Investigation of different pharmacological interventions and cardiotoxicity in the myocardium: the importance of autophagy

A szív- és érrendszeri megbetegedések világszerte növekvő és súlyosbodó egészségügyi problémát jelentenek. A kardiovaszkuláris betegségek következtében kialakult halálozások a mai napig vezetik a mortalitási statisztikát. A rizikófaktorok csökkentése és a prevenció fontossága vitathatatlan a szív- és érrendszeri betegségekben. A doxorubicin (DOX) egy széleskörűen alkalmazott, nem-szelektív antraciklin típusú antibiotikum, melynek leggyakoribb felhasználási területe az akut leukémiák, malignáns limfómák, és az emlő daganatok kezelése. A DOX egy nagyon hatékony antineoplasztikus szer, azonban klinikai felhasználásának limitáló tényezője a kumulatív és irreverzibilis kardiotoxicitás. A DOX-indukálta kardiotoxicitás multifaktoriális, hátterében számos bonyolult molekuláris mechanizmusok állnak, melyek a mai napig nem teljesen tisztázottak, azonban tudjuk, hogy mitokondriális diszfunkció, oxidatív stressz, lipidperoxidáció, apoptózis és az autofágia abnormális működése áll. Mivel a DOX kezelés során sérül a mitokondrium, és modulálódik az ATP termelődés, így az AMPK útvonal modulálódása várható: ez egyrészt hatással van az energia homeosztázisra, másrészt az autofágia folyamatára, hiszen az AMPK hatására indukálódhat az autofágia. Kutatócsoportunk célul tűzte ki egyrészt az autofágia szerepének vizsgálatát DOX-indukálta kardiotoxicitásban, ahol, mint kiegészítő terápiaként metformint adagoltunk. A legújabb tanulmányokban arról olvashatunk, hogy a MET számos esetben kardioprotektív hatással bír. Az irodalomban korábban már leírták, hogy a MET az AMPK útvonalon keresztül indukálni képes az autofágiát, azonban azt még nem írták le mindeddig, hogy a doxorubicinnal együttesen adagolva hogyan befolyásolja az autofágia folyamatát. További kísérleteinkben pedig a különböző dózisban alkalmazott ISO dózisfüggő szívkárosító hatásait, és a folyamatban résztvevő, különböző túlélési és halálozási útvonalak (apoptózis, nekrózis és autofágia), és azok kapcsolatát vizsgáltuk. A tanszéken korábban végzett ex vivo kísérletekből azt már tudjuk, hogy az ISO dózisfüggő toxicitást mutat, azonban a molekuláris útvonalak, s azok pontos szerepe nem teljesen tisztázott a β-receptorokra gyakorolt hatása folyamán.Doxorubicin (DOX) is a highly effective and widely-used non-selective class I anthracycline antibiotic which is frequently incorporated in the treatment of acute leukemia, malignant lymphoma, and several solid tumors. However, the efficacy of DOX is hindered due to the cumulative and irreversible cardiotoxicity. The molecular mechanisms underlying DOX-induced cardiotoxicity are multifactorial and are still unclear, but mitochondrial dysfunction, oxidative stress, apoptosis, and dysregulation of autophagy are involved. Furthermore, the heart is very susceptible to DOX-induced lipid peroxidation and toxicity because of its high energy requirement and mitochondrial density. Autophagy is a highly conserved process which is aimed to maintain cell and tissue homeostasis, and involves the elimination of damaged and long-lived organelles under both physiological and pathological conditions, including energy and oxygen status, nutrient starvation, and modification in metabolism. Several studies have found that DOX treatment affects autophagy, however, it is still not clearly elucidated how DOX alters this process. Previous studies on this matter have shown many controversial results. Recently reported studies have demonstrated that DOX induces autophagy; however, it causes dysregulation in the autophagic flux and the autophagic process cannot be completed. In addition, Tokarska et al. reported that DOX can cause dysregulation in most processes of myocardial energy metabolism, such as the AMP-activated protein kinase (AMPK) signaling pathway. AMPK is a major sensor of cell energetic homeostasis. Low cellular energy levels and increased reactive oxygen species (ROS) result in the phosphorylation and activation of AMPK, which is able to induce autophagic processes. Metformin (MET) is an orally used first-line anti-diabetic drug for the treatment of type 2 diabetes. Several studies reported that application of MET decreases mortality and cardiovascular end-points of type 2 diabetes and has protective effects in cardiac function. Recently, several studies have found that MET activates AMPK, and through the AMPK signaling pathway it induces cardiac autophagy and improves cardiac functions. Various studies have revealed that boosting or restoring autophagy could help the cardiomyocytes to survive during DOX therapy. In the present study, we co-administered DOX and MET in order to investigate the role of MET in the autophagic process and its cardioprotective properties in DOX-induced cardiotoxicity. Thus, our investigation may offer further understanding of the role of cardiac autophagy in DOX-treated animal subjects. In addition, our hypothesis was that MET could activate AMPK, restore autophagy, and improve cardiac function, which may consequently mean that DOX co-administered with MET help the cardiomyocytes to survive. To further study the importance of autophagy during cardiotoxicity we administered isoproterenol (ISO) at different doses (0.005 mg/kg ISO; 0.05 mg/kg ISO; 0.5 mg/kg ISO; 5 mg/kg ISO; 50 mg/kg ISO), and monitored the autophagic process and the two death pathways, including apoptosis and necrosis. ISO is a catecholamine and bearing a non-selective β-adrenergic agonist property. At low dose it can be used in heart block and cardiac arrest; however, at chronic or high doses, administration of ISO leads to the development of irreversible damage of the myocardium and ultimately causes infarct-like necrosis in heart muscle. β-adrenergic overstimulation by ISO upset the balance between the oxygen demand and supply of the myocardium leading to pathological alteration. The underlying mechanisms of ISO-induced cardiac injury are complex and multifactorial, but the major drawbacks of the ISO therapy are the generation of cytotoxic free radicals in myocytes followed by oxidative stress and lipid peroxidation, which leads to progressive mitochondrial damage, inflammatory cytokines production, ionic imbalance including intracellular Ca2+ overloading that result in cardiac injury. Previous studies on this matter showed that ISO-induced myocardial damage involves apoptosis, and necrosis, moreover apoptosis seems to be an important complicating factor of myocardial injuries increasing the degree of myocyte cell death, which eventually leads to irreversible damages. However, the role of autophagy, and its connection with apoptosis and necrosis under this condition remains to be elucidated. Taken together our aims were to investigate the autophagy and cell death pathways including apoptosis and necrosis in ISO-induced cardiac injury in a dose-dependent manner.N

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

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

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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

Basic Pharmacological Characterization of EV-34, a New H₂S-Releasing Ibuprofen Derivative

Background: Cardioprotective effects of H2S are being suggested by numerous studies. Furthermore, H2S plays a role in relaxation of vascular smooth muscle, protects against oxidative stress, and modulates inflammation. Long-term high-dose use of NSAIDs, such as ibuprofen, have been associated with enhanced cardiovascular risk. The goal of the present work is the synthesis and basic pharmacological characterization of a newly designed H2S-releasing ibuprofen derivative. Methods: Following the synthesis of EV-34, a new H2S-releasing derivative of ibuprofen, oxidative stability assays were performed (Fenton and porphyrin assays). Furthermore, stability of the molecule was studied in rat serum and liver lysates. H2S-releasing ability of the EC-34 was studied with a hydrogen sulfide sensor. MTT (3-(4,5-dimethylthiazol 2-yl)-2,5-(diphenyltetrazolium bromide)) assay was carried out to monitor the possible cytotoxic effect of the compound. Cyclooxygenase (COX) inhibitory property of EV-34 was also evaluated. Carrageenan assay was carried out to compare the anti-inflammatory effect of EV-34 to ibuprofen in rat paws. Results: The results revealed that the molecule is stable under oxidative condition of Fenton reaction. However, EV-34 undergoes biodegradation in rat serum and liver lysates. In cell culture medium H2S is being released from EV-34. No cytotoxic effect was observed at concentrations of 10, 100, 500 µM. The COX-1 and COX-2 inhibitory effects of the molecule are comparable to those of ibuprofen. Furthermore, based on the carrageenan assay, EV-34 exhibits the same anti-inflammatory effect to that of equimolar amount of ibuprofen (100 mg/bwkg). Conclusion: The results indicate that EV-34 is a safe H2S releasing ibuprofen derivative bearing anti-inflammatory properties

TNFα induces endothelial dysfunction in rheumatoid arthritis via LOX-1 and arginase 2: reversal by monoclonal TNFα antibodies

none25Aims: Rheumatoid arthritis (RA) is a chronic inflammatory disease affecting joints and blood vessels. Despite low levels of low-density lipoprotein cholesterol (LDL-C), RA patients exhibit endothelial dysfunction and are at increased risk of death from cardiovascular (CV) complications, but the molecular mechanism of action is unknown.We aimed in the present study to identify the molecular mechanism of endothelial dysfunction in a mouse model of RA and in patients with RA. Methods and results: Endothelium-dependent relaxations to acetylcholine were reduced in aortae of two TNFα transgenic mouse lines with either mild (Tg3647) or severe (Tg197) forms of RA in a time- and severity-dependent fashion as assessed by organ chamber myograph. In Tg197, TNFα plasma levels were associated with severe endothelial dysfunction. LOX-1 receptor was markedly upregulated leading to increased vascular oxLDL uptake and NFκB-mediated enhanced Arg2 expression via direct binding to its promoter resulting in reduced NO bioavailability and vascular cGMP levels as shown by ELISA and chromatin immunoprecipitation. Anti-TNFα treatment with infliximab normalized endothelial function together with LOX-1 and Arg2 serum levels in mice. In RA patients, soluble LOX-1 serum levels were also markedly increased and closely related to serum levels of C-reactive protein. Similarly, ARG2 serum levels were increased. Similarly, anti-TNFα treatment restored LOX-1 and ARG2 serum levels in RA patients. Conclusions: Increased TNFα levels not only contribute to RA, but also to endothelial dysfunction by increasing vascular oxLDL content and activation of the LOX-1/NFκB/Arg2 pathway leading to reduced NO bioavailability and decreased cGMP levels. Anti-TNFα treatment improved both articular symptoms and endothelial function by reducing LOX-1, vascular oxLDL and Arg2 levelsmixedAkhmedov, Alexander; Crucet, Margot; Simic, Branko; Kraler, Simon; Bonetti, Nicole R; Ospelt, Caroline; Distler, Oliver; Ciurea, Adrian; Liberale, Luca; Jauhiainen, Matti; Metso, Jari; Miranda, Melroy; Cydecian, Rose; Schwarz, Lena; Fehr, Vera; Zilinyi, Rita; Amrollahi-Sharifabadi, Mohammad; Ntari, Lydia; Karagianni, Niki; Ruschitzka, Frank; Laaksonen, Reijo; Vanhoutte, Paul M; Kollias, George; Camici, Giovanni G; Lüscher, Thomas FAkhmedov, Alexander; Crucet, Margot; Simic, Branko; Kraler, Simon; Bonetti, Nicole R; Ospelt, Caroline; Distler, Oliver; Ciurea, Adrian; Liberale, Luca; Jauhiainen, Matti; Metso, Jari; Miranda, Melroy; Cydecian, Rose; Schwarz, Lena; Fehr, Vera; Zilinyi, Rita; Amrollahi-Sharifabadi, Mohammad; Ntari, Lydia; Karagianni, Niki; Ruschitzka, Frank; Laaksonen, Reijo; Vanhoutte, Paul M; Kollias, George; Camici, Giovanni G; Lüscher, Thomas