Gabriel Doka

Enalaprilat increases PPARβ/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 PPARβ/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.

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.

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.

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

ABSTRACT 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.

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.

Combination treatment with valsartan and amlodipine intensifies evening suppression of Bmal1 clock gene in kidneys of spontaneously hypertensive rats

Abstract Blood pressure (BP) rhythm is exhibited in a circadian pattern regulated by complex system of endogenous factors. Administration of pharmacological treatment at the right time can influence the efficacy of treatment; but while kidneys play significant role in BP regulation, little is known about their role in chronopharmacotherapy. This study aimed to compare differences between morning and evening dosing with valsartan and amlodipine combination in both short-term and long-term settings and to elucidate the role of kidneys in chronopharmacology. Spontaneously hypertensive rats aged between 8 and 10 weeks were daily treated with 10mg/kg of valsartan and 4 mg/kg of amlodipine, either in the morning or in the evening with treatment duration of 1 and 6 weeks. After short-term treatment, only morning treatment group demonstrated significantly better outcomes in terms of BP control when compared to placebo. After long-term treatment, both treatment groups gained superior results in BP control against placebo; however, no significant difference was seen between morning and evening treatment. Interestingly, clock gene expression in kidney has been significantly modulated only in the evening-treated groups, with treatment intensifying the reduced Bmal1 levels, while Per2 expression was less altered. However, no direct relation with the outcomes of the therapy has been observed, suggesting that pharmacotherapy may serve as an independent modulator of peripheral circadian clock in the kidney.

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.