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.

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.

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.

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.

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.

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.

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.

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.

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.

Perinatally administered losartan augments renal ACE2 expression but not cardiac or renal Mas receptor in spontaneously hypertensive rats.

Since the identification of the alternative angiotensin converting enzyme (ACE)2/Ang-(1-7)/Mas receptor axis, renin-angiotensin system (RAS) is a new complex target for a pharmacological intervention. We investigated the expression of RAS components in the heart and kidney during the development of hypertension and its perinatal treatment with losartan in young spontaneously hypertensive rats (SHR). Expressions of RAS genes were studied by the RT-PCR in the left ventricle and kidney of rats: normotensive Wistar, untreated SHR, SHR treated with losartan since perinatal period until week 9 of age (20 mg/kg/day) and SHR treated with losartan only until week 4 of age and discontinued until week 9. In the hypertrophied left ventricle of SHR, cardiac expressions of Ace and Mas were decreased while those of AT1 receptor (Agtr1a) and Ace2 were unchanged. Continuous losartan administration reduced LV weight (0.43 ± 0.02; P < 0.05 versus SHR) but did not influence altered cardiac RAS expression. Increased blood pressure in SHR (149 ± 2 in SHR versus 109 ± 2 mmHg in Wistar; P < 0.05) was associated with a lower renal expressions of renin, Agtr1a and Mas and with an increase in ACE2. Continuous losartan administration lowered blood pressure to control levels (105 ± 3 mmHg; P < 0.05 versus SHR), however, only renal renin and ACE2 were significantly up-regulated (for both P < 0.05 versus SHR). Conclusively, prevention of hypertension and LV hypertrophy development by losartan was unrelated to cardiac or renal expression of Mas. Increased renal Ace2, and its further increase by losartan suggests the influence of locally generated Ang-(1-7) in organ response to the developing hypertension in SHRs.

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.

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.

Enalapril decreases cardiac mass and fetal gene expression without affecting the expression of endothelin-1, transforming growth factor ß-1, or cardiotrophin-1 in the healthy normotensive rat.

Angiotensin II can induce cardiac hypertrophy by stimulating the release of growth factors. ACE inhibitors reduce angiotensin II levels and cardiac hypertrophy, but their effects on the healthy heart are largely unexplored. We hypothesized that ACE inhibition decreases left ventricular mass in normotensive animals and that this is associated with altered expression of cardiac fetal genes, growth factors, and endothelial nitric oxide synthase (eNOS). Wistar rats (n = 7 per group) were orally administered with enalapril twice daily for a total daily dose of 5 mg·kg(-1)·d(-1) (ENAP5) or 15 mg·kg(-1)·d(-1) (ENAP15) or vehicle. Systolic blood pressure was measured by the tail-cuff method. Left ventricular expression of cardiac myosin heavy chain-α (MYH6) and -ß (MYH7), atrial natriuretic peptide (ANP), endothelin-1 (ET-1), transforming growth factor ß-1 (TGFß-1), cardiotrophin-1 (CT-1), and renal renin were examined by real-time PCR, and eNOS using Western blot. Blood pressure was decreased only in ENAP15 animals (p < 0.05 vs. Control), whereas left ventricular mass decreased after both doses of enalapril (p < 0.05 vs. Control). MYH7 and ANP were reduced in ENAP15, while no changes in ET-1, TGFß-1, CT-1, and MYH6 mRNA or eNOS protein were observed. Renal renin dose-dependently increased after enalapril treatment. Enalapril significantly decreased left ventricular mass even after 1 week treatment in the normotensive rat. This was associated with a decreased expression of the fetal genes MYH7 and ANP, but not expression of ET-1, CT-1, or TGFß-1.

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.

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.

Pycnogenol improves left ventricular function in streptozotocin-induced diabetic cardiomyopathy in rats.

We studied whether Pycnogenol (PYC) may attenuate the development of experimental streptozotocin-induced diabetic cardiomyopathy in rat. In addition, we aimed to study whether PYC affects cardiac oxidative stress and the protein expression of reactive oxygen species (ROS)-producing molecules (gp91(phox)-containing NADPH oxidase and NO-signalling proteins). Experimental diabetes mellitus was manifested by hyperglycaemia and impaired cardiac function estimated using left ventricular catheterisation in vivo. PYC lowered fasting plasma glucose and normalized basal cardiac function. Excessive oxidative stress in streptozotocin (STZ) hearts, evidenced by 40% increase (P < 0.05) of thiobarbituric acid reactive substances (TBARS) concentration, was associated with increased expression of gp91(phox) (by 75%, P < 0.05), iNOS (by 40%, P < 0.05) and alpha-tubulin (by 49%, P < 0.05), but unchanged expression of eNOS and its alosteric regulators, as compared to CON. PYC failed to affect these expression abnormalities. Our study shows that PYC corrects diabetic cardiac dysfunction, probably by its metabolic and direct radical scavenging activity without affecting the molecular maladaptations of ROS-producing enzymes and cytoskeletal components.