Rapid changes of mRNA expressions of cardiac ion channels affected by Torsadogenic drugs influence susceptibility of rat hearts to arrhythmias induced by Beta-Adrenergic stimulation.

Drug-induced long QT syndrome (LQTS) and Torsades de Pointes (TdP) are serious concerns in drug development. Although rats are a useful scientific tool, their hearts, unlike larger species, usually do not respond to torsadogenic drugs. Consequently, their resistance to drug-induced arrhythmias is poorly understood. Here, we challenged rats with rapid delayed rectifier current (Ikr)-inhibiting antibiotic clarithromycin (CLA), loop diuretic furosemide (FUR) or their combination (CLA + FUR), and examined functional and molecular abnormalities after stimulation with isoproterenol. Clarithromycin and furosemide were administered orally at 12-h intervals for 7 days. To evaluate electrical instability, electrocardiography (ECG) was recorded either in vivo or ex vivo using the Langendorff-perfused heart method under basal conditions and subsequently under beta-adrenergic stimulation. Gene expression was measured using real-time quantitative PCR in left ventricular tissue. Indeed, FUR and CLA + FUR rats exhibited hypokalemia. CLA and CLA + FUR treatment resulted in drug-induced LQTS and even an episode of TdP in one CLA + FUR rat. The combined treatment dysregulated gene expression of several ion channels subunits, including KCNQ1, calcium channels and Na+/K + -ATPase subunits, while both monotherapies had no impact. The rat with recorded TdP exhibited differences in the expression of ion channel genes compared to the rest of rats within the CLA + FUR group. The ECG changes were not detected in isolated perfused hearts. Hence, we report rapid orchestration of ion channel reprogramming of hearts with QT prolongation induced by simultaneous administration of clarithromycin and furosemide in rats, which may account for their ability to avoid arrhythmias triggered by beta-adrenergic stimulation.

Pioglitazone restores phosphorylation of downregulated caveolin-1 in right ventricle of monocrotaline-induced pulmonary hypertension.

BACKGROUND: Caveolin-1 (cav-1) plays a role in pulmonary arterial hypertension (PAH). Monocrotaline (MCT)-induced PAH is characterized by a loss of cav-1 in pulmonary arteries; however, less is known regarding its role in the hypertrophied right ventricle (RV). We aimed to characterize the role of cav-1 and Hsp90 in the RV of MCT-induced PAH and their impact on endothelial nitric oxide synthase (eNOS). Additionally, we focused on restoration of cav-1 expression with pioglitazone administration. METHODS: Male 12-week-old Wistar rats were injected subcutaneously with monocrotaline (60 mg/kg). Selected proteins (cav-1, eNOS, pSer1177eNOS, Hsp90) and mRNAs (cav-1α, cav-1ß, eNOS) were determined in the RV and left ventricle (LV) 4 weeks later. In a separate MCT-induced PAH study, pioglitazone (10 mg/kg/d, orally) administration started on day 14 after MCT. RESULTS: MCT induced RV hypertrophy and lung enlargement. Cav-1 and pTyr14cav-1 were decreased in RV. Caveolin-1α (cav-1α) and caveolin-1ß (cav-1ß) mRNAs were decreased in both ventricles. Hsp90 protein was increased in RV. eNOS and pSer1177eNOS proteins were unchanged in the ventricles. eNOS mRNA was reduced in RV. Pioglitazone treatment increased oxygen saturation and pTyr14cav-1 vs. MCT group. CONCLUSIONS: Restoration of pTyr14cav-1 did not lead to amelioration of the disease, nor did it prevent RV hypertrophy and fibrosis, which was indicated by an increase in Acta2, Nppb, Col3a1, and Tgfß1 mRNA.

Hematocrit-Related Alterations of Circulating microRNA-21 Levels in Heart Failure Patients with Reduced Ejection Fraction: A Preliminary Study.

Aim: Circulating microRNA-21 (miR-21) has been utilized as a diagnostic tool in the assessment of heart failure (HF). Blood constitution may be altered when HF occurs and miR-21 may affect hematopoiesis. Sample hemolysis may influence the determination of circulating miRNAs, challenging the diagnostic use of miRNAs. Methods: We examined the relationship between blood measurements and miR-21 levels in ambulant chronic HF patients with reduced ejection fraction (HFrEF; n = 19). Healthy volunteers (n = 11) served as controls. Serum miR-21 levels were measured through quantitative reverse transcription polymerase chain reaction (RT-qPCR) and we calculated the hemolysis score (H-score). Study was approved by an Institutional Review Board (EK FaF UK 02/2018). Results: MiR-21 serum levels were reduced in HFrEF patients compared with the controls (p < 0.05), without relationship to New York Heart Association class, left ventricular ejection fraction or N-terminal prohormone of brain natriuretic peptide levels. MiR-21 levels decreased markedly in anemic patients, compared with those with normal hematocrits (p < 0.05). We found a significant relationship between miR-21 to hematocrit (p < 0.05) and hemoglobin concentration (p < 0.05). Importantly, we found a correlation between hematocrit and sample H-score (p < 0.05) in the cohort of HFrEF patients; however, there was no correlation between hemolysis and miR-21. Conclusion: Circulating miR-21 levels were decreased in HFrEF patients and hematocrit was identified as a factor associated with this abnormality. This suggests that miR-21 mirrors other characteristics of HFrEF patients rather than the standard identifiers of HF severity and progression.

Isolated downregulation of HCN2 in ventricles of rats with streptozotocin-induced diabetic cardiomyopathy.

BACKGROUND: In spite of disrupted repolarization of diabetic heart, some studies report less tendency of diabetic heart to develop ventricular arrhythmias suggesting effective compensatory mechanism. We hypothesized that myocardial alterations in HCN2 and HCN4 channels occur under hyperglycaemia. METHODS: Diabetes was induced in rats using a single injection of streptozotocin (STZ; 55 mg/kg body weight, i.p.). Basal ECG was measured. Expression of mRNA for HCN channels, potassium channels and microRNA 1 and 133a were measured in ventricular tissues. Protein expression of HCN2 channel isoform was assessed in five different regions of the heart by western blotting. Differentiated H9c2 cell line was used to examine HCN channels expression under hyperglycaemia in vitro. RESULTS: Six weeks after STZ administration, heart rate was reduced, QRS complex duration, QT interval and T-wave were prolonged in diabetic rats compared to controls. mRNA and protein expressions of HCN2 decreased exclusively in the ventricles of diabetic rats. HCN2 expression levels in atria of STZ rats and H9c2 cells treated with excess of glucose were not changed. MicroRNA levels were stable in STZ rat hearts. We found significantly decreased mRNA levels of several potassium channels participating in repolarization, namely Kcnd2 (Ito1), Kcnh2 (IKr), Kcnq1 (IKs) and Kcnj11 (IKATP). CONCLUSIONS: This result together with downregulated HCN2 channels suggest that HCN channels might be an integral part of ventricular electric remodelling and might play a role in cardiac repolarization projected in altered arrhythmogenic profile of diabetic heart.

The tyrosine kinase inhibitor crizotinib influences blood glucose and mRNA expression of GLUT4 and PPARs in the heart of rats with experimental diabetes.

Tyrosine kinases inhibitors (TKIs) may alter glycaemia and may be cardiotoxic with importance in the diabetic heart. We investigated the effect of multi-TKI crizotinib after short-term administration on metabolic modulators of the heart of diabetic rats. Experimental diabetes mellitus (DM) was induced by streptozotocin (STZ; 80 mg·kg-1, i.p.), and controls (C) received vehicle. Three days after STZ, crizotinib (STZ+CRI; 25 mg·kg-1 per day p.o.) or vehicle was administered for 7 days. Blood glucose, C-peptide, and glucagon were assessed in plasma samples. Receptor tyrosine kinases (RTKs), cardiac glucose transporters, and peroxisome proliferator-activated receptors (PPARs) were determined in rat left ventricle by RT-qPCR method. Crizotinib moderately reduced blood glucose (by 25%, P < 0.05) when compared to STZ rats. The drug did not affect levels of C-peptide, an indicator of insulin secretion, suggesting altered tissue glucose utilization. Crizotinib had no impact on cardiac RTKs. However, an mRNA downregulation of insulin-dependent glucose transporter Glut4 in the hearts of STZ rats was attenuated after crizotinib treatment. Moreover, crizotinib normalized Ppard and reduced Pparg mRNA expression in diabetic hearts. Crizotinib decreased blood glucose independently of insulin and glucagon. This could be related to changes in regulators of cardiac metabolism such as GLUT4 and PPARs.

Disease severity-related alterations of cardiac microRNAs in experimental pulmonary hypertension.

Right ventricular (RV) failure is the primary cause of death in pulmonary arterial hypertension (PAH). We hypothesized that heart-relevant microRNAs, that is myomiRs (miR-1, miR-133a, miR-208, miR-499) and miR-214, can have a role in the right ventricle in the development of PAH. To mimic PAH, male Wistar rats were injected with monocrotaline (MCT, 60 mg/kg, s.c.); control group received vehicle. MCT rats were divided into two groups, based on the clinical presentation: MCT group terminated 4 weeks after MCT administration and prematurely terminated group (ptMCT) displaying signs of terminal disease. Myocardial damage genes and candidate microRNAs expressions were determined by RT-qPCR. Reduced blood oxygen saturation, breathing disturbances, RV enlargement as well as elevated levels of markers of myocardial damage confirmed PH in MCT animals and were more pronounced in ptMCT. MyomiRs (miR-1/miR-133a/miR-208a/miR-499) were decreased and the expression of miR-214 was increased only in ptMCT group (P < 0.05). The myomiRs negatively correlated with Fulton index as a measure of RV hypertrophy in MCT group (P < 0.05), whereas miR-214 showed a positive correlation (P < 0.05). We conclude that the expression of determined microRNAs mirrored the disease severity and targeting their pathways might represent potential future therapeutic approach in PAH.

Hepatocyte growth factor plays a particular role in progression of overall cardiac damage in experimental pulmonary hypertension.

Background: HGF/MET pathway may have a role in pulmonary hypertension (PH). However, the link between the pathway and development of target organ damage in PH remains elusive. We aimed to demonstrate the relation between plasma HGF and HGF/MET tissue expressions in affected organs during PH progression. Methods: 12 weeks old male Wistar rats were injected with monocrotaline (MCT, 60 mg/kg, s.c.) to induce PH and sacrificed after 1, 2 and 4 weeks. Controls received saline. mRNA levels of HGF regulatory complex (Hgf, Met, Hgfa, Hai-1, Hai-2) were determined in right and left ventricles (RV, LV), lungs, pulmonary artery and liver by RT-qPCR. HGF protein levels in plasma were analysed by ELISA. Results: PH development was associated with a progressive elevation of HGF plasma levels that correlated with relative RV mass. Furthermore, Hgf mRNA expressions at week 4 were upregulated solely in the cardiac ventricles while being downregulated in a. pulmonalis, lungs and liver. Met and Hai-1/Hai-2 followed a similar pattern and were upregulated in cardiac ventricles, where Hgfa remained unchanged, but downregulated in lungs. Conclusion: We suggest that cardiac overexpression of Hgf might contribute to increased plasma HGF in MCT-induced PH. HGF could be exploited as a cardiospecific biomarker and HGF/MET pathway as a target in drug discovery for PH.

Pegfilgrastim and linagliptin potentiate chemoattraction of Ccr2 and Cd44 stem cells accompanied by alterations of cardiac Hgf, Igf-1 and Mcp-1 in daunorubicin cardiomyopathy.

OBJECTIVE: Daunorubicin (DAU) downregulates cytokines promoting stem cell migration and homing into the heart, reducing cardiac regeneration after anticancer chemotherapy. Pegfilgrastim (PFIL) protects from DAU-induced neutropenia but its cardioprotective potential remains unclear. We tested whether pegfilgrastim and a dipeptidyl peptidase-4 inhibitor linagliptin, potential enhancers of stem cells migration and homing, would improve DAU-cardiomyopathy. METHODS: DAU (7.5 mg/kg, i.v.) was administered to male Wistar rats to induce cardiotoxicity. Pegfilgrastim (100 µg/kg, s.c.) was administered 24h after DAU, and linagliptin was administered orally for 8 weeks (5 mg/kg/day, LINA). Cardiac damage markers (Nppa, Myh6, Myh7, Gp91phox), cytokines (Sdf-1alpha, Mcp-1, Vegf, Hgf, Igf-1), stem cell markers (Cxcr4, Ccr2, Cd34, Cd133, Cd44, Cd105) were determined by qRT-PCR. KEY FINDINGS: Decreased Myh6, elevated Myh7 Nppa, and Gp91phox were not ameliorated by PFIL + LINA. Downregulated expressions of cytokines (Vegf, Sdf-1alpha) and stem cells markers (Cxcr4, Cd34, Cd133, and Cd105) remained decreased after PFIL + LINA. DAU-induced upregulation of Mcp-1, Ccr2 and Cd44 was further potentiated by PFIL + LINA. PFIL + LINA normalised expression of Hgf and Igf-1. CONCLUSIONS: Although PFIL + LINA failed in universal potentiation of stem cells migration and homing, the expression of stem cell markers Ccr2 and Cd44 in the heart potentially increased through the preservation of Hgf, Igf-1 and upregulation of Mcp-1.

Opposite alterations of endothelin-1 in lung and pulmonary artery mirror gene expression of bone morphogenetic protein receptor 2 in experimental pulmonary hypertension.

Aim of the Study: Endothelin-1 (ET-1) overexpression was suggested to play a role in pulmonary hypertension (PH). However, the roles of ET-1 in early stages of PH remain unexplored. We examined the expression of ET-1 and relevant disease progression markers in the pulmonary artery and the lungs during the development of PH induced by monocrotaline (MCT). Material and Methods: Male 12-weeks-old Wistar rats were administered with MCT (60 mg/kg, s.c.) or saline (CON). We measured right ventricular pressure (RVP) by catheterization under tribromoethanol anesthesia; hemoglobin oxygen saturation, breathing rate were measured by pulse oximetry in conscious animals. Rats were sacrificed 1, 2 or 4 weeks after MCT. mRNA levels of ET-1, its receptors, inflammatory markers IL-1beta, TNFalpha, IL-6 and genes related to VSMC proliferation or lung damage (Bmpr2, nestin, Pim1, PAI-1, TGFbeta-1) were analyzed by RT-qPCR. Results: RVP and breathing rate increased and hemoglobin oxygen saturation decreased after MCT only at week 4. Lung weight was increased at all time points. ET-1 was upregulated in the pulmonary artery at weeks 1 and 4, while being clearly suppressed in the lungs at all times. Bone morphogenetic protein receptor 2 followed a similar pattern to ET-1. PAI-1 markedly increased in the MCT lungs (but not pulmonary artery) from week 1 to 4. Nestin peaked at week 2 in both tissues. TGFbeta-1 increased in both tissues at week 4. ET-1 expression did not correlate with other genes, however, Bmpr2 tightly negatively correlated with PAI-1 in the lungs, but not pulmonary artery of MCT groups. Conclusions: ET-1 overexpression in the pulmonary artery preceded development of PH, but it was clearly and unexpectedly downregulated in the lungs of monocrotaline-treated rats and showed no correlation to disease progression markers. We speculate that endothelin-1 may play opposing roles in the lungs vs pulmonary artery in monocrotaline-induced PH.

mRNA levels of circadian clock components Bmal1 and Per2 alter independently from dosing time-dependent efficacy of combination treatment with valsartan and amlodipine in spontaneously hypertensive rats.

Chronopharmacological effects of antihypertensives play a role in the outcome of hypertension therapy. However, studies produce contradictory findings when combination of valsartan plus amlodipine (VA) is applied. Here, we hypothesized different efficacy of morning versus evening dosing of VA in spontaneously hypertensive rats (SHR) and the involvement of circadian clock genes Bmal1 and Per2. We tested the therapy outcome in short-term and also long-term settings. SHRs aged between 8 and 10 weeks were treated with 10 mg/kg of valsartan and 4 mg/kg of amlodipine, either in the morning or in the evening with treatment duration 1 or 6 weeks and compared with parallel placebo groups. After short-term treatment, only morning dosing resulted in significant blood pressure (BP) control (measured by tail-cuff method) when compared to placebo, while after long-term treatment, both dosing groups gained similar superior results in BP control against placebo. However, mRNA levels of Bmal1 and Per2 (measured by RT-PCR) exhibited an independent pattern, with similar alterations in left and right ventricle, kidney as well as in aorta predominantly in groups with evening dosing in both, short-term and also long-term settings. This was accompanied by increased cardiac mRNA expression of plasminogen activator inhibitor-1. In summary, morning dosing proved to be advantageous due to earlier onset of antihypertensive action; however, long-term treatment was demonstrated to be effective regardless of administration time. Our findings also suggest that combination of VA may serve as an independent modulator of circadian clock and might influence disease progression beyond the primary BP lowering effect.

Downregulation of myogenic microRNAs in sub-chronic but not in sub-acute model of daunorubicin-induced cardiomyopathy.

Cardiac muscle-related microRNAs play important roles in cardiac development and disease by translational silencing of mRNAs, the dominant mechanism of microRNA action. To test whether they could be involved in daunorubicin-associated cardiomyopathy (DACM), we determined expression patterns of myomiRs in two distinct models of DACM. We used 10-12 weeks old male Wistar rats. In the sub-acute model, rats were administered with six doses of daunorubicin (DAU-A, 3 mg/kg, i.p., every 48 h). Rats were sacrificed two days after the last dose. In the sub-chronic model, anaesthetized rats were administered a single dose of daunorubicin (15 mg/kg, i.v., DAU-C). Age-matched controls (CON) received vehicle. Rats were sacrificed eight weeks later. Left ventricular (LV) functions (LV pressure, rate of pressure development, +dP/dt and decline, -dP/dt) were measured using left ventricular catheterization. Expressions of myomiRs (miR-208a, miR-499, miR-1 and miR-133a), markers of cardiac failure (atrial and brain natriuretic peptides genes; Nppa and Nppb) and myosin heavy chain genes (Myh6, Myh7, Myh7b) in cardiac tissue were determined by RT-PCR. Protein expression of gp91phox NADPH oxidase subunit was detected by immunoblotting. Both DAU groups exhibited a similar depression of LV function, and LV weight reduction, accompanied by an upregulation of natriuretic peptides, and a decrease of Myh6 to total Myh ratio (-18% in DAU-A and - 25% in DAU-C, as compared to controls; both P < 0.05). DAU-C, but not DAU-A rats had a 35% mortality rate and exhibited a significantly increased gp91phox expression (DAU-C: 197 ± 33 versus CON-C: 100 ± 11; P < 0.05). Interestingly, myomiRs levels were only reduced in DAU-C compared to CON-C (miR-208: -45%, miR-499: -30%, miR-1: -29%, miR- and miR133a: -25%; all P < 0.05) but were unaltered in DAU-A. The lack of myomiRs expression, particularly in sub-chronic model, suggests the loss of control of myomiRs network on late progression of DACM. We suppose that the poor inhibition of mRNA targets might contribute to chronic DACM.

Ramipril restores PPARß/δ and PPARγ expressions and reduces cardiac NADPH oxidase but fails to restore cardiac function and accompanied myosin heavy chain ratio shift in severe anthracycline-induced cardiomyopathy in rat.

We hypothesized that peroxisome proliferator-activated receptors (PPARs) might be involved in a complex protective action of ACE inhibitors (ACEi) in anthracyclines-induced cardiomyopathy. For purpose of study, we compared effects of ramipril on cardiac dysfunction, cardiac failure markers and PPAR isoforms in moderate and severe chronic daunorubicin-induced cardiomyopathy. Male Wistar rats were administered with a single intravenous injection of daunorubicin: 5mg/kg (moderate cardiomyopathy), or 15mg/kg (severe cardiomyopathy) or co-administered with daunorubicin and ramipril (1mg/kg/d, orally) or vehicle for 8 weeks. Left ventricular function was measured invasively under anesthesia. Cardiac mRNA levels of heart failure markers (ANP, Myh6, Myh7, Myh7b) and PPARs (alpha, beta/delta and gama) were measured by qRT-PCR. Protein expression of NADPH subunit (gp91phox) was measured by Western blot. Moderate cardiomyopathy exhibited only minor cardiac dysfunction what was corrected by ramipril. In severe cardiomyopathy, hemodynamic dysfunction remained unaltered upon ramipril although it decreased the significantly up-regulated cardiac ANP mRNA expression. Simultaneously, while high-dose daunorubicin significantly decreased PPARbeta/delta and PPARgama mRNA, ramipril normalized these abnormalities. Similarly, ramipril reduced altered levels of oxidative stress-related gp91phox. On the other hand, ramipril was unable to correct both the significantly decreased relative abundance of Myh6 and increased Myh7 mRNA levels, respectively. In conclusion, ramipril had a protective effect on cardiac function exclusively in moderate chronic daunorubicin-induced cardiomyopathy. Although it normalized abnormal PPARs expression and exerted also additional protective effects also in severe cardiomyopathy, it was insufficient to influence impaired cardiac function probably because of a shift in myosin heavy chain isoform content.

Daunorubicin Down-Regulates the Expression of Stem Cell Markers and Factors Involved in Stem Cell Migration and Homing in Rat Heart in Subchronic but not Acute Cardiomyopathy.

We tested the hypothesis that daunorubicin (DAU) cardiotoxicity alters expression of cytokines involved in stem cell migration and homing. Male Wistar rats were treated with daunorubicin to induce acute DAU cardiomyopathy (6 × 3 mg/kg, i.p., every 48 hr, DAU-A) or subchronic DAU cardiomyopathy (15 mg/kg, i.v., DAU-C). The left ventricle was catheterized. The animals were killed 48 hr (DAU-A) and 8 weeks (DAU-C) after the last dose of DAU. Expression of foetal genes (Nppa, Nppb), isomyosins (Myh6, Myh7), sources of oxidative stress (Abcb8, gp91phox), cytokines (Sdf-1, Cxcr4, Scf, Vegf, Hgf, Igf-1), markers of cardiac progenitor (c-kit, Atnx-1), endothelial progenitor (CD34, CD133) and mesenchymal (CD44, CD105) stem cells were determined by qRT-PCR in left ventricular tissue. Reduced body-weight, decreased left ventricular weight and function, and elevated Nppa, Nppb, Myh7 were observed in both models. Myh6 decreased only in DAU-C, which had a 35% mortality. Up-regulated gp91phox and down-regulated Abcb8 in DAU were present only in DAU-C where we observed markedly decreased expressions of Scf and Vegf as well as expressions of stem cell markers. Down-regulation of cytokines and stem cell markers may reflect impaired chemotaxis, migration and homing of stem cells and tissue repair in the heart in subchronic but not acute model of DAU cardiomyopathy.

Unbalanced upregulation of ryanodine receptor 2 plays a particular role in early development of daunorubicin cardiomyopathy.

Calcium release channel on the sarcoplasmic reticulum of cardiomyocytes (ryanodine receptor type 2, RyR2) plays a critical role in the regulation of calcium and was identified as a crucial factor for development of chronic anthracycline cardiomyopathy. Its early stages are less well described although these determine the later development. Hence, we tested the effect of repeated, short-term anthracycline (daunorubicin) administration on cardiac performance, cardiomyocyte function and accompanied changes in calcium regulating proteins expression. Ten-twelve weeks old male Wistar rats were administered with 6 doses of daunorubicin (DAU, 3 mg/kg, i.p., every 48 h), controls (CON) received vehicle. Left ventricular function (left ventricular pressure, LVP; rate of pressure development, +dP/dt and decline, -dP/dt) was measured using left ventricular catheterization under tribromethanol anaesthesia (15 ml/kg b.w.). Cell shortening was measured in enzymatically isolated cardiomyocytes. The expressions of RyR2 and associated intracellular calcium regulating proteins, cytoskeletal proteins (alpha-actinin, alpha-tubul in) as well as oxidative stress regulating enzymes (gp91phox, MnSOD) were detected in ventricular tissue samples using immunoblotting. mRNA expressions of cardiac damage markers (Nppa and Nppb, atrial and brain natriuretic peptides; Myh6, Myh7 and Myh7b, myosin heavy chain alpha and beta) were detected using RT-PCR. Thiobarbituric acid reactive substances concentration was measured to estimate oxidative stress. DAU rats exhibited significantly depressed left ventricular features (LVP by 14%, +dP/dt by 36% and -dP/dt by 30%; for all P<0.05), in line with concomitant increase in Nppa and Nppb gene expressions (3.23- and 2.18-fold, for both P<0.05), and a 4.34-fold increase in Myh7 (P<0.05). Controversially, we observed increased cell shortening of isolated cardiac cells by 31% (p<0.05). DAU administration was associated with a twofold upregulation of RyR2 (P<0.05), but not of other examined Ca(2+) regulating proteins remained. In addition, we observed a significant reduction in alpha-tubulin (by 46% when compared to CON P<0.05). Indicators of oxidative injury were unaffected. In conclusion, unbalanced RyR2 overexpression plays a particular role in early development of daunorubicin cardiomyopathy characterized by discrepant in situ versus in vitro cardiac performance.

l-Arginine Attenuates Cardiac Dysfunction, But Further Down-Regulates α-Myosin Heavy Chain Expression in Isoproterenol-Induced Cardiomyopathy.

In view of previously reported increased capacity for nitric oxide production, we suggested that l-arginine (ARG), the nitric oxide synthase (NOS) substrate, supplementation would improve cardiac function in isoproterenol (ISO)-induced heart failure. Male Wistar rats were treated with ISO for 8 days (5 mg/kg/day, i.p.) or vehicle. ARG was given to control (ARG) and ISO-treated (ISO+ARG) rats in water (0.4 g/kg/day). ISO administration was associated with 40% mortality, ventricular hypertrophy, decreased heart rate, left ventricular dysfunction, fibrosis and ECG signs of ischaemia. RT-PCR showed increased mRNA levels of cardiac hypertrophy marker atrial natriuretic peptide, but not BNP, decreased expression of myosin heavy chain isoform MYH6 and unaltered expression of pathological MYH7. ISO increased the protein levels of endothelial nitric oxide synthase, but at the same time it markedly up-regulated mRNA and protein levels of gp91phox, a catalytical subunit of superoxide-producing NADPH oxidase. Fibrosis was markedly increased by ISO. ARG treatment moderately ameliorated left ventricular dysfunction, but was without effect on cardiac hypertrophy and fibrosis. Combination of ISO and ARG led to a decrease in cav-1 expression, a further increase in MYH7 expression and a down-regulation of MYH6 that inversely correlated with gp91phox mRNA levels. Although ARG, at least partially, improved ISO-impaired basal left ventricular systolic function, it failed to reduce cardiac hypertrophy, fibrosis, oxidative stress and mortality. The protection of contractile performance might be related to increased capacity for nitric oxide production and the up-regulation of MYH7 which may compensate for the marked down-regulation of the major MYH6 isoform.

Upregulation of SERCA2a following short-term ACE inhibition (by enalaprilat) alters contractile performance and arrhythmogenicity of healthy myocardium in rat.

Chronic angiotensin-converting enzyme inhibitor (ACEIs) treatment can suppress arrhythmogenesis. To examine whether the effect is more immediate and independent of suppression of pathological remodelling, we tested the antiarrhythmic effect of short-term ACE inhibition in healthy normotensive rats. Wistar rats were administered with enalaprilat (ENA, i.p., 5 mg/kg every 12 h) or vehicle (CON) for 2 weeks. Intraarterial blood pressure in situ was measured in A. carotis. Cellular shortening was measured in isolated, electrically paced cardiomyocytes. Standard 12-lead electrocardiography was performed, and hearts of anaesthetized open-chest rats were subjected to 6-min ischemia followed by 10-min reperfusion to examine susceptibility to ventricular arrhythmias. Expressions of calcium-regulating proteins (SERCA2a, cardiac sarco/endoplasmic reticulum Ca(2+)-ATPase; CSQ, calsequestrin; TRD, triadin; PLB, phospholamban; Thr(17)-PLB-phosphorylated PLB at threonine-17, FKBP12.6, FK506-binding protein, Cav1.2-voltage-dependent L-type calcium channel alpha 1C subunit) were measured by Western blot; mRNA levels of L-type calcium channel (Cacna1c), ryanodine receptor (Ryr2) and potassium channels Kcnh2 and Kcnq1 were measured by qRT-PCR. ENA decreased intraarterial systolic as well as diastolic blood pressure (by 20%, and by 31%, respectively, for both P < 0.05) but enhanced shortening of cardiomyocytes at basal conditions (by 34%, P < 0.05) and under beta-adrenergic stimulation (by 73%, P < 0.05). Enalaprilat shortened QTc interval duration (CON 78 ± 1 ms vs. ENA 72 ± 2 ms; P < 0.05) and significantly decreased the total duration of ventricular fibrillations (VF) and the number of VF episodes (P < 0.05). Reduction in arrhythmogenesis was associated with a pronounced upregulation of SERCA2a (CON 100 ± 20 vs. ENA 304 ± 13; P < 0.05) and complete absence of basal Ca(2+)/calmodulin-dependent phosphorylation of PLB at Thr(17). Short-term ACEI treatment can provide protection against I/R injury-induced ventricular arrhythmias in healthy myocardium, and this effect is associated with increased SERCA2a expression.

Enalaprilat increases PPARß/δ expression, without influence on PPARα and PPARγ, and modulate cardiac function in sub-acute model of daunorubicin-induced cardiomyopathy.

Anthracycline therapy is limited by a cardiotoxicity that may eventually lead to chronic heart failure which is thought to be prevented by ACE inhibitors (ACEi). However, the protective effect of ACEi in early stages of this specific injury remains elusive. Activated nuclear transcription factors peroxisome proliferator-activated receptors (PPAR) regulate cellular metabolism, but their involvement in anthracycline cardiomyopathy has not been investigated yet. For this purpose, Wistar rats were administered with daunorubicin (i.p., 3 mg/kg, in 48 h intervals) or co-administered with daunorubicine and enalaprilat (i.p., 5 mg/kg in 12 h intervals). Control animals received vehicle. Left ventricular function was measured invasively under anesthesia. Cell-shortening was measured by videomicroscopy in isolated cardiomyocytes. Expression of PPARs mRNA in cardiac tissue was measured by Real-Time PCR. Although the hemodynamic parameters of daunorubicin-treated rats remained altered upon ACEi co-administration, ACEi normalized daunorubicin-induced QT prolongation. On cellular level, ACEi normalized altered basal and isoproterenol-stimulated cardiac cell shortening in daunorubicine-treated group. Moreover, anthracycline administration significantly up-regulated heart PPARα mRNA and its expression remained increased after ACEi co-administration. On the other hand, the expression of cardiac PPARß/δ was not altered in anthracycline-treated animals, whereas co-administration of ACEi increased its expression. Conclusively, effect of ACEi can be already detected in sub-acute phase of anthracycline-induced cardiotoxicity. Altered expression of heart PPARs may suggest these nuclear receptors as a novel target in anthracycline cardiomyopathy.