Regeneration of the heart:f rom molecular mechanisms to clinical therapeutics

Heart injury such as myocardial infarction leads to cardiomyocyte loss,fibrotic tissue deposition,and scar formation.These changes reduce cardiac contractility,resulting in heart failure,which causes a huge public health burden.Military personnel,compared with civilians,is exposed to more stress,a risk factor for heart diseases,making cardiovascular health management and treatment innovation an important topic for military medicine.So far,medical intervention can slow down cardiovascular disease progression,but not yet induce heart regeneration.In the past decades,studies have focused on mechanisms underlying the regenerative capability of the heart and applicable approaches to reverse heart injury.Insights have emerged from studies in animal models and early clinical trials.Clinical interventions show the potential to reduce scar formation and enhance cardiomyocyte proliferation that counteracts the pathogenesis of heart disease.In this review,we discuss the signaling events controlling the regeneration of heart tissue and summarize current therapeutic approaches to promote heart regeneration after injury.

Single-cell analysis reveals an Angpt4-initiated EPDC-EC-CM cellular coordination cascade during heart regeneration

Mammals exhibit limited heart regeneration ability,which can lead to heart failure after myocardial infarction.In contrast,zebrafish exhibit remarkable cardiac regeneration capacity.Several cell types and signaling pathways have been reported to participate in this process.However,a comprehensive analysis of how different cells and signals interact and coordinate to regulate cardiac regeneration is unavailable.We collected major cardiac cell types from zebrafish and performed high-precision single-cell transcriptome analyses during both development and post-injury regeneration.We revealed the cellular heterogeneity as well as the molecular progress of cardiomyocytes during these processes,and identified a subtype of atrial cardiomyocyte exhibiting a stem-like state which may transdifferentiate into ventricular cardiomyocytes during regeneration.Furthermore,we identified a regeneration-induced cell(RIC)population in the epicardium-derived cells(EPDC),and demonstrated Angiopoietin 4(Angpt4)as a specific regulator of heart regeneration.angpt4 expression is specifically and transiently activated in RIC,which initiates a signaling cascade from EPDC to endocardium through the Tie2-MAPK pathway,and further induces activation of cathepsin K in cardiomyocytes through RA signaling.Loss of angpt4 leads to defects in scar tissue resolution and cardiomyocyte proliferation,while overexpression of angpt4 accelerates regeneration.Furthermore,we found that ANGPT4 could enhance proliferation of neonatal rat cardiomyocytes,and promote cardiac repair in mice after myocardial infarction,indicating that the function of Angpt4 is conserved in mammals.Our study provides a mechanistic understanding of heart regeneration at single-cell precision,identifies Angpt4 as a key regulator of cardiomyocyte proliferation and regeneration,and offers a novel therapeutic target for improved recovery after human heart injuries.

Induction of Wnt signaling antagonists and p21-activated kinase enhances cardiomyocyte proliferation during zebrafish heart regeneration

Heart regeneration occurs by dedifferentiation and proliferation of pre-existing cardiomyocytes(CMs).However,the signaling mechanisms by which injury induces CM renewal remain incompletely understood.Here,we find that cardiac injury in zebrafish induces expression of the secreted Wnt inhibitors,including Dickkopf 1(Dkkl),Dkk3,secreted Frizzled-related protein 1(sFrpl),and sFrp2,in cardiac tissue adjacent to injury sites.Experimental blocking of Wnt activity via Dkkl overexpression enhances CM proliferation and heart regeneration,whereas ectopic activation of Wnt8 signaling blunts injury-induced CM dedifferentiation and proliferation.Although Wnt signaling is dampened upon injury,the cytoplasmic β-catenin is unexpectedly increased at disarrayed CM sarcomeres in myocardial wound edges.Our analyses indicated that p21-activated kinase 2(Pak2)is induced at regenerating CMs,where it phosphorylates cytoplasmic β-catenin at Ser 675 and increases its stability at disassembled sarcomeres.Myocardial-specific induction of the phospho-mimeticβ-catenin(S675E)enhances CM dedifferentiation and sarcomere disassembly in response to injury.Conversely,inactivation of Pak2 kinase activity reduces the Ser 675-phosphorylatedβ-catenin(pS675-β-catenin)and attenuates CM sarcomere disorganization and dedifferentiation・Taken together,these findings demonstrate that coordination of Wnt signaling inhibition and Pak2/pS675-βYatenin signaling enhances zebrafish heart regeneration by supporting CM dedifferentiation and proliferation.

The roles and activation of endocardial Notch signaling in heart regeneration

As a highly conserved signaling pathway in metazoans,the Notch pathway plays important roles in embryonic development and tissue regeneration.Recently,cardiac injury and regeneration have become an increasingly popular topic for biomedical research,and Notch signaling has been shown to exert crucial functions during heart regeneration as well.In this review,we briefly summarize the molecular functions of the endocardial Notch pathway in several cardiac injury and stress models.Although there is an increase in appreciating the importance of endocardial Notch signaling in heart regeneration,the mechanism of its activation is not fully understood.This review highlights recent findings on the activation of the endocardial Notch pathway by hemodynamic blood flow change in larval zebrafish ventricle after partial ablation,a process involving primary cilia,mechanosensitive ion channel Trpv4 and mechanosensitive transcription factor Klf2.

Cardiac cell type-specific responses to injury and contributions to heart regeneration

Heart disease is the leading cause of mortality worldwide.Due to the limited proliferation rate of mature cardiomyocytes,adult mammalian hearts are unable to regenerate damaged cardiac muscle following injury.Instead,injured area is replaced by fibrotic scar tissue,which may lead to irreversible cardiac remodeling and organ failure.In contrast,adult zebrafish and neonatal mammalian possess the capacity for heart regeneration and have been widely used as experimental models.Recent studies have shown that multiple types of cells within the heart can respond to injury with the activation of distinct signaling pathways.Determining the specific contributions of each cell type is essential for our understanding of the regeneration network organization throughout the heart.In this review,we provide an overview of the distinct functions and coordinated cell behaviors of several major cell types including cardiomyocytes,endocardial cells,epicardial cells,fibroblasts,and immune cells.The topic focuses on their specific responses and cellular plasticity after injury,and potential therapeutic applications.

Overlapping Cardiac Programs in Heart Development and Regeneration

Gaining cellular and molecular insights into heart development and regeneration will likely provide new therapeutic targets and opportunities for cardiac regenerative medicine,one of the most urgent clinical needs for heart failure.Here we present a review on zebrafish heart development and regeneration,with a particular focus on early cardiac progenitor development and their contribution to building embryonic heart,as well as cellular and molecular programs in adult zebrafish heart regeneration.We attempt to emphasize that the signaling pathways shaping cardiac progenitors in heart development may also be redeployed during the progress of adult heart regeneration.A brief perspective highlights several important and promising research areas in this exciting field.

Primary cilia mediate Klf2-dependant Notch activation in regenerating heart

Unlike adult mammalian heart,zebrafish heart has a remarkable capacity to regenerate after injury.Previous study has shown Notch signaling activation in the endocardium is essential for regeneration of the myocardium and this activation is mediated by hemodynamic alteration after injury,however,the molecular mechanism has not been fully explored.In this study we demonstrated that blood flow change could be perceived and transmitted in a primary cilia dependent manner to control the hemodynamic responsive klf2 gene expression and subsequent activation of Notch signaling in the endocardium.First we showed that both homologues of human gene KLF2 in zebrafish,klf2a and klf2b,could respond to hemodynamic alteration and both were required for Notch signaling activation and heart regeneration.Further experiments indicated that the upregulation of klf2 gene expression was mediated by endocardial primary cilia.Overall,our findings reveal a novel aspect of mechanical shear stress signal in activating Notch pathway and regulating cardiac regeneration.

Postnatal state transition of cardiomyocyte as a primary step in heart maturation

Postnatal heart maturation Is the basis of normal cardiac function and provides critical insights into heart repair and regenerative medicine.While static snapshots of the maturing heart have provided much Insight into its molecular signatures,few key events during postnatal cardiomyocyte maturation have been uncovered.Here,we report that cardiomyocytes(CMs)experience epige-netic and transcriptional decline of cardiac gene expression immediately after birth,leading to a transi-tion state of CMs at postnatal day 7(P7)that was essential for CM subtype specification during heart maturation.Large-scale single-cell analysis and genetic lineage tracing confirm the presence of transition state CMs at P7 bridging immature state and mature states.Silencing of key transcription factor JUN In P1-hearts significantly repressed CM transition,resulting in per-turbed CM subtype proportions and reduced cardiac function in mature hearts.In addition,transplantation of P7-CMs into infarcted hearts exhibited cardiac repair potential superior to P1-CMs.Collectively,our data uncover CM state transition as a key event in postnatal heart maturation,which not only provides insights into molecular foundations of heart maturation,but also opens an avenue for manipulation of cardiomyocyte fate in disease and regenerative medicine.

The role of cellular senescence in cardiac development,regeneration and diseases

Background Cellular senescence,an irreversible state of cell-cycle arrest triggered by multiple stress factors,plays a key role in organ development and wound healing.Accumulated senescent cells also promote tissue inflammation and involve in various diseases including myocardial infarction,atherosclerosis,diabetes and nonalcoholic steatohepatitis.Understanding the mechanism and consequences of cellular senescence is crucial to develop new therapies for diseases.Here,we describe the characteristics of senescent cells and involvement of senescent cardiac cells in heart development,regeneration and diseases.We summarize the work in this area and provide directions and clues for future studies.

Advances in 3D bioprinting technology for cardiac tissue engineering and regeneration

Cardiovascular disease is still one of the leading causes of death in the world,and heart transplantation is the current major treatment for end-stage cardiovascular diseases.However,because of the shortage of heart donors,new sources of cardiac regenerative medicine are greatly needed.The prominent development of tissue engineering using bioactive materials has creatively laid a direct promising foundation.Whereas,how to precisely pattern a cardiac structure with complete biological function still requires technological breakthroughs.Recently,the emerging three-dimensional(3D)bioprinting technology for tissue engineering has shown great advantages in generating micro-scale cardiac tissues,which has established its impressive potential as a novel foundation for cardiovascular regeneration.Whether 3D bioprinted hearts can replace traditional heart transplantation as a novel strategy for treating cardiovascular diseases in the future is a frontier issue.In this review article,we emphasize the current knowledge and future perspectives regarding available bioinks,bioprinting strategies and the latest outcome progress in cardiac 3D bioprinting to move this promising medical approach towards potential clinical implementation.

The characterization of protein lactylation in relation to cardiac metabolic reprogramming in neonatal mouse hearts

In mammals,the neonatal heart can regenerate upon injury within a short time after birth,while adults lose this ability.Metabolic reprogramming has been demonstrated to be critical for cardiomyocyte proliferation in the neonatal heart.Here,we reveal that cardiac metabolic reprogramming could be regulated by altering global protein lactylation.By performing 4D label-free proteomics and lysine lactylation(Kla)omics analyses in mouse hearts at postnatal days 1,5,and 7,2297 Kla sites from 980 proteins are identified,among which 1262 Kla sites from 409 proteins are quantified.Functional clustering analysis reveals that the proteins with altered Kla sites are mainly involved in metabolic processes.The expression and Kla levels of proteins in glycolysis show a positive correlation while a negative correlation in fatty acid oxidation.Furthermore,we verify the Kla levels of several differentially modified proteins,including ACAT1,ACADL,ACADVL,PFKM,PKM,and NPM1.Overall,our study reports a comprehensive Kla map in the neonatal mouse heart,which will help to understand the regulatory network of metabolic reprogramming and cardiac regeneration.

CRISPR-CasRx knock-in mice for RNA degradation

The RNA editing tool CRISPR-CasRx has provided a platform for a range of transcriptome analysis tools and therapeutic approaches with its broad efficacy and high specificity.To enable the application of CasRx in vivo,we established a Credependent CasRx knock-in mouse.Using these mice,we specifically knocked down the expression of Meis1 and Hoxb13 in cardiomyocytes,which induced cardiac regeneration after myocardial infarction.We also knocked down the lnc RNA Mhrt in cardiomyocytes with the CasRx knock-in mice,causing hypertrophic cardiomyopathy.In summary,we generated a Credependent CasRx knock-in mouse that can efficiently knock down coding gene and lnc RNA expression in specific somatic cells.This in vivo CRISPR-CasRx system is promising for gene function research and disease modeling.

The regenerative response of cardiac interstitial cells

Understanding how certain animals are capable of regenerating their hearts will provide much needed insights into how this process can be induced in humans in order to reverse the damage caused by myocardial infarction.Currently,it is becoming increasingly evident that cardiac interstitial cells play crucial roles during cardiac regeneration.To understand how interstitial cells behave during this process,we performed single-cell RNA sequencing of regenerating zebrafish hearts.Using a combination of immunohistochemistry,chemical inhibition,and novel transgenic animals,we were able to investigate the role of cell type-specific mechanisms during cardiac regeneration.This approach allowed us to identify a number of important regenerative processes within the interstitial cell populations.Here,we provide detailed insight into how interstitial cells behave during cardiac regeneration,which will serve to increase our understanding of how this process could eventually be induced in humans.

Molecular barriers to direct cardiac reprogramming

Myocardial infarction afflicts close to three quarters of a million Americans annually, resulting in reduced heart function, arrhythmia, and frequently death. Cardiomy- ocyte death reduces the heart＇s pump capacity while the deposition of a non-conductive scar incurs the risk of arrhythmia. Direct cardiac reprogramming emerged as a novel technology to simultaneously reduce scar tissue and generate new cardiomyocytes to restore cardiac function. This technology converts endogenous cardiac fibroblasts directly into induced cardiomyocyte-like cells using a variety of cocktails including transcription factors, microRNAs, and small molecules. Although promising, direct cardiac reprogramming is still in its fledging phase, and numerous barriers have to be overcome prior to its clinical application. This review discusses current findings to optimize reprogramming efficiency, including reprogramming factor cocktails and stoichiometry, epigenetic barriers to cell fate reprogramming, incomplete conversion and residual fibroblast identity, requisite growth factors, and environmental cues. Finally, we address the current challenges and future directions for the field.