The Role of Autophagy and Death Pathways in Dose-dependent Isoproterenolinduced Cardiotoxicity.

BACKGROUND: Isoproterenol (ISO) is a non-selective ß-adrenergic agonist. Our aims were to investigate the autophagy and cell death pathways including apoptosis and necrosis in ISO-induced cardiac injury in a dosedependent manner. METHODS: Male Sprague-Dawley rats were treated for 24 hours with I. vehicle (saline); II. 0.005 mg/kg ISO; III. 0.05 mg/kg ISO; IV. 0.5 mg/kg ISO; V. 5 mg/kg ISO; VI. 50 mg/kg ISO, respectively. Hearts were isolated and infarct size was measured. Serum levels of Troponin T (TrT), lactate dehydrogenase (LDH), creatine kinase isoenzyme MB (CK-MB) were measured. TUNEL assay was carried out to monitor apoptotic cell death and Western blot was performed to evaluate the level of autophagic and apoptotic markers. RESULTS: Survival rate of animals was dose-dependently decreased by ISO. Serum markers and infarct size revealed the development of cardiac toxicity. Level of Caspase-3, and results of TUNEL assay, demonstrated that the level of apoptosis was dose-dependently increased. They reached the highest level in ISO 5 and it decreased slightly in ISO 50 group. Focusing on autophagic proteins, we found that level of Beclin-1 was increased in a dose-dependent manner, but significantly increased in ISO 50 treated group. Level of LC3B-II and p62 showed the same manner, but the elevated level of p62 indicated that autophagy was impaired in both ISO 5 and ISO 50 groups. CONCLUSION: Taken together these results suggest that at smaller dose of ISO autophagy may cope with the toxic effect of ISO; however, at higher dose apoptosis is initiated and at the highest dose substantial necrosis occurs.

Electrically-Induced Ventricular Fibrillation Alters Cardiovascular Function and Expression of Apoptotic and Autophagic Proteins in Rat Hearts.

BACKGROUND: The pathological heart contractions, called arrhythmias, especially ventricular fibrillation (VF), are a prominent feature of many cardiovascular diseases leading to sudden cardiac death. The present investigation evaluates the effect of electrically stimulated VF on cardiac functions related to autophagy and apoptotic mechanisms in isolated working rat hearts. METHODS: Each group of hearts was subjected to 0 (Control), 1, 3, or 10 min of spacing-induced VF, followed by 120 min of recovery period and evaluated for cardiac functions, including aortic flow (AF), coronary flow (CF), cardiac output (CO), stroke volume (SV), and heart rate (HR). Hearts were also evaluated for VF effects on infarcted zone magnitude and Western blot analysis was conducted on heart tissue for expression of the apoptotic biomarker cleaved-caspase-3 and the autophagy proteins: p62, P-mTOR/mTOR, LC3BII/LC3BI ratio, and Atg5-12 complexes. RESULTS: Data revealed that VF induced degradation in AF, CF, CO, and SV, which prominently included-variable post-VF capacity for recovery of normal heart rhythm; increased extent of infarcted heart tissue; altered expression of cleaved-caspase-3 suggesting potential for VF-mediated amplification of apoptosis. VF influence on expression of p62, LC3BII/LC3BI, and Atg5-12 proteins was complex, possibly due to differential effects of VF-induced expression on proteins comprising the autophagic program. CONCLUSIONS: VF was observed to cause time-dependent changes in autophagy processes, which with additional analysis under ongoing investigations, likely to yield novel therapeutic targets for the prevention of VF and sudden cardiac death.

Fluconazole is not inferior than caspofungin, micafungin or amphotericin B in the presence of 50% human serum against Candida albicans and Candida parapsilosis biofilms.

Biofilm formation is a relevant risk factor for mortality in candidemia. Data about serum-based susceptibility testing against Candida biofilms are scant; therefore, the activity of fluconazole, amphotericin B, caspofungin and micafungin was determined against Candida albicans and C. parapsilosis biofilms with or without 50% human serum using XTT-based assays. Serum caused a remarkable adverse effect regarding biofilm structure for both species. Additionally, the ratio of nonviable cells increased for C. parapsilosis biofilms, as confirmed by fluorescent microscopy and flow cytometry. Despite impaired biofilm development, traditionally biofilm-active antifungals, surprisingly, showed decreased activity against C. albicans biofilms in serum at concentrations ranging from 0.5 to 1 mg/l and from 0.015 to 1 mg/l for amphotericin B and echinocandins, respectively (P < .01-.05). However, C. parapsilosis showed higher susceptibility to these antifungals due to reduced biofilm mass and the fungicidal effect of serum at concentrations ranging from 0.015 to 1 mg/l and from 0.015 to 512 mg/l for amphotericin B and echinocandins, respectively (P < .01-.05). Fluconazole exerted better antifungal activity in serum than traditionally biofilm-active antifungals against both examined biofilms. For fluconazole, significant differences were observed in susceptibility between serum-treated and serum-free biofilms at concentrations ranging from 0.015 to 8 mg/l and from 0.03 to 512 mg/l for C. albicans and C. parapsilosis isolates, respectively (P < .01-.05). The high antifungal activity of fluconazole in 50% serum both against C. albicans and C. parapsilosis biofilms supports the utility of fluconazole prophylaxis to reduce the risk of catheter-associated fungal infections.

The Cardioprotective Effect of Metformin in Doxorubicin-Induced Cardiotoxicity: The Role of Autophagy.

The molecular mechanisms underlying doxorubicin-induced cardiotoxicity are still being investigated, but are known to involve oxidative stress, mitochondrial dysfunction, and the dysregulation of autophagy. The objective of the current study was to examine the protective role of metformin and its effect on autophagy in doxorubicin-induced cardiotoxicity. Sprague⁻Dawley rats were divided into four groups at random. The doxorubicin-treated group received doxorubicin (3 mg/kg every second day) intraperitoneally. The metformin-treated group received 250 mg/kg/day metformin via gavage. The doxorubicin + metformin-treated group received both at the above-mentioned doses. The control group received vehicle only. Following the two-week treatment, the hearts were isolated, and cardiac functions were registered. Serum levels of lactate dehydrogenase (LDH), creatine kinase iso-enzyme MB (CK-MB) enzyme, Troponin T, and cardiac malondialdehyde (MDA) were also measured. Heart tissue samples were histopathologically examined by using Masson's trichrome staining and Western blot analysis was conducted for evaluating the expression level of AMP-activated protein kinase (AMPK) and autophagy-associated proteins beclin-1, LC3B-II, and p62, respectively. The results revealed that treatment with metformin conferred increased cardiac protection against the development of cardiotoxicity manifested by a significant decrease in serum Troponin T and cardiac MDA levels, and remarkable improvement in heart function in connection with histopathological features. Furthermore, by focusing on the contribution of autophagic proteins, it was found that metformin normalised autophagy, which may help cardiomyocytes survive doxorubicin-induced toxicity. These results promote the use of metformin, which would be a preferable drug for patients receiving doxorubicin.

Cardioprotection afforded by sour cherry seed kernel: the role of heme oxygenase-1.

Cardiovascular diseases are primary cause of death worldwide, particularly among populations with sedentary lifestyles and diets rich in animal products and processed foods. Currently, public health countermeasures to these disorders focus on costly and often marginally effective interventions administered only after the development of disease. These countermeasures are mainly palliative and fail to address the underlying causes of cardiac pathologies. Previously, the authors of this report have demonstrated that sour cherry seed kernel extract (SCSE), a nontoxic low-cost plant material, strongly preserves tissues through induction of heme oxygenase-1 (HO-1), a critical host antioxidant defense enzyme. This investigation seeks to characterize underlying mechanisms of SCSE-mediated tissue protection. Isolated hearts from Sprague-Dawley rats fed 30 mg·kg·d SCSE for 8 weeks, and untreated controls were mounted in a "working heart" apparatus and subjected to ischemia and reperfusion. A panel of cardiac functional evaluations was conducted on each heart. Infarct size assessments were made along with Western blot and immunohistochemical analysis for selected proteins involved in cardiovascular homeostasis. SCSE treatment was observed to improve postischemic cardiac functions and suppress infarct size. Analysis of the outcomes produced by this study is consistent with SCSE cardioprotection that involve interaction of Bcl-2 and HO-1.

The cellular autophagy markers Beclin-1 and LC3B-II are increased during reperfusion in fibrillated mouse hearts.

Autophagy is an intracellular bulk degradation process for elimination of damaged macromolecules and organelles. In the past decades, the scientific community has gained increasingly detailed understanding of the role of autophagy in myocardial homeostasis, although still many controversies remain. In the ischemic myocardium, autophagy appears to be beneficial for survival, whereas upon reperfusion the process may induce cell death. However, the overall effect of autophagy seems to depend on the duration and intensity of stress, as along with the extent of autophagy within myocardial tissue. Reperfusion of an ischemic heart maybe harmful, but it is an essential process for myocardial survival. One of the major adverse consequences of reperfusion is the occurrence of ventricular fibrillation (VF). In the present study, we investigated the possible connection between autophagy and VF. Isolated mouse hearts were subjected to ischemia/reperfusion (I/R) and divided into two groups based on the development of VF at the beginning of reperfusion. Western blot analysis was conducted for autophagy-associated proteins LC3B, ATG-5, ATG-7, ATG-12, Bcl-2 and Beclin-1 proteins. Significantly higher level of Beclin-1 and LC3B-II/LC3B-I ratio (both definitive autophagy biomarkers) was observed in the fibrillated myocardium, versus tissue from the nonfibrillated hearts. Interestingly, although Bcl-2 is a major regulator of Beclin-1, level of this protein was not significantly altered in tissue from fibrillated, versus non-fibrillated hearts. Moreover, Atg7 expression showed a trend, albeit nonsignificant, towards elevation in fibrillated versus non-fibrillated hearts. Results of the present investigation demonstrate a possible link between VF and autophagy. Studies by authors of this report to evaluate potential etiologic relationships between the two processes are ongoing.