Induction Of Cardiomyocyte Proliferation Research Articles

Abstract Tu071: Computational Modeling to Predict Mechanisms of DYRK1A-mediated Inhibition of Cardiomyocyte Proliferation

Introduction and Background: Adult cardiomyocytes lack strong proliferative capability with only a small subset being able to undergo mitosis. This can lead to poor functional recovery after heart injuries such as myocardial infarctions (MI). We have previously demonstrated that inhibition of DYRK1A, a regulator of the DREAM complex and cell quiescence, increases cardiomyocyte cycling. However, the mechanism of DYRK1A inhibition that induces this increased cycling has yet to be elucidated. Therefore, we built a network model that encapsulates potential components that can lead to cardiomyocyte proliferation. Methods: We developed a logic-based differential equations model to simulate DYRK1A-mediated contributors of cardiomyocyte proliferation and G1/S phase transition. Various knock-down/knock-out and overexpression simulations were performed to predict targets of cell cycle regulation. Literature validation was performed to compare experiments across cancer, cardiomyocytes, and broader cell types to independent model simulations. A sensitivity analysis was used to identify the influence of particular proteins/genes on cardiomyocyte proliferation. Results: Our model simulations validated against (n = # of primary literature articles) 84.6% of literature experiments in cancer cell lines (n = 13), 88.8% in broader cell types (n = 9), and 100% in cardiomyocytes (n = 5). Simulated knockouts of DYRK1A and overexpression of Cyclin-D, Cyclin-E, and B-MyB increased DNA replication, indicating exit from G 0 phase. Sensitivity analysis showed that a DYRK1A knockout was the most influential in terms of increasing DNA replication. Conclusion: Our model’s predictions were successfully validated against literature in several different types of cell lines. Furthermore, it gives valuable insight on possible targets that can induce cardiomyocyte proliferation in addition to DYRK1A. Further in vitro and in vivo experiments are necessary to fully evaluate our simulations in cardiomyocytes.

Abstract We035: PTMA-MBD3 axis is a core heart regenerative driver in mammals

Introduction: The adult mammalian heart has an extremely limited ability for regeneration after injury, whereas the fetal heart can steadily regenerate. Uncovering the molecular underpinnings of proliferative cardiomyocytes and manipulating the core genes activate cardiomyocyte proliferation may provide new insight into adult heart regrowth and functional repair post-injury. Methods and Results: We performed single-cell RNA-sequencing of mouse embryonic hearts from various developmental stages (E8.5-E17.5), and identified a cardiomyocyte population in proliferative phase with 120 highly expressed feature genes. Combining with our bulk RNA-seq data on isolated cardiomyocytes from different heart developmental periods (E14.5, P1-P56), we screened and demonstrated that Ptma is the core conservative factor in promoting mouse, rat and human induced pluripotent stem cell-derived cardiomyocyte (hiPSC-CM) proliferation. Cardiac specifically knocking out Ptma blocked cardiomyocyte proliferation and neonatal mouse heart regeneration after apical resection injury. AAV9-delivered Ptma overexpression extended neonatal heart regenerative time window and showed therapeutic benefit in driving adult cardiomyocyte proliferation and cardiac repair. As PTMA is a polypeptide, we further validated that PTMA derived conservative peptide Tα1 displayed more effective in treating adult cardiac injury compared to Decapeptide. PTMA is an unstructured, intrinsically disordered nuclear protein that functions by binding to target proteins. Using Co-IP, IP and Native PAGE, we found PTMA promoted cardiomycyte proliferaton by binding to MBD3, which decreases the formation of MBD3-HDAC1 NuRD complex . We further decreased MBD3-HDAC1 NuRD complex formation by inhibiting MBD3, boosting both rat and mouse cardiomyocyte proliferation. Conclusions: We identified the PTMA-MBD3 axis as an effective target for promoting mammalian heart regeneration and provided a new insight into cardiomyocyte proliferation through blocking MBD3-HDAC1 NuRD complex.

Abstract We021: Connexin 43 mediates macrophage induced cardiomyocyte proliferation and heart regeneration

Background: Heart regeneration is a potential strategy to recover pumping function after cardiac injury and protect against heart failure. Macrophages have been demonstrated to promote heart regeneration, while its mechanism has not been addressed. Modulating the interaction between macrophages and cardiomyocytes through connexin 43 (CX43) may provide a clinical therapy for preventing further deterioration of patients with heart injury and restoring the lost myocardium. Hypothesis: We hypothesize that macrophages can interact with cardiomyocytes via CX43, leading to promotion of cardiac proliferation and heart regeneration. Aims: Our study aims to provide the evidence of macrophages contacting cardiomyocytes through CX43 and promoting heart regeneration. Methods and Results: Through cell co-culture experiments, we found that macrophages attache to cardiomyocytes via CX43, subsequently promoting myocyte proliferation as evidenced by proliferative markers. Blocking the CX43 connection by SiRNA between two cells suspended cardiomyocyte proliferation in vitro. We generated macrophage-specific CX43 deficient mice (CX43 Mφ/- ) by crosing CD11b-Cre mice and CX43-flox mice. We performed apical resection operation on 1 day-old CX43 Mφ/- mice and harvested the hearts at 21 day-post-resection. CX43 Mφ/- mice demonstrated either the heart regeneration ability nor pumping function. Applying single-cell RNA sequencing in CX43 Mφ/- hearts, we illustrated the unique transcriptional signature of macrophage and cardiomyocyte in macrophage-specific CX43 deficient mice. Conclusion: Our study provides the evidence that macrophages attache to cardiomyocyte via CX43 after cardiac injury, promote cardiomyocyte proliferation and heart regeneration. This discovery may offer a new target for clinical treatment of myocardial infarction.

Enhancing miR-19a/b induced cardiomyocyte proliferation in infarcted hearts by alleviating oxidant stress and controlling miR-19 release

Fully restoring the lost population of cardiomyocytes and heart function remains the greatest challenge in cardiac repair post myocardial infarction. In this study, a pioneered highly ROS-eliminating hydrogel was designed to enhance miR-19a/b induced cardiomyocyte proliferation by lowering the oxidative stress and continuously releasing miR-19a/b in infarcted myocardium in situ. In vivo lineage tracing revealed that ∼20.47 % of adult cardiomyocytes at the injected sites underwent cell division in MI mice. In MI pig the infarcted size was significantly reduced from 40 % to 18 %, and thereby marked improvement of cardiac function and increased muscle mass. Most importantly, our treatment solved the challenge of animal death--all the treated pigs managed to live until their hearts were harvested at day 50. Therefore, our strategy provides clinical conversion advantages and safety for healing damaged hearts and restoring heart function post MI, which will be a powerful tool to battle cardiovascular diseases in patients.

Identification of FDA-approved drugs that induce heart regeneration in mammals.

Targeting Meis1 and Hoxb13 transcriptional activity could be a viable therapeutic strategy for heart regeneration. In this study, we performd an in silico screening to identify FDA-approved drugs that can inhibit Meis1 and Hoxb13 transcriptional activity based on the resolved crystal structure of Meis1 and Hoxb13 bound to DNA. Paromomycin (Paro) and neomycin (Neo) induced proliferation of neonatal rat ventricular myocytes in vitro and displayed dose-dependent inhibition of Meis1 and Hoxb13 transcriptional activity by luciferase assay and disruption of DNA binding by electromobility shift assay. X-ray crystal structure revealed that both Paro and Neo bind to Meis1 near the Hoxb13-interacting domain. Administration of Paro-Neo combination in adult mice and in pigs after cardiac ischemia/reperfusion injury induced cardiomyocyte proliferation, improved left ventricular systolic function and decreased scar formation. Collectively, we identified FDA-approved drugs with therapeutic potential for induction of heart regeneration in mammals.

Runx1 is upregulated by STAT3 and promotes proliferation of neonatal rat cardiomyocytes.

Though it is well known that mammalian cardiomyocytes exit cell cycle soon after birth, the mechanisms that regulate proliferation remain to be fully elucidated. Recent studies reported that cardiomyocytes undergo dedifferentiation before proliferation, indicating the importance of dedifferentiation in cardiomyocyte proliferation. Since Runx1 is expressed in dedifferentiated cardiomyocytes, Runx1 is widely used as a dedifferentiation marker of cardiomyocytes; however, little is known about the role of Runx1 in the proliferation of cardiomyocytes. The purpose of this study was to clarify the functional significance of Runx1 in cardiomyocyte proliferation. qRT-PCR analysis and immunoblot analysis demonstrated that Runx1 expression was upregulated in neonatal rat cardiomyocytes when cultured in the presence of FBS. Similarly, STAT3 was activated in the presence of FBS. Interestingly, knockdown of STAT3 significantly decreased Runx1 expression, indicating Runx1 is regulated by STAT3. We next investigated the effect of Runx1 on proliferation. Immunofluorescence microscopic analysis using an anti-Ki-67 antibody revealed that knockdown of Runx1 decreased the ratio of proliferating cardiomyocytes. Conversely, Runx1 overexpression using adenovirus vector induced cardiomyocyte proliferation in the absence of FBS. Finally, RNA-sequencing analysis revealed that Runx1 overexpression induced upregulation of cardiac fetal genes and downregulation of genes associated with fatty acid oxidation. Collectively, Runx1 is regulated by STAT3 and induces cardiomyocyte proliferation by juvenilizing cardiomyocytes.

Networks that Govern Cardiomyocyte Proliferation to Facilitate Repair of the Injured Mammalian Heart.

Cardiovascular diseases are the number one cause of death worldwide and in the United States (US). Cardiovascular diseases frequently progress to end-stage heart failure, and curative therapies are extremely limited. Intense interest has focused on deciphering the cascades and networks that govern cardiomyocyte proliferation and regeneration of the injured heart. For example, studies have shown that lower organisms such as the adult newt and adult zebrafish have the capacity to completely regenerate their injured heart with restoration of function. Similarly, the neonatal mouse and pig are also able to completely regenerate injured myocardium due to cardiomyocyte proliferation from preexisting cardiomyocytes. Using these animal models and transcriptome analyses, efforts have focused on the definition of factors and signaling pathways that can reactivate and induce cardiomyocyte proliferation in the adult mammalian injured heart. These studies and discoveries have the potential to define novel therapies to promote cardiomyocyte proliferation and repair of the injured, mammalian heart.

Massive expansion of functional human iPSC-derived cardiomyocytes by concomitant glycogen synthase kinase-3 beta inhibition and removal of cell-cell contact

Abstract Background Adult mammalian heart has limited capacity for regeneration. Cardiac injuries such as myocardial infarction lead to significant cardiomyocyte (CM) loss and subsequent heart failure with significant morbidity and mortality. Cell-based therapeutic approaches such as injection of stem cell-derived cells or transplantation of engineered cardiac tissue patches have shown promise to re-muscularize the damaged myocardium and enhance cardiac functions. Rationale: Modulating signaling pathways including Wnt and Hippo can induce cardiomyocyte proliferation in vivo. Applying these signaling modulators to human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) in vitro can expand CMs modestly (< 5-fold). Results Here, we demonstrate massive expansion of hiPSC-CMs in vitro (i.e. 100 to 250-fold) by glycogen synthase kinase-3β (GSK-3β) inhibition using CHIR99021 and concurrent removal of cell-cell contact. We show GSK-3β inhibition suppresses CM maturation while contact removal prevents CMs from cell cycle exit. Remarkably, contact removal enabled 10- to 25-times greater expansion beyond GSK-3β inhibition alone. Mechanistically, persistent CM proliferation required both LEF/TCF activity and AKT phosphorylation but was independent from Yes associated protein (YAP) signaling. Engineered heart tissues from expanded hiPSC-CMs showed comparable contractility to those from unexpanded hiPSC-CMs, demonstrating uncompromised cellular functionality after expansion. Conclusion In summary, we uncovered a molecular interplay that enables massive hiPSC-CMs expansion for large-scale drug screening and tissue engineering applications.

A surgical mouse model of neonatal right ventricular outflow tract obstruction by pulmonary artery banding

Diseased animal models play an extremely important role in preclinical research. Lacking the corresponding animal models, many basic research studies cannot be carried out, and the conclusions obtained are incomplete or even incorrect. Right ventricular (RV) outflow tract (RVOT) obstruction leads to RV pressure overload (PO) and reduced pulmonary blood flow (RPF), which are two of the most important pathophysiological characteristics in pediatric cardiovascular diseases and seriously affect the survival rate and long-term quality of life of many children. Due to the lack of a neonatal mouse model for RVOT obstruction, it is largely unknown how RV PO and RPF regulate postnatal RV and pulmonary development. The aim of this study was to construct a neonatal RVOT obstruction mouse model. Here, we first introduced a neonatal mouse model of RVOT obstruction by pulmonary artery banding (PAB) on postnatal day 1. PAB induced neonatal RVOT obstruction, RV PO, and RPF. Neonatal RV PO induced cardiomyocyte proliferation, and neonatal RPF induced pulmonary dysplasia, the two features that are not observed in adult RVOT obstruction. As a result, PAB neonates exhibited overall developmental dysplasia, a sign similar to that of children with RVOT obstruction. Because many pediatric cardiovascular diseases are associated with RV PO and RPF, the introduction of a neonatal mouse model of RVOT obstruction may greatly enhance our understanding of these diseases and eventually improve or save the lives of many children.

Abstract P3161: Engineering Mesenchymal Stem Cell-derived Extracellular Vesicles To Enhance Their Therapeutic Efficacy In Cardiovascular Disease

Background: Extracellular vesicles derived from cardiosphere-derived cells (CDC-EVs) have demonstrated the ability to regulate cardiomyocyte apoptosis and induce cardiomyocyte proliferation in pre-clinical models of acute MI. While CDC-EVs exhibit cardioprotective effects, they require invasive intramyocardial injections to achieve therapeutic efficacy, detracting from their translatability. Mesenchymal stem cells (MSC) also generate cardioprotective EVs (MSC-EVs), which have demonstrated therapeutic efficacy in models of cardiac disease following intravenous administration. However, MSC-EVs lack several of the cardioprotective miRs expressed by CDC-EVs, including miR146a, which has been shown to modulate cardiomyocyte apoptosis. The objective of our current study was to determine the effect of enriching MSC-EVs with cardioprotective CDC-EV miRNA on cardiomyocyte apoptosis. Methods: MSCs were transfected with miR-146a-5p mimics using Trans-IT TKO in serum free growth media. EVs were isolated using a combination of ultrafiltration and size exclusion chromatography (SEC) and characterized by size, morphology and expression of surface markers. RT-PCR was performed on EV samples to quantify miR-146a expression. Primary neonatal mouse cardiomyocytes were treated with MSC-EVs, miR-146a loaded MSC-EVs, CDC-EVs or matched volume vehicle control. Cardiomyocyte apoptosis was assessed by a TUNEL assay. Results: Treatment with miR-146a loaded MSC EVs reduced cardiomyocyte apoptosis compared with cardiomyocytes treated with unmodified MSC exosomes (14±1% vs. 31±3%) (mean ± SEM, p=0.001, one-way ANOVA). miR-146a loaded MSC-EVs and CDC-EVs demonstrated a similar level of reduction in cardiomyocyte apoptosis (14±1% vs. 15±1%). There was no difference in the level of apoptosis between cardiomyocytes treated with unmodified MSC exosomes and cardiomyocytes treated with vehicle control (31±3% vs. 27±1%) (mean ± SEM, p=0.366, one-way ANOVA). Conclusion: Engineering MSC-EVs to express miR-146a enhances their ability to reduce cardiomyocyte apoptosis in vitro . This highlights the potential of cargo engineering as a means to improve the therapeutic efficacy of EVs for cardiovascular disease.

Abstract P3080: Macrophages Synthesize Bioactive Lipids To Modulate Cardiomyocyte Metabolism And Proliferation During Cardiac Regeneration

Introduction: Once thought to be a quiescent process, elimination of dying cells by macrophages actively reprograms macrophage metabolism to support downstream functions. Previously, we discovered that the phagocytic receptor MerTK is necessary for regeneration in the neonatal murine heart. Single-cell RNA sequencing and bulk-tissue lipidomics revealed prioritization of arachidonic acid metabolism towards the synthesis of Thromboxane A 2 (TXA 2 ) in regenerating hearts, yet the molecular mechanisms directly connecting macrophage-derived TXA 2 to regeneration remained undefined. Therefore, we sought to determine if TXA 2 and activation of its receptor ( Tbxa2r ) on cardiomyocytes is necessary for cardiac regeneration. Results: We created a novel mouse to conditionally knockout Tbxa2r in cardiomyocytes using the CRE recombinase under the Myh6 promoter. Loss of Tbxa2r did not affect normal cardiac development but significantly impaired cardiac regeneration as seen by significant fibrotic scarring, reduced cardiac output, and decreased cardiomyocyte proliferation. We next asked how Tbxa2r initiates intracellular signaling to support cardiomyocyte proliferation. Knowing that the YAP/TAZ pathway has been previously described to induce cardiomyocyte proliferation during regeneration and is regulated by GPCRs, we found that TXA 2 mimetics activate TBXA2R leading to the stabilization of YAP/TAZ in a time- and dose-dependent manner. Furthermore, YAP was translocated into the nucleus of cardiomyocytes in the border zone 3 days post-MI. However, deletion of Tbxa2r in cardiomyocytes completely prevents YAP translocation into the nucleus. Lastly, we have found that a TXA 2 mimetic increases the glycolytic capacity of cardiomyocytes further supporting anabolic processes needed for proliferation. Excitingly, we also observed an increase in glycolytic enzyme transcripts at the border zone of the infarct using publicly available spatial transcriptomic data. Conclusions: Our findings advance our understanding of how cell death, removal and recycling regulate macrophage metabolism, resulting in the production of bioactive lipids that further regulate the metabolism of neighboring cardiomyocytes during regeneration.

Abstract P3193: Cardiac Glutamine Metabolism As NAD+ Generating Pathway During Chronic Hypoxia

Cardiovascular disease morality relies in the inability of the adult mammalian heart to regenerate damaged tissue due to the inability of the adult cardiomyocytes to proliferate. This lack of proliferation in the adult heart is due to an increased DNA damage and activation of the DNA Damage response upon an increase of mitochondrial ROS generation. We have previously shown that modulation of mitochondrial metabolism towards glucose oxidation at expenses of fatty acid utilization can induce adult cardiomyocyte proliferation and support cardiac regeneration. Moreover, by modulating the oxygen levels and, thus, cardiomyocyte metabolism, by chronic hypoxia exposure, we were also able to induce cardiomyocyte proliferation and cardiac regeneration in adult mice. Remarkably, our metabolomic studies of the hypoxic hearts did not show increase of lactate production, a hallmark of hypoxic metabolism in order to regenerate NAD+ required for glycolysis and ATP production under low oxygen conditions. Interestingly, glutamine metabolism is one of the most compensatory pathways in these conditions. Glutamine can be metabolized to its conversion into glutamate, which can enter in the Krebs’ Cycle, but also feed the malate-aspartate shuttle. This shuttle has the ability to generate cytoplasmic NAD+. Here, we hypothesize that, during chronic hypoxia exposure, the heart uses glutamine as a fuel to regenerate cytoplasmic NAD+ as an alternative to lactate production. By means of carbon labeling and metabolic flux studies in vivo, we have proven that glutamine contribution to the malate-aspartate shuttle is enhanced during hypoxia in the heart. Moreover, we have determined that glutamine supplementation during hypoxia in cell culture increases cytoplasmid NAD+ levels, confirming the contribution of glutamine catabolism to NAD/NADH balance during hypoxia. Overall, we show that glutamine metabolism is an important adaptation to hypoxic metabolism in the heart and describe a new mechanism of metabolic rewiring under hypoxia beyond the classic lactate production upon glycolysis upregulation.

Regulation of Cardiomyocyte Division During Cardiac Regeneration.

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. 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. 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.

Systemic Hypoxemia Induces Cardiomyocyte Hypertrophy and Right Ventricular Specific Induction of Proliferation.

A recent study suggests that systemic hypoxemia in adult male mice can induce cardiac myocytes to proliferate. The goal of the present experiments was to confirm these results, provide new insights on the mechanisms that induce adult cardiomyocyte cell cycle reentry, and to determine if hypoxemia also induces cardiomyocyte proliferation in female mice. EdU-containing mini pumps were implanted in 3-month-old, male and female C57BL/6 mice. Mice were placed in a hypoxia chamber, and the oxygen was lowered by 1% every day for 14 days to reach 7% oxygen. The animals remained in 7% oxygen for 2 weeks before terminal studies. Myocyte proliferation was also studied with a mosaic analysis with double markers mouse model. Hypoxia induced cardiac hypertrophy in both left ventricular (LV) and right ventricular (RV) myocytes, with LV myocytes lengthening and RV myocytes widening and lengthening. Hypoxia induced an increase (0.01±0.01% in normoxia to 0.11±0.09% in hypoxia) in the number of EdU+ RV cardiomyocytes, with no effect on LV myocytes in male C57BL/6 mice. Similar results were observed in female mice. Furthermore, in mosaic analysis with double markers mice, hypoxia induced a significant increase in RV myocyte proliferation (0.03±0.03% in normoxia to 0.32±0.15% in hypoxia of RFP+ myocytes), with no significant change in LV myocyte proliferation. RNA sequencing showed upregulation of mitotic cell cycle genes and a downregulation of Cullin genes, which promote the G1 to S phase transition in hypoxic mice. There was significant proliferation of nonmyocytes and mild cardiac fibrosis in hypoxic mice that did not disrupt cardiac function. Male and female mice exhibited similar gene expression following hypoxia. Systemic hypoxia induces a global hypertrophic stress response that was associated with increased RV proliferation, and while LV myocytes did not show increased proliferation, our results minimally confirm previous reports that hypoxia can induce cardiomyocyte cell cycle activity in vivo.

ER stress induces upregulation of transcription factor Tbx20 and downstream Bmp2 signaling to promote cardiomyocyte survival

In the mammalian heart, fetal cardiomyocytes proliferate prior to birth; however, they exit the cell cycle shortly after birth. Recent studies show that adult cardiomyocytes re-enters the cell cycle postinjury to promote cardiac regeneration. The endoplasmic reticulum (ER) orchestrates the production and assembly of different types of proteins, and a disruption in this machinery leads to the generation of ER stress, which activates the unfolded protein response. There is a very fine balance between ER stress-mediated protective and proapoptotic responses. T-box transcription factor 20 (Tbx20) promotes embryonic and adult cardiomyocyte proliferation postinjury to restore cardiac homeostasis. However, the function and regulatory interactions of Tbx20 in ER stress-induced cardiomyopathy have not yet been reported. We show here that ER stress upregulates Tbx20, which activates downstream bone morphogenetic protein 2 (Bmp2)-pSmad1/5/8 signaling to induce cardiomyocyte proliferation and limit apoptosis. However, augmenting ER stress reverses this protective response. We also show that increased expression of tbx20 during ER stress is mediated by the activating transcription factor 6 arm of the unfolded protein response. Cardiomyocyte-specific loss of Tbx20 results in decreased cardiomyocyte proliferation and increased apoptosis. Administration of recombinant Bmp2 protein during ER stress upregulates Tbx20 leading to augmented proliferation, indicating a feed-forward loop mechanism. In in vivo ER stress, as well as in diabetic cardiomyopathy, the activity of Tbx20 is increased with concomitant increased cardiomyocyte proliferation and decreased apoptosis. These data support a critical role of Tbx20-Bmp2 signaling in promoting cardiomyocyte survival during ER stress-induced cardiomyopathies.

Extracellular vesicle-derived CircWhsc1 promotes cardiomyocyte proliferation and heart repair by activating TRIM59/STAT3/Cyclin B2 pathway

IntroductionExtracellular vesicles (EVs)-mediated cell-to-cell communication is crucial for hypoxia-induced cell proliferation and tissue repair, but its function in endogenous cardiac regeneration is still unknown. ObjectivesHerein, we aimed to determine whether hypoxia-inducible circWhsc1 in endothelial EVs promoted cardiomyocyte (CM) proliferation and cardiac regeneration. MethodsRNA-sequence data was used to identify EV circRNAs that were involved into endogenous cardiac regeneration. Quantitative polymerase chain reactions were conducted to determine circRNA expression in tissue, cells and EVs. Gain- and loss-of-function assays were performed to explore the function of EV-derived circWhsc1 during cardiac regeneration. Western blotting and RNA pulldown assays were used to investigate its underlying mechanism. ResultsWe found that circWhsc1 was enriched in neonatal mouse hearts, particularly in cardiac ECs, and was further upregulated both in ECs and EC-derived EVs under hypoxic conditions. When cocultured with hypoxia-preconditioned neonatal ECs or their secreted EVs, both neonatal and adult CMs exhibited an increased proliferation rate and G2/M ratio, which could be attenuated by knockdown of circWhsc1 in ECs. In vivo, EC-restricted overexpression of circWhsc1 and EV-mediated delivery of circWhsc1 induced CM proliferation, alleviated cardiac fibrosis and restored cardiac function following myocardial infarction in adult mice. Mechanistic studies revealed that EV-derived circWhsc1 activated TRIM59 by enhancing its phosphorylation, thereby reinforcing the binding of TRIM59 to STAT3, phosphorylating STAT3 and inducing CM proliferation. ConclusionThe current study demonstrated that hypoxia-inducible circWhsc1 in EC-derived EVs induces CM proliferation and heart regeneration. EC-CM communication mediated by EV-derived circWhsc1 might represent a prospective therapeutic target for inducing cardiac repair post-myocardial infarction.

Retinoic acid released from self-assembling peptide activates cardiomyocyte proliferation and enhances repair of infarcted myocardium

The limited cardiomyocyte proliferation is insufficient for repair of the myocardium. Therefore, activating cardiomyocyte proliferation might be a reasonable option for myocardial regeneration. Here, we investigated effect of retinoic acid (RA) on inducing adult cardiomyocyte proliferation and assessed efficacy of self-assembling peptide (SAP)-released RA in activating regeneration of the infarcted myocardium. Effect of RA on inducing cardiomyocyte proliferation was examined with the isolated cardiomyocytes. Expression of the cell cycle-associated genes and paracrine factors in the infarcted myocardium was examined at one week after treatment with SAP-carried RA. Cardiomyocyte proliferation, myocardial regeneration and improvement of cardiac function were assessed at four weeks after treatment. In the adult rat myocardium, expression of RA synthetase gene Raldh2 and RA concentration were decreased significantly. After treatment with RA, the proliferated cardiomyocytes were increased. The formulated SAP could sustainedly release RA. After treatment with SAP-carried RA, expression of the pro-proliferative genes in cell cycle and paracrine factors in the infarcted myocardium were up-regulated. Myocardial regeneration was enhanced, and cardiac function was improved significantly. These results demonstrate that RA can induce adult cardiomyocytes to proliferate effectively. The sustained release of RA with SAP is a promise strategy to enhance repair of the infarcted myocardium.

Live cell screening identifies glycosides as enhancers of cardiomyocyte cell cycle activity.

Promoting cardiomyocyte proliferation is a promising strategy to regenerate the heart. Yet, so far, it is poorly understood how cardiomyocyte proliferation is regulated, and no factor identified to promote mammalian cardiomyocyte proliferation has been translated into medical practice. Therefore, finding a novel factor will be vital. Here, we established a live cell screening based on mouse embryonic stem cell-derived cardiomyocytes expressing a non-functional human geminin deletion mutant fused to Azami Green (CM7/1-hgem-derived cardiomyocytes). We screened for a subset of compounds of the small molecule library Spectrum Collection and identified 19 potential inducers of stem cell-derived cardiomyocyte proliferation. Furthermore, the pro-proliferative potential of identified candidate compounds was validated in neonatal and adult rat cardiomyocytes as well as human induced pluripotent stem cell-derived cardiomyocytes. 18 of these compounds promoted mitosis and cytokinesis in neonatal rat cardiomyocytes. Among the top four candidates were two cardiac glycosides, peruvoside and convallatoxin, the flavonoid osajin, and the selective α-adrenoceptor antagonist and imidazoline I1 receptor ligand efaroxan hydrochloride. Inhibition of PTEN and GSK-3β enhanced cell cycle re-entry and progression upon stimulation with cardiac glycosides and osajin, while inhibition of IP3 receptors inhibited the cell cycle-promoting effect of cardiac glycosides. Collectively, we established a screening system and identified potential compounds to promote cardiomyocyte proliferation. Our data suggest that modulation of calcium handling and metabolism promotes cardiomyocyte proliferation, and cardiac glycosides might, besides increasing myocardial contraction force, contribute to cardiac repair by inducing cardiomyocyte proliferation.

Abstract P1014: Cardiomyocyte Energetic Capacity Governs Responses To Cell Cycle Stimulation

Background: Induction of cardiomyocyte (CM) proliferation is a promising strategy for cardiac regeneration. We previously showed that the overexpression of four cell cycle factors (4F), i.e. cyclin B1, CDK1, cyclin D1 and CDK4, induces CM proliferation. Temporal profiling of the single cell transcriptomes of 4F- and LacZ-overexpressing CMs showed a specific population with higher levels of CD36 that was more responsive to the 4F cell cycle induction. In addition, P7 primary CMs isolated from CD36 KO mice demonstrated 50% less response to the 4F compared with the WT controls (Abouleisa et al., Circulation, 2022). These data indicate that CD36 may be required to prime CMs to proliferate. CD36 is known to internalize fatty acids to be utilized for energy production. Here, we aim to investigate the direct effect of CM energetic capacity on their response to cell cycle stimulation. Methods and Results: Carnitine palmitoyl transferase -1 (CPT-1) is a rate limiting enzyme for the β-oxidation of fatty acids. Knockdown of CPT-1 using siRNA significantly reduced mitochondrial ATP production by 10% and led to a significant reduction in cell cycle markers (PHH3, EDU) in response to the 4F (PHH3; control siRNA+4F:24±1% vs CPT1 siRNA+4F:15±1%, EDU; control siRNA+4F:22±2% vs CPT1siRNA+4F:16±1%, P<0.01, n=4). Small molecule inhibition of CPT1 using etomoxir significantly decreased mitochondrial ATP production and suppressed CM responses to the 4F. Furthermore, overexpression of CPT-1 using viral vectors resulted in significant increase in ATP production and increased response to cell cycle stimulation using the 4F. Supplementing the culture medium with (1μmol) L-carnitine significantly increased mitochondrial ATP production and enhanced the CM response to the 4F. Finally, to increase ATP availability in CMs directly, we overexpressed a light-activated mitochondrial proton pump (MT-ON). CMs expressing MT-ON showed a significant increase in mitochondrial ATP levels and enhanced cell cycle induction in CMs transduced with 4F (PHH3; 4F:18±2% vs 4F+MT-ON:27±3%, EDU; 4F:18±1% vs 4F+MT-ON:30±4 %, P<0.01, n=4). Conclusions: These findings suggest that an energy threshold is required for CMs to progress into the cell cycle.

Abstract P1003: Mechanical Loading Controls Cardiomyocyte Proliferation

Introduction: Mammal cardiomyocytes (CMs) lose their proliferative capacity early after birth and switch to an adult phenotype, characterized by organized sarcomeres. The mechanisms that control this switch remain largely elusive. Among the changes experienced by the heart at birth is a sudden increase in mechanical load. Hypothesis: This study tests the hypothesis that variations in mechanical loading may regulate both the proliferation and the maturation of cardiomyocytes. Methods: Engineered heart tissues (EHTs) were generated using neonatal rat heart cells, with a modified protocol allowing to alter mechanical load. Unloading is obtained by reducing the distance between the silicon posts that anchor the extremities of the EHT, while overloading is induced by changing Young’s module of posts using metal braces. Adult heart unloading in vivo was achieved by heterotopically transplanting a mouse heart into the neck of syngeneic recipient mice, which were injected with EdU twice per week. Both hearts, native and transplanted, were harvested at day 30. Results: In line with our hypothesis, mechanical unloading of developed EHTs significantly increased the percentage of EdU-, Ki67- and pH3-positive CMs at 72 hours. In contrast, increasing afterload induced CM maturation, characterized by increased cross-sectional area and force of contraction, associated with reduced proliferation. Consistently, heterotopically transplanted hearts showed a significant increase in the the number of proliferating CMs in both ventricles, which were almost absent in native hearts. Conclusions: Our data indicate that mechanical unloading induce CM proliferation in both rat EHTs and an in vivo in a mouse model of heterotopic heart transplantation. These results support our initial hypothesis and indicate that mechanical loading is a master regulator of cardiomyocytes proliferation and maturation. Ongoing experiments aim at identifying the molecular mechanisms that regulate this process.