Injured human hearts are fibrotic, whereas zebrafish hearts functionally regenerate following myocardial injury. The unique regeneration niche microenvironment has been extensively studied in zebrafish hearts. However whether this can be extrapolated to humans remains unclear owing to significant species differences. We xenografted human induced pluripotent stem cell-derived cardiomyocytes (hiCMs) into the cardiac region of one-day post-fertilized zebrafish embryos and established a zebrafish xenograft model of hiCMs. This model can be used to explore the behavior of hiCMs transplanted into zebrafish hearts. Fluctuations in the fluorescence intensity of the genetically encoded calcium indicator protein GCaMP indicated that the donor hiCMs were beating. We analyzed the synchronization of the GCaMP + hiCMs transplanted into the zebrafish heart. We found synchronous beating between the host and 40 % of the zebrafish hearts with beating GCaMP-hiPSCs. Our chimeric heart model has the potential to bridge the regeneration capacity gap between zebrafish and humans and has proming future applications.

Human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) have attracted significant interest for use in disease modeling, drug discovery and potential therapeutic applications. However, conventional hiPSC-CM cryopreservation protocols largely use dimethyl sulfoxide (DMSO) as the cryoprotectant (CPA), which is linked with a loss of post-thaw recovery and function for various cell types and is not ideal for therapeutic protocols. Additionally, the effect of freezing parameters such as cooling rate and nucleation temperature on post-thaw recovery of hiPSC-CMs has not been explored.

hiPSC-CMs were generated by Wnt pathway inhibition, followed by sodium l-lactate purification. Subsequently, biophysical characterization of the cells was performed. A differential evolution (DE) algorithm was utilized to determine the optimal composition of a mixture of a sugar, sugar alcohol and amino acid to replace DMSO as the CPA. The hiPSC-CMs were subjected to controlled-rate freezing at different cooling rates and nucleation temperatures. The optimum freezing parameters were identified by post-thaw recoveries and the partitioning ratio obtained from low temperature Raman spectroscopy studies. The post-thaw osmotic behavior of hiPSC-CMs was studied by measuring diameter of cells resuspended in the isotonic culture medium over time. Immunocytochemistry and calcium transient studies were performed to evaluate post-thaw function.

hiPSC-CMs were found to be slightly larger than hiPSCs and exhibited a large osmotically inactive volume. The best-performing DMSO-free solutions enabled post-thaw recoveries over 90%, which was significantly greater than DMSO (69.4 ± 6.4%). A rapid cooling rate of 5 °C/min and a low nucleation temperature of -8 °C was found to be optimal for hiPSC-CMs. hiPSC-CMs displayed anomalous osmotic behavior post-thaw, dropping sharply in volume after resuspension. Post-thaw function was preserved when hiPSC-CMs were frozen with the best-performing DMSO-free CPA or DMSO and the cells displayed similar cardiac markers pre-freeze and post-thaw.

It was shown that a CPA cocktail of naturally-occurring osmolytes could effectively replace DMSO for preserving hiPSC-CMs while preserving morphology and function. Understanding the anomalous osmotic behavior and managing the excessive dehydration of hiPSC-CMs could be crucial to improve post-thaw outcomes. Effective DMSO-free cryopreservation would accelerate the development of drug discovery and therapeutic applications of hiPSC-CMs.

The adult mammalian heart has limited regenerative capacity. Cardiac injury, such as a myocardial infarction (MI), leads to permanent scarring and impaired heart function. In contrast, neonatal mice and zebrafish possess the ability to repair injured hearts. Cardiac regeneration is driven by profound transcriptional changes, which are controlled by gene regulatory elements, such as tissue regeneration enhancer elements (TREEs). Here, we review recent studies on cardiac injury/regeneration enhancers across species. We further explore regulatory mechanisms governing TREE activities and their associated binding regulators. We also discuss the potential of TREE engineering and how these enhancers can be utilized for heart repair. Decoding the regulatory logic of cardiac regeneration enhancers presents a promising avenue for understanding heart regeneration and advancing therapeutic strategies for heart failure.

Rationale: Improving heart regeneration through reactivating cardiomyocyte proliferation holds a great potential for repairing diseased hearts. We recently reported that LSD1-dependent epigenetic repression of Cend1 transcription is prerequisite for cardiomyocyte proliferation and mouse heart development. This study interrogates the potential role of this LSD1-CEND1 axis in heart regeneration and repair. Methods: The cardiomyocyte-specific Lsd1 knockout or overexpression mice, Cend1 null mice and cardiomyocyte-specific Cend1 overexpression mice were used to determine the role of LSD1-CEND1 axis in heart regeneration after experimental injuries. Neonatal and adult mice were subjected to apical resection or left anterior descending coronary artery ligation, respectively, to establish cardiac injury models. Echocardiography and Masson staining were employed to assess cardiac function and histopathology, respectively. The molecular changes were determined using RNA sequencing, quantitative RT-PCR, Western blotting and immunostaining. Results: Cardiomyocyte-specific deletion impeded neonatal heart regeneration, while overexpression of Lsd1 had the opposite effect. RNA sequencing revealed that Cend1, a crucial suppressor of cardiomyocyte cycling, was the most significantly elevated gene induced by Lsd1 loss during heart regeneration. Cardiomyocyte-specific Cend1 overexpression hindered neonatal heart regeneration, while Cend1 loss in nullizygous mice had the opposite effect. Cend1 deletion resulted in gene expression alterations associated with enhanced cardiomyocyte proliferation, neovascularization, and macrophage activation. Furthermore, the cardiac regeneration defect caused by Lsd1 loss was not observed when experiments were performed with mice that were nullizyogus for Cend1. Moreover, we found that either Lsd1 overexpression or Cend1 deletion could promote heart regeneration and repair, and improve cardiac function following experimental myocardial infraction in adult mice. Conclusion: Our results demonstrate that LSD1-dependent suppression of CEND1 is crucial for heart regeneration in neonatal and adult mice after experimental injury. These findings suggest LSD1 activation and CEND1 inhibition as promising therapeutic strategies to enhance endogenous cardiac repair in humans.

Somatic polyploidy, a non-inheritable form of genome multiplication, plays cell-type specific and context-dependent roles in organ development and regeneration. In the mammalian heart, embryonic cardiomyocytes are primarily diploid, which lose their ability to complete cell division and become polyploid as they mature. Unlike lower vertebrates like zebrafish, polyploid cardiomyocytes are commonly found across mammals, including humans. Intriguingly, the degree, timing, and modes of cardiomyocyte polyploidization vary greatly between species. In addition to the association with cardiomyocyte development and maturation, recent studies have established polyploidy as a barrier against cardiomyocyte proliferation and heart regeneration following cardiac injury. Hence, a thorough understanding of how and why cardiomyocyte become polyploid will provide insights into heart development and may help develop therapeutic strategies for heart regeneration. Here, we review the dynamics of cardiomyocyte polyploidization across species and how cardiomyocyte-intrinsic, -extrinsic, and environmental factors regulate this process as well as the impact of cardiomyocyte polyploidization on heart development and regeneration.

The adult mammalian heart is a terminally differentiated organ in which the majority of cardiomyocytes are in a state of cell cycle arrest, rendering them incapable of effectively proliferating to replace damaged cells. ALDH2, an enzyme known for alleviating oxidative stress, has been demonstrated to play a critical role in cardiac protection. However, whether ALDH2 regulates cardiomyocyte proliferation has not been conclusively established. We found that activation of ALDH2 activity significantly promotes cardiomyocyte proliferation and extends the proliferation window during early postnatal development in neonatal mice. Furthermore, administration of Alda-1 to activate ALDH2 in adult mice subjected to transverse aortic constriction markedly enhanced cardiomyocyte proliferation and delayed the onset of pressure overload-induced heart failure. In summary, our findings identify ALDH2 as a potential target for regulating cardiomyocyte proliferation and offer a novel therapeutic approach for treating heart failure.

Acute myocardial infarction (AMI) induces significant myocardial damage, ultimately leading to heart failure as the surrounding healthy myocardial tissue undergoes progressive deterioration due to excessive mechanical stress.

This study aimed to investigate myocardial regeneration in a porcine model of AMI using an acellular amniotic membrane with fibrin-termed an amnion bilayer (AB) or heart patch-as a cellular delivery system using porcine amniotic stem cells (pASCs) and autologous porcine cardiomyocytes (pCardios). Fifteen pigs (aged 2-4 months, weighing 50-60 kg) were randomly assigned to three experimental groups (n = 5): control group (AMI induction only), pASC group (pASC transplantation only), and coculture group (pASC and pCardio transplantation). AMI was induced via posterior left ventricular artery ligation and confirmed through standard biomarkers. After eight weeks, histological and molecular analyses were conducted to assess myocardial regeneration.

Improvement in regional wall motion abnormality (RWMA) was observed in 60% of the coculture group, 25% of the pASC group, and none in the control group. Histological analysis of the control group revealed extensive fibrosis with pronounced lipomatosis, particularly at the infarct center. In contrast, pASC and coculture groups exhibited minimal fibrotic scarring at both the infarct center and border regions. Immunofluorescence analysis demonstrated positive α-actinin expression in both the pASC and coculture groups, with the coculture group displaying sarcomeric structures-an organization absent in control group. RNA expression levels of key cardiomyogenic markers, including cardiac troponin T (cTnT), myosin heavy chain (MHC), and Nkx2.5, were significantly elevated in the treatment groups compared to the controls, with the coculture group exhibiting the highest MHC expression. The expression of c-Kit was also increased in both treatment groups relative to the control. Conversely, apoptotic markers p21 and Caspase-9 were highest in the control group, while coculture group exhibited the lowest p53 expression.

Epicardial transplantation of an acellular amniotic heart patch cocultured with cardiomyocytes and pASCs demonstrated superior cardiomyogenesis after eight weeks compared to pASC transplantation alone or control conditions. The coculture system was found to enhance the cardiac regeneration process, as evidenced by improved RWMA, distinct sarcomeric organization, reduced fibrotic scarring, and lower apoptotic gene expression.

Understanding and exploiting bioelectric signaling pathways and physicochemical properties of materials that interface with living tissues is central to advancing tissue regeneration. In particular, the emerging field of bioelectronics leverages these principles to develop personalized, minimally invasive therapeutic strategies tailored to the dynamic demands of individual patients. By integrating sensing and actuation modules into flexible, biocompatible devices, clinicians can continuously monitor and modulate local electrical microenvironments, thereby guiding regenerative processes without extensive surgical interventions. This review provides a critical examination of how fundamental bioelectric cues and physicochemical considerations drive the design and engineering of next-generation bioelectronic platforms. These platforms not only promote the formation and maturation of new tissues across neural, cardiac, musculoskeletal, skin, and gastrointestinal systems but also precisely align therapies with the unique structural, functional, and electrophysiological characteristics of each tissue type. Collectively, these insights and innovations represent a convergence of biology, electronics, and materials science that holds tremendous promise for enhancing the efficacy, specificity, and long-term stability of regenerative treatments, ushering in a new era of advanced tissue engineering and patient-centered regenerative medicine.

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 to nTg 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.

The prepartum rise in cortisol promotes cardiac development and maturation. Here, we investigated the impact of elevated circulating cortisol during mid-late gestation on cardiac growth and metabolism in fetal sheep. Saline or cortisol (2-3 mg in 4.4 mL/24 h) was infused into the fetal jugular vein from 109 to 116 days gestation (dG, term = 150 dG), and fetal heart tissue was collected at 116 dG. Glucocorticoid concentrations, gene and protein expression were measured in fetal left ventricle (LV) tissue. Intrafetal cortisol infusion increased cardiac cortisol concentration but downregulated the protein abundance of glucocorticoid receptor (GR) isoforms (GRα-A, GR-P, GR-A, GRα-D2 and GRα-D3). The gene and protein expression of markers of cardiac hyperplastic growth (IGF1, IGF-1R, TGFβ and AGT) were downregulated, while a protein marker of DNA replication (proliferating cell nuclear antigen) was upregulated by cortisol infusion. Cardiac protein and/or gene expression of complex I of the electron transport chain, SOD2, GLUT-4 (gene and protein), and phosphorylated IRS-1, were upregulated in response to elevated fetal cortisol concentration. Intrafetal cortisol infusion downregulated gene expression of PDK4, which mediates the metabolic switch from glucose to fatty acid metabolism. Cardiac expression of molecular markers involved in cardiovascular protection (SIRT-1, HO1, LAMP1 and SK1) were also downregulated in the cortisol group. In conclusion, these findings suggest that chronic cortisol exposure in preterm fetuses alters heart development, promoting cardiac maturation and potentially increasing the risk of cardiovascular disease later in life if these changes persist into adulthood.

The limited capacity of adult mammalian cardiomyocytes to undergo cell division and proliferation is one of the key factors contributing to heart failure. In newborn mice, cardiac proliferation occurs during a brief window, but this proliferative capacity diminishes by 7 days after birth. Current studies on cardiac regeneration focused on elucidating changes in regulatory factors within the heart before and after this proliferative window, aiming to determine whether potential association between these factors and cell cycle arrest in cardiomyocytes. Facilitating the re-entry of cardiomyocytes into the cell cycle or reversing their exit from it represents a critical strategy for cardiac regeneration. This paper provides an overview of the role of cell cycle arrest in cardiac regeneration, briefly describes cardiomyocyte proliferation and cardiac regeneration, and systematically summarizes the regulation of the cell cycle arrest in cardiomyocytes, and the potential metabolic mechanisms underlying cardiomyocyte cycle arrest. Additionally, we highlight the development of cardiovascular disease drugs targeting cardiomyocyte cell cycle regulation and their status in clinical treatment. Our goal is to outline strategies for promoting cardiac regeneration and repair following cardiac injury, while also pointing toward future research directions that may offer new technologies and prospects for treating cardiovascular diseases, such as myocardial infarction, arrhythmia and heart failure.

Diabetic cardiomyopathy (DCM) is a complex metabolic disorder resulting from chronic hyperglycemia and lipid toxicity, which leads to cardiac dysfunction, fibrosis, inflammation, and mitochondrial impairment. Traditional two-dimensional (2D) cell cultures and animal models have limitations in replicating human cardiac physiology and pathophysiology. In this study, we successfully developed a three-dimensional (3D) model of DCM using cardiac organoids generated from human induced pluripotent stem cells (hiPSCs). These organoids were treated with varying concentrations of glucose and sodium palmitate to mimic the high-glucose and high-lipid environment associated with diabetes. At lower concentrations, glucose and sodium palmitate enhanced cell viability, while higher concentrations induced significant cardiotoxic effects, including apoptosis, oxidative stress, and mitochondrial dysfunction. The cardiac organoids also exhibited increased expression of cardiac injury markers, fibrosis-related genes, and inflammatory cytokines under high-glucose and high-lipid conditions. Treatment with metformin, a widely used antidiabetic drug, mitigated these adverse effects, indicating the model's potential for drug testing and evaluation. Our findings demonstrate that this human-derived 3D cardiac organoid model provides a more physiologically relevant platform for studying DCM and can effectively complement traditional models. This model holds promise for advancing the understanding of diabetic heart disease and for assessing the efficacy of potential therapeutic interventions.

Stem cell-based therapies are an emerging treatment modality aimed at replenishing lost cardiomyocytes and improving myocardial function after cardiac injury. This review examines the current state of research on injectable stem cell therapies in the setting of cardiovascular disease given their relative simplicity and ability for deep myocardial tissue penetration. Various methods of cell delivery, ranging in level of invasiveness and procedural complexity, have been developed, and numerous cell types have been studied as potential sources of stem cells, each with distinct advantages and disadvantages. We discuss key challenges associated with this approach, including low stem cell retention after transplantation and the innovative biomolecular strategies that have been explored to address this issue. Overall, investigations into the application of stem cells toward cardiac regeneration remain predominantly in the preclinical stage with a number of small, early-phase clinical trials. However, continued scientific advancements in stem cell technology may provide transformative treatment options for patients with heart failure, offering improved survival and quality of life.

Adult zebrafish robustly regenerate injured hearts through a complex orchestration of molecular and cellular activities. However, this remarkable process, which is largely non-existent in humans, remains incompletely understood. Here, we utilize integrated spatial transcriptomics (Stereo-seq) and single-cell RNA-sequencing (scRNA-seq) to generate a spatially-resolved molecular and cellular atlas of regenerating zebrafish heart across eight stages. We characterize the cascade of cardiomyocyte cell states responsible for producing regenerated myocardium and explore a potential role for tpm4a in cardiomyocyte re-differentiation. Moreover, we uncover the activation of ifrd1 and atp6ap2 genes as a unique feature of regenerative hearts. Lastly, we reconstruct a 4D "virtual regenerating heart" comprising 569,896 cells/spots derived from 36 scRNA-seq libraries and 224 Stereo-seq slices. Our comprehensive atlas serves as a valuable resource to the cardiovascular and regeneration scientific communities and their ongoing efforts to understand the molecular and cellular mechanisms underlying vertebrate heart regeneration.

Human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) have attracted significant interest for use in disease modeling, drug discovery and potential therapeutic applications. However, conventional hiPSC-CM cryopreservation protocols largely use dimethyl sulfoxide (DMSO) as the cryoprotectant (CPA), which is linked with a loss of post-thaw recovery and function for various cell types and is not ideal for therapeutic protocols. Additionally, the effect of freezing parameters such as cooling rate and nucleation temperature on post-thaw recovery of hiPSC-CMs has not been explored.

hiPSC-CMs were generated by Wnt pathway inhibition, followed by sodium I-lactate purification. Subsequently, biophysical characterization of the cells was performed. A differential evolution (DE) algorithm was utilized to determine the optimal composition of a mixture of a sugar, sugar alcohol and amino acid to replace DMSO as the CPA. The hiPSC-CMs were subjected to controlled-rate freezing at different cooling rates and nucleation temperatures. The optimum freezing parameters were identified by post-thaw recoveries and the partitioning ratio obtained from low temperature Raman spectroscopy studies. The post-thaw osmotic behavior of hiPSC-CMs was studied by measuring diameter of cells resuspended in the isotonic culture medium over time. Immunocytochemistry and calcium transient studies were performed to evaluate post-thaw function.

hiPSC-CMs were found to be slightly larger than hiPSCs and exhibited a large osmotically inactive volume. The best-performing DMSO-free solutions enabled post-thaw recoveries over 90%, which was significantly greater than DMSO (69.4 ± 6.4%). A rapid cooling rate of 5°C/min and a low nucleation temperature of -8°C was found to be optimal for hiPSC-CMs. hiPSC-CMs displayed anomalous osmotic behavior post-thaw, dropping sharply in volume after resuspension. Post-thaw function was preserved when hiPSC-CMs were frozen with the best-performing DMSO-free CPA or DMSO and the cells displayed similar cardiac markers pre-freeze and post-thaw.

It was shown that a CPA cocktail of naturally-occurring osmolytes could effectively replace DMSO for preserving hiPSC-CMs while preserving morphology and function. Understanding the anomalous osmotic behavior and managing the excessive dehydration of hiPSC-CMs could be crucial to improve post-thaw outcomes. Effective DMSO-free cryopreservation would accelerate the development of drug discovery and therapeutic applications of hiPSC-CMs.

Cardiac regeneration studies have been plagued by technical challenges in unequivocally identifying cardiomyocyte (CM) nuclei in cardiac sections, crucial for accurate identification of cycling CMs. The use of antibodies to sarcomeric proteins is error-prone, the CM specificity of common nuclear markers is controversial, and utilizing genetically modified mouse models poses risk of inducing unintended cardiac phenotypes. The application of RNAscope intronic probes overcomes the above shortcomings. Intronic probes label intronic RNAs within nuclei and can therefore be utilized as a method for nuclear localization. A Tnnt2 intronic RNAscope probe highly colocalized with Obscurin-H2B-GFP in adult mouse hearts, demonstrating CM specificity. Studies in embryos demonstrated that the Tnnt2 intronic RNAscope probe labeled CM nuclei that had undergone DNA replication, and remained closely associated with CM chromatin at all stages of mitosis, even with nuclear envelope breakdown. The efficiency, accuracy, and perdurance of the Tnnt2 intronic RNAscope probe even with nuclear envelope breakdown facilitated reliable investigation of dynamics of DNA synthesis and potential mitoses in CMs in both border and infarct zones after myocardial infarction (MI). Furthermore, we designed Myl2 and Myl4 intronic RNAscope probes, which labeled ventricular and atrial CM nuclei, respectively, and may help identify CM subtypes generated in vitro.

Despite recent efforts to stimulate endogenous cardiomyocyte proliferation for cardiac regeneration, the lack of reliable in vivo methods for monitoring cardiomyocyte replication has hindered our understanding of its mechanisms. Thymidine analogs, used to label proliferating cells, are unsuitable for long-term cardiac regeneration studies as their DNA incorporation elicits a damage response, leading to their elimination. Here we present CycleTrack, a genetic strategy based on the transcriptional activation of Cre recombinase from a temporally regulated cyclin B2 promoter segment, for permanent labeling of cardiomyocytes passing through the G2/M phase. Using CycleTrack, we visualized cardiomyocyte turnover in neonatal and adult mice under various conditions, including pregnancy, increased ventricular afterload, and myocardial infarction. CycleTrack also provided visual and quantitative evidence of ventricular remuscularization following treatment with pro-regenerative microRNAs. We identify the subendocardium as a key site of mitotic activity and provide a mode of cardiomyocyte division along their short axis. CycleTrack is a powerful tool to monitor cardiomyocyte renewal during regenerative interventions.

Cardiovascular disease (CVD) remains the leading cause of death globally, with myocardial infarction (MI) being a major contributor. The current therapeutic approaches are limited in effectively regenerating damaged cardiac tissue. Up-to-date strategies for heart regeneration/reconstitution aim at cardiac remodeling through repairing the damaged tissue with an external cell source or by stimulating the existing cells to proliferate and repopulate the compromised area. Cell reprogramming is addressed to this challenge as a promising solution, converting fibroblasts and other cell types into functional cardiomyocytes, either by reverting cells to a pluripotent state or by directly switching cell lineage. Several strategies such as gene editing and the application of miRNA and small molecules have been explored for their potential to enhance cardiac regeneration. Those strategies take advantage of cell plasticity by introducing reprogramming factors that regress cell maturity in vitro, allowing for their later differentiation and thus endorsing cell transplantation, or promote in situ cell proliferation, leveraged by scaffolds embedded with pro-regenerative factors promoting efficient heart restoration. Despite notable advancements, important challenges persist, including low reprogramming efficiency, cell maturation limitations, and safety concerns in clinical applications. Nonetheless, integrating these innovative approaches offers a promising alternative for restoring cardiac function and reducing the dependency on full heart transplants.

The extracellular matrix (ECM) plays a crucial role in providing structural integrity and regulating cell communication essential for organ development, homeostasis, and regeneration, including hearts. Evidence indicates that disruptions in the spatiotemporal expression or alterations in ECM components lead to cardiac malformations, including a wide range of congenital heart diseases (CHDs). Furthermore, research on injured hearts across various vertebrate species, some of which show effective regeneration while others experience irreversible fibrosis, underscores the significance of ECM molecules in cardiac regeneration. This review presents an overview of heart development and the dynamics of ECM during cardiac morphogenesis, beginning with the formation of the contractile heart tube and advancing to the development of distinct chambers separated by valves to facilitate unidirectional blood flow. Furthermore, we discuss research emphasizing the multifaceted roles of secreted molecules in mediating fibrosis and regeneration following myocardial injury.

Promoting endogenous cardiomyocyte proliferation is a promising strategy for cardiac repair. Identifying key factors that regulate cardiomyocyte proliferation can advance the development of novel therapies for heart regeneration. Here, we identify Foxk1 and Foxk2 as key regulators of cardiomyocyte proliferation, whose expression declines during postnatal heart development. Cardiomyocyte-specific knockout of Foxk1 or Foxk2 impairs neonatal heart regeneration after myocardial infarction (MI) injury. AAV9-mediated Foxk1 or Foxk2 overexpression extends the postnatal cardiomyocyte proliferative window and enhances cardiac repair in adult mice after MI. Mechanistically, Foxk1 and Foxk2 drive cardiomyocyte cell cycle progression by directly activating CCNB1 and CDK1 expression, forming the CCNB1/CDK1 complex that facilitates G2/M transition. Moreover, Foxk1 and Foxk2 promote cardiomyocyte proliferation by upregulating HIF1α expression, which enhances glycolysis and the pentose phosphate pathway (PPP), which further favors cardiomyocyte proliferation. These findings establish Foxk1 and Foxk2 as promising therapeutic targets for cardiac injury.

Cardiac tissue lacks regenerative capacity, making heart transplantation the primary treatment for end-stage heart failure. Engineered cardiac tissues developed through three-dimensional bioprinting (3DBP) offer a promising alternative. However, reproducing the native structure, cellular diversity, and functionality of cardiac tissue requires advanced cardiac bioinks. Major obstacles in CTE (cardiac tissue engineering) include accurately characterizing bioink properties, replicating the cardiac microenvironment, and achieving precise spatial organization. Optimizing bioink properties to closely mimic the extracellular matrix (ECM) is essential, as deviations may result in pathological effects. This review encompasses the rheological and electromechanical properties of bioinks and the function of the cardiac microenvironment in the design of functional cardiac constructs. Furthermore, it focuses on improving the rheological characteristics, printability, and functionality of bioinks, offering valuable perspectives for developing new bioinks especially designed for CTE.

While heart function is broadly conserved across vertebrates, the cellular phenotype of muscle cells (cardiomyocytes) varies across taxa and throughout ontogeny. Emerging evidence suggests that some attributes may correlate with the capacity for spontaneous cardiomyocyte replacement following injury. For example, among non-regenerating taxa like adult mammals and birds, cardiomyocytes are polyploid, rarely proliferate, and are large in size. In contrast, in regeneration-competent zebrafish and amphibians, cardiomyocytes are diploid, spontaneously proliferate, and are comparatively small. For other species, less is known.

Here, we investigate these attributes in the squamate Eublepharis macularius, the leopard gecko. Using the nuclear counterstain DAPI to measure fluorescence intensity as a proxy for DNA content, we found that >90% of adult cardiomyocytes are diploid. Using serial histology and immunostaining for markers of DNA synthesis and mitosis, we determined that adult gecko cardiomyocytes spontaneously proliferate, albeit at significantly lower levels than previously reported in subadults. Furthermore, using wheat germ agglutinin, we found that the cross-sectional area is maintained across ontogeny and that gecko cardiomyocytes are 10× smaller than those of mice.

Taken together, our data show that gecko cardiomyocytes share several key cellular attributes with regeneration-competent species and that postnatal ventricular growth occurs via cardiomyocyte hyperplasia.

The Forkhead box O3 (FoxO3) transcription factor is crucial to controlling heart growth in adulthood, but its exact role in cardiac repair and regeneration in postnatal mice remains unclear. Here, we show that FoxO3 deficiency promotes cardiomyocyte proliferation in postnatal mice and improves cardiac function in homeostatic adult mice. Moreover, FoxO3 deficiency accelerates heart regeneration following injury in postnatal mice at the regenerative and non-regenerative stages. We reveal that FoxO3 directly promotes the expression of secreted frizzled-related protein 2 (Sfrp2) and suppresses the activation of canonical Wnt/β-catenin signaling during heart regeneration. The increased activation of β-catenin in FoxO3-deficient cardiomyocytes can be blocked by Sfrp2 overexpression. In addition, Sfrp2 overexpression suppressed cardiomyocyte proliferation and heart regeneration in FoxO3-deficient mice. These findings suggest that FoxO3 negatively controls cardiomyocyte proliferation and heart regeneration in postnatal mice at least in part by promoting Sfrp2 expression, which leading to the inactivation of canonical Wnt/β-catenin signaling.

High-sensitive (hs-) cardiac troponin assays provide prognostic information in atrial fibrillation (AF) patients. Few studies have explored the impact of long-term rate control therapy on levels of troponin in AF patients without coronary heart disease and heart failure. This substudy of the RATe control in Atrial Fibrillation (RATAF) II study aimed to compare the effects of six months' treatment with diltiazem and metoprolol on hs-troponin I (TnI) levels both at rest and during exercise testing in patients with permanent AF.

This was a parallel-group, randomized, investigator-blinded clinical trial. The cohort consisted of 93 patients (28 women, mean age 71 ± 7 years) with symptomatic, permanent AF with preserved left ventricular systolic function and no coronary heart disease. Participants were randomized in a 1:1 ratio to receive either diltiazem 360 mg (n = 49) or metoprolol 100 mg (n = 44) once daily for six months. Blood tests were drawn at rest and during peak exercise testing at baseline, one month and six months' treatment. This research has been supported by grants from the South-Eastern Norway Regional Health Authority and Vestre Viken Hospital Trust.

Six months' treatment with diltiazem and metoprolol significantly lowered the heart rate at rest and peak exercise. Both treatment groups exhibited a decrease in hs-TnI levels at rest (diltiazem p = 0.008, metoprolol p = 0.03) and peak exercise (diltiazem p < 0.001, metoprolol p = 0.004) at six months compared to baseline levels, with no significant differences observed between the groups.

In patients with permanent AF, six months of rate control therapy with diltiazem or metoprolol lowered levels of hs-TnI. Further research is warranted to determine whether this reduction translates into an improved prognosis.

NCT02695992. Registration date: 2015-04-28.

Vascular endothelial growth factor B186 (VEGF-B186), a ligand for VEGF receptor 1 (VEGFR1) and neuropilin (NRP), promotes vascular growth in healthy and ischemic myocardium. However, the mechanisms and signaling of VEGF-B186 to support angiogenesis have remained unclear. We studied the effects of VEGF-B186 and its variant, VEGF-B186R127S, which cannot bind to NRPs, using VEGFR1 tyrosine kinase knockout (TK-/-) mice to explore the mechanism of VEGF-B186 in promoting vascular growth. Ultrasound-guided adenoviral VEGF-B186, VEGF-B186R127S, and control vector gene transfers were performed into VEGFR1 TK-/- mice hearts. In vitro studies in cardiac endothelial cells and further validation in normal and ischemic pig hearts, as well as in wild-type mice, were conducted. Both VEGF-B186 forms promoted vascular growth in VEGFR1 TK-/- mouse heart and increased the expression of proangiogenic and hematopoietic factors. Unlike VEGF-A, VEGF-B186 forms induced endoplasmic reticulum (ER) stress via the upregulation of Binding immunoglobulin Protein (BiP) as well as ER stress sensors (ATF6, PERK, IRE1α) through ITGAV and ITGA5 integrins, newly identified receptors for VEGF-B, activating the unfolded protein response (UPR) through XBP1. VEGFR1 and NRP are not essential for VEGF-B186-induced vascular growth. Instead, VEGF-B186 can stimulate cardiac regeneration through RGD-binding integrins and ER stress, suggesting a novel mechanism of action for VEGF-B186.

Myocardial infarction remains a leading cause of death worldwide due to the heart's limited regenerative capability and the current lack of viable therapeutic solutions. Therefore, there is an urgent need to develop effective treatment options to restore cardiac function after a heart attack. Stem cell-derived cardiac cells have been extensively utilised in cardiac tissue regeneration studies. However, the use of Matrigel as a substrate for the culture and maturation of these cells has been a major limitation for the translation of this research into clinical application. Hydrogels are emerging as a promising system to overcome this problem. They are biocompatible and can provide stem cells with a supportive scaffold that mimics the extracellular matrix, which is essential for repairing damaged tissue in the myocardium after an infarction. Thus, hydrogels provide an alternative and reproducible option in addressing myocardial infarction due to their unique potential therapeutic benefits. This review explores the different types of natural and synthetic polymers used to create hydrogels and their various delivery methods, the most common being via injection and cardiac patches and other applications such as bioprinting. Many challenges remain before hydrogels can be used in a clinical setting, but they hold great promise for the future of cardiac tissue regeneration.

Following injury, many organs are capable of rapid regeneration of necrotic tissue to regain normal function. In contrast, the damaged heart typically replaces tissue with a collagen-rich scar, due to the limited regenerative capacity of its functional contractile cardiomyocytes (CMs). However, this regenerative capacity varies dramatically during development and between species. Furthermore, studies have shown that cardiac regeneration can be enhanced to return contractile function to the damaged heart following myocardial infarction (MI). In this review, we outline the proliferative capacity of CMs in utero, postnatally and in adulthood. We also describe the regenerative capacity of the heart following MI injury. Finally, we focus on the various therapeutic strategies that aim to augment cardiac regeneration in preclinical animal models. These include altering transcripts, microRNAs, extracellular matrix proteins and inducing metabolic rewiring. Together, these therapies aim to return function to the damaged heart and potentially improve the lives of the millions of heart failure patients currently suffering worldwide.

Biological accelerator mass spectrometry (AMS) provides ultrasensitive carbon-14 isotopic analysis enabling a deeper understanding of human health concerns by enabling quantification of pharmacokinetics and other molecular endpoints directly in humans. It enables environmentally and human relevant studies of metabolic pathways through the use of very low concentrations of labeled metabolic substrates in cells and organisms. Here, we discuss why AMS is an important tool for the biosciences, the development and evolution of biological AMS at Livermore and discuss technical refinements that will improve the efficiency of operation for the measurement of ultra-trace levels of 14C, which, long term, will enable greater ease of use and sample throughput.

Forty first-trimester human hearts were studied to lay groundwork for further studies of the mechanisms underlying congenital heart defects. We first sampled 49,227 cardiac nuclei from three fetuses at 8.6, 9.0, and 10.7 post-conceptional weeks (pcw) for single-nucleus RNA sequencing, enabling the distinction of six classes comprising 21 cell types. Improved resolution led to the identification of previously unappreciated cardiomyocyte populations and minority autonomic and lymphatic endothelial transcriptomes, among others. After integration with 5-7 pcw heart single-cell RNA-sequencing data, we identified a human cardiomyofibroblast progenitor preceding the diversification of cardiomyocyte and stromal lineages. Spatial transcriptomic analysis (six Visium sections from two additional hearts) was aided by deconvolution, and key spatial markers validated on sectioned and whole hearts in two- and three-dimensional space and over time. Altogether, anatomical-positional features, including innervation, conduction and subdomains of the atrioventricular septum, translate latent molecular identity into specialized cardiac functions. This atlas adds unprecedented spatial and temporal resolution to the characterization of human-specific aspects of early heart formation.

Cell fate changes play a crucial role in the processes of natural development, disease progression, and the efficacy of therapeutic interventions. The definition of the various types of cell fate changes, including cell expansion, differentiation, transdifferentiation, dedifferentiation, reprogramming, and state transitions, represents a complex and evolving field of research known as cell lineage tracing. This review will systematically introduce the research history and progress in this field, which can be broadly divided into two parts: prospective tracing and retrospective tracing. The initial section encompasses an array of methodologies pertaining to isotope labeling, transient fluorescent tracers, non-fluorescent transient tracers, non-fluorescent genetic markers, fluorescent protein, genetic marker delivery, genetic recombination, exogenous DNA barcodes, CRISPR-Cas9 mediated DNA barcodes, and base editor-mediated DNA barcodes. The second part of the review covers genetic mosaicism, genomic DNA alteration, TCR/BCR, DNA methylation, and mitochondrial DNA mutation. In the final section, we will address the principal challenges and prospective avenues of enquiry in the field of cell lineage tracing, with a particular focus on the sequencing techniques and mathematical models pertinent to single-cell genetic lineage tracing, and the value of pursuing a more comprehensive investigation at both the spatial and temporal levels in the study of cell lineage tracing.

Stress arises in response to threats or challenges, affecting both physical and mental health. While its harmful effects on the heart are widely recognized, cellular-level investigations remain limited. Antidepressants, including vortioxetine (VOR), are known to impact the cardiovascular system. VOR, used to treat major depressive disorder, is considered a promising option for patients with heart disease due to its anti-inflammatory and antioxidant properties, which may reduce cardiac damage.

This study aimed to assess the effects of chronic unpredictable mild stress (CUMS) on rat hearts and evaluate VOR's potential protective effects against stress-induced cardiac damage.

Twenty-eight male Wistar Albino rats were divided into four groups. The CUMS group experienced random daily stress for 6 weeks, while the CUMS+VOR group received VOR treatment alongside stress. VOR and control groups were not exposed to stress. Heart samples were examined histopathologically and immunohistochemically.

The CUMS group showed increased hyperemia, hemorrhage, edema, vacuolar degeneration, and mononuclear cell infiltrations, with reduced troponin and IL-10 and increased caspase-3 and NF-κB expressions compared to the control group (p≤0.001). VOR treatment improved these findings, normalizing histopathological and immunohistochemical results.

CUMS caused significant cardiac damage in rats, while VOR treatment showed protective effects by alleviating these pathological changes.

Mitochondria are pivotal in the modulation of macrophage activation, differentiation, and survival. Furthermore, macrophages are instrumental in the onset and progression of cardiovascular diseases. Hence, it is imperative to investigate the role of mitochondria within macrophages in the context of cardiovascular disease. In this review, we provide an updated description of the origin and classification of cardiac macrophages and also focused on the relationship between macrophages and mitochondria in cardiovascular diseases with respect to (1) proinflammatory or anti-inflammatory macrophages, (2) macrophage apoptosis, (3) macrophage pyroptosis, and (4) macrophage efferocytosis. Clarifying the relationship between mitochondria and macrophages can aid the exploration of novel therapeutic strategies for cardiovascular disease.

In advancing cardiac tissue engineering (CTE), the development of injectable hydrogels mirroring myocardial properties is pivotal. The designed hydrogels must not only support cardiac cell growth but also have to be conductive to properly promote the functionalities of cardiac cells. Here, a facile approach is developed to incorporate gold nanoparticles (AuNPs) into an injectable hydrogel composed of Alginate (Alg) and Gelatin (Gel). The resultant nanocomposite hydrogel boasts a porous interconnected network and superior conductivity (2.04 × 10-4 S cm-1) compared to the base Alg/Gel hydrogel. Hydrogel hydration and in vitro degradation profiles affirm their suitability as carriers for cardiac cells. Importantly, Alg/Gel+AuNPs hydrogels exhibit no toxicity to mouse Embryonic Cardiac Cells (mECCs) over 7 days, elevating connexin 43 (Cx43) and cardiac troponin T (CTnT) gene expression compared to controls. Then, the Alg/Gel+AuNPs hydrogel is used as a carrier for intramyocardial delivery of mECCs in rats with myocardial infarction. The significant increase in α-Smooth Muscle Actin (α-SMA) and cardiac troponin T (CTnT) expression along with the increase in ejection fraction (EF), smaller infarction size, less fibrosis area confirmed that the hydrogel efficiently promoted the transmission of mechanical and electrical signals between transplanted cells and surrounding tissue.

Atrial fibrillation (AF) substrate progresses with the advancement of atrial structural remodeling, resulting in AF perpetuation and recurrence. Although fibrosis is considered a hallmark of atrial structural remodeling, the histological background has not been fully elucidated because obtaining atrial specimens is difficult, especially in patients not undergoing open-heart surgery. Bipolar voltage reduction evaluated using electroanatomic mapping during AF ablation is considered a surrogate marker for the progression of structural remodeling; however, histological validation is lacking. We developed an intracardiac echocardiography-guided endomyocardial atrial biopsy technique to evaluate atrial structural remodeling in patients undergoing catheter ablation for nonvalvular AF. The histological factors associated with a decrease in bipolar voltage were interstitial fibrosis, as well as an increase in myocardial intercellular space preceding fibrosis, myofibrillar loss, and a decrease in cardiomyocyte nuclear density, which is a surrogate marker for cardiomyocyte density. Cardiomyocyte hypertrophy is closely associated with a decrease in cardiomyocyte nuclear density, suggesting that hypertrophic changes compensate for cardiomyocyte loss. Electron microscopy also revealed that increased intercellular spaces indicated the leakage of plasma components owing to increased vascular permeability. Additionally, amyloid deposition was observed in 4 % of biopsy cases. Only increased intercellular space and interstitial fibrosis were significantly higher for long-standing persistent AF than for paroxysmal AF and associated with recurrence after AF ablation, suggesting that this interstitial remodeling is the AF substrate. An increase in intercellular space that occurs early in AF formation is a therapeutic target for the AF substrate, which prevents irreversible interstitial degeneration due to collagen accumulation. This endomyocardial atrial biopsy technique will allow the collection of atrial tissue from a wide variety of patients and significantly facilitate the elucidation of the mechanisms of atrial cardiomyopathy, structural remodeling, and AF substrates.

Organoids, exhibiting the capability to undergo differentiation in specific in vitro growth environments, have garnered significant attention in recent years due to their capacity to recapitulate human organs with resemblant in vivo structures and physiological functions. This groundbreaking technology offers a unique opportunity to study human diseases and address the limitations of traditional animal models. Cardiovascular diseases (CVDs), a leading cause of mortality worldwide, have spurred an increasing number of researchers to explore the great potential of human cardiovascular organoids for cardiovascular research. This review initiates by elaborating on the development and manufacture of human cardiovascular organoids, including cardiac organoids and blood vessel organoids. Next, we provide a comprehensive overview of their applications in modeling various cardiovascular disorders. Furthermore, we shed light on the prospects of cardiovascular organoids in CVDs therapy, and unfold an in-depth discussion of the current challenges of human cardiovascular organoids in the development and application for understanding and treating CVDs.

Myocardial infarction (MI) causes the loss of millions of cardiomyocytes, and current treatments do not address this root issue. New therapies focus on stimulating cardiomyocyte division in the adult heart, inspired by the regenerative capacities of lower vertebrates and neonatal mice. This review explores strategies for heart regeneration, offers insights into cardiomyocyte proliferation, evaluates in vivo models, and discusses integrating in vitro human cardiac models to advance cardiac regeneration research.

Cardiomyocytes in the adult human heart show a regenerative capacity, with an annual renewal rate of ≈0.5%. Whether this regenerative capacity of human cardiomyocytes is employed in heart failure has been controversial.

We determined cardiomyocyte renewal in 52 patients with advanced heart failure, 28 of whom received left ventricular assist device support. We measured the concentration of nuclear bomb test-derived 14C in cardiomyocyte genomic DNA and performed mathematical modeling to establish cardiomyocyte renewal in heart failure with and without LVAD unloading.

We show that cardiomyocyte generation is minimal in end-stage heart failure patients at rates 18 to 50× lower compared with the healthy heart. However, patients receiving left ventricle support device therapy, who showed significant functional and structural cardiac improvement, had a >6-fold increase in cardiomyocyte renewal relative to the healthy heart.

Our findings reveal a substantial cardiomyocyte regeneration potential in human heart disease, which could be exploited therapeutically.