Adding SGLT2 Cotransporter Inhibitor to PPARγ Activator Does Not Provide an Additive Effect in the Management of Diabetes-Induced Vascular Dysfunction.

INTRODUCTION: Endothelial dysfunction (ED) plays a key role in the pathogenesis of diabetic vascular complications. In monotherapy, dapagliflozin (Dapa) as well as pioglitazone (Pio) prevent the progression of target organ damage in both type 1 (T1DM) and type 2 diabetes. We investigated whether the simultaneous PPAR-γ activation and SGLT2 cotransporter inhibition significantly alleviate ED-related pathological processes and thus normalize vascular response in experimental T1DM. METHODS: Experimental diabetes was induced by streptozotocin (STZ; 55 mg/kg, i.p.) in Wistar rats. Dapa (10 mg/kg), Pio (12 mg/kg), or their combination were administrated to the STZ rats orally. Six weeks after STZ administration, the aorta was excised for functional studies and real-time qPCR analysis. RESULTS: In the aorta of diabetic rats, impaired endothelium-dependent and independent relaxation were accompanied by the imbalance between vasoactive factors (eNos, Et1) and overexpression of inflammation (Tnfα, Il1b, Il6, Icam, Vcam) and oxidative stress (Cybb) markers. Pio monotherapy normalized response to vasoactive substances and restored balance between Et1-eNos expression, while Dapa treatment was ineffective. Nevertheless, Dapa and Pio monotherapy significantly reverted inflammation and oxidative stress markers to normal values. The combination treatment exhibited an additive effect in modulating Il6 expression, reaching the effect of Pio monotherapy in other measured parameters. CONCLUSION: Particularly, Pio exerts a vasoprotective character when used in monotherapy. When combined with Dapa, it does not exhibit an expected additive effect within modulating vasoreactivity or oxidative stress, though having a significant influence on IL6 downregulation.

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.

Physical activity enhances fecal lactobacilli in rats chronically drinking sweetened cola beverage.

Overweight and obesity have been linked with increased intake of sugar-sweetened beverages. On the other hand, physical activity has been known to lead to weight loss. Therefore, we hypothesized that exercise might influence the Lactobacillus population in fecal microbiota as their changed abundance is often associated with shifts in the physical activity and diet. In our experiment, Wistar rats were allocated into groups with normal feed or added sugar-sweetened beverages with or without access to a running wheel. Interestingly, only a combination of physical activity and sweetened beverage intake was associated with a significant increase in fecal lactobacilli abundance, suggesting a connection between exercise and a rise in lactobacilli abundance. Moreover, physical activity has improved weight-related parameters and led to increased plasma and mRNA adiponectin levels. Ghrelin and leptin plasma levels were unaltered. Taken together, our results demonstrate that effect of physical activity on adiposity even during unhealthy feeding patterns is accompanied by increased lactobacilli abundance in the fecal microbiota population.

Analysis of necroptosis and its association with pyroptosis in organ damage in experimental pulmonary arterial hypertension.

In this study, a role of cell loss due to necroptosis and its linkage with pyroptosis in organ damage under the conditions of pulmonary arterial hypertension (PAH) was examined. Monocrotaline (MCT) was used to induce PAH in Wistar rats, and depending on the severity of the disease progression, they were further divided into two subgroups: MCT group-sacrificed 4 weeks after MCT administration and ptMCT group-prematurely sacrificed due to rapid deterioration in vital functions (on Day 24,11 ± 0,7). The elevation of respiratory rate and right ventricular (RV) hypertrophy were more evident in ptMCT group, while the heart rate and cardiac haemodynamic stress markers were comparably higher in both diseased groups. Detailed immunoblotting analysis revealed that the upregulation of pThr231 /Ser232 -RIP3 proceeded into necroptosis execution in the RVs, unlike in the lungs of both PAH stages. The elevated pulmonary pThr231 /Ser232 -RIP3 levels in both PAH subgroups were associated rather with GSDMD-mediated pyroptosis. On the contrary, other inflammasome forms, such as AIM2 and NLRC4, were higher in the RV, unlike in the lungs, of diseased groups. The PAH-induced increase in the plasma RIP3 levels was more pronounced in ptMCT group, and positively correlated with RV hypertrophy, but not with haemodynamic stress. Taken together, we indicated for the first time that pThr231 /Ser232 -RIP3 upregulation resulting in two different necrosis-like cell death modes might underlie the pathomechanisms of PAH and that the plasma RIP3 might serve as an additional diagnostic and prognostic marker of cardiac injury under these conditions.

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.

Dapagliflozin elevates plasma high-density lipoprotein levels and influences visceral fat gene expression in streptozotocin-induced diabetes mellitus.

OBJECTIVES: Dapagliflozin (Dapa) could potentially be used to treat type 1 diabetes mellitus. We tested the hypothesis that it would influence blood lipid levels and visceral fat accumulation in a rodent diabetic model. METHODS: We used three groups of male Wistar rats: Controls, streptozotocin (STZ)-treated rats and STZ-treated orally with Dapa (STZ+Dapa), 10 mg/kg/day for six weeks. Blood glucose and serum lipids levels were determined. Plasma levels of lipases (hormone-sensitive lipase, HSL and lipoprotein lipase, LPL), adipokines (leptin and adiponectin) and proinflammatory cytokines [tumour necrosis factor-alpha (TNFα) and interleukin-6 (IL-6)] were determined by ELISA assays. mRNA levels in the perirenal fat were determined by real-time PCR. KEY FINDINGS: Dapa suppressed STZ-related hyperglycemia by 20% (P < 0.05) and increased serum HDL when compared to the controls and the STZ-only treated rats (both P < 0.05). STZ treatment caused elevations of other serum lipids that were resistant to Dapa treatment. Dapa treatment also increased both plasma and visceral fat mRNA levels of leptin, LPL and IL-6, while decreasing plasma and fat expressions of HSL and TNFα compared to the STZ-only treated rats (all P < 0.05). CONCLUSIONS: Our results suggest that Dapa, in addition to its antidiabetic effect, also influences the function of adipose tissue which could be beneficial in the treatment of diabetes.

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.

Effects of inorganic nitrate in a rat model of monocrotaline-induced pulmonary arterial hypertension.

The nitrate-nitrite-nitric oxide (NO) pathway represents an alternative source of NO generation, which is independent of NO synthase and potentiated by hypoxia. Augmentation of this pathway by dietary nitrate has proven favourable effects in several cardiovascular disease models. However, less is known regarding its potential value in pulmonary arterial hypertension (PAH). The aim of this study was to assess the effects of oral inorganic nitrate administration in monocrotaline (MCT)-induced PAH. Male 12-week-old Wistar rats were injected subcutaneously with monocrotaline (MCT, 60 mg/kg). Nitrate treatment (0.3 or 1 mmol/kg/d; drinking water) commenced on day 12 following the MCT injection and continued for 16 days. Nitrate administration did not attenuate right ventricular (RV) hypertrophy, increased lung weight and up-regulated mRNA expression of brain natriuretic peptide. Plasma nitrate and nitrite levels were significantly increased as well as lung nitrate level, whereas nitrite lung level was decreased following nitrate treatment (1 mmol/kg/d). MCT-induced PAH resulted in an increased MnSOD protein level, which was not observed following nitrate treatment. MCT-associated up-regulation of nNOS in the lung appeared to be dose-dependently prevented by nitrate treatment. Western blot analysis did not reveal any differences in eNOS, iNOS, XO or gp91phox expression in the lungs among the groups. In conclusion, nitrate treatment did not significantly attenuate pathological RV and lung remodelling in the rat MCT model of PAH. The suppression of MnSOD and nNOS expression by nitrate could be interpreted as reduced demand of endogenous antioxidant defence in this model.

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.

High glucose induces HGF-independent activation of Met receptor in human renal tubular epithelium.

CONTEXT: The role of hepatocyte growth factor (HGF) in diabetic kidney damage remains controversial. OBJECTIVE: To test the hypothesis that high glucose levels activate pathways related to HGF and its receptor Met and that this could participate in glucose-induced renal cell damage. MATERIALS AND METHODS: HK2 cells, a human proximal tubule epithelial cell line, were stimulated with high glucose for 48 hours. Levels of pMet/Met, pEGFR/EGFR, pSTAT3/STAT3, pAkt/Akt and pERK1/2/ERK1/2 were studied by immunoblotting. Absence of HGF was verified by qRT-PCR and ELISA. RESULTS: High glucose level activated Met and its downstream pathways STAT3, Akt and ERK independently of HGF. High glucose induced an integrin ligand fibronectin. HGF-independent Met phosphorylation was prevented by inhibition of integrin α5ß1, Met inhibitor crizotinib, Src inhibitors PP2 and SU5565, but not by EGFR inhibitor AG1478. High glucose increased the expression of TGFß-1, CTGF and the tubular damage marker KIM-1 and increased apoptosis of HK2 cells, effects inhibited by crizotinib. CONCLUSION: High glucose activated Met receptor in HK2 cells independently of HGF, via induction of integrin α5ß1 and downstream signaling. This mode of Met activation was associated with tubular cell damage and apoptosis and it may represent a novel pathogenic mechanism and a treatment target in diabetic nephropathy.

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.

Local and systemic renin-angiotensin system participates in cardiopulmonary-renal interactions in monocrotaline-induced pulmonary hypertension in the rat.

Renin-angiotensin system (RAS) is one of the pathophysiological mechanisms in heart failure. Recently, involvement of the kidney in the disease progression has been proposed in patients with pulmonary arterial hypertension (PAH). We hypothesized that local and systemic RAS could be the central regulators of cardiopulmonary-renal interactions in experimental monocrotaline-induced pulmonary hypertension (PH) in rats. Male 12-week-old Wistar rats were injected subcutaneously with monocrotaline (60 mg/kg). The experiment was terminated 4 weeks after monocrotaline administration. Using RT-PCR, we measured the expression of RAS-related genes in right and left ventricles, lungs and kidneys, together with indicators of renal dysfunction and damage. We observed a significantly elevated expression of angiotensin-converting enzyme (ACE) in both left and right ventricles and kidneys (P < 0.05), but a significantly decreased ACE in the lungs (P < 0.05). Kidneys showed a significant 2.5-fold increase in renin mRNA (P < 0.05) along with erythropoietin, TGFß1, COX-2, NOS-1 and nephrin. Expression of erythropoietin correlated inversely with hemoglobin oxygen saturation and positively with renin expression. In conclusion, monocrotaline-induced PH exhibited similar alterations of ACE expression in the left and right ventricles, and in the kidney, in contrast to the lungs. Increased renal renin was likely a consequence of renal hypoxia/hypoperfusion, as was increased renal erythropoietin expression. Alterations in RAS in the monocrotaline model are probably a result of hypoxic state, and while they could serve as a compensatory mechanism at a late stage of the disease, they could be viewed also as an indicator of multiorgan failure in PAH.

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.