The effects of mitoquinone pretreatment on doxorubicin-induced acute cardiac dysfunction

Introduction: Doxorubicin (DOX) is a widely used anti-cancer drug notorious for its irreversible cardiac toxicity. Currently, Dexrazoxane is the only FDA-approved treatment for this toxicity. However, Dexrazoxane still bears some serious adverse events, and developing new strategies to mitigate DOX-induced heart damage is critical. Our lab has shown that pretreatment of the H9c2 myoblast cells with mitoquinone (MitoQ), a mitochondrial-targeted antioxidant, and significantly improved cells’ resiliency to DOX. This study aimed to determine if MitoQ pretreatment can preserve cardiac function against DOX-induced damage in isolated rat hearts. Objectives: The effects of DOX and MitoQ on cardiac function were evaluated in isolated rat hearts. Moreover, the benefits of MitoQ pretreatment on DOX-induced cardiac dysfunction were also assessed. Methods: Langendorff heart preparation was performed after anesthesia of male SD rats (275-325 g). Hearts were isolated and retrograde perfused with Krebs’ buffer at a constant pressure of 80 mmHg with 37 ⁰C and pH of 7.35-7.45. Cardiac parameters, including left ventricle end-systolic pressure (LVESP), left ventricle end-diastolic pressure (LVEDP), left ventricular developed pressure (LVDP=LVESP-LVEDP), maximal rate of rise of LVP (dP/dt(max)), and heart rate (HR), were measured by a pressure transducer placed in the left ventricle of the rat heart. After obtaining a stable initial cardiac function, DOX (20 µM or 25 µM) or MitoQ (0.1-0.5 or 1-2.5 µM) were infused into the heart for 60 min. to determine the individual drug\u27s effects on the cardiac function. Moreover, another set of hearts was pretreated with MitoQ (0.25-0.5 or 1-2.5 µM) for 10-15 min before giving DOX (25 µM) to evaluate if MitoQ pretreatment would mitigate DOX-induced cardiac dysfunction. Cardiac functions were recorded every 5 min. throughout the experiments. The ratio between the final and initial recordings was calculated and compared among experimental groups. Results: Acute infusion of DOX into the isolated hearts dose-dependently reduced some cardiac parameters. Higher dose DOX (25 μM, n=5) induced a higher reduction in the ratios of LVESP, LVDP, and dP/dt(max) to 0.39±0.05, 0.35±0.06, and 0.26±0.05 than those of lower dose DOX infusion (20 μM, n=2; 0.77±0.01, 0.75±0.01, and 0.57±0.01), respectively. DOX had no effects on LVEDP and HR. Moreover, lower doses of MitoQ (0.1-0.5 μM, n=6) only slightly reduced HR to 0.77±0.01 without affecting other parameters. By contrast, higher doses of MitoQ (1-5 μM, n=4) reduced the ratios of LVESP, LVDP, dP/dt(max), and HR to 0.72±0.12, 0.51±0.18, and 0.45±0.17 0.65±0.07, respectively. Interestingly, MitoQ pretreatment before DOX (25 µM) exhibited better cardiac function accompanied by reduced HR than DOX alone. Higher MitoQ (1-2.5 µM) pretreatment improved the ratios of cardiac LVESP, LVDP, and dP/dt(max) to 0.67±0.14, 0.65±0.16, and 0.40±0.09, which were higher than those of lower dose MitoQ (0.25-0.5 μM, n=3; 0.49±0.11, 0.44±0.11, and 0.36±0.08), respectively. Conclusion: The preliminary data suggest that infusion of DOX into the heart acutely attenuated cardiac systolic function. Higher doses of MitoQ, not lower doses, also suppressed cardiac function. MitoQ pretreatment mitigated DOX-induced heart dysfunction. Acknowledgement: The project is funded by CCDA at PCOM

The effects of Mitoquinone on simulated ischemia/reperfusion injuries in H9c2 cells

Introduction: Reperfusion to an ischemic myocardium could result in damage termed myocardial ischemia/reperfusion (I/R) injury. Mitochondrial dysfunction is a major factor in I/R injury, producing less ATP and generating more reactive oxygen species (ROS). Mitoquinone (MitoQ) is an antioxidant that highly accumulates in the mitochondria. However, the dose-response effects and underlying mechanisms of MitoQ on simulated I/R injury have not been well established. Objectives: We hypothesized that H9c2 myoblast cells would be damaged by simulated I/R. Moreover, MitoQ would attenuate myocardial injury, characterized by increased cell viability, compared to non-treated control. Methods: The H9c2 myoblast cells (less than 20 passages) were treated with or without various concentrations of MitoQ (0.005, 0.05, 0.1, 0.5, 1, 2, 5 μM) under 3 different mediums: normal (containing 4.5 g glucose and pyruvate), low glucose (containing 1 g glucose and pyruvate), and no glucose/pyruvate medium. Three different experiments were conducted on the cells. The first experiment aimed to determine if MitoQ alone exerts different effects under different medium conditions by treating the cells with MitoQ for 24 hrs in a normal incubator. The second experiment aimed to determine if MitoQ increased cell viability under simulated ischemia conditions after MitoQ pretreatment. The third experiment aimed to determine if MitoQ increased cell viability under simulated I/R conditions after MitoQ pretreatment. Cell viability was measured by absorbance at 450 nm after adding a cell counting agent. The change in cell viability was expressed as ratios relative to the untreated controls. Results: Low concentrations of MitoQ alone slightly increased cell viability in all three mediums. The maximum increased cell viability was 1.25 ± 0.07 (n=9) at 0.005 μM MitoQ in the normal medium, 1.35 ± 0.23 (n=5, p MitoQ pretreatment exerts protection to cells in simulated ischemia conditions at certain MitoQ concentrations. The maximum increased cell viability was 1.37 ± 0.3 (n=4) at 0.01 μM MitoQ in normal medium, 1.20 ± 0.13 (n=4) at 1.0 μM MitoQ in low glucose medium, and 1.45 ± 0.24 (n=3) at 0.1 μM MitoQ in no glucose medium compared to the untreated control. MitoQ effects on simulated I/R injury will be reported in the future. Discussion: Preliminary data shows the effects of MitoQ alone and MitoQ pretreatment in ischemic conditions on cell viability is influenced by different mediums and concentrations of MitoQ

Comparison of the inhibition of an OCT3 transporter inhibitor, Nilotinib, on Doxorubicin’s effects on cardiac and cancer cell lines

Introduction Doxorubicin (DOX)-induced cardiotoxicity remains a significant barrier limiting its clinical application due to a lack of effective resolution. Targeting how DOX enters cardiac and cancer cells is a promising new strategy. Research suggests that an OCT3 transporter significantly contributes to DOX entry into the heart tissue. By contrast, it expresses much lower on breast cancer cell lines. Moreover, Nilotinib (NIB) can suppress OCT3 transporter function by 80%. Therefore, exploring the impact of NIB on the DOX’s effects on cardiac and cancer cell lines by altering DOX intracellular accumulation is intriguing. Objective First, we would establish a dose-response curve of DOX and NIB alone to assess their individual effects on cell viability. Secondly, we would record the impact of NIB on DOX entry within cardiac myoblasts (H9C2) and breast cancer cells (MCF7) through OCT3 transporter antagonism to assess if NIB can exert cardioprotective effects while maintaining DOX’s anticancer effect. Methods H9C2 myoblast and MCF7 breast cancer cells were seeded in 96-well black plates. Cells were treated with only DOX or NIB to establish a dose-response curve. Moreover, NIB was combined with DOX as a cotreatment or pretreatment regimen to evaluate the impacts of NIB on DOX’s effect. Titrated combinations of NIB (10 nM, 50 nM, 100 nM, 500 nM, 1 µM, 2 µM, 5 µM) and DOX (10 µM and 40µM) were used. Bioassays were conducted after cells were treated for 24 hours. Intracellular DOX fluorescence intensity was measured at 488/590 nm by fluoroskan. Subsequently, cell viability was detected by measuring absorbance at 450 nm after adding a cell counting reagent. The data were expressed as a ratio relative to untreated or the DOX control. Results DOX dose-dependently reduced viability of H9c2 and MCF7 cells. H9c2 cell showed significantly lower cell viability at 1 µM (0.86±0.04, n=10, p\u3c0.05) and 40 µM (0.40±0.02, n=10, p\u3c0.05) when compared to those of MCF7 cells (1.07±0.05 and 0.68±0.08 for 1 µM and 40 µM, respectively, n=7). By contrast, NIB (10 nM-2 µM) only slightly increased cell viability to 1.13±0.05 (n=11) in H9c2 cells and to 1.16±0.13 (n=7) in MCF7 cells, respectively, when compared to untreated control. The highest tested dose of NIB (5 µM) showed a similar reduction of cell viability to 0.83±0.07 in H9c2 cells and to 0.81±0.10 in MCF7 cells. Furthermore, NIB cotreatment mitigated DOX-induced damages in H9c2 by increasing cell viability to 1.28±0.07 (n=5) and 1.26±0.11 (n=7) when compared to the DOX controls (10 µM and 40µM), respectively. Interestingly, NIB cotreatment enhanced DOX’s anti-cancer effects in by decreasing MCF7 cell viability to 0.66±0.10 (n=7) and 0.70±0.09 (n=6) when compared to the DOX controls (10 µM and 40µM), respectively. The intracellular DOX fluorescence data and NIB pretreatment results are still being gathered. Conclusion DOX, not NIB, dose-dependently induced H9c2 and MCF7 cell death. Moreover, DOX-induced damage was more potent in H9c2 cells than in MCF7 cells. NIB cotreatment mildly protected H9c2 cells against DOX, whereas it increased DOX’s anti-cancer effects in MCF7 cells

Cardioprotective effects of naltrindole in rat myocardial ischemia/reperfusion injury are concentration-dependent

Myocardial infarction remains a major cause of mortality and morbidity globally and infarct size is a major determinant of prognosis. Early and successful restoration of myocardial reperfusion is effective to reduce final infarct size and improve clinical outcomes. However, reperfusion induces further damage to the myocardium, hence the need for adjunctive therapy. Cardioprotective therapies to limit myocardial hypercontracture that occurs during prolonged ischemia and is associated with ischemia-reperfusion (I/R) injury are an important clinical goal. Previously, naltrindole (NTI, 5µM), when given prior to ischemia, exerted cardioprotective effects in ex-vivo, rat myocardial I/R, in part, by attenuating ischemic hypercontracture that occurred 20 min into global ischemia. The aim of this study was to determine the concentration-dependent relationship of NTI on infarct size and cardiac function in rat myocardial I/R. Male Sprague Dawley rats (~300g) were anesthetized with heparin (1000U), xylazine (30mg/kg), and ketamine solution (90mg/kg). Hearts were excised and placed in a Langendorff apparatus. A pressure transducer was placed into the left ventricle (LV) to measure LV end diastolic pressure (LVEDP) during infusion prior to ischemia, ischemia (ischemic peak pressure; IPP) and reperfusion. Global ischemia was induced by stopping Krebs buffer infusion for 30 min, followed by reperfusion (45min). Hearts were treated with NTI at concentrations of 1.25µM (n=5), 2.5µM (n=6), 5.0µM (n=5), or Krebs buffer (i.e. control; n=7) at a rate of 1 mL/min for 5 min prior to ischemia (preconditioning; PC) and during the first 5 min of reperfusion. After reperfusion, hearts were frozen, sectioned (2mm), and stained with 1% triphenyltetrazolium chloride. Infarct size was determined (weight of infarcted vs. total tissue at risk). All data were evaluated using ANOVA Student Newman Keuls post-hoc analysis. NTI 5µM (4.8±1%) and 2.5µM (6.8±0.8%), but not NTI 1.25 µM (12.2±3%), significantly reduced infarct size compared to control (14.1±1%, p Only NTI 2.5µM reduced infarct size without a negative inotropic effect (e.g. increased LVEDP during pretreatment administration). Collectively, NTI concentration-dependently decreased infarct size by 66% (5μM), 52%, (2.5µM), 13% (1.25 μM). Data suggest that NTI (2.5μM) can be given prophylactically to high-risk MI patients. Future studies will investigate the effect of NTI on cardiac hypercontracture and intracellular calcium content during ischemia

Protein Kinase C Beta II Inhibitor combined with Conjugated Myristic Acid and Trans-Activator of Transcription achieves cardiovascular protection in porcine myocardial ischemia/reperfusion model

Introduction While timely reperfusion is critical during resuscitation of ischemic myocardium, oxidative stress still leads to ischemia/reperfusion (I/R) injury and ultimately, cardiomyocyte death. The major targets for mitigating oxidative stress are NADPH oxidase (NOX-2) and mitochondria, which are both activated by Protein Kinase C Beta II (PKCβII). In previous studies with an ex-vivo rat heart I/R model, Myristic Acid (Myr) and Trans-Activator Transcription (Tat) conjugated PKCβII inhibitor (Myr-Tat-PKCβII-; N-Myr-Tat-CC-SLNPEWNET) showed cardioprotective effects and a decrease in infarct area. In this study, we investigated the cardioprotective effects of Myr-Tat-PKCβII- in comparison with a scrambled peptide control. Methods Regional I(1 hour)/R(3 hours) was induced in Male Yorkshire pigs (38-50kg) using balloon-assisted occlusion of the second diagonal branch of the Left Anterior Descending Artery (LAD) which is responsible for supplying 40% of the anterior myocardium. At the end of ischemia, the balloon was deflated to allow for reperfusion and the LAD was treated immediately with a bolus of either Myr-Tat-PKCβII- or scrambled peptide. Cardiac function was measured by monitoring changes in ejection fraction (EF). Cardiac injury was assessed through routine measurements of serum creatine phosphokinase (CPK), troponin I, and myoglobin. After I/R, the hearts were stained with Evans Blue dye to identify the area at risk (AR) and 1% triphenyltetrazolium chloride to determine the area of necrosis (AN). Infarct size was then quantified (AN/AR) and was analyzed via Student’s t-test, along with EF and cardiac marker measurements. Results Analysis showed that Myr-Tat-PKCβII- significantly restored EF to within 1.40.7% of baseline compared to controls which only restored EF to within 6.42.1% (p\u3c0.05) of baseline. Myr-Tat-PKCβII- showed a significant decrease in serum myoglobin levels at 1 hr of reperfusion (135132 ng/mL, n=4) compared to scrambled control (1022346 ng/mL, n=3 p\u3c0.05). Myr-Tat-PKCβII- reduced infarct size to 10.0±2.8%; n=4; compared to scrambled control hearts (28.5±8.3%; n=6; *p\u3c0.05). CPK and Troponin I levels were comparable in both groups. These results suggest that Myr-Tat-PKCβII- can help prevent cardiac injury when given immediately after an ischemic event. Discussion Data from ex-vivo rat heart I/R model, coupled with data from this in-vivo porcine I/R model indicate the efficacy of Myr-Tat-PKCβII- in preventing oxidative stress-induced cardiac injury. These findings suggest that Myr-Tat-PKCβII- could be useful in the clinical setting when administered immediately after cardiac resuscitation following an ischemic event. Future studies include the treatment of human umbilical vein endothelial cells with Myr-Tat-PKCβII- prior to hypoxia and at the beginning of reperfusion conditions, followed by cell viability assays in comparison with the untreated control. This additional data can help determine the optimal dose to use in an 8 week survival study using the same porcine myocardial I/R protocol for Myr-Tat-PKCβII-

In vitro naltrindole mitigates phorbol 12-myristate 13-acetate induced superoxide release in polymorphonuclear leukocytes independent of delta opioid receptor antagonism

Introduction: Naltrindole (NTI) is a selective delta opioid receptor antagonist that recently has been shown to be cardioprotective in both in vivo and ex vivo studies. In vivo, NTI showed cardioprotective effects that were dose-dependent significantly reducing infarct size up to 82% compared to control (p\u3c0.05). In ex vivo isolated rat hearts, NTI significantly reduced infarct size compared to controls (p\u3c0.01). NTI restored cardiac left ventricular end diastolic pressure to near baseline values compared to controls or other general opioid receptor antagonists in ex vivo hearts, suggesting a tissue salvaging mechanism independent of opioid delta receptor antagonism. Polymorphonuclear leukocytes (PMNs) do not express opioid receptors. In this study, we investigated NTI attenuation of PMN superoxide (SO) release in vitro. We hypothesized that NTI would attenuate SO release in PMNs via a calcium handling mechanism, a similar mechanism that explained cardioprotective effects of NTI pretreatment that mitigated ischemic hypercontracture that is due to an increase in intracellular calcium. Methods: Rat PMNs (5x 106) were incubated for 15 min at 37oC in the presence or absence (dH2O vehicle control) of NTI (10μM, 50μM, or 200 μM). PMN SO release was calculated by the change in absorbance at 550 nm over 420 sec via ferricytochrome c reduction after phorbal 12-myristate-13 acetate (PMA) stimulation (100nM). The cell viability was determined microscopically by 0.2% trypan blue exclusion at the end of the assay. Data were analyzed using ANOVA Fisher’s PLSD post-hoc test. Results: NTI significantly decreased PMN SO release at 420 sec at 200 μM (0.244±0.07, n=8, p\u3c0.05) and 50μM (0.293±0.06, n=4, p\u3c0.05), compared to control (0.487±0.12, n=9). NTI 10μM (0.412±0.07, n=5) was not significant compared to control. Cell viability was not significantly different compared to PMA across all NTI concentrations. Discussion: These results suggest that NTI reduces I/R injury and PMN SO release by a mechanism independent of delta opioid receptor antagonism. Future studies will assess the effects of NTI on ROS attenuation in human umbilical vein endothelial cells. We will use different concentrations to investigate optimal concentration response. Support or Funding Information This research was supported by the Division of Research, Department of Biomedical Sciences, and the Center for Chronic Disorders of Aging at Philadelphia College of Osteopathic Medicine. Current research license is supported by Young Therapeutics, LLC

Myristoylated Protein Kinase C Epsilon Peptide Inhibitor (Myr-PKC ε-) mitigates renal ischemia-reperfusion injury and PKCε translocation to epithelial cell membranes in vivo

Introduction: Delayed graft function (DGF), a post-transplant acute kidney injury, occurs in one-third (~7000) of the total number of kidney transplant recipients (~20,000) in the U.S. annually. It is characterized by prolonged ischemia (~20 mins) and subsequent restoration of blood flow to the previously ischemic renal tissue. Collectively, this is described as renal ischemia-reperfusion (I/R) injury. During reperfusion of blood to the previously ischemic renal tissue, generation of reactive oxygen species (ROS) from mitochondrial and uncoupled endothelial nitric oxide synthase (eNOS) is known to contribute to increased ROS-induced infarct size during reperfusion in myocardial I/R. Previously, Myr-PKCε- when given i.v. at the onset of reperfusion, decreased infarct size in both in vivo and ex vivo myocardial I/R models. Myr-PKCε- also decreased serum H2O2 in vivo in both renal extracorporeal shock wave lithotripsy and in a hindlimb I/R model. Myr-PKCε-(N-myr-EAVSLKPT) is known to have a protective effect by inhibiting superoxide production from uncoupled eNOS and mitochondrial ATP-sensitive K+ channels. We hypothesized that Myr-PKCε- would mitigate murine renal I (19min bilateral)/R(96 hrs.) injury and PKCε expression in renal tubular epithelium when given i.v. at the onset of reperfusion compared to scrambled control peptide (N-myr-LSETKPAV; Myr-PKCε-scram). Methods: Renal pedicles of anesthetized male C57BL/6J mice (25–30g) were clamped bilaterally for 19 mins. One minute before unclamping, 1.6 mg/kg (~20 µM serum concentration) Myr-PKCε- (n=6) or Myr-PKCε-scram (n=7) was administered by tail vein injection. Serum creatinine (Cr) (mg/dL) was measured at baseline, 24hrs, 72hrs, and 96hrs post-injury. Glomerular filtration rate (GFR) (µl/min) was measured via fluorescein-isothiocyanate (FITC)-Sinistrin. At the conclusion of the experiment, mice kidneys were removed, sectioned, formalin fixed and paraffin embedded. Immunohistochemistry (IHC) was performed using a PKCε antibody to evaluate PKCε localization and samples were analyzed using Aperio ImageScope. Results: Myr-PKCε- (n=6) significantly improved both GFR and Cr throughout reperfusion compared to Myr-PKCε-scram control (n=7, p\u3c0.05). Myr-PKCε- significantly improved both GFR and Cr throughout the 96 hrs reperfusion period compared to myr-PKCε-scram control. Myr-PKCε- restored final GFR and Cr to 52% and 54% vs. myr-PKCε-scram 29% and 18% respectively, compared to initial baseline values. IHC staining of kidney sections following I/R, diaminobenzidine chromogen reaction resulted in a brown precipitate indicating detection of PKCε and was defined to be a positive signal. All other signals were defined to be negative. Myr-PKCε- (1.77x108 ± 3.14x107) resulted in a significant decrease in the number of positive signals, in whole-kidney samples compared to Myr-PKCε-scram (3.58x10-1±5.03x10-2) (p \u3c 0.05). There was no difference in the number of negative signals or total number of signals between Myr-PKCε- and Myr-PKCε-scram. Discussion: Results suggest that Myr-PKCε- improved post-reperfused kidney function following bilateral renal I(19min)/R(96 hrs) ischemia and attenuated PKCε localization in tubular epithelium compared to Myr-PKCε-scram. In future studies, PKCε IHC staining and analysis will be performed on pig heart samples that have undergone I/R injury