Mettl14 Regulates Cardiac Ishemia/Reperfusion Injury

N6-methyladenosine (m6A) methylation in RNA is a dynamic and reversible modification regulated by methyltransferases and demethylases, which has been reported to participate in many pathological processes of various diseases, including cardiac disorders. This study was designed to investigate an m6A writer Mettl14 on cardiac ischemia–reperfusion (I/R) injury and uncover the underlying mechanism. The m6A and Mettl14 protein levels were increased in I/R hearts and neonatal mouse cardiomyocytes upon oxidative stress. Mettl14 knockout (Mettl14+/−) mice showed pronounced increases in cardiac infarct size and LDH release and aggravation in cardiac dysfunction post-I/R. Conversely, adenovirus-mediated overexpression of Mettl14 markedly reduced infarct size and apoptosis and improved cardiac function during I/R injury. Silencing of Mettl14 alone significantly caused a decrease in cell viability and an increase in LDH release and further exacerbated these effects in the presence of H2O2, while overexpression of Mettl14 ameliorated cardiomyocyte injury in vitro. Mettl14 resulted in enhanced levels of Wnt1 m6A modification and Wnt1 protein but not its transcript level. Furthermore, Mettl14 overexpression blocked I/R-induced downregulation of Wnt1 and β-catenin proteins, whereas Mettl14+/− hearts exhibited the opposite results. Knockdown of Wnt1 abrogated Mettl14-mediated upregulation of β-catenin and protection against injury upon H2O2. Our study demonstrates that Mettl14 attenuates cardiac I/R injury by activating Wnt/β-catenin in an m6A-dependent manner, providing a novel therapeutic target for ischemic heart disease.

Cox-2 Antagonizes the Protective Effect of Sevoflurane on Hypoxia/Reoxygenation-Induced Cardiomyocyte Apoptosis through Inhibiting the Akt Pathway

Objective. To uncover the protective role of sevoflurane on hypoxia/reoxygenation-induced cardiomyocyte apoptosis through the protein kinase B (Akt) pathway. Methods. An in vitro hypoxia/reoxygenation (H/R) model was established in cardiomyocyte cell line H9c2. Sevoflurane (SEV) was administrated in H9c2 cells during the reoxygenation period. Viability, layered double hydroxide (LDH) release, and apoptosis in H9c2 cells were determined to assess H/R-induced cell damage. Relative levels of apoptosis-associated genes were examined. Moreover, phosphorylation of Akt was determined. Results. H/R injury declined viability and enhanced LDH release and apoptotic rate in H9c2 cells. Cyclooxygenase-2 (Cox-2) was upregulated following H/R injury, which was partially reversed by SEV treatment. In addition, SEV treatment reversed changes in viability and LDH release owing to H/R injury in H9c2 cells, which were further aggravated by overexpression of Cox-2. The Akt pathway was inhibited in H9c2 cells overexpressing Cox-2. Conclusions. Sevoflurane protects cardiomyocyte damage following H/R via the Akt pathway, and its protective effect was abolished by overexpression of Cox-2.

TBHQ attenuates ferroptosis against 5-fluorouracil-induced intestinal epithelial cell injury and intestinal mucositis via activation of Nrf2

Background Intestinal mucositis is a common side effect of chemotherapy and radiotherapy. Very few drugs can efficiently ameliorate it. Tertiary butylhydroquinone (TBHQ) is a widely used food preservative with known immunomodulatory activity. Whether it has an effect on intestinal mucositis remains unknown. In this study, we investigated the role and mechanism of action of TBHQ on 5-fluorouracil-induced (5-FU-induced) human intestinal epithelial cell (HIEC) injury and intestinal mucositis in mice. Methods We established a cell model of HIEC injury and a mouse model of intestinal mucositis via treatment with 5-FU. Cell death, Cell Counting Kit-8, and lactate dehydrogenase (LDH) release were assessed for the HIECs. Diarrhea, body weight, intestinal length, mucosal damage, and the levels of IL-6, TNF-α, IL-1β, glutathione, reactive oxygen species, and malondialdehyde were determined for the mice. Additionally, we performed immunohistochemical analysis, immunofluorescence, western blotting, quantitative real-time PCR, and ELISA to examine the effects of TBHQ. Finally, HIECs were transfected with an Nrf2 gene silencer to verify its role in ferroptosis. All data were analyzed using one-way analysis of variance or paired t-tests. Results TBHQ markedly decreased LDH release and cell death and improved the proliferative ability of 5-FU-treated HIECs. The TBHQ-treated mice showed reduced weight loss, a lower diarrhea score, and longer colons than the 5-FU-treated mice. The in vivo expressions of IL-1β, IL-6, and TNF-α were suppressed by TBHQ treatment. Ferroptosis was shown to be involved in 5-FU-induced intestinal mucositis, and TBHQ markedly hampered its activation. Mechanistically, TBHQ activated Nrf2 effectively and selective Nrf2 knockdown significantly reduced the anti-ferroptotic functions of TBHQ in 5-FU-treated HIECs. Conclusions TBHQ attenuates ferroptosis in 5-FU-induced intestinal mucositis, making it a potential novel protective agent against intestinal mucositis.

Abstract 11710: Poloxamer 188 Protects Coronary Artery Endothelial Cells After Extended Hypoxia and Reoxygenation in an in vitro Model of Simulated Ischemia/Reperfusion Injury

Reperfusion restores blood flow after myocardial ischemia but can cause additional cellular injury by the sudden reintroduction of oxygen and nutrients. There is still no effective remedy for myocardial ischemia/reperfusion (IR) injury. Our previous study using cardiomyocytes (CMs) found that, after 3 hrs hypoxia followed by 2 hrs reoxygenation, viability decreased, and release of lactate dehydrogenase (LDH), calcium influx, membrane leakage (insertion of fluorescent probe FM1-43) significantly increased, indicating that cell membrane function was negatively affected. This was attenuated by the triblock copolymer Poloxamer (P)188. Here, we first hypothesized that endothelial cells are also susceptible to simulated IR injury, albeit requiring longer hypoxia times. We further hypothesized that P188 can also attenuate simulated IR injury in endothelial cells when given upon reoxygenation. Mouse coronary artery endothelial cells (MCAECs) were exposed to different durations of hypoxia (2, 3, 12 and 24 hrs) in serum- and glucose-free media +/- reoxygenation for 2 hrs in regular media. P188 was administered upon reoxygenation at 0, 100, 300 or 1,000 μM in experiments of 24 hrs hypoxia / 2 hrs reoxygenation. LDH release was measured and compared to appropriately timed normoxic control experiments. Reoxygenation and hypoxia times significantly longer than 3 hrs were required to elicit sufficient injury (panel A). When P188 was given upon reoxygenation after 24 hrs hypoxia, it dose-dependently attenuated LDH release (panel B). These findings contrast to the higher susceptibility of CMs to IR injury that only allowed shorter hypoxia durations. They also confirm a protective effect of P188 on the endothelium, not just on CMs. These findings have important implications for co-culture models with MCAECs and CMs to elucidate the interplay of both cell types on each other when studying mechanisms of cardioprotective strategies and compounds like P188.

SAMHD1 Performs Tumor-Promoting Effects in Diffuse Large B-Cell Lymphoma By Inhibiting DNA Damage and Sting-Mediated Cell Pyroptosis

Sterile alpha motif and HD domain-containing protein 1 (SAMHD1), a mammalian triphosphohydrolase, plays a critical role in regulating DNA replication and damage repair. Dysregulation of SAMHD1 facilitates DNA damage-mediated cell proliferation, anti-tumor immune response and chemoresistance of cancer cells. Stimulator of interferon genes (STING) is a critical regulator of the innate immune response through the perception of DNA damage. Here, we investigated the functional significance of SAMHD1 and its regulatory effect on STING signaling. We first elucidated the expression level of SAMHD1 in DLBCL. Upregulation of SAMHD1 mRNA were identified in DLBCL cells (Figure 1A). High protein levels of SAMHD1 was validated in a cohort of newly diagnosed DLBCL patients (n=80). To further identify the biological functions of SAMHD1, SAMHD1-knockdown model was constructed in vitro. Loss-of SAMHD1 resulted not only in impaired proliferation but also in increased cell apoptosis and G0/G1 blockage (Figure 1B). To explore the function of SAMHD1 in vivo, xenograft DLBCL mice model was established. Mice bearing tumors with silenced SAMHD1 revealed delayed tumor growth and diminished tumor activity (Figure 1C). To further explore the functional mechanism of SAMHD1 in DLBCL, we performed RNA-sequencing (RNA-seq) in LY1 cells with and without SAMHD1-knockdown. Gene set enrichment analysis (GSEA) revealed significant enrichment of the cytosolic DNA-sensing pathway, which included an elevation of STING (Figure 2A). Reactome analysis identified the enrichment of STING-mediated introduction of host immune response, suggesting the potential regulation of SAMHD1 on STING (Figure 2B). Nervetheless, co-immunoprecipitation analysis couldn't found the direct interactions between SAMHD1 and STING (Figure 2C). As a DNA damage sensor, STING activation responses to the presence of double stranded DNA (dsDNA). Our results then showed that SAMHD1-knockdown significantly induced nuclear DNA damage and cytosolic dsDNA accumulation (Figure 2D and E), thereby triggering STING activation in DLBCL cells (Figure 2F). Next, we investigated the biological roles of STING in DLBCL. Genetic activation of STING impaired cell viability and enhanced cell death (Figure 3A-3B). To identify the form of cell death, morphological observation was performed and revealed that cell pyroptosis, represented by cell swelling with large bubbles, could be induced by elevated STING (Figure 3C). High levels of LDH were detected in STING overexpressed DLBCL cells, further verifying the emergence of cell pyroptosis (Figure 3D). To validate the involvement of STING in pyroptosis, we deleted STING by CRISPR/Cas9 genomic-editing system (Figure 3E). STING deletion not only alleviated proliferation inhibition (Figure 3F) but also restrained pyroptosis-related morphological changes and LDH release (Figure 3G and H). Expression of pyroptotic effectors were then detected to investigated the molecular mechanisms of pyroptosis. It was worth noting that the Bak/Bax-Caspase-3 mediated gasdermin E (GSDME), rather than ASC/NLRP3/Caspase-1 mediated GSDMD, was activated in STING overexpressed DLBCL cells (Figure 4A and B). Furthermore, silencing of GSDME significantly inhibited pyroptosis-induced morphological changes and LDH release (Figure 4C). The anti-tumor effects of STING agonists in DLBCL were further explored. Treatment of DMXAA, a STING agonist, significantly promoted pyroptosis in DLBCL cells (Figure 5A-5B), further resulting in suppressed proliferation in time- and dose-dependent manners in vitro (Figure 5C). Besides, DMXAA also contributes to the upregulation of immune checkpoints, especially programmed cell death protein 1 (PD-1). We further detected the synergistic effect of DMXAA and PD-1 inhibitor (BMS1166). Of note, combination of DMXAA and BMS1166 remarkably produced an enhanced anti-tumor effect on DLBCL cells in vitro (Figure 5D). In summary, our present study firstly demonstrates that SAMHD1 functions as an oncogene in DLBCL by inhibiting DNA damage induced STING activation. Intrinsic STING induces cell pyroptosis in DLBCL cells via activating the Bak/Bax-Caspase3-GSDME pathway. Moreover, investigation of drug combination highlights the synergistic effect of STING agonist and PD-1 inhibitor, providing a novel option for improving therapeutic effects of anti-PD-1 treatment in DLBCL. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

Siegesbeckiae Herba Extract and Chlorogenic Acid Ameliorate the Death of HaCaT Keratinocytes Exposed to Airborne Particulate Matter by Mitigating Oxidative Stress

Airborne particulate matter with a size of 10 μm or less (PM10) can cause oxidative damages and inflammatory reactions in the skin. This study was conducted to discover natural products that are potentially useful in protecting the skin from PM10. Among the hot water extracts of a total of 23 medicinal plants, Siegesbeckiae Herba extract (SHE), which showed the strongest protective effect against PM10 cytotoxicity, was selected, and its mechanism of action and active constituents were explored. SHE ameliorated PM10-induced cell death, lactate dehydrogenase (LDH) release, lipid peroxidation, and reactive oxygen species (ROS) production in HaCaT cells. SHE decreased the expression of KEAP1, a negative regulator of NRF2, and increased the expression of NRF2 target genes, such as HMOX1 and NQO1. SHE selectively induced the enzymes involved in the synthesis of GSH (GCL-c and GCL-m), the regeneration of GSH (GSR and G6PDH), and GSH conjugation of xenobiotics (GSTκ1), rather than the enzymes that directly scavenge ROS (SOD1, CAT, and GPX1). SHE increased the cellular content of GSH and mitigated the oxidation of GSH to GSSG caused by PM10 exposure. Of the solvent fractions of SHE, the n-butyl alcohol (BA) fraction ameliorated cell death in both the absence and presence of PM10. The BA fraction contained a high amount of chlorogenic acid. Chlorogenic acid reduced PM10-induced cell death, LDH release, and ROS production. This study suggests that SHE protects cells from PM10 toxicity by increasing the cellular antioxidant capacity and that chlorogenic acid may be an active phytochemical of SHE.

289 Cadonilimab, an anti-PD1/CTLA4 bi-specific antibody with Fc effector null backbone

BackgroundTumor infiltrating lymphocytes co-express PD-1 and CTLA-4 at much higher levels compared to normal tissues and peripheral blood cells, thus anti-PD1/CTLA4 bi-specific antibody with a preferential tumor tissue enrichment over normal tissue would contribute to enhanced efficacy and safety. Currently available anti-PD1 and anti-CTLA4 antibodies used in combination therapy are of residual bindings to FcγRs, which mediate antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), leading to compromise on efficacy and safety. Moreover, activated macrophage in tumor microenvironment plays key role in mediating immune suppression by secreting proinflammatory cytokines, such as IL-6. Cadonilimab, also known as AK104, is an IgG1 scaffold Fc-engineered antibody, which is designed to eliminate binding to FcγRs and C1q, and subsequently minimize lymphocyte loss, and antibody dependent cytokine release from macrophage, which associate with irAEs and poor prognosis in immunotherapy.1MethodsPD-1 and CTLA4 antigen co-binding activity of Cadonilimab was determined by Fortebio and assay of co-culture Cadonilimab with Hoechst33342-labelled Jurkat cells expressing PD-1 and CHO-K1-CTLA4 cells. Binding kinetics of Cadonilimab to C1q, FcγRIa, FcγRIIa_H131, FcγRIIa_R131, FcγRIIIa_V158 and FcγRIIIa_F158 were measured by Fortebio. ADCC, ADCP and CDC activities were determined in cellular assays. IL-6 and IL-8 from macrophage were detected in a assay of human macrophage and CHO-K1-PD1-CTLA4 cells co-culture.ResultsCadonilimab binds to the antigens PD-1 and CTLA-4 simultaneously, and as shown in figure.1, Cadonilimab cross-links cells expressing CTLA-4 with those expressing PD-1 (figure 1). Cadonilimab exhibited no binding to FcγRIa, FcγRIIa_H131, FcγRIIIa_V158, FcγRIIIa_F158 or C1q (table 1), eliciting no apparent ADCC, ADCP or CDC (figure 2). Cadonilimab induced no remarkable IL-6 or IL-8 release by human macrophage compared with combination of nivolumab and ipilimumab (figure 3). Clinical trials of Cadonilimab as monotherapy, in combination with chemotherapy or tyrosine kinase inhibitor, such as Lenvatinib and Anlotinib, to treat metastatic cervical cancer (NCT04380805), gastric adenocarcinoma/gastroesophageal junction adenocarcinoma (NCT04728321), non-small cell lung cancer (NCT04647344) and hepatocellular carcinoma (NCT04444167) are ongoing, and a promising efficacy and acceptable safety profile were observed.Abstract 289 Figure 1AK104 crosslinks cells expressing CTLA-4 and PD-1. AK104 can crosslink cells expressing CTLA-4 with cells expressing PD-1. CTLA-4-expressing CHO-K1 cells were plated into the plates. Then the mixture of AK104 or control antibodies with Hoechst 33342 labelled PD-1-expressing Jurkat cells were added into the plates and incubated for 20min. After the incubation, suspended Jurkat cells were removed, and the crosslinking between PD-1 and CTLA-4 expressing cells was analyzed microscopically.Abstract 289 Figure 2ADCC, CDC and ADCP activities of AK104. (A) Antibody-dependent cell-mediated cytotoxicity (ADCC) activities of AK104 (hG1), a version of Cadonilimab with wildtype IgG1 scaffold and Cadonilimab were determined by measuring lactase dehydrogenase (LDH) release from 293T-CTLA4-PD1 cells. (B) Complementary-dependent cell-mediated cytotoxicity (CDC) activities of AK104 (hG1) and Cadonilimab were determined by measuring LDH release from 293T-CTLA4-PD1 cells. (C) Antibody-dependent cellular phagocytosis (ADCP) activities of AK104 (hG1) and Cadonilimab were studied by examining phagocytosis of CHO-K1-PD1-CTLA4 cells by murine bone marrow derived macrophages. Data are expressed as mean±SEM of two independent experiments.Abstract 289 Figure 3IL-8 and IL-6 secretion induced by AK104. Effects of Fc engineering of cadonilimab on the release of inflammatory cytokines. (A) IL-8 and (B) IL-6 by HPMMs in the presence of IFN-γ. Data are expressed as mean ±SEM of two independent experiments.Abstract 289 Table 1Affinity of AK104 to FcγRs and C1q. Affinity of cadonilimab to FcγRIa, FcγRIIa_H131, FcγRIIa_R131, FcγRIIIa_V158, FcγRIIIa_F158 and C1q.ConclusionsCadonilimab, an IgG1 antibody with Fc-engineering, exhibits neither Fc effector functions including ADCC, ADCP, CDC, nor activating macrophage to secret IL-6 or IL-8. Possible tumor tissue preferential retention of Cadonilimab over conventional anti-PD-1 and anti-CTLA-4 antibodies noted above could potentially lead to better safety profile.ReferenceYang F, He Z, Duan H, Zhang D, Li J, Yang H, Dorsey JF, Zou W, Ali Nabavizadeh S, Bagley SJ, Abdullah K, Brem S, Zhang L, Xu X, Byrne KT, Vonderheide RH, Gong Y, Fan Y. Synergistic immunotherapy of glioblastoma by dual targeting of IL-6 and CD40. Nat Commun 2021 June 8;12(1):3424.

Study on the effects of Polyphyllin I and Curcumin on liver cancer based on the cross-action of ferroptosis and energy metabolism

Background: To investigate the effect and mechanism of Polyphyllin I(PPI) and Curcumin(Cur) on human liver cancer HepG2 and HepG2.2.15 cells. Methods: Download the hepatocellular carcinoma specimens and normal control specimens from the TCGA database, take the intersection with ferroptosis-related genes, and screen the differentially expressed ferroptosis genes; again, make the intersection with the selected differential genes related to energy metabolism; conduct survival analysis; construct prognosis Risk scoring model and evaluation of model performance; through molecular docking to verify the binding effect of PPI, Cur and Ribonucleoside-diphosphate reductase subunit M2(RRM2), SRC(SRC), Acetyl-CoA carboxylase alpha(ACACA) and other genes. Human hepatocellular carcinoma cells were cultured in vitro, PPI and Cur intervened, and Cell Counting Kit-8(CCK-8) was used to detect cell inhibition rate; FeRhoNox-1 fluorescent probe staining to observe the intracellular Fe 2+ status; lactate dehydrogenase (LDH) release Experiment to detect cell LDH release rate; JC-1 staining to detect mitochondrial membrane potential; reactive oxygen species(ROS) kit to detect ROS level;Western blotting (WB) to detect RRM2 and SRC , ACACA and other genes protein expression levels. Results: Through screening, 25 differential genes related to ferroptosis and energy metabolism in liver cancer were obtained;; through survival analysis, three ferroptosis-related genes, such as RRM2, SRC, and ACACA, were obtained，the results showed that these three genes showed high expression and predicted poor overall survival(OS) and disease-free survival(DFS); molecular docking results showed that PPI, Cur It has good affinity with RRM2, SRC and ACACA. The results of in vitro experiments show that PPI and Cur inhibit cell proliferation in a concentration-dependent manner ( P <0.01). PPI and Cur can significantly increase the intracellular Fe2+ concentration, LDH release rate and intracellular ROS levels of HepG2, HepG2.2.15 ( P <0.01), and the effect on mitochondrial membrane potential was significantly lower than that of the blank group ( P <0.01), and significantly down-regulated the protein expression levels of RRM2, SRC, and ACACA ( P <0.01). Conclusion: The high expression of RRM2, SRC, ACACA and other three genes related to ferroptosis and energy metabolism in liver cancer indicate poor OS and DFS; PPI and Cur can up-regulate the LDH release rate, ROS and Fe 2+ levels of liver cancer cells, and down-regulate the cell mitochondrial membrane potential and other methods to inhibit the proliferation of liver cancer cells; and down-regulate the expression of RRM2, SRC, ACACA and other proteins to affect the prognosis of liver cancer.

Expression and roles of N-type Ca channel in cardiaomyocytes

Background Voltage dependent Ca channels are divided to L-, T-, N-, P/Q-, and R-types, and N-type Ca channel (NCC) is mainly expressed in nerve terminal and regulates neurotransmitter release. Recently, NCC has been reported to express in adrenal gland and renal tubular cells. We examined whether NCC is expressed in cardiac myocytes and if so, the roles of this channel. Methods Expression of NCC mRNA and protein in cardiomyocytes were assessed by quantitative real time PCR and Western blot analysis using neonatal rat cultured cardiomyocytes, infant, and adult rat hearts. Expression site of NCC in cardiomyocytes was examined by confocal imaging of immunofluorescent staining. The roles of NCC in physiological Ca transient in neonatal myocytes were examined using fluorescence imaging of Fluo4, an intracellular Ca indicator. To examine the effects of pathological condition, such as heart failure and ischemia-reperfusion, on NCC expression, cultured cardiomyocytes were treated with norepinephrine (10 μmol/L, 24 hours) or subjected to 5 hours of hypoxia followed by 30 minutes of reoxygenation. In addition, adult rats were subjected to myocardial infarction by ligating the left anterior coronary artery. Lethal myocyte injury was examined by LDH activity in culture medium and myocyte apoptosis was examined by nuclear staining with DAPI and caspase 3 activity. To clarify the roles of NCC in neonatal myocytes in these pathological conditions, we examine the effect of ω-conotoxin, a selective NCC blocker. Results NCC mRNA and protein were expressed in neonatal cardiomyocytes. Immunocytochemical staining showed NCC was expressed in myocyte plasma membrane. During physiological spontaneous beating, ω-conotoxin did not affect beating rate and intra cellular Ca transient, suggesting that the roles of NCC on physiological beating are little. After birth level of NCC mRNA expression in cardiac tissue gradually decreased within 2 weeks and low level of mRNA expressed continuously in adult cardiac tissue. However, in pathological condition, mRNA and protein levels of NCC in non-infarcted region were increased 4 weeks after myocardial infarction. In addition, hypoxia-reoxygenation and norepinephrine administration increased LDH release and myocyte apoptosis in association with increase in NCC expression in neonatal cultured myocytes. ω-conotoxin significantly suppressed hypoxia/reoxygenation- and norepinephrine-induced LDH release and caspase 3 activation. Conclusion NCC is expressed in neonatal cardiac myocytes and the expression level was decreased after birth. Pathological condition, such as ischemic heart disease and heart failure, upregulated NCC expression in cardiomyocytes and NCC exacerbated lethal myocyte injury, while roles of NCC in physiological beating are little. FUNDunding Acknowledgement Type of funding sources: None.

Abstract P343: Physiologic Mechanical Stress In An Induced Pluripotent Stem Cell Derived Cardiomyocyte Model Of Duchene Muscular Dystrophy-related Cardiomyopathy Treated With A Membrane Resealant

Heart failure is leading cause of morbidity and mortality in the X-linked disease Duchenne muscular dystrophy (DMD). DMD is due to mutations the gene encoding dystrophin. Dystrophin localizes to the costamere in skeletal and cardiac muscle and is part of the larger dystrophin complex, which forms a critical connection linking the sarcomere to extracellular matrix. Disruptions in this complex lead to membrane fragility and multiple forms of muscular dystrophy, most of which have significant cardiac involvement. Therapeutic strategies for DMD include FDA-approved agents for exon skipping, as well as micro-dystrophins gene therapy, which is currently in clinical trials. Despite this progress, there is inadequate information as to how these and other novel agents will affect the DMD heart. Given the critical importance of cardiac muscle efficacy for any therapeutic for DMD and importance of membrane fragility in the disease phenotype, we assessed the susceptibility of patient-derived induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) harboring an out-of-frame exon 46-47 DMD deletion. iPSC-CMs were reprogrammed via a standard approach and differentiated, expanded, and purified by published methods. Troponin T flow cytometry was performed to and a minimum troponin T positivity >85% positivity was set for inclusion. DMD and control cells were plated onto flexible silicone membranes and subjected to equibiaxial strain as physiologic mechanical stressor, consistent with the inciting pathologic insult in DMD. Troponin and LDH release were assessed as clinically-relevant biomarkers of injury. Physiologic stress parameters were defined using troponin and LDH release relative to unstressed conditions. A membrane resealant being developed for treating DMD was shown to reduce troponin and LDH release in DMD iPSC-CMs, and also showed benefit in control cells. This work provides a ready platform for assessing therapeutics that target not only DMD-related cardiomyopathy, but other forms of cardiomyopathy and myocardial injury.