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Background: Idiopathic Hypereosinophilic Syndrome (iHES) is a rare disease with varied clinical presentations including acute heart failure. Case: A 44-year-old man who had been treated for asthma 9 months ago was admitted with worsening exertional dyspnea. EKG demonstrated sinus tachycardia with anterolateral ST depressions. Labs revealed troponin-I 44.95 ng/ml, BNP 405 pg/mL, and white blood cell count 21.2k with 50% eosinophils. Transthoracic echocardiogram showed normal left ventricular size with globally reduced ejection fraction (30-35%). Coronary angiography found normal coronary arteries. An extensive infectious and auto-immune workup was unrevealing, and bone marrow biopsy was negative for monoclonal proliferation (Fig 1A). He received 1 gram of IV methylprednisolone for 2 days lowering eosinophil count to zero and was discharged on heart failure GDMT. Two weeks later, he was readmitted from clinic for ventricular tachycardia (VT) near 120bpm (Fig 1B). Cardiac MRI and endomyocardial biopsy confirmed active eosinophilic myocarditis (Fig 1C/D). Patient was diagnosed with iHES and treated with IV methylprednisolone (500mg BID x 3 days) with an oral prednisone taper. Amiodarone and lidocaine infusions were temporarily needed to suppress his VT and an ICD was placed. After clinical stabilization and immunology consultation, an interleukin-5 inhibitor was used for further treatment of iHES. Discussion: Only 6% of patients with iHES present with cardiac symptoms, however one in five eventually develop cardiac manifestations that are often rapidly progressive. Prompt treatment with glucocorticoids is indicated for severe presentations, and subsequent use of targeted therapies can dramatically reduce morbidity and mortality. Recognition of severe peripheral eosinophilia should prompt a broad hematologic, infectious, and auto-immune workup to guide treatment and prevent end-organ damage.

Introduction: In vitro studies showed that activation of β-catenin suppresses Na V 1.5 expression by inhibiting SCN5a promoter activity, leading to a decrease of Na + channel activity. How β-catenin regulates cardiac electrophysiological phenotype is unknown. Hypothesis: We hypothesized that cardiac activation of β-catenin regulates electrophysiological phenotype by suppressing Na V 1.5 expression. Methods: Adult mice with cardiac-specific, tamoxifen-induced deletion of β-catenin exon3 , leading to cardiac activation of β-catenin (β-catenin exon3 -/- ) were generated, and the effects of cardiac activation of β-catenin on the electrophysiological remodeling were assessed by electrocardiogram (ECG) recording. Class Ic antiarrhythmic reagent, flecainide, was administered to evaluate susceptibility to ventricular tachycardia (VT). Cardiac structure and function were evaluated by histologic and echocardiographic examinations, respectively. Western blot and qRT-PCR were performed to determine the levels of Na V 1.5 and β-catenin expression in mouse hearts. Whole-cell recording technique was utilized to record Na + currents and action potentials (APs) from ventricular myocytes. Results: Histologic and echocardiographic examinations showed that β-catenin exon3 -/- mice had normal cardiac structure and function. Compared to wild type (WT) mice, the ratio of heart/body weight was not changed and the duration of QRS was significantly prolonged in β-catenin exon3 -/- mice. VT was induced by flecainide in 60% of β-catenin exon3 -/- mice but not in WT mice. Western blot and qRT-PCR showed that Na V 1.5 protein and mRNA were significantly decreased in β-catenin exon3 -/- hearts, compared to WT hearts. Maximal upstroke velocity and amplitude of APs and Na + currents were significantly decreased in β-catenin exon3 -/- ventricular myocytes, compared to WT cells. Conclusion: Cardiac activation of β-catenin leads to prolongation of QRS and susceptibility to VT by decreasing Na V 1.5 expression and Na + channel activity.

Background: Cardiovascular disease is the leading cause of death in the world with a dearth of effective therapies. Heart undergoes a complex differentiation and maturation process throughout embryonic and post-natal stages. Intense efforts have been made in the study of cardiomyocyte differentiation, maturation and pathological remodeling. With the aid of stem cells, investigators are able to recapitulate events in early cardiac development and gain valuable insight in transcriptional regulatory networks directing early cardiomyocyte differentiation. However, the molecular network that determine myocyte maturation in postnatal development, which are especially important for understanding diseases and developing cell based clinical applications, are far less well characterized. Human induced pluripotent stem cell derived cardiomyocytes (hIPSC-CMs) are lineage committed but remain immature and fetal-like in molecular, morphological and functional characteristics. Their application in cell based therapy or serving as a disease model for heart failure is limited, in part due to the lack of sufficient insight to promote their maturation into adult myocytes. Results: We find from previous studies that postnatal cardiomyocyte development is marked by global alternative splicing (AS) programming. In mouse heart, we find RNA splicing regulator RBFox1 is markedly induced in heart only in post-natal period and functions as a key regulator to post-natal global RNA alternative splicing reprogramming during cardiomyocyte maturation. Transcriptome analysis revealed that exogenous expression of RBFox1 promotes gene expression in neonatal ventricular myocytes reminiscent of mature adult heart, and results in cellular maturation based on sarcomere organization and calcium cycling characteristics. Furthermore, ectopic expression of rodent RBFox1 in human iPSC derived CMs resulted in similar maturation effects, implicating a conserved mechanism in human and rodent myocytes. Conclusion: RBFox1 mediated RNA splicing is an important contributor to post-natal myocyte maturation and it can be manipulated to promote cardiomyocyte maturation for cell-based therapy or disease modeling.

T-cell factor (TCF) and lymphoid enhancer factor (LEF) family transcriptional factors are important nuclear mediators of Wnt/β-catenin signaling. Their roles in cardiac development and diseases remain to be determined. We found that these TCF/LEF family members, including LEF1, TCF7, TCF7L1, and TCF7L2 had unique expression pattern during mouse heart development. At embryonic day (E) 13.5, LEF1 was one of the most abundant TCF/LEF members at mRNA level in the murine heart. By immunohistochemical staining, mesenchymal cells in valvular regions had much stronger intensity of LEF1 than cardiomyocytes in ventricles. Cardiomyocytes with weak to moderate nuclear staining for LEF1 were scattered in compact and trabecular layers. At E17.5, LEF1 mRNA levels dropped dramatically and its protein was no longer detected in cardiomyocytes. TCF7L1 mRNA levels also decreased from E13.5 to E17.5. Weak cytoplasmic, but not nuclear TCF7L1 signal was detected in cardiomyocytes at E13.5 by immunochemistry. TCF7 mRNA was the lowest among all four TCF/LEF members at E13.5 and it further decreased at E17.5. TCF7 protein was only detected by immunohistochemistry in mesothelial and endothelial cells, but not in cardiomyocytes or mesenchymal cells. TCF7L2 mRNA became the most abundant TCF/LEFs at E17.5. Immunolocalization revealed that TCF7L2 formed an intensity gradient with the highest levels at the inner trabecular layer, opposite to the distribution of β-catenin. Wnt signaling activation by cardiac deletion of adenomatosis polyposis coli ( Apc ), a negative Wnt regulator, led to a dramatic increase in cyclin D2, Bmp4, LEF1, and TCF7. BMP mediators, SMAD1/5/8, were increasingly phosphorylated. Chromatin immunoprecipitation revealed that TCF7L2 was removed from and replaced by LEF1 and TCF7 in the promoter of cyclin D2 and Bmp4 to promote β-catenin recruitment. Furthermore, there was co-occupancy of TCF/LEF and SMAD in the adjacent TCF/LEF and SMAD binding sites of Wnt target genes. Finally, we proved that TCF7L2 was a transcriptional suppressor of cyclin D2 and Bmp 4 in a cardiac cell line by overexpression and knockdown experiments. Our results indicate that TCF7L2 is transcriptional repressor of Wnt/β-Catenin pathway in the heart.

Background— How canonical Wnt/β-catenin signals in adult hearts, especially in different diseased states, remains unclear. The proto-oncogene, c-Myc, is a Wnt target and an early response gene during cardiac stress. It is not clear whether c-Myc is activated or how it is regulated during heart failure. Methods and Results— We investigated canonical Wnt/β-catenin signaling and how it regulated c-Myc expression in failing hearts of human ischemic heart disease, idiopathic dilated cardiomyopathy, and murine desmin–related cardiomyopathy. Our data demonstrated that canonical Wnt/β-catenin signaling was activated through nuclear accumulation of β-catenin in idiopathic dilated cardiomyopathy, ischemic heart disease, and murine desmin–related cardiomyopathy when compared with nonfailing controls and transcription factor 7-like 2 (TCF7L2) was the main β-catenin partner of the T-cell factor (TCF) family in adult hearts. We further revealed that c-Myc mRNA and protein levels were significantly elevated in failing hearts by real-time reverse transcription polymerase chain reaction, Western blotting, and immunohistochemical staining. Immunoprecipitation and confocal microscopy further showed that β-catenin interacted and colocalized with TCF7L2. More importantly, chromatin immunoprecipitation confirmed that β-catenin and TCF7L2 were recruited to the regulatory elements of c-Myc. This recruitment was associated with increased histone H3 acetylation and transcriptional upregulation of c-Myc. With lentiviral infection, TCF7L2 overexpression increased c-Myc expression and cardiomyocyte size, whereas shRNA-mediated knockdown of TCF7L2 suppressed c-Myc expression and cardiomyocyte growth in cultured neonatal rat cardiomyocytes. Conclusions— This study indicates that TCF7L2 mediates canonic Wnt/β-catenin signaling and c-Myc upregulation during abnormal cardiac remodeling in heart failure and suppression of Wnt/β-catenin to c-Myc axis can be explored for preventing and treating heart failure.

Global profiling of histone changes in some human cancers demonstrated that loss of histone H4 acetylation at lysine16 (H4KA16) and trimethylation at lysine 20 (H4KM20) was a common hallmark of cancer. It is not clear whether these epigenetic changes also exist in neuroendocrine carcinomas. We semiquantitatively analyzed 32 cases of lung neuroendocrine tumors (LNETs) immunohistochemically stained with H4KA16, H4KM20, and Ki67 antibodies by calculating cumulative scores based on the sum of the product of nuclear stain intensity (1-3) and percentages of positive cells in each category. H4KA16 and H4KM20 levels were compared among typical carcinoid (TC, 11), atypical carcinoid (AC, 6), large cell neuroendocrine carcinoma (LCNEC, 8), and small cell lung cancer (SCLC, 7) and correlated with histologic types and Ki67 labeling. Data were presented as mean±standard error of the mean and statistically analyzed by 1-way analysis of variance and Holm-Sidak method. Normal bronchiolar epithelium had relatively uniform and strong +3 positivity of H4KM20 and H4KA16, which was considered as internal positive controls. This uniformity, however, was gradually lost from low to high grades of LNETs. Semiquantitative analysis revealed that there were significant differences in cumulative scores of H4KA16 (TC, 2.36±0.03; AC, 2.04±0.08; LCNEC, 1.58±0.07; SCLC 1.32±0.05) among LNETs. For H4KM20, significant differences were only observed between low grade (TC, 2.49±0.05 and AC, 2.24±0.09) and high grade (LCNEC, 1.58±0.10 and SCLC 1.68±0.11) LNETs, but not within low or high grade LNETs. The Ki67 cumulative scores (TC, 0.06±0.02; AC, 0.41±0.08; LCNEC, 1.29±0.09; SCLC 1.83±0.06) were inversely correlated with both cumulative H4KA16 and H4KM20 scores by Pearson correlation. We conclude that progressive loss of H4KA16 and H4KM20 from low to high grade LNETs reflects the degree of differentiation and proliferative activity. These histone modifications may serve as tumor biomarkers that may aid in diagnosis and prediction of clinical outcome.

Approximately 5% to 15% of central nervous system metastases by solid tumors spread to the leptomeninges. Transmembrane MUC16 is thought to facilitate anchoring of metastases to other tissues particularly those expressing mesothelin such as pleura and peritoneum. Recently, we have demonstrated that mesothelin is also expressed in the leptomeninges. Mesothelin has a high affinity for MUC16 and mesothelin expressed by some adenocarcinomas. In this study, we evaluated MUC16 and mesothelin immunoreactivity by immunohistochemistry in 11 adenocarcinomas to the leptomeninges, 24 to mesothelin-negative brain, 8 metastases to mesothelin-expressing peritoneum/pleura, 22 to mesothelin-negative peripheral tissues, and 24 with no metastases. MUC16 was detected in 36% of leptomeningeal metastases and 10% of metastases to the brain. Adenocarcinoma metastases to mesothelin-expressing peritoneum or pleura exhibited extensive MUC16 in 75% of cases. In adenocarcinomas with local metastases to lymph nodes or no metastases, 53% or 38% had MUC16 immunoreactivity, respectively. Mesothelin-immunoreactivity was detected in 9% of metastases to the leptomeninges, 17% to the brain, none of the metastases to pleura/peritoneum, 50% of adenocarcinomas with local metastases to lymph nodes, etc, and 33% of adenocarcinomas without metastases. Mesothelin expression was significantly more common in nonmetastatic adenocarcinomas than in metastases to the pleura/peritoneum or leptomeninges. Our findings suggest that adenocarcinomas with MUC16 expression may have an increased risk for metastases to pleura/peritoneum but not the leptomeninges or brain.

Malignant solitary fibrous tumor (MSFT) is a rare neoplasm. Three cases of MSFT with unusual features, including 1 pleural and 2 extrapleural, are reported. A 64-year-old woman with a large right thoracic MSFT and episodes of severe hypoglycemia experienced resolution of her hypoglycemia immediately after resection of the MSFT. A 27-year-old woman with primary retroperitoneal MSFT had pulmonary metastases 10 months after resection of the primary tumor. A 54-year-old man with an intracranial solitary fibrous tumor suffered from multiple pulmonary metastases and local recurrence 21 and 28 months after resection of the primary tumor, respectively. All 3 cases of solitary fibrous tumor displayed malignant features. The tumor cells in each case were positive for CD34 and Bcl-2, but negative for cytokeratin, smooth muscle actin, S-100, and c-kit. In addition, the tumor cells in the case with concomitant hypoglycemia were strongly positive for insulin-like growth factor-II. The histopathologic diagnostic criteria for MSFT, the differential diagnosis with other spindle cell tumors, and the mechanism of MSFT-derived hypoglycemia via insulin-like growth factor-II are discussed.

It has been shown that tyrosine kinase oncoprotein c-kit and antiapoptotic molecule bcl-2 are overexpressed in several types of malignancy, including small cell carcinoma (SCLC) and large cell neuroendocrine carcinoma (LCNEC) of the lung. Whether these 2 molecules are coexpressed in lung neuroendocrine tumors has not been investigated. Here, we analyzed immunohistochemical results to determine expression and coexpression patterns of c-kit and bcl-2 in the spectrum of lung neuroendocrine tumors. Using a polyclonal antibody against c-kit and a monoclonal antibody against bcl-2, our data demonstrated that all 7 cases (100%) of SCLC included in this study were positive for both c-kit and bcl-2. Among 14 LCNECs, 7 (50%) stained positive for c-kit and 9 (64%) for bcl-2. All cases of high grade neuroendocrine carcinomas (SCLCs and LCNECs) that showed positive staining for c-kit coexpressed bcl-2. In contrast, all typical and atypical carcinoids (TC and AC) were negative for c-kit, and only 1 of 16 (6.3%) TCs and 1 of 6 (16.7%) ACs stained positive for bcl-2. These results indicate a progressive increase in the frequency of c-kit and bcl-2 expression and coexpression, from carcinoid tumors (TC and AC) to LCNEC and to SCLC. High grade neuroendocrine carcinomas are more likely to coexpress c-kit and bcl-2 when compared with carcinoid tumors. The high frequency of coexpression of these 2 molecules in high grade neuroendocrine carcinomas of the lung suggests that they may be involved in the carcinogenic pathway, given their important roles in carcinogenesis. Therapeutic targeting on both c-kit and bcl-2 molecules might be beneficial in the management of patients with high grade neuroendocrine carcinomas of the lung in the future.

Substantial evidence suggests that the progressive loss of cardiomyocytes caused by apoptosis significantly contributes to the development of heart failure. beta-Adrenergic receptor activation and subsequent persistent phosphodiesterase 3A (PDE3A) downregulation and concomitant inducible cAMP early repressor (ICER) upregulation (PDE3A/ICER feedback loop) has been proposed to play a key role in the pathogenesis of cardiomyocyte apoptosis. In contrast, insulin-like growth factor-1 can activate cell survival pathways, providing protection against cell death and restoring muscle function. In this study, we found that insulin-like growth factor-1 activates extracellular signal-regulated kinase 5 (ERK5) and inhibits PDE3A/ICER feedback loop. Insulin-like growth factor-1 normalized isoproterenol-mediated PDE3A downregulation and ICER upregulation via ERK5/MEF2 activation, and also inhibited isoproterenol-induced myocyte apoptosis. To determine the physiological relevance of ERK5 activation in regulating PDE3A/ICER feedback loop, we investigated the PDE3A/ICER expression and cardiomyocyte apoptosis in transgenic mice with cardiac specific expression of a constitutively active form of mitogen-activated protein (MAP)/extracellular signal-regulated protein kinase (ERK) kinase 5alpha (MEK5alpha) (CA-MEK5alpha-Tg). In wild-type mice, pressure overload- or doxorubicin-induced significant reduction of PDE3A expression and subsequent ICER induction. Cardiac specific expression of CA-MEK5alpha rescued pressure overload- or doxorubicin-mediated PDE3A downregulation and ICER upregulation and inhibited myocyte apoptosis as well as subsequent cardiac dysfunction in vivo. These data suggest that preventing the feedback loop of PDE3A/ICER by ERK5 activation could inhibit progression of myocyte apoptosis as well as cardiac dysfunction. These data suggest a new therapeutic paradigm for end stage of heart failure by inhibiting the PDE3A/ICER feedback loop via activating ERK5.

Hepatoid carcinomas are tumors that display, at least focally, cytologic and/or architectural features of hepatocellular carcinoma. They have been described in several organs, most notably in the stomach and ovary. We report a case of hepatoid carcinoma of the pancreas that developed in a 41-year-old woman in association with a pancreatic endocrine carcinoma. The fine needle aspiration material was characterized by the presence of monotonous, small-to-medium sized tumor cells with round nuclei and finely granular chromatin, intermixed with more atypical tumor cells displaying larger nuclei with coarse clumped chromatin, prominent nucleoli, and moderate amounts of foamy cytoplasm. The excised specimen displayed a poorly differentiated pancreatic endocrine carcinoma associated with well-defined islands of larger tumor cells growing in a perisinusoidal pattern which, based on their immunohistochemical profile and the demonstration of bile, proved to represent a hepatoid component. This case and prior examples in the literature suggest that hepatoid carcinomas of the pancreas appear to be a heterogeneous group of tumors (pure or associated with another histologic component) that are often associated with early liver metastasis and a short survival, although those arising as a component of endocrine tumors seem to fare slightly better. Hepatoid carcinoma of the pancreas should be included in the differential diagnosis of pancreatic tumors composed of large eosinophilic cells.

D2-40 is a new monoclonal antibody recognizing podoplanin and it is selective for lymphatic endothelium. Several studies have confirmed the reliability of D2-40 in the diagnosis of pleural malignant mesothelioma, but the evaluation of this antibody in other pleural neoplasms is limited. The aim of this study was to determine the diagnostic value of D2-40 in segregation of malignant mesothelioma from other pleural or lung neoplasms. Sixty-seven cases, including 36 pleural malignant mesotheliomas, 15 solitary fibrous tumors, 13 pleomorphic carcinomas, and 3 synovial sarcomas, were immunohistochemically studied using a D2-40 monoclonal antibody. Twenty-five (21 epithelioid and 4 biphasic) of 36 (69%) malignant mesotheliomas and 2 of 15 (13%) solitary fibrous tumors were positive for D2-40. The difference of D2-40 positivity between these 2 types of tumor was significant (P

Parathyroid adenoma with prominent lymphocytic infiltrates is very rare, with only 8 previously reported cases in the English scientific literature. Cases with prominent lymphocytic infiltrates of ectopic (mediastinal) parathyroid adenomas have not been reported. The prominent lymphocytic infiltration may make the diagnosis difficult on frozen sections, even on permanent histologic sections. We herein describe a case of ectopic (mediastinal) parathyroid adenoma with prominent lymphocytic infiltration in a 29-year-old man with hyperparathyroidism for 4 years. Immunohistochemical studies were important in making such a diagnosis.

Lu Xiao, Haiqing Bai, James Boyer, Bo Ye, Ning Hou, Haodong Xu, and Faqian Li Department of Pathology and Laboratory Medicine and Cardiovascular Research Institute, University of Rochester Medical Center, Rochester, NY, USA Backgrounds: Canonical Wnt signaling appears to have multiphasic and often antagonistic roles in cardiac development. The molecular mechanism for these opposing actions is not clear. We hypothesized that alternative splicing of TCF7L2, a nuclear interaction partner of beta-catenin is involved in the specificity of canonical Wnt signaling. Methods: RT-PCR were performed on embryonic (E16.5) and neonatal (day 8) hearts with primers spanning the end of first exon and the beginning of last exon and the products were cloned and sequenced. Result: There are totally 18 exons identified so far in TCF7L2. We sequenced 56 clones and 53 clones (29 from day 8) and (24 from E16.5) contained TCF7L2 sequences. No exon 6 or exon 17 was found in TCF7L2 transcripts of mouse hearts. Most clones (more than 80%) from E16.5 and day 8 hearts excluded exon 4. Both E16.5 and day 8 hearts had one clone with exon 9 deletion which does not change reading frame and another with alterations in exon 3 that lead to reading frame shift and premature stop codon. As reported in other organs, there were extensive alternative splicing in the C-terminal exons 14, 15 and 16. The inclusion of exon 14 was more frequently in day 8 (18 of 29, 62%) than in E16.5 (8 of 24, 33%) hearts. The peptide encoded by exon 14 has conserved functional motif. Additionally, this alternative exon usage can change the C-terminus of TCF7L2 to include or exclude the so-called E tail with two binding motifs for C-terminal binding protein. Conclusion: The isoform switch of TCF7L2 occurs in neonatal mouse hearts and may have a role in the terminal differentiation of cardiac myocytes during this period.

Backgrounds: Genetic variations of TCF7L2, a nuclear transcriptional factor in canonical Wnt signaling are associated with increased risks of Diabetes Mellitus. How TCF7L2 is regulated by diabetic conditions in the heart is poorly understood. Methods: TCF7L2 expression was investigated by Western blotting in HL-1 cells treated with 14 mM mannitol (M), 25 mM glucose (G), 10 nM insulin (I), and G plus I (G+I). In addition, TCF7L2 levels were examined by Western blotting in the heart of B6.Cg-m Leprdb/+ +/J (db) and C57Bl/6J (C57) mice 4 hours after intraperitoneal injections of 1.5 IU/kg insulin. Result: The expression of TCF7L2 was higher in db than C57 hearts. Insulin treatment decreased TCF7L2 in C57 hearts. Paradoxically, TCF7L2 was further increased by insulin in db hearts. In HL-l cells, TCF7L2 started to decrease at one hour and remained lower up to 24 hours after insulin treatment compared to untreated controls. In contrast, G increased TCF7L2 expression at 1, 6 and 24 hours after treatment relative to M. Insulin could decrease TCF7L2 at 1 and 6 hours, but failed to do so at 24 hours in G+I. At 48 hours, no difference in TCF7L2 expression was observed among different treatment groups. Conclusion: Insulin regulation of TCF7L2 is interfered by high glucose in vitro and misregulation of TCF7L2 in the heart of diabetic mice may contribute to the pathogenesis of diabetic cardiomyopathy.

Backgrounds: DNA damage occurs in cardiomyocytes during normal cellular metabolism and is significantly increased under cardiac stresses. How cardiomyocytes repair their DNA damage, especially DNA double strand breaks (DSBs), remains undetermined. We assessed DSBs caused by oxidative stress. More importantly, we investigated the spatiotemporal dynamics of DNA repair protein assembly/disassembly in DNA damage sites. Methods: Cultured neonatal rat cardiomyocytes were treated with different doses of hydrogen peroxide (H2O2) for 30 minutes to assess DNA damage response (DDR). To investigate the dynamics of DDR, cells were treated with 200 uM H2O2 and followed up to 72 hours. DSBs were evaluated by counting DNA damage foci after staining with antibody against histone H2AX phosphorylation at serine 139 (g-H2AX). The dynamics and posttranslational modification of DNA repair proteins were determined by Western blotting, immunolabeling, and confocal microscopy. Result: g-H2AX was proportionally increased to H2O2 dosage. Discrete nuclear g-H2AX foci were seen 30 minutes after hydrogen peroxide treatment with 50 uM, but became pannuclear when H2O2 was above 400 uM. At 200 uM of hydrogen peroxide, g-H2AX started to increase at 15 minutes and reached to highest levels at 60 minutes with up to 70 nuclear foci, started to decline at 2 hours, and returned to basal levels at 24 hours. DDR transducer kinase, ataxia telangiectasia mutated (ATM) was activated at 5 minutes with increased phosphorylation at serine 1981 (pATM) which started to decrease at 24 hours, but remained elevated up to 48 hours. Another DDR transducer kinase, ATM and Rad3-related (ATR) showed a biphasic activation at 30 minutes and 8 hours. ATM and ATR colocalized with g-H2AX. DNA damage mediator proteins such as MRN complex and p53BP1 were also recruited to sites of DNA damage at g-H2AX foci. Conclusions: DSBs and their repair have emerged as a new frontier of stress responses. Newly developed methods for studying g-H2AX and DNA repair protein dynamics can be explored to investigate DDR to oxidative stress in cardiomyocytes.