E. Malikova

Ramipril restores PPARβ/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 (60xa0mg/kg). The experiment was terminated 4xa0weeks 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 (Pxa0<xa00.05), but a significantly decreased ACE in the lungs (Pxa0<xa00.05). Kidneys showed a significant 2.5-fold increase in renin mRNA (Pxa0<xa00.05) along with erythropoietin, TGFβ1, 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.

INCREASED HEPATOCYTE GROWTH FACTOR IN MONOCROTALINE-INDUCED PULMONARY HYPERTENSION IN RATS

Objective: Right-heart catheterization remains the only reliable diagnostic method of right ventricular damage in pulmonary hypertension (PH) and search for a specific biomarker still continues. The aim of this project is to evaluate the significance of plasma levels of hepatocyte growth factor (HGF) and related mRNA expressions in rat model of PH. Design and method: To induce pulmonary hypertension, 12 weeks old male Wistar rats were subcutaneously injected with monocrotaline (60u200amg/kg). They were divided into groups based on experiment duration (1, 2 and 4 weeks) and type of treatment (CON, MCT). Right ventricular pressure was measured in anesthesized rats during catheterisation. Samples of left ventricles (LV), right ventricles (RV), a. pulmonalis, lungs, liver were analysed by RT-qPCR and plasma by ELISA. Results: MCT-induced PH was characterized by significant progressive increase of right ventricular pressure (2-fold, pu200a<u200a0,05), breathing frequency (by 40%, pu200a<u200a0,05) and decrease of oxygen saturation (by 3%, pu200a<u200a0,05). These were accompanied by increased RV and lungs weights (by 70%, pu200a<u200a0,05) while weights of other measured organs remained unaffected. In the fourth week after PH induction in MCT group, RV as well as LV mRNA levels of HGF were significantly increased (by 60%, pu200a<u200a0.05) and significantly reduced in a. pulmonalis, lungs and liver (by 20%, pu200a<u200a0.05). This was accompanied by an identical pattern of MET receptor expression except for no change in a. pulmonalis and liver. Finally, plasma levels of HGF were significantly elevated (6-fold, pu200a<u200a0.05) in MCT group when compared to controls after 2 as well as 4 weeks of PH induction. Conclusions: During the progress of pulmonary hypertension, plasma levels of HGF rise and gene expression of HGF is enhanced selectively in heart ventricles. Consequently, the source of elevated levels of plasma HGF originates from the heart and therefore can present a potential specific biomarker of PH.

SERUM NEPRILYSIN CONCENTRATION AS A DIAGNOSTICS TOOL IN HEART FAILURE PATIENTS IN ROUTINE CLINICAL PRACTICE

Objective: Neprilysin is a zinc metallopeptidase that cleaves and inactivates several signalling peptides and peptide hormones including natriuretic peptides (NPs). Plasma concentration of NT-proBNP (which is not cleaved by neprilysin) is one of principal diagnostic tests in heart failure (HF) diagnostics. We hypothesized that neprilysin levels might provide additional diagnostic performance in treated chronic HF patients when a mineralocorticoid antagonist (MR) is added. Design and method: Patients treated for chronic HF with addition of MR (duration of therapy 37 months in average) because still symptomatic following angiotensin-converting enzyme inhibitor (or angiotensin receptor blocker) and beta-blocker therapy were enrolled (nu200a=u200a19) and compared to healthy individuals (nu200a=u200a11). Besides standard diagnostic tools (left ventricular ejection fraction - LVEF, laboratory biochemical diagnostic tests, NT-proBNP concentrations), we analysed serum levels of neprilysin by using commercially available ELISA kit. Additionally, we tested the diagnostic performance of other experimental markers including hepatocyte growth factor, stromal cell derived factor-1, angiotensin (1–7) and angiotensin II by using ELISA kits and cardiospecific microRNAs (miR-208a and miR-208b) by using qRT-PCR technique. Results: Serum neprilysin concentrations were significantly elevated in HF patients (5.56u200a±u200a1.25u200ang/ml) when compared to controls (2.43u200a±u200a0.70u200ang/ml; Pu200a<u200a0.05) while none of the other tested markers was able to discriminate between healthy controls and HF patients. Moreover, HF patients with LVEF equal or less than 35% (nu200a=u200a10) tended to have higher neprilysin concentration (6.03u200a±u200a1.29u200ang/ml) when compared to those with LVEFu200a>u200a35% (5.04u200a±u200a1.47u200ang/ml; nu200a=u200a9; NS) but we found a lack of relationship between neprilysin levels and clinical markers of cardiac dysfunction in overall group of HF patients. Conclusions: Conclusively, neprilysin concentrations were significantly elevated in treated HF when compared to controls and tended to be higher in HF patients with LVEF equal or less than 35% when compared to patients with LVEFu200a>u200a35%. In contrast to other experimental markers, neprilysin levels showed certain diagnostic performance but its prognostic information must be proven in further studies.

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, 3xa0mg/kg, i.p., every 48xa0h). Rats were sacrificed two days after the last dose. In the sub-chronic model, anaesthetized rats were administered a single dose of daunorubicin (15xa0mg/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 −u200925% in DAU-C, as compared to controls; both Pu2009<u20090.05). DAU-C, but not DAU-A rats had a 35% mortality rate and exhibited a significantly increased gp91phox expression (DAU-C: 197u2009±u200933 versus CON-C: 100u2009±u200911; Pu2009<u20090.05). 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 Pu2009<u20090.05) 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.

[PP.22.02] EFFECTS OF NITRATE THERAPY IN MONOCROTALINE-INDUCED PULMONARY HYPERTENSION IN RATS

Objective: Nitric oxide is one of fundamental regulators of vascular tone and is also a crucial factor influencing target organ damage. Multiple organ dysfunction has been described in monocrotaline (MCT)-induced pulmonary hypertension (PH) in rats. Here, we hypothesized that nitrate administration attenuates target organ injury in this experimental PH. Design and method: Male Wistar rats were subcutaneously injected either with 60u200amg/kg MCT or with vehicle (CON). Twelve days after MCT injection, 0.3u200amM and 1u200amM NaNO3 was administered to rats in drinking water for part of the MCT group (MCT+N0.3, MCT+N1), while the rest received corresponding dose of natrium in 0.08% solution of NaCl. One month after the MCT injection, right ventricular pressure (RVP) was measured invasively. Afterwards, the animals were sacrificed in CO2 and their organs were harvested for further analysis. Protein expression of endothelial nitric oxide synthase (eNOS), pSer1177eNOS, Hsp90 and caveolin-1 (Cav-1) in right ventricle and lung was evaluated by western blot. Results: MCT injection led to right ventricular hypertrophy, a significant increase in the mass of lung and interestingly, a significant decrease in the mass of kidney compared to CON. However, these changes were absent in the MCT+N1 when compared to CON, while administration of 0.3u200amM NaNO3 was inefficient to prevent organ remodelling. RVP was significantly elevated in all groups that received MCT injection and nitrate therapies were unable to prevent this increase. Protein expression of Hsp90 in right ventricle was increased by 53% in MCT and by 54% in MCT+N0.3 group when compared to controls (P<0.05), but not in MCT+N1. Protein expression of Cav-1 was significantly decreased in MCT+N0.3 group by 63% in right ventricle and by 68% in lung compared to controls (Pu200a<u200a0.01). Expressions of other studied proteins remained stable. Conclusions: After 1u200amM NaNO3 administration, we noted the attenuation of MCT-induced increase of right ventricular mass and lung, and the preservation of kidney weight, compared to control group suggesting positive effect of nitrates on tissue remodelling. Interestingly, this was in accordance with the attenuation of increased Hsp90 expression in right ventricles of MCT+N1 rats.

[PP.31.07] CARDIAC UPREGULATION OF ATRIAL NATRIURETIC PEPTIDE IN METABOLIC SYNDROME INDUCED BY CONSUMPTION OF COLA BEVERAGE IN WISTAR RATS

Objective: The present study was designed to investigate the influence of long-term consumption of cola beverage on expression of cardiac atrial natriuretic peptide (ANP) gene. Natriuretic peptides, aside from their classic hemodynamic effects, have been associated with regulation of numerous physiological functions controlling energy metabolism. In rat, the effects of the consumption of cola beverage on cardiac ANP expression have not been described yet. Design and method: Male Wistar rats received a standard diet. Additionally, a group of rats received a commercially available cola beverage to induce experimental metabolic syndrome conditions (MetS, nu200a=u200a12) while controls (CON, nu200a=u200a7) received standard drinking water. We measured weight gain weekly. After six months of administration, blood glucose was determined by conventional oral glucose tolerance test (oGTT). In addition, heart rate (HR), systolic and diastolic blood pressures (sBP and dBP) were measured by using tail-cuff method. Cardiac gene expression was determined by quantitative real-time polymerase chain reaction (RT-PCR) in left ventricles. Results: We observed a significantly increased body weight in MetS rats (541u200a±u200a12u200ag; Pu200a<u200a0.01) when compared to controls (443u200a±u200a23u200ag). This was accompanied with significantly increased HR (CC: 383u200a±u200a9 mmHg vs. CON: 312u200a±u200a15 mmHg; Pu200a<u200a0.01), significantly increased sBP (MetS: 135u200a±u200a3 mmHg vs. CON: 120u200a±u200a4 mmHg; Pu200a<u200a0.01) but unaltered dBP (MetS: 86u200a±u200a1 mmHg vs. CON: 85u200a±u200a1 mmHg; NS). Postprandial glycaemia was significantly increased after 60 minutes (MetS: 9.3u200a±u200a0.5u200ammol/L vs. CON: 6.6u200a±u200a0.6u200ammol/L; Pu200a<u200a0.01) as well as after 90 minutes following oGTT induction (MetS: 8.3u200a±u200a0.4u200ammol/L vs. CON: 6.6u200a±u200a0.5u200ammol/L; Pu200a<u200a0.01). Left ventricles from MetS rats exhibited significant upregulation of ANP (MetS: 244u200a±u200a0.5 % vs. CON: 100u200a±u200a0.2 %; Pu200a<u200a0.01). Conclusions: Experimental metabolic syndrome induced by cola beverage administration is characterised by increased sBP and impaired glucose utilisation and is accompanied by upregulation of cardiac expression of ANP. The data indicate that the altered expression profile in the heart could contribute to the increased incidence of hypertension, cardiovascular and also metabolic alterations observed in this animal model.

[PP.31.04] INVOLVEMENT OF RENIN-ANGIOTENSIN SYSTEM IN MONOCROTALINE INDUCED PULMONARY HYPERTENSION

Objective: Pulmonary arterial hypertension (PAH) is a rare and complex disease, where renin-angiotensin system (RAS) is known to be activated. Monocrotaline induced PAH is well known experimental animal model. We aimed to measure RAS components, including angiotensin converting enzyme (ACE) and its receptors in various tissues. Additionally, we hypothesized, that kidneys might pose as a source of RAS activation, as they excrete renin, which is a rate limiting step of angiotensin II formation. Design and method: Group of 15 male Wistar rats was injected with monocrotaline (60u200amg/kg) and 7 control rats (CON) received vehicle. Separate group of 20 (MCT) and 10 (CON) rats was used for hemodynamic measurements. Animals were weighted frequently and vital functions were measured using MouseOx meter. Rats were sacrificed after 4 weeks or immediately if showing dyspnea, lethargy and significant weight loss. Results: MCT-treated rats exhibited significant increase in the right ventricular systolic pressure (MCT: 50.65u200a±u200a6.28 vs. CON: 21.52u200a±u200a2.49, Pu200a<u200a0.01). Right ventricular (RV) weight was significantly increased (MCT: 0.29u200a±u200a0.02 vs. CON: 0.17u200a±u200a0.01, Pu200a<u200a0.05), whereas left ventricular (LV) weight was not significantly changed (MCT: 0.69u200a±u200a0.03 vs. CON: 0.70u200a±u200a0.05). Expression of ACE mRNA was significantly increased in the RV (MCT: 2.31u200a±u200a0.41 vs. CON: 1.00u200a±u200a0.16, Pu200a<u200a0.05), also in the LV (MCT: 2.37u200a±u200a0.26 vs. CON: 1.00u200a±u200a0.06, Pu200a<u200a0.01), as was in the kidney (MCT: 1.66u200a±u200a0.25 vs. CON: 1.00u200a±u200a0.15, Pu200a<u200a0.05), but in lungs was significantly decreased (MCT: 0.47u200a±u200a0.03 vs. CON: 1.00u200a±u200a0.06, Pu200a<u200a0.01). Expression of angiotensin II receptor type 1 was significantly increased in the RV (MCT: 2.1u200a±u200a0.21 vs. CON: 1.00u200a±u200a0.13, Pu200a<u200a0.05), while in the left ventricle, lungs and kidney was unchanged. There was no significant alteration in the mRNA of angiotensin II receptor type 2 in the heart, lungs or kidney. Renin mRNA expression was significantly increased in the kidney (MCT: 2.46u200a±u200a0.69 vs. CON: 1.00u200a±u200a0.13, Pu200a<u200a0.01). Conclusions: Renin-angiotensin system is activated in the heart and kidneys, for which the increased kidney renin might be responsible. Interestingly, the opposite effect is present in lungs, where ACE is decreased.

Voluntary exercise and testosterone therapy caused increase in percentage of Myh6 and expression of oxidative stress marker Cybb in left ventricles of rats

Abstract Aim: The aim of this study is to identify a possible damage to heart ventricles caused by supraphysiological doses of testosterone, voluntary physical activity or their combination. Methods: In the 8-week long experiment, 10-12 weeks old male Wistar rats were administered testosterone depot in dose of 100 mg/kg (TES, n = 15) or vehiculum (CON, n = 12) once a week subcutaneously. Next groups injected with testosterone (SPOTES, n = 12) or vehiculum (SPO, n = 12) were running in exercise wheels ad libitum. Gene expressions in left and right ventricles of the heart were measured by quantitative reverse transcription polymerase chain reaction method. Results:ln left ventricles of the testosterone groups, we observed a mild but significant increase in the percentage of Myh6 myosin heavy chain isoform and higher expression of NADPH oxidase subunit Cybb (*p < 0.05). Conclusions:Testosterone affected the expression of genes related to contractile apparatus and oxidative stress in the left ventricle but not in right ventricle of the heart of rats. The observed level of physical activity did not have a compelling effect on the expression of measured genes.