = 0·42; P < 0·05) and LVEF (r = -0·73; P < 0·01). Serum creatinine correlated negatively with LVEF (r = -0·50, P < 0·01). CONCLUSIONS: This is the first demonstration that (i) serum nitrotyrosine correlates with MMP-9 activity, (ii) lipid parameters correlate with nitrotyrosine and MMP-2 activity, (iii) myocardial function correlates with creatinine, nitrotyrosine and MMP-9 activity, and (iv) creatinine correlates with nitrotyrosine and urea nitrogen with MMP-9 activity in patients with CAD. Studying the biomarkers of peroxynitrite-MMP pathway in large prospective trials may reveal their diagnostic avails.

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.

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.

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.

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.

An Observational Study on the Pharmacokinetics of Oseltamivir in Lactating Influenza Patients.

Influenza infection may lead to serious complications in the postpartum period, therefore, oseltamivir treatment in these patients and their breastfed infants is of great importance. However, the pharmacokinetics of oseltamivir in postpartum lactating women with acute influenza infection, and the consequent infant exposure to oseltamivir are still unknown, and these data would help in assessing risk and the need for dose adjustment in breastfed infants. Six lactating women with influenza-like symptoms, at a standard dose of 75 mg oral oseltamivir twice daily for 5 days, were recruited in this phase IV clinical study during the 2011/2012 H1N1 pandemic seasons. Breast milk/colostrum and venous blood samples were taken at multiple timepoints, maternal urine samples were obtained from total output within the 12-hour observational period following the seventh dose of oseltamivir. Oseltamivir phosphate (OP) reached a maximum 69.5 ± 29.4 ng/mL concentration in breast milk, higher than that found in the plasma, and showed elimination within ~ 8 hours. Oseltamivir carboxylate (active metabolite of OP) showed a lower, nearly steady-state concentration in breast milk during the observational period (maximum plasma concentration (Cmax ) = 38.4 ± 12.9 ng/mL). Based on estimated daily milk consumption of exclusively breastfed infants, their calculated daily exposure is < 0.1% of the infant dose of oseltamivir for treatment of influenza as per marketing authorization. Here, we provide the first maternal breast milk pharmacokinetic data for oral multiple-dose oseltamivir in lactating patients with influenza and showed that its concentration in the breast milk is not sufficient to reach a therapeutic dose for breastfed infants.

Identification of New, Translatable ProtectomiRs against Myocardial Ischemia/Reperfusion Injury and Oxidative Stress: The Role of MMP/Biglycan Signaling Pathways.

INTRODUCTION: Ischemic conditionings (ICon) were intensively investigated and several protective signaling pathways were identified. Previously, we have shown the role of matrix metalloproteinases (MMP) in myocardial ischemia/reperfusion injury (MIRI) and the cardioprotective role of biglycan (BGN), a small leucine-rich proteoglycan in vitro. Here, we hypothesized that cardiac MMP and BGN signaling are involved in the protective effects of ICon. METHODS: A reverse target-microRNA prediction was performed by using the miRNAtarget™ 2.0 software to identify human microRNAs with a possible regulatory effect on MMP and BGN, such as on related genes. To validate the identified 1289 miRNAs in the predicted network, we compared them to two cardioprotective miRNA omics datasets derived from pig and rat models of MIRI in the presence of ICons. RESULTS: Among the experimentally measured miRNAs, we found 100% sequence identity to human predicted regulatory miRNAs in the case of 37 porcine and 24 rat miRNAs. Upon further analysis, 42 miRNAs were identified as MIRI-associated miRNAs, from which 24 miRNAs were counter-regulated due to ICons. CONCLUSIONS: Our findings highlight 24 miRNAs that potentially regulate cardioprotective therapeutic targets associated with MMPs and BGN in a highly translatable porcine model of acute myocardial infarction.