Cardiomyocyte Toxicity Research Articles

A recombinant fragment antigen-binding (Fab) of trastuzumab displays low cytotoxic profile in adult human cardiomyocytes: first evidence and the key implication of FcγRIIA receptor.

Fragment crystallizable gamma receptors (FcγRs) mediate various cellular responses with significant cardiovascular implications. They contribute to the anticancer activity of trastuzumab (TRZ), a recombinant humanized monoclonal antibody that interferes with human epidermal growth factor receptor 2 (HER2), thereby blocking its physiological function in cardiac cells. This is responsible for cardiac complications that hamper TRZ clinical application. In this study we investigated the involvement of FcγRs in the TRZ cardiotoxicity. We used a recombinant antigen-binding fragment (Fab) of TRZ (rFab-HER2) to examine whether the absence of the Fc region resulted in fewer cardiomyocyte toxicity while preserving TRZ's ability to inhibit HER2. When exposed to rFab-HER2, AC16 human adult ventricular cardiomyocytes were less vulnerable to damage and death, than to TRZ. Specifically, TRZ exhibited cytotoxicity at a lower concentration (150 µg/mL, corresponding to ~1 µM) compared to rFab-HER2 (250 µg/mL, corresponding to ~5 µM). Like TRZ, rFab-HER2 negatively modulated HER2 levels in cardiomyocyte (without inducing cytotoxic activity in BJ human fibroblast cells that either did not express or express very low levels of HER2) and inhibited the downstream ERK/AKT cascades. But rFab-HER2 did not alter cardiomyocyte mitochondrial dynamic balance, and affect apoptosis and inflammation, while it limited cytosolic and mitochondrial ROS indicators. On contrary, the Fc region (50-250 μg/mL) exerted direct cytotoxic action on cardiomyocytes (but not on human fibroblasts that lacked Fc receptors). TRZ (150 μg/mL) markedly upregulated the expression level of FcγRIIA (a FcγRs strongly involved in TRZ-induced antibody-dependent cellular toxicity) in cardiomyocytes, whereas the Fab fragment (150 μg/mL) had no effect. Our results demonstrate that Fc region plays an important pathogenic role in TRZ-induced cardiomyocyte toxicity. In addition, targeting FcγRIIA might contribute to the off-target effects of TRZ therapy.

Metformin-mediated protection against doxorubicin-induced cardiotoxicity

BackgroundA phase II clinical trial of metformin (MET) for the treatment of doxorubicin (DOX)-induced cardiotoxicity (NCT02472353) failed. ObjectivesThe aims of this study were to confirm MET-mediated protection against DOX-induced cardiotoxicity and its mechanism using H9C2 cells, and to establish a Wistar rat model of DOX-induced cardiotoxicity. Subsequently, Wistar rats were utilized to identify clinically relevant indicators for evaluating MET-mediated protection against DOX-induced cardiotoxicity, thereby facilitating early transition towards successful clinical trials. MethodsMET-mediated protection was assessed using cell viability and cytotoxicity experiments. Additionally, intramitochondrial reactive oxygen species (ROS) levels were measured using an ROS fluorescent probe (dihydroethidium) to confirm the oxidative stress mechanism. Eighteen Wistar rats were randomly allocated to the control, DOX, and DOX+MET groups; and the body weight, adverse drug reactions (ADRs), myocardial injury, cardiac function, oxidative stress, and histopathology of heart tissues were compared between groups. ResultsH9C2 cells treated with MET/Dexrazoxane demonstrated dose-dependent protection against DOX-induced cardiotoxicity. The fluorescence intensity of H9C2 cells suggested DOX-induced cardiomyocyte toxicity and MET-mediated protection against DOX-induced cardiotoxicity. In vivo experiments confirmed that a rat model of DOX-induced cardiotoxicity was successfully established, but MET-mediated protection against DOX-induced cardiotoxicity was not demonstrated. This was attributed to insufficient energy intake because of ADRs, such as vomiting. ConclusionsWe confirmed the MET-mediated protection against DOX-induced cardiomyocyte toxicity and its mechanism involving the inhibition of oxidative stress in vitro experiments. It is imperative to investigate the optimal conditions for MET-mediated protection against DOX-induced cardiotoxicity in vivo or clinical trials.

12343 Cardiac Function Recovery Following Left Adrenalectomy For A Pheochromocytoma With Ectopic ACTH Syndrome

Abstract Disclosure: T.D. Williams: None. D. Lovre: None. R.C. Galagan: None. Background: Pheochromocytoma and Cushing’s Syndrome are implicated in Non-ischemic Cardiomyopathy/Heart Failure with reduced Ejection Fraction (NICM/HfrEF) due to catecholamine induced hypertension and cardiomyocyte toxicity. Subsequent hypoxia and calcium overload in cardiomyocytes results in oxidative stress and cell death. Excess cortisol secretion increases angiotensin II, causing hypertrophy. Subsequent increases in atrogin I lead to myo-fibrinolysis and fibrosis. We present a patient with NICM/HFrEF due to both catecholamine excess and ectopic ACTH syndrome from an adrenal pheochromocytoma. Following adrenalectomy, systolic cardiac function returned to normal. Clinical Case A 42-year-old female was referred to endocrinology after a hypertensive emergency concurrent with NSTEMI, NICM, and HFrEF (EF 45%). Initial work up revealed: Troponin I 0.49 ng/ml; (0 - 0.04 ng/ml), Na+ 140 mmol/L; (136 - 144 mmol/L), K+ 2.3 mmol/L; ​​(3.7 - 5.2 mmol/L), CO2 41 mmol/L; (23 - 29 mmol/L), Glucose 53 mg/dL; (64 - 100 mg/dL), Cortisol 57.2 ug/dL; (5 - 25 ug/dL), ACTH 363.7 pg/mL; (7.2 - 63.3 pg/mL). She failed both 1 mg and 8 mg overnight dexamethasone suppression tests: 0800 cortisol 48.5 ug/dL (< 1.8 ug/dL) & 56.8 ug/dL (> 50% decrease in baseline cortisol level of 57.2 ug/dL) consistent with ectopic ACTH syndrome. Elevated plasma normetanephrine 0.98 nmol/L; (0 - 0.89 nmol/L) and chromogranin A 250 ng/mL; (0 - 103 ng/mL) suggested a neuroendocrine tumor. Pituitary MRI revealed a normal pituitary gland. Abdominal MRI detected a 3.6 x 2.5 cm left adrenal nodule and PET/CT CU-64 showed increased activity (Krenning 4) consistent with a pheochromocytoma. Notwithstanding Ketoconazole (200 mg/day) for 7 months, her EF fell to a low of 15-20%. A left adrenalectomy was performed and post-op 0800 cortisol was 12.3 ug/dL (6.0 - 26.0 ug/dL). Oral hydrocortisone was initiated at 20 mg QAM/10 mg QPM for steroid withdrawal syndrome, tapered over 8 months and withdrawn when a cosyntropin stimulation test 60-min cortisol was 19.3 ug/dL; (>18.0 ug/dL). Eleven months post op, her 0800 cortisol was 8.8 ug/dL and ACTH 27.4 pg/mL. One month post-op, TTE showed EF: 15-20%, six months post-op EF: 25-30%, eight months post-op EF: 35-40% and 15 months post-op EF was normal at 50%. Her heart failure symptoms resolved, and her activity returned to normal. Conclusion This case illustrates a remarkable restoration of cardiac function following resection of a pheochromocytoma. Our patient's case is unique due to the restoration of cardiac function despite the dual impact of excessive catecholamines and cortisol. Our patient’s clinical course contributes to an evolving understanding of cardiomyopathy associated with pheochromocytoma and ectopic ACTH syndrome and highlights the potential for cardiomyopathy reversal with targeted endocrine surgical and medical interventions. Presentation: 6/1/2024

Aculeaquamides B and C, two new paraherquamides from the co-culture of Aspergillus aculeatinus WHUF0198 and Penicillium sp. DM27

Two new paraherquamides (PHQs) namely aculeaquamides B and C (1 and 2), along with four known PHQs (3–6), were isolated from the co-culture of marine fungus Aspergillus aculeatinus WHUF0198 and mangrove-associated fungus Penicillium sp. DM27. Compound 1 represents the first PHQ derivative featuring an uncommon 7/6/5/5/6/5 hexacyclic system. The structures of the isolated compounds were elucidated based on exhaustive NMR spectroscopy measurement and HRESIMS data. The absolute configurations of new compounds were determined by TDDFT-ECD calculations. Compound 3 demonstrated suppression of AngII-induced cardiac hypertrophy while exhibiting relatively low cardiomyocyte toxicity.

Characterization of ferroptosis-triggered pyroptotic signaling in heart failure

Pressure overload–induced cardiac hypertrophy is a common cause of heart failure (HF), and emerging evidence suggests that excessive oxidized lipids have a detrimental effect on cardiomyocytes. However, the key regulator of lipid toxicity in cardiomyocytes during this pathological process remains unknown. Here, we used lipidomics profiling and RNA-seq analysis and found that phosphatidylethanolamines (PEs) and Acsl4 expression are significantly increased in mice with transverse aortic constriction (TAC)–induced HF compared to sham-operated mice. In addition, we found that overexpressing Acsl4 in cardiomyocytes exacerbates pressure overload‒induced cardiac dysfunction via ferroptosis. Notably, both pharmacological inhibition and genetic deletion of Acsl4 significantly reduced left ventricular chamber size and improved cardiac function in mice with TAC-induced HF. Moreover, silencing Acsl4 expression in cultured neonatal rat ventricular myocytes was sufficient to inhibit hypertrophic stimulus‒induced cell growth. Mechanistically, we found that Acsl4-dependent ferroptosis activates the pyroptotic signaling pathway, which leads to increased production of the proinflammatory cytokine IL-1β, and neutralizing IL-1β improved cardiac function in Acsl4 transgenic mice following TAC. These results indicate that ACSL4 plays an essential role in the heart during pressure overload‒induced cardiac remodeling via ferroptosis-induced pyroptotic signaling. Together, these findings provide compelling evidence that targeting the ACSL4-ferroptosis-pyroptotic signaling cascade may provide a promising therapeutic strategy for preventing heart failure.

Adenosine protects cardiomyocytes against acrolein-induced cardiotoxicity by enhancing mitochondrial homeostasis, antioxidant defense, and autophagic flux via ERK-activated FoxO1 upregulation

Acrolein is a ubiquitous gaseous air pollutant and endogenous toxicant, which poses strong risk for oxidative stress-related diseases such as cardiovascular disease. Adenosine has been identified as potential therapeutic agent for age-related cardiovascular disease, while the molecular mechanisms underlying its cardioprotection remain elusive. In the present study, we investigated the myocardial protective effects and the mechanism of adenosine on acrolein-induced toxicity in H9c2 cells and primary neonatal rat cardiomyocytes. We found that acrolein caused apoptosis of cardiomyocytes resulting from oxidative damage, autophagy defect, and mitochondrial dysfunction, as evidenced by loss of mitochondrial membrane potential, impairment of mitochondrial biogenesis, dynamics, and oxidative phosphorylation, decrease of mitochondrial deoxyribonucleic acid (mtDNA) copy number and adenosine 5’-triphosphate (ATP) production. Adenosine pretreatment protected against acrolein-induced cardiotoxicity by maintaining mitochondrial homeostasis, activating the phase II detoxifying enzyme system, promoting autophagic flux, and alleviating mitochondrial-dependent apoptosis. We further demonstrated that the up-regulation of forkhead box protein O1 (FoxO1) mediated by extracellular regulated protein kinases (ERK) activation contributes to the cardioprotection of adenosine. These results expand the application of adenosine in cardioprotection to preventing myocardial damages induced by environmental pollutant acrolein exposure, and uncover the adenosine-ERK-FoxO1 axis as the underlying mechanism mediating the protection of mitochondrial homeostasis, Nrf2-mediated antioxidant defense and autophagic flux, shedding light on the better understanding about the pathological mechanism of cardiovascular disease caused by environmental pollutants and applications of adenosine in cardioprotection.

ATP Citrate Lyase Supports Cardiac Function and NAD+/NADH Balance And Is Depressed in Human Heart Failure.

ATP-citrate lyase (ACLY) converts citrate into acetyl-CoA and oxaloacetate in the cytosol. It plays a prominent role in lipogenesis and fat accumulation coupled to excess glucose, and its inhibition is approved for treating hyperlipidemia. In RNAseq analysis of human failing myocardium, we found ACLY gene expression is reduced; however the impact this might have on cardiac function and/or metabolism has not been previously studied. As new ACLY inhibitors are in development for cancer and other disorders, such understanding has added importance. Cardiomyocytes, ex-vivo beating hearts, and in vivo hearts with ACLY inhibited by selective pharmacologic (BMS303141, ACLYi) or genetic suppression, were studied. Regulation of ACLY gene/protein expression, and effects of ACLYi on function, cytotoxicity, tricarboxylic acid (TCA)-cycle metabolism, and redox and NAD+/NADH balance were assessed. Mice with cardiac ACLY knockdown induced by AAV9-acly-shRNA or cardiomyocyte tamoxifen-inducible Acly knockdown were studied. Acly gene expression was reduced more in obese patients with heart failure and preserved EF (HFpEF) than HF with reduced EF. In vivo pressure-overload and in vitro hormonal stress increased ACLY protein expression, whereas it declined upon fatty-acid exposure. Acute ACLYi (1-hr) dose-dependently induced cytotoxicity in adult and neonatal cardiomyocytes, and caused substantial reduction of systolic and diastolic function in myocytes and ex-vivo beating hearts. In the latter, ATP/ADP ratio also fell and lactate increased. U13C-glucose tracing revealed an ACLYdependent TCA-bypass circuit in myocytes, where citrate generated in mitochondria is transported to the cytosol, metabolized by ACLY and then converted to malate to re-enter mitochondria,bypassing several NADH-generating steps. ACLYi lowered NAD+/NADH ratio and restoring this balance ameliorated cardiomyocyte toxicity. Oxidative stress was undetected with ACLYi. Adult hearts following 8-weeks of reduced cardiac and/or cardiomyocyte ACLY downregulation exhibited ventricular dilation and reduced function that was prevented by NAD augmentation. Cardiac dysfunction from ACLY knockdown was worse in hearts subjected to sustained pressureoverload, supporting a role in stress responses. ACLY supports normal cardiac function through maintenance of the NAD+/NADH balance and is upregulated by hemodynamic and hormonal stress, but depressed by lipid excess. ACLY levels are most reduced in human HFpEF with obesity potentially worsening cardio-metabolic reserve.

Antiapoptotic Effect of Target Protein for Xklp2 on Diabetic Rat Cardiomyocytes

This study investigates the potential of TPX2 in mitigating diabetic cardiomyopathy by modulating the Akt/FoxO1 signaling pathway. Diabetic cardiomyopathy was induced in rats using streptozotocin, and heart structure and function were assessed through echocardiography and HE staining. TPX2 expression in myocardial tissue was measured using qRT-PCR. Lentivirus was used to overexpress TPX2 specifically in myocardial tissue, followed by reassessment of cardiac structure and function. Immunohistochemical staining was employed to evaluate Caspase3 expression. In the diabetic cardiomyopathy group, rat hearts exhibited evident damage in structure and function, with increased Caspase3 expression in myocardial tissue. Conversely, TPX2 overexpression effectively improved heart structure and function while inhibiting Caspase3 upregulation. In H9C2 cells cultured with high glucose, TPX2 overexpression counteracted reduced cell activity and increased apoptosis rates. It also downregulated the mRNA levels of apoptosis-related factors (Bax, Caspase3, Caspase9) and promoted anti-apoptotic factor Bcl-2 mRNA. TPX2 overexpression inhibited Caspase3 and Caspase9 activities, upregulated Akt mRNA and protein expression, and inhibited FoxO1 mRNA and protein expression. This suggests that TPX2 may regulate the Akt/FoxO1 signaling pathway, reducing apoptosis and cardiomyocyte toxicity induced by high glucose. These findings offer potential drug targets and therapeutic strategies for diabetic cardiomyopathy treatment.

Perfluoroalkyl sulfonate induces cardiomyocyte apoptosis via endoplasmic reticulum stress activation and autophagy flux inhibition

Perfluoroalkyl sulfonate (PFOS) is a commonly used chemical compound that often found in materials such as waterproofing agents, food packaging, and fire retardants. Known for its stability and persistence in the environment, PFOS can enter the human body through various pathways, including water and the food chain, raising concerns about its potential harm to human health. Previous studies have suggested a cardiac toxicity of PFOS, but the specific cellular mechanisms remained unclear. Here, by using AC16 cardiomyocyte as a model to investigate the molecular mechanisms potential the cardiac toxicity of PFOS. Our findings revealed that PFOS exposure reduced cell viability and induces apoptosis in human cardiomyocyte. Proteomic analysis and molecular biological techniques showed that the Endoplasmic Reticulum (ER) stress-related pathways were activated, while the cellular autophagy flux was inhibited in PFOS-exposed cells. Subsequently, we employed strategies such as autophagy activation and ER stress inhibition to alleviate the PFOS-induced apoptosis in AC16 cells. These results collectively suggest that PFOS-induced ER stress activation and autophagy flux inhibition contribute to cardiomyocyte apoptosis, providing new insights into the mechanisms of PFOS-induced cardiomyocyte toxicity.

Integrated virtual screening and in vitro studies for exploring the mechanism of triterpenoids in Chebulae Fructus alleviating mesaconitine-induced cardiotoxicity via TRPV1 channel.

Background: In traditional Mongolian or Tibetan medicine in China, Chebulae Fructus (CF) is widely used to process or combine with aconitums to decrease the severe toxicity of aconitums. Researches in this area have predominantly focused on tannins, with few research on other major CF components for cardiotoxicity mitigation. The present study aimed to clarify whether triterpenoids can attenuate the cardiotoxicity caused by mesaconitine (MA) and investigate the mechanism of cardiotoxicity attenuation. Methods: Firstly, the pharmacophore model, molecular docking, and 3D-QSAR model were used to explore the mechanism of CF components in reducing the toxicity of MA mediated by the TRPV1 channel. Then three triterpenoids were selected to verify whether the triterpenoids had the effect of lowering the cardiotoxicity of MA using H9c2 cells combined with MTT, Hoechst 33258, and JC-1. Finally, Western blot, Fluo-3AM, and MTT assays combined with capsazepine were used to verify whether the triterpenoids reduced H9c2 cardiomyocyte toxicity induced by MA was related to the TRPV1 channel. Results: Seven triterpenoids in CF have the potential to activate the TRPV1 channel. And they exhibited greater affinity for TRPV1 compared to other compounds and MA. However, their activity was relatively lower than that of MA. Cell experiments revealed that MA significantly reduced H9c2 cell viability, resulting in diminished mitochondrial membrane potential and nuclear pyknosis and damage. In contrast, the triterpenoids could improve the survival rate significantly and counteract the damage of MA to the cells. We found that MA, arjungenin (AR), and maslinic acid (MSA) except corosolic acid (CRA) upregulated the expression of TRPV1 protein. MA induced a significant influx of calcium, whereas all three triterpenoids alleviated this trend. Blocking the TRPV1 channel with capsazepine only increased the cell viability that had been simultaneously treated with MA, and AR, or MSA. However, there was no significant difference in the CRA groups treated with or without capsazepine. Conclusion: The triterpenoids in CF can reduce the cardiotoxicity caused by MA. The MSA and AR function as TRPV1 agonists with comparatively reduced activity but a greater capacity to bind to TRPV1 receptors, thus antagonizing the excessive activation of TRPV1 by MA.

Exploring the effects of topoisomerase II inhibitor XK469 on anthracycline cardiotoxicity and DNA damage.

Anthracyclines, such as doxorubicin (adriamycin), daunorubicin, or epirubicin, rank among the most effective agents in classical anticancer chemotherapy. However, cardiotoxicity remains the main limitation of their clinical use. Topoisomerase IIβ has recently been identified as a plausible target of anthracyclines in cardiomyocytes. We examined the putative topoisomerase IIβ selective agent XK469 as a potential cardioprotective and designed several new analogs. In our experiments, XK469 inhibited both topoisomerase isoforms (α and β) and did not induce topoisomerase II covalent complexes in isolated cardiomyocytes and HL-60, but induced proteasomal degradation of topoisomerase II in these cell types. The cardioprotective potential of XK469 was studied on rat neonatal cardiomyocytes, where dexrazoxane (ICRF-187), the only clinically approved cardioprotective, was effective. Initially, XK469 prevented daunorubicin-induced toxicity and p53 phosphorylation in cardiomyocytes. However, it only partially prevented the phosphorylation of H2AX and did not affect DNA damage measured by Comet Assay. It also did not compromise the daunorubicin antiproliferative effect in HL-60 leukemic cells. When administered to rabbits to evaluate its cardioprotective potential in vivo, XK469 failed to prevent the daunorubicin-induced cardiac toxicity in either acute or chronic settings. In the following in vitro analysis, we found that prolonged and continuous exposure of rat neonatal cardiomyocytes to XK469 led to significant toxicity. In conclusion, this study provides important evidence on the effects of XK469 and its combination with daunorubicin in clinically relevant doses in cardiomyocytes. Despite its promising characteristics, long-term treatments and in vivo experiments have not confirmed its cardioprotective potential.

Antioxidant properties of acteoside against biological systems: Hemoglobin and cardiomyocyte as potential models

Oxidative stress has been shown to be the main cause of protein denaturation and cell death. However, the exact mechanism of antioxidants against oxidative stress-mediated protein damage and cell cytotoxicity remains largely unknown. Therefore, in this paper, the protective effects of acteoside against H2O2-inudced hemoglobin structural changes and cardiomyocyte toxicity as models were determined by different techniques. Fluorescence quenching as well as molecular docking simulation studies showed that acteoside potentially interacted with hemoglobin in a one: one binding mode mediated mainly by the involvement of hydrophobic forces. It was also shown that H2O2 caused significant changes in the structure of hemoglobin as well as heme degradation, reversed by acteoside. Additionally, UV–visible studies exhibited that acteoside decreased the generation of methemoglobin triggered by H2O2. Cellular assays displayed that acteoside could mitigate the cardiomyocyte toxicity induced by H2O2 through regulation of LDH release, generation of reactive oxygen species (ROS) and 3,4-methylenedioxyamphetamine (MDA), mitochondrial membrane potential (MMP) collapse, as well as superoxide dismutase (SOD), catalase (CAT), caspase-9, and caspase-3 activities. In conclusion, acteoside may hold great promise for the control of protein-related disorders as well as cardiovascular diseases.

Rev-erbα attenuates diabetic myocardial injury through regulation of ferroptosis

Diabetes is a widespread disease that threatens the life and health of human beings, and diabetic cardiomyopathy (DCM) is one of the major complications of diabetic patients. The pathological mechanisms of DCM are complex, including inflammation, endoplasmic reticulum stress, and oxidative stress that have been reported previously. Although recent studies suggested that ferroptosis is also involved in the progression of DCM, the exact mechanism remains unclear. Rev-erbα cardiac conditional knockout mice were generated and type 2 diabetes were induced by high fat diet (HFD) and intraperitoneal injection of streptozotocin (STZ) in in vivo experiments. In parallel, our in vitro experiments entailed the introduction of elevated levels of glucose (HG) and palmitic acid (PA) to induce glycolipid toxicity in H9c2 cardiomyocytes. Further deterioration of cardiac function was detected by echocardiography after the clock gene rev-erbα was knocked out. This was accompanied by significant elevations in markers of inflammation, myocardial fibrosis, and oxidative stress. In addition, iron content, transmission electron microscopy (TEM), and RT-PCR assays confirmed significantly increased levels of ferroptosis in rev-erbα-deficient DCM. Intriguingly, Co-Immunoprecipitation (Co-IP) data uncovered an interaction between rev-erbα and nuclear factor E2-related factor 2 (NRF2) in diabetic myocardial tissues. It is worth highlighting that ferroptosis within cardiomyocytes witnessed significant mitigation upon the administration of sulforaphane (SFN), an NRF2 agonist, to HG + PA-incubated H9c2 cells. Our study demonstrates for the first time that knockdown of the clock gene rev-erbα exacerbates myocardial injury and ferroptosis in type 2 diabetic mice, which can be reversed by activating NRF2.

Abstract 18503: Endogenously Released Factors From Doxorubicin-Treated Breast Cancer Spheroids Contribute to Toxicity in Human Cardiomyocytes: Protective Effect of Combination Therapy With PDE5-mTOR Inhibitors

Background: We previously demonstrated that a novel combination therapy with the phosphodiesterase 5 (PDE5) inhibitor (sildenafil, Sild) and mTOR inhibitor (rapamycin, Rapa) potentiates doxorubicin (DOX)-induced breast cancer cell killing, but attenuates cardiotoxicities. However, it remains unclear whether there are any factors released from cancer cells treated with DOX that contribute to the injury in cardiomyocytes, which can be modified by Sild and Rapa in combination. Therefore, we designed experiments using spheroid cultures of breast cancer and human AC-16 cardiomyocytes to determine the role of endogenously released factors following treatment with DOX. Methods & Results: Spheroid cultures of breast cancer MDA-MB-231 cells were treated with DOX (1 μμ), and/or Sild (10 μμ) and Rapa (100 nμ) for 48 hours. The medium was replaced and spheroids were further incubated for 48 hours in the fresh medium. Thereafter, the AC-16 cardiomyocytes were treated with the medium from drugs-treated spheroids for 48 hours. Our results showed that treatment with DOX and/or Sild and Rapa was highly effective in reducing the size of spheroids as compared to the control ( Fig. A ). Moreover, Western blots showed that p-mTOR/mTOR and p-S6/S6 were induced and p-AKT/AKT was suppressed after DOX treatment in cancer cells, which were normalized following treatment with Sild and Rapa ( Fig. B ). The medium from DOX-treated spheroids caused increase of cardiomyocytes death as assessed by trypan blue exclusion assay, which was attenuated by co-treatment with the medium from Sild and Rapa treated spheroids ( Fig. C ). Conclusion: These results suggest that cardiotoxicity of chemotherapeutic agents could be partly contributed by the endogenously released unknown factors from tumors, which can be modulated by a novel combination treatment with Sild and Rapa via regulating mTOR-signaling pathways.

Oxygen consumption rate to evaluate mitochondrial dysfunction and toxicity in cardiomyocytes.

The increase in the types and complexity of diseases has led to significant advances in diagnostic techniques and the availability of effective therapies. Recent studies have focused on the role of mitochondrial dysfunction in the pathogenesis of cardiovascular diseases (CVDs). Mitochondria are important organelles in cells that generate energy. Besides the production of adenosine triphosphate (ATP), the energy currency of cells, mitochondria are also involved in thermogenesis, control of intracellular calcium ions (Ca2+), apoptosis, regulation of reactive oxygen species (ROS), and inflammation. Mitochondrial dysfunction has been implicated in several diseases including cancer, diabetes, some genetic diseases, and neurogenerative and metabolic diseases. Furthermore, the cardiomyocytes of the heart are rich in mitochondria due to the large energy requirement for optimal cardiac function. One of the main causes of cardiac tissue injuries is believed to be mitochondrial dysfunction, which occurs via complicated pathways which have not yet been completely elucidated. There are various types of mitochondrial dysfunction including mitochondrial morphological change, unbalanced levels of substances to maintain mitochondria, mitochondrial damage by drugs, and mitochondrial deletion and synthesis errors. Most of mitochondrial dysfunctions are linked with symptoms and diseases, thus we focus on parts of mitochondrial dysfunction about fission and fusion in cardiomyocytes, and ways to understand the mechanism of cardiomyocyte damage by detecting oxygen consumption levels in the mitochondria.

An integrated strategy to explore the potential role of melatonin against copper-induced adrenaline toxicity in rat cardiomyocytes: Insights into oxidative stress, inflammation, and apoptosis

AimsCircumstantial anxiety as well as chronic stress may stimulate the release of stress hormones including catecholamines. Adrenaline toxicity has been implicated in many cardiovascular conditions. Considering previous literature that suggests the oxidative potential of the adrenaline-copper entity, we have investigated its potential nocuous role in isolated adult rat cardiomyocytes, the underlying molecular mechanism, and its possible protection by melatonin. Main methodsGiven the mechanistic congruity of adrenaline-copper (AC) with the well-established H2O2-copper-ascorbate (HCA) system of free radical generation, we have used the latter as a representative model to study the cytotoxic nature of AC. We further investigated the cardioprotective efficacy of melatonin in both the stress models through scanning electron microscopy, immunofluorescence, flow cytometry, and western blot analysis. Key findingsResults show that melatonin significantly protects AC-treated cardiomyocytes from ROS-mediated membrane damage, disruption of mitochondrial membrane potential, antioxidant imbalance, and distortion of cellular morphology. Melatonin protects cardiomyocytes from inflammation by downregulating pro-inflammatory mediators viz., COX-2, NF-κB, TNF-α, and upregulating anti-inflammatory IL-10. Melatonin significantly ameliorated cardiomyocyte apoptosis in AC and HCA-treated cells as evidenced by decreased BAX/BCL-2 ratio and subsequent suppression of caspase-9 and caspase-3 levels. The isothermal calorimetric study revealed that melatonin inhibits the binding of adrenaline bitartrate with copper in solution, which fairly explains the rescue potential of melatonin against AC-mediated toxicity in cardiomyocytes. SignificanceFindings suggest that the multipronged strategy of melatonin that includes its antioxidant, anti-inflammatory, anti-apoptotic, and overall cardioprotective ability may substantiate its potential therapeutic efficacy against adrenaline-copper-induced damage and death of adult rat cardiomyocytes.

Three tyrosine kinase inhibitors cause cardiotoxicity by inducing endoplasmic reticulum stress and inflammation in cardiomyocytes

BackgroundTyrosine kinase inhibitors (TKIs) are anti-cancer therapeutics often prescribed for long-term treatment. Many of these treatments cause cardiotoxicity with limited cure. We aim to clarify molecular mechanisms of TKI-induced cardiotoxicity so as to find potential targets for treating the adverse cardiac complications.MethodsEight TKIs with different levels of cardiotoxicity reported are selected. Phenotypic and transcriptomic responses of human cardiomyocytes to TKIs at varying doses and times are profiled and analyzed. Stress responses and signaling pathways that modulate cardiotoxicity induced by three TKIs are validated in cardiomyocytes and rat hearts.ResultsToxicity rank of the eight TKIs determined by measuring their effects on cell viability, contractility, and respiration is largely consistent with that derived from database or literature, indicating that human cardiomyocytes are a good cellular model for studying cardiotoxicity. When transcriptomes are measured for selected TKI treatments with different levels of toxicity in human cardiomyocytes, the data are classified into 7 clusters with mainly single-drug clusters. Drug-specific effects on the transcriptome dominate over dose-, time- or toxicity-dependent effects. Two clusters with three TKIs (afatinib, ponatinib, and sorafenib) have the top enriched pathway as the endoplasmic reticulum stress (ERS). All three TKIs induce ERS in rat primary cardiomyocytes and ponatinib activates the IRE1α-XBP1s axis downstream of ERS in the hearts of rats underwent a 7-day course of drug treatment. To look for potential triggers of ERS, we find that the three TKIs induce transient reactive oxygen species followed by lipid peroxidation. Inhibiting either PERK or IRE1α downstream of ERS blocks TKI-induced cardiac damages, represented by the induction of cardiac fetal and pro-inflammatory genes without causing more cell death.ConclusionsOur data contain rich information about phenotypic and transcriptional responses of human cardiomyocytes to eight TKIs, uncovering potential molecular mechanisms in modulating cardiotoxicity. ER stress is activated by multiple TKIs and leads to cardiotoxicity through promoting expression of pro-inflammatory factors and cardiac fetal genes. ER stress-induced inflammation is a promising therapeutic target to mitigate ponatinib- and sorafenib-induced cardiotoxicity.Graphical

Protective Effects of Epigallocatechin-3-gallate (EGCG) against the Jellyfish Nemopilema nomurai Envenoming.

Jellyfish stings are the most common marine animal injuries worldwide, with approximately 150 million envenomation cases annually, and the victims may suffer from severe pain, itching, swelling, inflammation, arrhythmias, cardiac failure, or even death. Consequently, identification of effective first aid reagents for jellyfish envenoming is urgently needed. Here, we found that the polyphenol epigallocatechin-3-gallate (EGCG) markedly antagonized the hemolytic toxicity, proteolytic activity, and cardiomyocyte toxicity of the jellyfish Nemopilema nomurai venom in vitro and could prevent and treat systemic envenoming caused by N. nomurai venom in vivo. Moreover, EGCG is a natural plant active ingredient and widely used as a food additive without toxic side effects. Hence, we suppose that EGCG might be an effective antagonist against systemic envenoming induced by jellyfish venom.

Proarrhythmic toxicity of low dose bisphenol A and its analogs in human iPSC-derived cardiomyocytes and human cardiac organoids through delay of cardiac repolarization

Bisphenol A (BPA) and its analogs are common environmental chemicals with many potential adverse health effects. The impact of environmentally relevant low dose BPA on human heart, including cardiac electrical properties, is not understood. Perturbation of cardiac electrical properties is a key arrhythmogenic mechanism. In particular, delay of cardiac repolarization can cause ectopic excitation of cardiomyocytes and malignant arrhythmia. This can occur as a result of genetic mutations (i.e., long QT (LQT) syndrome), or cardiotoxicity of drugs and environmental chemicals. To define the impact of low dose BPA on electrical properties of cardiomyocytes in a human-relevant model system, we examined the rapid effects of 1 nM BPA in human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) using patch-clamp and confocal fluorescence imaging. Acute exposure to BPA delayed repolarization and prolonged action potential duration (APD) in hiPSC-CMs through inhibition of the hERG K+ channel. In nodal-like hiPSC-CMs, BPA acutely increased pacing rate through stimulation of the If pacemaker channel. Existing arrhythmia susceptibility determines the response of hiPSC-CMs to BPA. BPA resulted in modest APD prolongation but no ectopic excitation in baseline condition, while rapidly promoted aberrant excitations and tachycardia-like events in myocytes that had drug-simulated LQT phenotype. In hiPSC-CM-based human cardiac organoids, the effects of BPA on APD and aberrant excitation were shared by its analog chemicals, which are often used in “BPA-free” products, with bisphenol AF having the largest effects. Our results reveal that BPA and its analogs have repolarization delay-associated pro-arrhythmic toxicity in human cardiomyocytes, particularly in myocytes that are prone to arrhythmias. The toxicity of these chemicals depends on existing pathophysiological conditions of the heart, and may be particularly pronounced in susceptible individuals. An individualized approach is needed in risk assessment and protection.

Protective effects of protocatechuic acid against doxorubicin- and arsenic trioxide-induced toxicity in cardiomyocytes.

Some chemotherapeutic drugs are associated with an increased risk of cardiotoxicity in patients. Protocatechuic acid (PCA) is a phenolic acid with valuable cardiovascular, chemo-preventive, and anticancer activities. Recent studies have shown the cardioprotective effects of PCA in several pathological conditions. This investigation aimed to assess the possible protective effects of PCA on cardiomyocytes against toxicities caused by anti-neoplastic agents, doxorubicin (DOX), and arsenic trioxide (ATO). H9C2 cells were exposed to DOX (1 μM) or ATO (35 μM) after 24 h pretreatment with PCA (1-100 μM). MTT and lactate dehydrogenase (LDH) tests were used to define cell viability or cytotoxicity. Total oxidant and antioxidant capacities were evaluated by measuring hydroperoxides and ferric-reducing antioxidant power (FRAP) levels. Expression of the TLR4 gene was also quantitatively estimated by real-time polymerase chain reaction. PCA showed a proliferative effect on cardiomyocytes and significantly enhanced cell viability and reduced cytotoxicity of DOX and ATO during MTT and LDH assays. Pretreatment of cardiomyocytes with PCA significantly decreased hydroperoxide levels and elevated FRAP value. Moreover, PCA meaningfully decreased TLR4 expression in DOX-and ATO-treated cardiomyocytes. In conclusion, antioxidant and cytoprotective activities were found for PCA versus toxicities caused by DOX and ATO in cardiomyocytes. However, further in vivo investigations are recommended to assess its clinical value for the prevention and treatment of cardiotoxicity induced by chemotherapeutic agents.