Mayel Gharanei

Attenuation of Doxorubicin-Induced Cardiotoxicity by mdivi-1: A Mitochondrial Division/Mitophagy Inhibitor

Doxorubicin is one of the most effective anti-cancer agents. However, its use is associated with adverse cardiac effects, including cardiomyopathy and progressive heart failure. Given the multiple beneficial effects of the mitochondrial division inhibitor (mdivi-1) in a variety of pathological conditions including heart failure and ischaemia and reperfusion injury, we investigated the effects of mdivi-1 on doxorubicin-induced cardiac dysfunction in naïve and stressed conditions using Langendorff perfused heart models and a model of oxidative stress was used to assess the effects of drug treatments on the mitochondrial depolarisation and hypercontracture of cardiac myocytes. Western blot analysis was used to measure the levels of p-Akt and p-Erk 1/2 and flow cytometry analysis was used to measure the levels p-Drp1 and p-p53 upon drug treatment. The HL60 leukaemia cell line was used to evaluate the effects of pharmacological inhibition of mitochondrial division on the cytotoxicity of doxorubicin in a cancer cell line. Doxorubicin caused a significant impairment of cardiac function and increased the infarct size to risk ratio in both naïve conditions and during ischaemia/reperfusion injury. Interestingly, co-treatment of doxorubicin with mdivi-1 attenuated these detrimental effects of doxorubicin. Doxorubicin also caused a reduction in the time taken to depolarisation and hypercontracture of cardiac myocytes, which were reversed with mdivi-1. Finally, doxorubicin caused a significant elevation in the levels of signalling proteins p-Akt, p-Erk 1/2, p-Drp1 and p-p53. Co-incubation of mdivi-1 with doxorubicin did not reduce the cytotoxicity of doxorubicin against HL-60 cells. These data suggest that the inhibition of mitochondrial fission protects the heart against doxorubicin-induced cardiac injury and identify mitochondrial fission as a new therapeutic target in ameliorating doxorubicin-induced cardiotoxicity without affecting its anti-cancer properties.

Doxorubicin induced myocardial injury is exacerbated following ischaemic stress via opening of the mitochondrial permeability transition pore.

Chemotherapeutic agents such as doxorubicin are known to cause or exacerbate cardiovascular cell death when an underlying heart condition is present. However, the mechanism of doxorubicin-induced cardiotoxicity is unclear. Here we assess the cardiotoxic effects of doxorubicin in conditions of myocardial ischaemia reperfusion and the mechanistic basis of protection, in particular the role of the mitochondrial permeability transition pore (mPTP) in such protection. The effects of doxorubicin (1μM)±cyclosporine A (CsA, 0.2μM; inhibits mPTP) were investigated in isolated male Sprague-Dawley rats using Langendorff heart and papillary muscle contraction models subjected to simulated ischaemia and reperfusion injury. Isolated rat cardiac myocytes were used in an oxidative stress model to study the effects of drug treatment on mPTP by confocal microscopy. Western blot analysis evaluated the effects of drug treatment on p-Akt and p-Erk 1/2 levels. Langendorff and the isometric contraction models showed a detrimental effect of doxorubicin throughout reperfusion/reoxygenation as well as increased p-Akt and p-Erk levels. Interestingly, CsA not only reversed the detrimental effects of doxorubicin, but also reduced p-Akt and p-Erk levels. In the sustained oxidative stress assay to study mPTP opening, doxorubicin decreased the time taken to depolarization and hypercontracture, but these effects were delayed in the presence of CsA. Collectively, our data suggest for the first that doxorubicin exacerbates myocardial injury in an ischaemia reperfusion model. If the inhibition of mPTP ameliorates the cardiotoxic effects of doxorubicin, then more selective inhibitors of mPTP should be further investigated for their utility in patients receiving doxorubicin.

Predictivity of in vitro non-clinical cardiac contractility assays for inotropic effects in humans--A literature search.

Adverse drug effects on the cardiovascular system are a major cause of compound attrition throughout compound discovery and development. There are many ways by which drugs can affect the cardiovascular system, including effects on the electrocardiogram, vascular resistance, heart rate and the force of contraction of the heart (inotropy). Compounds that increase the force of contraction of the heart can be harmful in patients with ischemic heart disease, whilst negative inotropes can induce symptoms of heart failure. There is a range of non-clinical in vitro and in vivo assays used to detect inotropic effects of drugs. We have conducted a literature review of the in vitro assays and compared the findings from these with known effects on cardiac contractility in man. There was a wide variety of assays used, ranging from perfuse whole hearts to isolated regions of the heart (papillary muscle, ventricle and atria), which were removed from a number of species (cat, guinea pig, rabbit and rat). We conducted two analyses. The first was investigating the concordance of the findings from the in vitro assays at any concentration with those observed in man (an assessment of hazard identification) and the second was the concordance of the in vitro findings at concentrations tested up to 10-fold higher than those tested in the clinic. We found that when used as a hazard identification tool, the available assays had good sensitivity (88%), although the specificity was not so good (60%), but when used as a risk management tool the sensitivity was considerably reduced (sensitivity 58-70% and specificity 60%). These data would suggest that the available in vitro assays can be used as hazard identification tools for adverse drug effects on cardiac contractility, but there is a need for new assays to better predict the exposures in man that may cause a change in cardiac contractility and therefore better predict the likely therapeutic index of compounds prior to nomination of compounds for clinical development.

Investigation into the cardiotoxic effects of doxorubicin on contractile function and the protection afforded by cyclosporin A using the work-loop assay

Doxorubicin is known to cause cardiotoxicity through multiple routes including the build-up of reactive oxygen species and disruption of the calcium homeostasis in cardiac myocytes, but the effect of drug treatment on the associated biomechanics of cardiac injury remains unclear. Detecting and understanding the adverse effects of drugs on cardiac contractility is becoming a priority in non-clinical safety pharmacology assessment. The work-loop technique enables the assessment of force-length work-loop contractions, which mimic those of the pressure-volume work-loops experienced by the heart in vivo. During this study we evaluated whether the work-loop technique could potentially provide improved insight into the biomechanics associated with drug-induced cardiac dysfunction. In order to do this we investigated the cardiotoxic effects of doxorubicin and characterised the protection afforded by the co-administration of cyclosporin A (CsA). This study provides detailed biomechanical in vitro insight into the cardiac dysfunction associated with Doxorubicin treatment, including reduction in peak force, force during shortening and power output. These effects were significantly abrogated in doxorubicin-CsA co-treatment studies. Closely mimicking the in vivo pressure-volume muscle mechanics, this assay provides a quick and easy technique to gain a better understanding of the detailed biomechanics of drug-induced cardiac dysfunction.

15 Mitochondrial Division Inhibitor-1 Protects against Doxorubicin-Induced Cardiotoxicity

Doxorubicin is one of the most effective anti-cancer agents. However, its use is associated with adverse cardiac effects, including cardiomyopathy and progressive heart failure. Given the multiple beneficial effects of the mitochondrial division inhibitor (mdivi-1) in a variety of pathological conditions including heart failure and ischaemia-reperfusion injury (IRI), we investigated the effects of mdivi-1 on doxorubicin-induced cardiac dysfunction in naï and stressed conditions. Drug-induced effects were assessed using the Langendorff system, oxidative stress model using isolated cardiomyocytes, western blot and flow cytometry analysis to measure the levels of p-Akt, p-Erk ½, p-Drp1 and p-p53 upon drug-treatment. The HL60 leukaemia cell line was used to evaluate the effects of combined treatment of doxorubicin and mdivi-1 on the cytotoxicity of doxorubicin in a cancer cell line. Doxorubicin caused a significant impairment of cardiac function and increased the infarct size to risk-ratio in both naï and IRI conditions. Interestingly, co-treatment of doxorubicin with mdivi-1 attenuated these detrimental effects of doxorubicin. Doxorubicin also caused a reduction in the time taken to depolarisation and hypercontracture of cardiac myocytes, which were prevented with mdivi-1. Finally, doxorubicin caused a significant elevation in the levels of signalling proteins p-Akt, p-Erk 1/2, p-Drp1 and p-p53. Co-incubation of mdivi-1 with doxorubicin did not reduce the cytotoxicity of doxorubicin against HL60 cells. These data suggest that the inhibition of mitochondrial fission protects the heart against doxorubicin-induced cardiac injury. We have identified for the first time mitochondrial fission as a new therapeutic target in ameliorating doxorubicin-induced cardiotoxicity without affecting its anti-cancer properties.

20 Cardioprotection During Chemotherapy: A Case Study to Understand Intracellular Mechanisms to Combat the Cardiotoxicity of Sunitinib

Tyrosine Kinase inhibitor Sunitinib mediates its apoptotic anti-cancer effects by inhibiting intracellular signalling involved in tumour angiogenesis and cell proliferation. Unfortunately Sunitinib has revealed unwanted cardiotoxicity side-effects and investigations into cardioprotective adjunct therapy agents are critical. A3 adenosine receptor agonists (A3AR) have been shown to have powerful cardioprotective effects against myocardial injury (MI). We studied the effect of A3AR agonist IB-MECA in preventing MI following Sunitinib treatment and assessed whether IB-MECA jeopardized the anti-cancer/apoptotic effect of Sunitinib in cancer cells. The associated intracellular signalling pathway through Protein Kinase C α/β (PKC α/β) and microRNA expression signatures were investigated. HL60 cells were incubated with increasing concentrations of Sunitinib (0.1-10 μM) ± IB-MECA (1 nM) for 24h and cell viability (CV) was assessed. Langendorff hearts underwent Sunitinib (0.3 μM) ± IB-MECA (1 nM) treatment and MI assessment. Phosphorylated expression levels of Protein Kinase C α/β were assessed by Western Blot analysis and rtPCR analysis revealed the microRNA expression signature. Sunitinib decreased HL60 cell viability and increased MI compared to vehicle (Vehicle: CV = 102.0 ± 1.4%; MI = 8.2 ± 3.6%; Sunitinib: CV(10 μM) = 43.2 ± 6.3%, p = 0.02; MI(0.3 μM) = 30.1 ± 1.5%, p < 0.001). Addition of IB-MECA did not alter the apoptotic effect of Sunitinib, however, IB-MECA attenuated the Sunitinib-induced MI (Sunitinib + IB-MECA: CV(10 μM) = 37.8 ± 6.4%; MI(0.3 μM) = 20.4 ± 2.8%, p < 0.01). The IC50-values of Sunitinib ± IB-MECA did not change significantly (Sunitinib IC50-value = 8.4 ± 1.3; Sunitinib + IB-MECA IC50-value = 7.0 ± 1.6) (n = 5–6). This study reveals for the first time that A3AR activation improves myocardial survival by attenuating Sunitinib induced MI without interfering with the anti-tumour efficacy of Sunitinib. A3AR associated signalling pathways could be important in the development of adjunctive chemotherapy treatment.