Hina W. Chaudhry

Cyclin A2 (CCNA2), a master cell cycle regulator silenced in postnatal cardiomyocytes, promotes cardiac repair in animal models. However, its effect on cytokinesis in adult human cardiomyocytes was previously unknown. We engineered a replication-deficient adenoviral vector encoding human CCNA2 under the cardiac Troponin T promoter and delivered it to freshly isolated cardiomyocytes from adult human hearts. Time-lapse live imaging revealed the induction of complete cytokinesis with preservation of sarcomeres and calcium mobilization in redifferentiated daughter cardiomyocytes. Single-nucleus transcriptomic profiling of CCNA2-transgenic and non-transgenic mouse hearts uncovered a cardiomyocyte subpopulation characterized by enrichment of cytokinesis, proliferation, and reprogramming gene signatures. Ultra-deep bulk RNA sequencing of adult and fetal human hearts further highlighted reprogramming pathways relevant to CCNA2-induced effects. Together, these findings demonstrate that CCNA2 can reinitiate cytokinesis in adult human cardiomyocytes, illuminating conserved molecular programs that support its promise as a regenerative gene therapy for the heart.

Cyclin A2 (CCNA2), a master cell cycle regulator, is silenced in postnatal mammalian cardiomyocytes. We have previously demonstrated its ability to promote cardiac repair in small and large animals when delivered to the heart via a viral vector. However, the effect of CCNA2 gene delivery on cytokinesis in isolated cardiomyocytes from adult human hearts has not been explored. We designed a human gene therapy vector featuring a replication-deficient adenovirus encoding human CCNA2 driven by the cardiac Troponin T promoter to enable the expression of CCNA2 in freshly isolated human cardiomyocytes. Time-lapse live imaging of adult human primary cardiomyocytes from a 21-year-old male, a 41-year-old female, and a 55-year-old male demonstrated the induction of complete cytokinesis in human adult cardiomyocytes with preservation of sarcomere integrity in the resulting daughter cells with active calcium mobilization in redifferentiated cardiomyocytes. To elucidate the transcriptional mechanisms underlying this response, we conducted single-nucleus transcriptomics analysis of hearts isolated from adult transgenic mice that constitutively express CCNA2 in cardiomyocytes (CCNA2-Tg) and non- transgenic mice (nTg). This revealed a cardiomyocyte subpopulation enriched with cytokinesis, proliferative, and reprogramming genes in hearts obtained from CCNA2-Tg mice as compared to41nTg mice. Ultra-deep bulk RNA sequencing of human adult and fetal hearts identified key reprogramming genes relevant to understanding the mechanisms of CCNA2-induced effects observed in our experimental models. These findings provide a promising path for the clinical development of CCNA2-based cardiac regenerative therapy.

Myocardial infarction (MI) in mice is a widely used surgical model in preclinical cardiac repair studies to recapitulate human cardiovascular ischemic disease. Induction of reproducible infarct size is crucial for quantitative and analytical purpose. Here we describe a quick and reliable method to induce consistent infarct size in mice in less than a minute.

Cyclin A2 (CCNA2), a master cell cycle regulator silenced in postnatal cardiomyocytes, promotes cardiac repair in animal models. However, its effect on cytokinesis in adult human cardiomyocytes remains unknown. We engineered a replication-deficient adenoviral vector encoding human CCNA2 under the cardiac Troponin T promoter and delivered it to freshly isolated cardiomyocytes from adult human hearts. Time-lapse live imaging revealed induction of complete cytokinesis with preservation of sarcomeres and calcium mobilization in redifferentiated daughter cardiomyocytes. To uncover underlying transcriptional mechanisms, single-nucleus transcriptomics of CCNA2-transgenic versus non-transgenic mouse hearts identified a cardiomyocyte subpopulation enriched for cytokinesis, proliferative, and reprogramming genes. Ultra-deep bulk RNA sequencing of adult and fetal human hearts further highlighted reprogramming pathways relevant to CCNA2-induced effects. Together, these findings demonstrate that CCNA2 can reinitiate cytokinesis in adult human cardiomyocytes and illuminate conserved molecular programs, supporting its promise as a regenerative gene therapy for the heart. Graphical CCNA2 Induces Cytokinesis and Drives Proliferation and Reprogramming of Adult Cardiomyocytes: An Integrative Transcriptomic Analysis across Human and Mouse Models.

It may be possible to treat cardiac hypertrophy and injury by using drugs that inhibit a protein called SIRT2.

We identify a population of multipotent CDX2 cells from term human placentas with clonal expansion, migratory capacity, and immune-privileged transcriptional profiles. Isolated from 180 healthy pregnancies, these cells differentiate into cardiomyocyte and vascular lineages in vitro and in vivo . Single-cell RNA sequencing uncovers distinct cardiogenic and vasculogenic subpopulations, along with immune-modulatory and chemotactic programs, providing a blueprint for precision-guided cardiovascular cell therapy. In a NOD/SCID myocardial infarction model, CDX2 cells restore cardiac function, and clonal propagation preserves their cardiovascular differentiation potential. These findings position placental CDX2 cells as an ethically accessible, regenerative platform for targeted treatment of cardiovascular disease.

Purpose of Review This review explores efforts made over the previous three decades to determine mechanisms of cardiomyocyte cell division. Many investigators have explored cell therapy strategies in animal models and clinical trials over the past 2 decades with marginal results thus far in clinical testing. Hence, there is a greater focus now on strategies to induce cardiomyocyte proliferation. Recent Findings Reports to induce reactivation of the cardiomyocyte cell cycle predated the focus on cell therapy, and we summarize the literature on this topic, which began with the very first transgenic mouse studies in cardiovascular science. Summary These earlier studies form the foundation for the use of cell cycle manipulation in cardiac repair and should inform current and future investigations with respect to rigor of assessment in the degree of cardiomyocyte cell division and gold standard measures of cardiac functional improvement.

Background: Our understanding of catheter-based pulsed field ablation (PFA) of the ventricular myocardium is limited. We conducted a series of exploratory evaluations of ventricular PFA in swine ventricles. Methods: A focal lattice-tip catheter was used to deliver proprietary biphasic monopolar PFA applications to swine ventricles under general anesthesia, with guidance from electroanatomical mapping, fluoroscopy, and intracardiac echocardiography. We conducted experiments to assess the impact of (1) delivery repetition (2×, 3×, or 4×) at each location, (2) epicardial PFA delivery, and (3) confluent areas of shallow healed endocardial scar created by prior PFA (4 weeks earlier) on subsequent endocardial PFA. Additional assessments included PFA optimized for the ventricle, lesion visualization by intracardiac echocardiography imaging, and immunohistochemical insights. Results: Experiment no. 1: lesions (n=49) were larger with delivery repetition of either 4× or 3× versus 2×: length 17.6±3.9 or 14.2±2.0 versus 12.7±2.0 mm ( P P =0.22), width 13.4±1.8 or 10.6±1.3 versus 10.5±1.1 mm ( P P =1.00), and depth 6.1±2.1 or 5.1±1.3 versus 4.2±1.0 mm ( P P =0.21). Experiment no. 2: epicardial lesions (n=18) were reliably created and comparable to endocardial lesions: length 24.6±9.7 mm (n=5), width 15.6±4.6 mm, and depth 4.5±3.7 mm. Experiment no. 3: PFA (n=16) was able to penetrate to a depth of 4.8 (interquartile range, 4.5–5.4) mm in healthy myocardium versus 5.6 (interquartile range, 3.6–6.6) mm in adjacent healed endocardial scar ( P =0.79), suggesting that superficial scar does not significantly impair PFA. Finally, we demonstrate, PFA optimized for the ventricle yielded adequate lesion dimensions, can result in myocardial activation, can be visualized by intracardiac echocardiography, and have unique immunohistochemical characteristics. Conclusions: This in vivo evaluation offers insights into the behavior of endocardial or epicardial PFA delivered using the lattice-tip catheter to normal or scarred porcine ventricular myocardium, thereby setting the stage for future clinical studies.

Importance: SARS-CoV-2 infection directly causes severe acute respiratory illness, leading to systemic tissue hypoxia and ischemia including the heart. Myocardial cytopathy associated with hypoxic response has been largely overlooked in COVID-19 patients. Additionally, histology analysis and cardiac function of COVID-19 cases are often reported separately, rendering an incomplete understanding of COVID-19 cardiac symptoms. Objective: To examine the relationship between myocardial cellular responses to hypoxic stress versus cardiac functional alterations within the same COVID-19 patients. Design, Setting, and Participants: Cellular hypoxia Inducible Factor 1 alpha (HIF1α) expression was analyzed by immunohistochemistry using post-mortem COVID-19 heart and lung tissues with known cardiac echocardiography records from a total of 8 patients. Clinical echocardiography data were obtained from Mount Sinai Heart between March to December, 2020. All gender and age groups were considered as long as cardiac involvement meets the preserved (EF > 50%) or moderate to severe (EF < 45%) criteria with confirmed SARS-CoV-2 infection. Cell-type specific subcellular localization of HIF1α expression and nuclear stability was examined by immunohistochemistry and transmission electronic microscopy (TEM). Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) was used to quantify apoptosis. Main Outcomes and Measures: No planned outcomes of this study as this is a retrospective analysis based on post-mortem specimens exclusively. Results: Cardiac HIF1α expression was found to be significantly higher in patients with preserved EF levels than it was in the low EF group. In the preserved EF group, HIF1α is protective against apoptosis predominantly in endothelial cells and cardiac fibroblasts. In the low EF group, HIF1α protects cardiomyocyte nuclear integrity as evident by its nuclear accumulation with nuclear envelope preservation. Conclusions and Relevance: This study establishes a direct link of cardiac cellular responses to hypoxic stress with matching functional and histological data, serving as one of the first studies to bridge previous stand-alone clinical data and cellular data. The protective role of HIF1α in hearts may help predict cardiac involvement in not only COVID-19 patients, but also decipher the underlying mechanisms in other forms of viral cardiomyopathy. KEY POINTS: Question: Are hypoxic signaling pathways associated with cardiac functional alterations in COVID-19 patients? Findings: Cardiac HIF1α expression of COVID-19 patients with EF>50% or EF+ cells were found in patients with higher EF. HIF1α ⁺ endothelial cells are resistant to apoptosis, and HIF1α ⁺ cardiomyocytes are able to retain nuclear envelope under hypoxic stress. Meaning: HIF1α is cardioprotective in hearts of COVID-19 patients.