Modeling Cardiotoxicity in Pediatric Oncology Patients Using Patient-Specific iPSC-Derived Cardiomyocytes Reveals Downregulation of Cardioprotective microRNAs.

Anthracyclines are widely used in the treatment of many solid cancers, but their efficacy is limited by cardiotoxicity. As the number of pediatric cancer survivors continues to rise, there has been a concomitant increase in people living with anthracycline-induced cardiotoxicity. Accordingly, there is an ongoing need for new models to better understand the pathophysiological mechanisms of anthracycline-induced cardiac damage. Here we generated induced pluripotent stem cells (iPSCs) from two pediatric oncology patients with acute cardiotoxicity induced by anthracyclines and differentiated them to ventricular cardiomyocytes (hiPSC-CMs). Comparative analysis of these cells (CTX hiPSC-CMs) and control hiPSC-CMs revealed that the former were significantly more sensitive to cell injury and death from the anthracycline doxorubicin (DOX), as measured by viability analysis, cleaved caspase 3 expression, oxidative stress, genomic and mitochondrial damage and sarcomeric disorganization. The expression of several mRNAs involved in structural integrity and inflammatory response were also differentially affected by DOX. Functionally, optical mapping analysis revealed higher arrythmia complexity after DOX treatment in CTX iPSC-CMs. Finally, using a panel of previously identified microRNAs associated with cardioprotection, we identified lower levels of miR-22-3p, miR-30b-5p, miR-90b-3p and miR-4732-3p in CTX iPSC-CMs under basal conditions. Our study provides valuable phenotype information for cellular models of cardiotoxicity and highlights the significance of using patient-derived cardiomyocytes for studying the associated pathogenic mechanisms.

Reparative cell therapy for the heart: critical internal appraisal of the field in response to recent controversies.

The concept that cell-based repair of myocardial injury might be possible was introduced almost two decades ago; however, the field of cardiovascular reparative medicine has been criticized as translation to clinically effective approaches has been slow. The recent retraction of a series of papers has further impacted perception of this area of research. As researchers, clinicians, and teachers in this field, we felt it incumbent to critically appraise the current state of cardiac cell repair, determine what can be learned from past mistakes, and formulate best practices for future work. This special communication summarizes an introspective assessment of what has fallen short, how to prevent similar issues, and how the field might best move forward in the service of science and patients.

Clinical Characteristics and Electrophysiological Mechanisms Underlying Brugada ECG in Patients With Severe Hyperkalemia.

Background Several metabolic conditions can cause the Brugada ECG pattern, also called Brugada phenotype (BrPh). We aimed to define the clinical characteristics and outcome of BrPh patients and elucidate the mechanisms underlying BrPh attributed to hyperkalemia. Methods and Results We prospectively identified patients hospitalized with severe hyperkalemia and ECG diagnosis of BrPh and compared their clinical characteristics and outcome with patients with hyperkalemia but no BrPh ECG. Computer simulations investigated the roles of extracellular potassium increase, fibrosis at the right ventricular outflow tract, and epicardial/endocardial gradients in transient outward current. Over a 6-year period, 15 patients presented severe hyperkalemia with BrPh ECG that was transient and disappeared after normalization of their serum potassium. Most patients were admitted because of various severe medical conditions causing hyperkalemia. Six (40%) patients presented malignant arrhythmias and 6 died during admission. Multiple logistic regression analysis revealed that higher serum potassium levels (odds ratio, 15.8; 95% CI, 3.1-79; P=0.001) and male sex (odds ratio, 17; 95% CI, 1.05-286; P=0.045) were risk factors for developing BrPh ECG in patients with severe hyperkalemia. In simulations, hyperkalemia yielded BrPh by promoting delayed and heterogeneous right ventricular outflow tract activation attributed to elevation of resting potential, reduced availability of inward sodium channel conductance, and increased right ventricular outflow tract fibrosis. An elevated transient outward current gradient contributed to, but was not essential for, the BrPh phenotype. Conclusions In patients with severe hyperkalemia, a BrPh ECG is associated with malignant arrhythmias and all-cause mortality secondary to resting potential depolarization, reduced sodium current availability, and fibrosis at the right ventricular outflow tract.

Myocardial commitment from human pluripotent stem cells: Rapid production of human heart grafts.

Genome editing on human pluripotent stem cells (hPSCs) together with the development of protocols for organ decellularization opens the door to the generation of autologous bioartificial hearts. Here we sought to generate for the first time a fluorescent reporter human embryonic stem cell (hESC) line by means of Transcription activator-like effector nucleases (TALENs) to efficiently produce cardiomyocyte-like cells (CLCs) from hPSCs and repopulate decellularized human heart ventricles for heart engineering. In our hands, targeting myosin heavy chain locus (MYH6) with mCherry fluorescent reporter by TALEN technology in hESCs did not alter major pluripotent-related features, and allowed for the definition of a robust protocol for CLCs production also from human induced pluripotent stem cells (hiPSCs) in 14 days. hPSCs-derived CLCs (hPSCs-CLCs) were next used to recellularize acellular cardiac scaffolds. Electrophysiological responses encountered when hPSCs-CLCs were cultured on ventricular decellularized extracellular matrix (vdECM) correlated with significant increases in the levels of expression of different ion channels determinant for calcium homeostasis and heart contractile function. Overall, the approach described here allows for the rapid generation of human cardiac grafts from hPSCs, in a total of 24 days, providing a suitable platform for cardiac engineering and disease modeling in the human setting.

Acellular human heart matrix: A critical step toward whole heart grafts.

The best definitive treatment option for end-stage heart failure currently is transplantation, which is limited by donor availability and immunorejection. Generating an autologous bioartificial heart could overcome these limitations. Here, we have decellularized a human heart, preserving its 3-dimensional architecture and vascularity, and recellularized the decellularized extracellular matrix (dECM). We decellularized 39 human hearts with sodium-dodecyl-sulfate for 4-8 days. Cell removal and architectural integrity were determined anatomically, functionally, and histologically. To assess cytocompatibility, we cultured human cardiac-progenitor cells (hCPC), bone-marrow mesenchymal cells (hMSCs), human endothelial cells (HUVECs), and H9c1 and HL-1 cardiomyocytes in vitro on dECM ventricles up to 21 days. Cell survival, gene expression, organization and/or electrical coupling were analyzed and compared to conventional 2-dimensional cultures. Decellularization removed cells but preserved the 3-dimensional cardiac macro and microstructure and the native vascular network in a perfusable state. Cell survival was observed on dECM for 21 days. hCPCs and hMSCs expressed cardiocyte genes but did not adopt cardiocyte morphology or organization; HUVECs formed a lining of endocardium and vasculature; differentiated cardiomyocytes organized into nascent muscle bundles and displayed mature calcium dynamics and electrical coupling in recellularized dECM. In summary, decellularization of human hearts provides a biocompatible scaffold that retains 3-dimensional architecture and vascularity and that can be recellularized with parenchymal and vascular cells. dECM promotes cardiocyte gene expression in stem cells and organizes existing cardiomyocytes into nascent muscle showing electrical coupling. These findings represent a first step toward manufacturing human heart grafts or matrix components for treating cardiovascular disease.