Neonatal Cardiac Regeneration Research Articles

MBNL1 Regulates Programmed Postnatal Switching Between Regenerative and Differentiated Cardiac States.

Discovering determinants of cardiomyocyte maturity is critical for deeply understanding the maintenance of differentiated states and potentially reawakening endogenous regenerative programs in adult mammalian hearts as a therapeutic strategy. Forced dedifferentiation paired with oncogene expression is sufficient to drive cardiac regeneration, but elucidation of endogenous developmental regulators of the switch between regenerative and mature cardiomyocyte cell states is necessary for optimal design of regenerative approaches for heart disease. MBNL1 (muscleblind-like 1) regulates fibroblast, thymocyte, and erythroid differentiation and proliferation. Hence, we examined whether MBNL1 promotes and maintains mature cardiomyocyte states while antagonizing cardiomyocyte proliferation. MBNL1 gain- and loss-of-function mouse models were studied at several developmental time points and in surgical models of heart regeneration. Multi-omics approaches were combined with biochemical, histological, and in vitro assays to determine the mechanisms through which MBNL1 exerts its effects. MBNL1 is coexpressed with a maturation-association genetic program in the heart and is regulated by the MEIS1/calcineurin signaling axis. Targeted MBNL1 overexpression early in development prematurely transitioned cardiomyocytes to hypertrophic growth, hypoplasia, and dysfunction, whereas loss of MBNL1 function increased cardiomyocyte cell cycle entry and proliferation through altered cell cycle inhibitor transcript stability. Moreover, MBNL1-dependent stabilization of estrogen-related receptor signaling was essential for maintaining cardiomyocyte maturity in adult myocytes. In accordance with these data, modulating MBNL1 dose tuned the temporal window of neonatal cardiac regeneration, where increased MBNL1 expression arrested myocyte proliferation and regeneration and MBNL1 deletion promoted regenerative states with prolonged myocyte proliferation. However, MBNL1 deficiency was insufficient to promote regeneration in the adult heart because of cell cycle checkpoint activation. Here, MBNL1 was identified as an essential regulator of cardiomyocyte differentiated states, their developmental switch from hyperplastic to hypertrophic growth, and their regenerative potential through controlling an entire maturation program by stabilizing adult myocyte mRNAs during postnatal development and throughout adulthood. Targeting loss of cardiomyocyte maturity and downregulation of cell cycle inhibitors through MBNL1 deletion was not sufficient to promote adult regeneration.

Abstract P1065: Primitive Macrophages Enhance The Function Of Bioengineered Human Cardiac Microtissues

Human cardiac bioengineering has traditionally focused on cardiomyocyte differentiation and maturation. Unlike normal cardiac growth in the native human heart, human pluripotent stem cell (hPSC)- derived cardiomyocytes are typically grown in the absence of key supportive populations, which may negatively impact function due to a failure to recreate complex multicellular environments. In vivo murine studies have demonstrated that embryonic-derived resident cardiac macrophages promote neonatal cardiac regeneration, yet macrophages have not been incorporated in cardiac bioengineering approaches. We hypothesized that inclusion of human embryonic stem cell derived primitive macrophages (hESC-macrophages) into bioengineered human cardiac microtissues (“Biowires”) would improve cardiomyocyte functional properties. We found that addition of hESC-macrophages into Biowires led to enhanced contractile force, improved relaxation, reduced excitation threshold, increased maximum capture rate, and increased conduction velocity that mechanistically was correlated with improved calcium cycling, indicating hESC-macrophages improve a broad range of electromechanical properties. Moreover, hESC-macrophages enhanced microtissue function across cardiomyocyte cell sources and bioengineering platforms (Experimental N=6 independent cellular differentiations across 2 different platforms). These changes occurred in the absence of significant transcriptional shifts in cardiomyocytes. Rather, Biowires with hESC-macrophages had reduced mitochondrial DNA release, oxidative stress and cytotoxicity in the very early phases of tissue formation, thereby promoting a favorable environment for cardiac microtissue development. In conclusion, these data reveal a previously unappreciated, yet major beneficial role for macrophages specifically in human cardiac tissue engineering. In general, bioengineering human tissue continues to be a major research focus, and our data argue that inclusion of yolk sac-derived macrophages may improve early microtissue formation and modeling in vitro , and could enhance cell delivery approaches in vivo .

Abstract P1004: Macrophage Apoptotic Cell Receptor Mertk Triggers The Synthesis Of Arachidonic Acid Derivatives To Promote Cardiac Regeneration

Introduction: Macrophages (MΦs) are indispensable for regeneration of the mammalian neonatal heart. However, the underlying mechanisms by which neonatal MΦs sense the injured cardiac milieu and promote regeneration remain obscure. Utilizing single-cell transcriptomics, we previously observed a regenerative neonatal cardiac MΦ population that distinctly upregulates MerTK, an apoptotic cell receptor, compared to adult cardiac MΦs. Objective: We hypothesized that MerTK is required to program neonatal cardiac MΦs to secrete specialized pro-“regenerative” lipids to induce cardiomyocyte proliferation leading to heart regeneration. Methods: Postnatal day 1 and adult mice were subjected to cardiac injury via ligation of the left anterior descending artery. Single-cell transcriptomics, lipid-omics, and molecular approaches were utilized to determine the mechanisms by which neonatal MΦs regulate cardiac regeneration. Results: To determine the necessity of MΦ MerTK for cardiac regeneration, we employed a myeloid-specific knockout of MerTK ( mMertk -/- ) which failed to regain homeostatic cardiac function. We next performed single-cell transcriptomics on Mertk +/+ and Mertk -/- neonates to determine how MerTK activation reprograms neonatal MΦs for regeneration. Mertk +/+ MΦs significantly upregulated genes associated with arachidonic acid metabolism. Hypothesizing that arachidonic acid derivatives may be key signaling molecules for regeneration, we performed a targeted lipid-omics screen which revealed an increase in Thromboxane B 2 (TXB 2 ) in regenerating hearts but not within non-regenerative Mertk -/- hearts. Furthermore, addition of a TXA 2 agonist to neonatal cardiomyocytes in vitro significantly increases their proliferation by regulating the YAP/TAZ signaling. Alternatively, inhibition of thromboxane synthase in neonatal MΦs or thromboxane receptor on cardiomyocytes by small molecule inhibitors significantly abrogated the increase in cardiomyocyte proliferation by efferocytic Mertk +/+ neonatal MΦs. Conclusions: Altogether, our data suggest that MerTK programs neonatal cardiac MΦs to secrete TXA 2 to induce cardiomyocyte proliferation via the HIPPO-YAP/TAZ pathway prompting neonatal cardiac regeneration.

A focus on congenital heart disease and neonatal cardiac regeneration: an exciting future.

A focus on congenital heart disease and neonatal cardiac regeneration: an exciting future.

Natural cardiac regeneration conserves native biaxial left ventricular biomechanics after myocardial infarction in neonatal rats

After myocardial infarction (MI), adult mammals exhibit scar formation, adverse left ventricular (LV) remodeling, LV stiffening, and impaired contractility, ultimately resulting in heart failure. Neonatal mammals, however, are capable of natural heart regeneration after MI. We hypothesized that neonatal cardiac regeneration conserves native biaxial LV mechanics after MI. Wistar rat neonates (1 day old, n = 46) and adults (8–10 weeks old, n = 20) underwent sham surgery or permanent left anterior descending coronary artery ligation. At 6 weeks after neonatal MI, Masson's trichrome staining revealed negligible fibrosis. Echocardiography for the neonatal MI (n = 15) and sham rats (n = 14) revealed no differences in LV wall thickness or chamber diameter, and both groups had normal ejection fraction (72.7% vs 77.5%, respectively, p = 0.1946). Biaxial tensile testing revealed similar stress-strain curves along both the circumferential and longitudinal axes across a full range of physiologic stresses and strains. The circumferential modulus (267.9 kPa vs 274.2 kPa, p = 0.7847), longitudinal modulus (269.3 kPa vs 277.1 kPa, p = 0.7435), and maximum shear stress (3.30 kPa vs 3.95 kPa, p = 0.5418) did not differ significantly between the neonatal MI and sham groups, respectively. In contrast, transmural scars were observed at 4 weeks after adult MI. Adult MI hearts (n = 7) exhibited profound LV wall thinning (p < 0.0001), chamber dilation (p = 0.0246), and LV dysfunction (ejection fraction 45.4% vs 79.7%, p < 0.0001) compared to adult sham hearts (n = 7). Adult MI hearts were significantly stiffer than adult sham hearts in both the circumferential (321.5 kPa vs 180.0 kPa, p = 0.0111) and longitudinal axes (315.4 kPa vs 172.3 kPa, p = 0.0173), and also exhibited greater maximum shear stress (14.87 kPa vs 3.23 kPa, p = 0.0162). Our study is the first to show that native biaxial LV mechanics are conserved after neonatal heart regeneration following MI, thus adding biomechanical support for the therapeutic potential of cardiac regeneration in the treatment of ischemic heart disease.

Type 2 innate immunity drives distinct neonatal immune profile conducive for heart regeneration.

Aims: Neonatal immunity is functionally immature and skewed towards a TH2-driven, anti-inflammatory profile. This neonatal immunotolerance is partly driven by the type 2 cytokines: interleukin-4 (IL-4) and interleukin-13 (IL-13). Studies on neonatal cardiac regeneration reveal the beneficial role of an anti-inflammatory response in restoring cardiac function after injury. However, the role of an imbalanced immune repertoire observed in neonates on tissue regeneration is poorly understood; specifically, whether IL-4 and IL-13 actively modulate neonatal immunity during cardiac injury.Methods and results: Neonatal mice lacking IL-4 and IL-13 (DKOs) examined at 2 days after birth exhibited reduced anti-inflammatory immune populations with basal cardiac immune populations like adult mice. Examination of neonates lacking IL-4 and IL-13 at 2 days post cardiac ischemic injury, induced on the second day after birth, showed impaired cardiac function compared to their control counterparts. Treatment with either IL-4 or IL-13 cytokine during injury restored both cardiac function and immune population profiles in knockout mice. Examination of IL-4/IL-13 downstream pathways revealed the role of STAT6 in mediating the regenerative response in neonatal hearts. As IL-4/IL-13 drives polarization of alternatively activated macrophages, we also examined the role of IL-4/IL-13 signaling within the myeloid compartment during neonatal cardiac regeneration. Injury of IL-4Rα myeloid specific knockout neonates 2 days after birth revealed that loss of IL-4/IL-13 signaling in macrophages alone was sufficient to impair cardiac regeneration.Conclusions: Our results confirm that the TH2 cytokines: IL-4 and IL-13, which skews neonatal immunity to a TH2 profile, are necessary for maintaining and mediating an anti-inflammatory response in the neonatal heart, in part through the activation of alternatively activated macrophages, thereby permitting a niche conducive for regeneration.

IL4Rα signaling promotes neonatal cardiac regeneration and cardiomyocyte cell cycle activity

Neonatal heart regeneration depends on proliferation of pre-existing cardiomyocytes, yet the mechanisms driving regeneration and cardiomyocyte proliferation are not comprehensively understood. We recently reported that the anti-inflammatory cytokine, interleukin 13 (IL13), promotes neonatal cardiac regeneration; however, the signaling pathway and cell types mediating this regenerative response remain unknown. Here, we hypothesized that expression of the type II heterodimer receptor for IL13, comprised of IL4Rα and IL13Rα1, expressed directly on cardiomyocytes mediates cardiomyocyte cell cycle and heart regeneration in neonatal mice. Our data demonstrate that indeed global deletion of one critical subunit of the type II receptor, IL4Rα (IL4Rα−/−), decreases cardiomyocyte proliferation during early postnatal development and significantly impairs cardiac regeneration following injury in neonatal mice. While multiple myocardial cell types express IL4Rα, we demonstrate that IL4Rα deletion specifically in cardiomyocytes mediates cell cycle activity and neonatal cardiac regeneration. This demonstrates for the first time a functional role for IL4Rα signaling directly on cardiomyocytes in vivo. Reciprocally, we examined the therapeutic benefit of activating the IL4Rα receptor in non-regenerative hearts via IL13 administration. Following myocardial infarction, administration of IL13 reduced scar size and promoted cardiomyocyte DNA synthesis and karyokinesis, but not complete cytokinesis, in 6-day old non-regenerative mice. Our data demonstrate a novel role for IL4Rα signaling directly on cardiomyocytes during heart regeneration and suggest the potential for type II receptor activation as one potential therapeutic target for promoting myocardial repair.

Identification of Novel and Potent Modulators Involved in Neonatal Cardiac Regeneration.

Neonatal mammalian heart has been shown to possess the capacity to regenerate substantially after an injury. This remarkable regenerative capacity is lost in a week. This transition has been marked with cardiomyocyte cell cycle arrest and induction of fibrotic response similar to what occurs after myocardial infarction in adult hearts. Recent studies outlined the function of several cardiogenic factors that play a pivotal role in neonatal cardiac regeneration. However, underlying molecular mechanisms of neonatal cardiac regeneration and other cardiogenic factors remained elusive. Here, we investigated the involvement of novel putative cardiogenic factors in neonatal cardiac regeneration and cardiomyocyte cell cycle withdrawal. We have shown that Cbl, Dnmt3a, and Itch are significantly downregulated during neonatal cardiac regeneration process after cardiac injury in vivo. Intriguingly, several of studied factors are upregulated in non-regenerative period of 7-day-old mice after cardiac injury. Knockdown of Cbl, Dnmt3a and Itch in rat neonatal cardiomyocytes lead to the induction of cardiomyocyte proliferation. Cardiomyocyte proliferation accompanies upregulation of positive regulators of cardiomyocyte division and downregulation of CDKIs. Taken together, our findings suggest that Cbl, Dnmt3a, and Itch may be involved in the regulation of cardiomyocyte cell cycle withdrawal and may represent new targets for the induction of cardiac regeneration.

Cell Cycle Withdrawal Limit the Regenerative Potential of Neonatal Cardiomyocytes.

The neonatal mouse possesses a transient capacity for cardiac regeneration during the first few days of life. The regenerative response of neonatal mouse is primarily mediated by pre-existing cardiomyocyte (CM) proliferation, which has been identified as the primary source of myocardial regeneration. Postnatal 4-day-old (P4) mouse CMs appear to undergo a rapid transition from hyperplastic to hypertrophic growth and binucleation. By 7 days following birth this regenerative potential is lost which coincidently correspond with CM cell cycle arrest and binucleation. CCM2-like (Ccm2l) plays pivotal roles in cardiovascular development and cardiac growth, indicating a potential function in heart regeneration postnatally. The aim of this study was to determine the cardiac regeneration ability of P4 neonatal mouse using a novel and more reproducible injury model and to determine whether Ccm2l has any functional roles in heart repair following ischemic injury. We performed a modified left anterior descending artery (LAD) ligation procedure on P4 mice to examine cardiac regenerative responses at different time points. Additionally, we generated an endothelial-specific Ccm2l gain-of-function transgenic mouse to determine the role of Ccm2l in neonatal cardiac regeneration. We found that the P4 mouse heart harbor a robust regenerative response after injury that was through the proliferation of pre-existing CMs but cardiac hypertrophy and subsequent remodeling was still evident 60 days after LAD ligation. Furthermore, we show that endothelial-specific overexpression of Ccm2l does not promote CM proliferation and heart repair after LAD ligation. The neonatal heart at P4 harbors a robust but incomplete capacity for cardiac regeneration. Endothelial overexpression of Ccm2l has no effect on cardiac regeneration.

Lymphoangiocrine signals promote cardiac growth and repair.

Recent studies suggested a beneficial role of lymphatics in restoring heart function after cardiac injury1–6. Here we report that in mice lymphatics promote cardiac growth, repair and cardio-protection. We show that a lymphoangiocrine signal produced by lymphatic endothelial cells (LECs) controls cardiomyocyte (CM) proliferation and survival during heart development, improves neonatal cardiac regeneration and is cardioprotective after myocardial infarction (MI). Embryos devoid of LECs develop smaller hearts as a consequence of reduced CM proliferation and increased CM apoptosis. Culturing primary mouse CMs in LEC-conditioned media increases CM proliferation and survival, indicating that LECs produce lymphoangiocrine signals controlling CM homeostasis. Characterization of the LEC secretome identified Reelin as a key player responsible for such function. Moreover, we report that LEC-specific Reln-null embryos also develop smaller hearts, that Reelin is required for efficient heart repair and function following neonatal MI, and that cardiac delivery of REELIN using collagen patches improves adult heart function after MI through a cardioprotective effect. These results identify a lymphoangiocrine role of LECs during cardiac development and injury response, and Reelin as an important mediator of this function.

Abstract 15735: Neonatal Cardiac Regeneration Depends on IGF1R-signaling

Introduction: In contrast to other adult tissues myocardium cannot be sufficiently regenerated following significant myocardial infarction (MI). Efficient cardiac regeneration was demonstrated in neonatal mouse myocardial injury models. Similar observations were reported in other neonatal mammals including newborn human babies suffering from MI. The mechanisms of neonatal mammalian cardiac regeneration remain unclear. Here we show the crucial role of IGF1R which was unraveled in a time-course transcriptome analysis of neonatal mouse hearts. Methods: IGF1R was specifically knocked-down (KD) in cardiomyocytes of wildtype postnatal day one (P1) mice using adeno-associated virus (rAAV9) delivered shRNAmirs. KD of Renilla served as control (REN-CTRL). 5x10 13 viral genomes per kg bodyweight were injected intrathoracally. Three batches of rAAV9 with different shRNAmirs were used for the IGF1R-KD groups. Viral transduction was confirmed by bioluminescence imaging on luciferase containing rAAV9 copies and by immunofluorescence staining of rAAV9 delivered GFP reporter. KD efficiency was confirmed in vitro using a retrovirus system and in vivo. On P2 mice underwent either left anterior descending artery (LAD) ligation for induction of MI or SHAM surgery. Cardiac function was subsequently assessed by echocardiography 1 day post injury (dpi) and 21 dpi. Thereafter, hearts were harvested and left ventricular fibrosis was analyzed histologically. Results: LAD ligation resulted in significant MI in both, IGF1R-KD and REN-CTRL, LAD groups as proven by a markedly reduced ejection fraction (EF) 1 dpi. Importantly, 21 dpi IGF1R-KD and REN-CTRL SHAM groups displayed normal cardiac function proving no effect on neonatal cardiac growth and development of only KD of IGF1R without LAD ligation. In contrast, LAD ligation IGF1R-KD mice presented significantly reduced EF 21dpi compared to the other 3 groups. Histological analysis revealed significant fibrosis in the IGF1R-KD LAD hearts compared to the other 3 groups. Conclusions: Whereas IGF1R-KD or control rAAV9 does not alter physiological cardiac development, KD of IGF1R markedly impairs neonatal cardiac regeneration in neonatal mice after MI suggesting a crucial role in neonatal cardiac regeneration.

Abstract 15876: Tie1 is Essential for Cardiac Repair and Myocardial Regeneration

Introduction: Cardiac lymphatics are essential in resolving interstitial fluid and inflammatory infiltrate after a myocardial infarction in adults. Little is known about the role of the lymphatic system during neonatal cardiac regeneration following injury in the perinatal animal with an immature lymphatic system. Hypothesis: Our hypothesis is that TIE1, an endothelial specific receptor tyrosine kinase essential for lymphatic development in mice, is required for myocardial repair following infarction (MI) in both neonatal and adult mice. Methods: Using an inducible and lymphatic specific deletion model, Prox1Cre-ER T2 ;Tie1 fl/fl , both mutant and non-mutant control mice undergo myocardial MI with left anterior descending coronary ligation. Adult surgery is performed at 6-8 weeks of age. Conditional Tie1 deletion is induced by ingestion of tamoxifen enriched chow 48 hours prior to adult ligation and continued until dissection is performed; cardiac function is assessed throughout this time by echocardiogram. Neonatal surgery is performed at postnatal day 2 (P2) and conditional Tie1 deletion induced with intraperitoneal tamoxifen with functional assessment by echocardiogram at P4 and P21. Results: Adult control animals had a robust lymphangiogenic response within the scar infarct border zone at 6 weeks post-MI that was not seen in mutants at 6 weeks. Furthermore, Tie1 deletion following led to a an increase in maximal injury, severe depression of cardiac function, and a blunted myocardial repair response, noted by histological analysis of the scar area. Similarly, Tie1deletion in the neonatal period resulted in markedly attenuated regenerative response with persistent scar function at P21. Conclusions: Lymphatic expression of Tie1 in the adult is required for myocardial repair following injury. In addition, lymphatic expression of Tie1 is required for optimal myocardial regeneration in the neonate even though the cardiac lymphatic system is immature in the postnatal period. Further delineation of the mechanisms involved in Tie1 mediated repair and regeneration may provide novel opportunities for therapeutic intervention following cardiac injury in both neonates and adults.

Adult T-cells impair neonatal cardiac regeneration

Abstract Background Cardiac remodeling and subsequent heart failure remain critical issues after myocardial infarction (MI). Complete cardiac regeneration was shown recently in a neonatal mouse model of MI. This cardiac regenerative potential is limited to the first few postnatal days and its decline parallels with the maturation of the adaptive immune system. Purpose Herein, we hypothesized that the T-cell maturation status critically impacts the myocardial healing outcomes in neonates and contributes to the shift from regenerative to scarring phenotype observed shortly after birth. Methods The post-MI immune responses were characterized in postnatal day one (P1, regenerative) compared to seven-day old (P7, scarring) mice subjected to permanent left anterior descending artery (LAD) ligation. The myocardial leukocyte infiltrate was phenotyped by flow cytometry at 36 hours and five days after LAD ligation. Next, we studied neonatal post-MI repair in lymphocyte-deficient Rag2 knock-out (KO) mice subjected to LAD ligation. Moreover, we adoptively transferred syngeneic splenic Thy 1.1+ T-cells obtained from adult donors into P1 versus P7 recipients and then assessed their impact on post-MI healing. Results LAD ligation induced a robust early inflammatory response (36h post-MI) in both age groups. The in situ inflammation was, nevertheless, rapidly resolved in P1-, but not in P7-infarcted animals. The distinct age groups showed a similar profile of cardiac myeloid cell infiltration but showed remarkable differences in the lymphoid compartment. P1-infarcted mice showed an early recruitment of γδT-cells, whereas P7-infarcted mice exhibited a prominent infiltration of αβT-cells. Of note, neonatal cardiac regeneration was not altered in neonatal lymphocyte-deficient (Rag2 KO) animals. However, the adoptive transfer of adult T-cells had several consequences in neonatal and one week old mice subjected to ischemic injury. P1-infarcted mice transferred with adult T-cells showed an adult-like healing phenotype, marked by an irreversible cardiac functional impairment (assessed by echocardiography) and increased fibrosis. This is in sharp contrast to the regenerative phenotype typically observed in untreated age-matched controls. Furthermore, P7-infarcted mice transferred with adult T-cell showed significantly decreased survival rate after LAD ligation. Conclusion Neonatal hearts demonstrate rapid clearance of the ischemia-induced leukocyte infiltration, further reflecting the known fact of fast cardiac regeneration in newborn rodents. Of note, the adoptive transfer of adult T-cells into neonate recipients partially blocked cardiac regeneration and promoted an irreversible functional impairment. These data indicate that the cardiac repair process, and its related “regeneration vs. scarring” dichotomy, is critically impacted by the T-cell development status. Funding Acknowledgement Type of funding source: Public Institution(s). Main funding source(s): Innsbruck Medical University, Medizinischer Forschungsfonds Tirol

Abstract 548: Cellular Contributions to Neonatal Cardiac Regeneration

While the neonatal mouse heart possesses a remarkable ability to regenerate up to a week after birth, the adult mammalian heart is susceptible to irreversible scar formation that impedes cardiac function. Such a scar is formed by the precocious deposition of extracellular matrix (ECM) by resident cardiac fibroblasts (CFs). Unlike their adult counterparts, neonatal CFs in the regenerative widow may have a unique phenotype that contributes to cardiac regeneration and scar resolution. Indeed, the neonatal cardiac ECM secreted by CFs is reported to stimulate regeneration, yet the underlying mechanisms of CF-mediated cardiac repair in the neonate has not been examined. Here, we present a strategy to establish the role of tissue resident CFs in mouse neonatal cardiac regeneration through selective cell depletion and RNA sequencing. Through the initial analysis of published RNA sequencing data, we identified an enrichment of pro-regenerative molecules such as amphiregulin (Areg) during the neonatal regenerative window. Areg, an epidermal growth factor ligand, has been shown to paradoxically stimulate both cardiac repair and pathological fibrosis after adult cardiac injury. To assess its impact on the neonatal cardiac injury response, we developed an adeno-associated virus serotype 9 with the complete coding sequence of mouse Areg (AAV9:Areg) controlled by the cardiomyocyte-specific cardiac troponin T promoter. In vivo , AAV9:Areg treated mice accumulate BrdU+ (proliferative) non-myocytes adjacent to Areg-expressing cardiomyocytes. Ongoing studies are aimed at evaluating the contribution of CFs to neonatal cardiac repair, including whether Areg-dependent cellular changes impact CFs in the neonatal regenerative window in a manner distinct from that in the adult injury response.

Mechanisms of Neonatal Heart Regeneration.

This review provides an overview of the molecular mechanisms underpinning the cardiac regenerative capacity during the neonatal period and the potential targets for developing novel therapies to restore myocardial loss. We present recent advances in the understanding of the molecular mechanisms of neonatal cardiac regeneration and the implications for the development of new cardiac regenerative therapies. During the early postnatal period, several cell types and pathways are involved in cardiomyocyte proliferation including immune response, nerve signaling, extracellular matrix, mitochondria substrate utilization, gene expression, miRNAs, and cell cycle progression. The early neonatal mammalian heart has remarkable regenerative capacity, which is mediated by proliferation of endogenous cardiomyocytes, and is lost when cardiomyocytes stop dividing shortly after birth. A wide array of mechanisms that regulate this regenerative process have been proposed.

Natural Heart Regeneration in a Neonatal Rat Myocardial Infarction Model

Newborn mice and piglets exhibit natural heart regeneration after myocardial infarction (MI). Discovering other mammals with this ability would provide evidence that neonatal cardiac regeneration after MI may be a conserved phenotype, which if activated in adults could open new options for treating ischemic cardiomyopathy in humans. Here, we hypothesized that newborn rats undergo natural heart regeneration after MI. Using a neonatal rat MI model, we performed left anterior descending coronary artery ligation or sham surgery in one-day-old rats under hypothermic circulatory arrest (n = 74). Operative survival was 97.3%. At 1 day post-surgery, rats in the MI group exhibited significantly reduced ejection fraction (EF) compared to shams (87.1% vs. 53.0%, p < 0.0001). At 3 weeks post-surgery, rats in the sham and MI groups demonstrated no difference in EF (71.1% vs. 69.2%, respectively, p = 0.2511), left ventricular wall thickness (p = 0.9458), or chamber diameter (p = 0.7801). Masson’s trichome and picrosirius red staining revealed minimal collagen scar after MI. Increased numbers of cardiomyocytes positive for 5-ethynyl-2′-deoxyuridine (p = 0.0072), Ki-67 (p = 0.0340), and aurora B kinase (p = 0.0430) were observed within the peri-infarct region after MI, indicating ischemia-induced cardiomyocyte proliferation. Overall, we present a neonatal rat MI model and demonstrate that newborn rats are capable of endogenous neocardiomyogenesis after MI.

Signaling of IL13 through IL4Ra Promotes Cardiac Regeneration

IntroductionDuring the first week of life, neonatal mice are able to regenerate their hearts after injury with minimal scarring. Our lab has previously published that the global knockout of anti‐inflammatory cytokine Interleukin 13 (IL13) inhibits neonatal cardiac regeneration in mice. IL13 primarily acts through the IL4Ra/IL13Ra1 heterodimer receptor to promote cell cycle activity. However, how IL13 specifically signals in the heart to promote this phenotype remains unexplored. We hypothesize that the deletion of the IL4Ra component of IL13's receptor complex will lead to a similar lack of regeneration in the mouse heart.MethodsTo delineate IL13 signaling mechanisms in the mouse, we utilized various knockouts of IL13 signaling—IL13 global knockout mice, IL4Ra global knockout mice, and C57/BL6 wildtype mice. With cardiomyocytes (CMs) isolated from these models, we analyzed the dynamics of downstream factors of recombinant IL13 (rIL13) in vitro. Additionally, at postnatal day 1 (P1), our mouse models were given apical resections to remove up to 10% of the apex of the heart. After resection, we investigated mRNA and protein expression of downstream markers, as well as CM proliferation at different time points. We also analyzed overall regenerative potential through echocardiography and Gömöri trichrome staining.ResultsIn vitro administration of rIL13 to wildtype neonatal mouse CMs resulted in strong, but transient activation of AKT and ERK1/2, which have been shown to be pro‐survival and pro‐proliferative respectively. In our IL4Ra KO mice, we see an attenuation of this signaling. At 7 days post resection (dpr), we see a significant decrease in proliferating CMs from IL13 KO mice when compared to wildtype. Additionally, at 21dpr in vivo, we observe significant fibrotic apical scars in IL13 KO mice, indicating lack of cardiac regeneration.ConclusionFrom these results, our data suggests that depletion of IL13 and its receptor, IL4Ra, inhibits regenerative pathways in the heart.Support or Funding InformationAmerican Heart AssociationThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Ezh2 is not required for cardiac regeneration in neonatal mice.

The neonatal mouse heart has the remarkable capacity to regenerate lost myocardium within the first week of life. Neonatal cardiomyocytes re-express fetal genes that control cell proliferation after injury to promote regeneration. The loss of regenerative capacity of the heart one week after birth coincides with repression of a fetal transcriptional program coordinated by epigenetic regulators. The histone methyltransferase enhancer of zeste homolog 2 (Ezh2) is a repressor of fetal cardiac transcriptional programs and suppresses cardiomyocyte cell proliferation, suggesting a potential function in heart regeneration. However, it was recently demonstrated that Ezh2 is dispensable for heart regeneration in the neonatal heart. Here, we provide evidence supporting this finding and demonstrate that Ezh2 deficiency does not affect regeneration of the neonatal heart. We inactivated Ezh2 in differentiating embryonic cardiomyocytes, which led to depletion of histone H3 trimethylated at lysine 27 (H3K27me3). Ezh2 deficiency in cardiomyocytes did not affect clearance of the fibrotic scar in myocardial infarction (MI) and apical resection models of cardiac injury at post-natal day 1 (P1). Similarly, cardiomyocyte-specific loss of Ezh2 did not affect fibrotic scar size after MI or apical resection at P7, suggesting that it does not extend the regenerative time window. Our results demonstrate that Ezh2 is not required for innate neonatal cardiac regeneration.

10 Macrophage-derived wnts are required for scar-free regeneration of the neonatal mouse myocardium

Objective In contrast to the adult, neonatal mice regenerate their myocardium following injury, at least during the first week after birth. 1 Macrophages (Mx) contribute to vessel formation and scar removal following neonatal myocardial infarction (MI) 2 . In the kidney 3 liver 4,7 and gut 5 Mx-derived WNTs are required for scar free regeneration following injury. Secretion of WNTs is dependent on acylation by Porcupine (PORCN). In the present study it was hypothesised that neonatal cardiac regeneration would be impaired in mice with Csf1r-Cre driven Mx specific Porcn deletion. 5 Methods Csf1r-EGFP(MacGreen), Porcn fl/ Csf1r Cre-ve and Porcn fl /Csf1r Cre+ve mice underwent coronary artery ligation at post-natal day 1 (P1). Functional loss 1 day after MI, and recovery by P21 were assessed by high-resolution ultrasound. Heart sections were stained with isolectin B4 (vessel density) and picrosirius res (fibrosis). Myocardial gene expression was determined by PCR array in wild-type (WT) mice after injury. Results At day 1 and day 7 post-MI, Csf1r-expressing cells accumulated within the injured myocardium, consistent with a role in regeneration. 2 At day 1 post-MI, fractional area change (FAC) decreased from 40.9% ± 1.6 to 18.0% ± 2.4% (p Porcn fl/ Csf1r Cre-ve and Porcnfl/Csf1rCre-ve mice respectively. By 21 days after MI, FAC had recovered to 47.4% ± 2.5% (p Porcn fl/ Csf1r Cre-ve and 45.8% ± 1.9% (p Porcn fl/ Csf1r Cre-ve littermates. Coronary vascularisation was restored in the infarct area by 21 days in both lines, but interstitial fibrosis was significantly higher in Porcn fl/ Csf1r Cre-ve (6.0 ± 0.9% LV) compared to Porcn fl/ Csf1r Cre-ve (3.8 ± 0.5% LV, p Wnt5b and Fzd2 , genes associated with regulation of fibrosis. 8 Conclusion Mx-derived WNTs are not required for re-vascularisation or restoration of myocardial function after neonatal myocardial injury, but are necessary for scar removal during regeneration. References Porrello et al . PNAS 2013; 110 :p187–92. Aurora et al . JCI 2014; 124 :p1382–92. Lin et al . PNAS 2010; 107 :p4194–99. Boulter et al . Nat Med 2012; 18 :572–9. Saha et al . Nat Com 2016; 7 :1–16. Derlindati et al . PLoS ONE 2015; 10 :1–17. Irvine et al . Fibrogenesis Tissue Repair 2015; 8 :19–32. Laeremans et al . Cardiovascular Research 2010; 87 :514–523.

Underlying mechanisms and prospects of heart regeneration

Findings in the last decade suggest that there is a considerable amount of cardiomyocyte turnover in the human heart throughout life, albeit not sufficient for heart regeneration following myocardial infarctions. Only a few species are known to be remarkably efficient in cardiac regeneration. They restore lost cardiomyocytes via a process of cardiomyocyte dedifferentiation, which is followed by robust proliferation of cardiomyocytes and incorporation into the myocardium. Similarly, neonatal mice have been recently shown to regenerate their heart following myocardial injuries. Studies with a neonatal cardiac regeneration mouse model suggest that the major source of new cardiomyocytes is likely to be of cardiomyocyte origin, with the possibility of involvement of cardiac stem cells. To this end, numerous studies have been conducted on the induction of cardiac regeneration to shed light on the underlying mechanisms. This review covers studies on the renewal of cardiomyocytes, the utilization of stem cells in myocardial therapies, and their future applications.