RESUMEN
BACKGROUND: The neonatal mammalian heart exhibits considerable regenerative potential following injury through cardiomyocyte proliferation, whereas mature cardiomyocytes withdraw from the cell cycle and lose regenerative capacities. Therefore, investigating the mechanisms underlying neonatal cardiomyocyte proliferation and regeneration is crucial for unlocking the regenerative potential of adult mammalian heart to repair damage and restore contractile function following myocardial injury. METHODS: The Tudor staphylococcal nuclease (Tudor-SN) transgenic (TG) or cardiomyocyte-specific knockout mice (Myh6-Tudor-SN -/-) were generated to investigate the role of Tudor-SN in cardiomyocyte proliferation and heart regeneration following apical resection (AR) surgery. Primary cardiomyocytes isolated from neonatal mice were used to assess the influence of Tudor-SN on cardiomyocyte proliferation in vitro. Affinity purification and mass spectrometry were employed to elucidate the underlying mechanism. H9c2 cells and mouse myocardia with either overexpression or knockout of Tudor-SN were utilized to assess its impact on the phosphorylation of Yes-associated protein (YAP), both in vitro and in vivo. RESULTS: We previously identified Tudor-SN as a cell cycle regulator that is highly expressed in neonatal mice myocardia but downregulated in adults. Our present study demonstrates that sustained expression of Tudor-SN promotes and prolongs the proliferation of neonatal cardiomyocytes, improves cardiac function, and enhances the ability to repair the left ventricular apex resection in neonatal mice. Consistently, cardiomyocyte-specific knockout of Tudor-SN impairs cardiac function and retards recovery after injury. Tudor-SN associates with YAP, which plays important roles in heart development and regeneration, inhibiting phosphorylation at Ser 127 and Ser 397 residues by preventing the association between Large Tumor Suppressor 1 (LATS1) and YAP, correspondingly maintaining stability and promoting nuclear translocation of YAP to enhance the proliferation-related genes transcription. CONCLUSION: Tudor-SN regulates the phosphorylation of YAP, consequently enhancing and prolonging neonatal cardiomyocyte proliferation under physiological conditions and promoting neonatal heart regeneration after injury.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales , Animales Recién Nacidos , Proliferación Celular , Miocitos Cardíacos , Regeneración , Proteínas Señalizadoras YAP , Animales , Miocitos Cardíacos/metabolismo , Miocitos Cardíacos/fisiología , Miocitos Cardíacos/citología , Fosforilación , Proteínas Señalizadoras YAP/metabolismo , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Ratones , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/genética , Corazón/fisiología , Ratones Noqueados , RatasRESUMEN
The neonatal heart can efficiently regenerate within a short period after birth, whereas the adult mammalian heart has extremely limited capacity to regenerate. The molecular mechanisms underlying neonatal heart regeneration remain elusive. Here, we revealed that as a coreceptor of Wnt signalling, low-density lipoprotein receptor-related protein 5 (LRP5) is required for neonatal heart regeneration by regulating cardiomyocyte proliferation. The expression of LRP5 in the mouse heart gradually decreased after birth, consistent with the time window during which cardiomyocytes withdrew from the cell cycle. LRP5 downregulation reduced the proliferation of neonatal cardiomyocytes, while LRP5 overexpression promoted cardiomyocyte proliferation. The cardiac-specific deletion of Lrp5 disrupted myocardial regeneration after injury, exhibiting extensive fibrotic scars and cardiac dysfunction. Mechanistically, the decreased heart regeneration ability induced by LRP5 deficiency was mainly due to reduced cardiomyocyte proliferation. Further study identified AKT/P21 signalling as the key pathway accounting for the regulation of cardiomyocyte proliferation mediated by LRP5. LRP5 downregulation accelerated the degradation of AKT, leading to increased expression of the cyclin-dependent kinase inhibitor P21. Our study revealed that LRP5 is necessary for cardiomyocyte proliferation and neonatal heart regeneration, providing a potential strategy to repair myocardial injury.
Asunto(s)
Corazón , Proteína-5 Relacionada con Receptor de Lipoproteína de Baja Densidad , Miocitos Cardíacos , Regeneración , Animales , Proliferación Celular , Corazón/fisiología , Proteína-5 Relacionada con Receptor de Lipoproteína de Baja Densidad/genética , Ratones , Miocitos Cardíacos/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Vía de Señalización WntRESUMEN
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.
Asunto(s)
Lesiones Cardíacas , Interleucina-13 , Animales , Inmunidad Innata , Interleucina-13/metabolismo , Interleucina-13/farmacología , Interleucina-4/metabolismo , Macrófagos/metabolismo , Ratones , Miocitos Cardíacos/metabolismoRESUMEN
Unlike adult cardiomyocytes, neonatal cardiomyocytes can readily proliferate that contributes to a transient regenerative potential after myocardial injury in mice. We have recently reported that CD4+ regulatory T-cells promote this process; however, the role of other CD4+ T-cell subsets as well as CD8+ T-cells in postnatal heart regeneration has been less studied. Methods: by comparing the regenerating postnatal day (P) 3 and the non-regenerating P8 heart after injury, we revealed the heterogeneity of CD4+ and CD8+ T-cells in the myocardium through single cell analysis. We also specifically ablated CD4+ and CD8+ T-cells using the lytic anti-CD4 and -CD8 monoclonal antibodies, respectively, in juvenile mice at P8 after myocardial injury. Results: we observe significantly more CD4+FOXP3- conventional T-cells in the P8 heart when compared to that of the P3 heart within a week after injury. Surprisingly, such a difference is not seen in CD8+ T-cells that appear to have no function as their depletion does not reactivate heart regeneration. On the other hand, specific ablation of CD4+ T-cells contributes to mitigated cardiac fibrosis and increased cardiomyocyte proliferation after injury in juvenile mice. Single-cell transcriptomic profiling reveals a pro-fibrotic CD4+ T-cell subset in the P8 but not P3 heart. Moreover, there are likely more Th1 and Th17 cells in the P8 than P3 heart. We further demonstrate that cytokines of Th1 and Th17 cells can directly reduce the proliferation and increase the apoptosis of neonatal cardiomyocytes. Moreover, ablation of CD4+ T-cells can directly or indirectly facilitate the polarization of macrophages away from the pro-fibrotic M2-like signature in the juvenile heart. Nevertheless, ablation of CD4+ T-cells alone does not offer the same protection in the adult heart after myocardial infarction, suggesting a developmental change of immune cells including CD4+ T-cells in the regulation of age-related mammalian heart repair. Conclusions: our results demonstrate that ablation of CD4+ but not CD8+ T-cells promotes heart regeneration in juvenile mice; and CD4+ T-cells play a distinct role in the regulation of heart regeneration and repair during development.
Asunto(s)
Linfocitos T CD4-Positivos/inmunología , Infarto del Miocardio/inmunología , Daño por Reperfusión Miocárdica/inmunología , Regeneración/inmunología , Subgrupos de Linfocitos T/inmunología , Envejecimiento/fisiología , Animales , Animales Recién Nacidos , Anticuerpos Monoclonales/administración & dosificación , Antígenos CD4/antagonistas & inhibidores , Antígenos CD8/antagonistas & inhibidores , Linfocitos T CD8-positivos/efectos de los fármacos , Linfocitos T CD8-positivos/inmunología , Células Cultivadas , Modelos Animales de Enfermedad , Femenino , Regulación del Desarrollo de la Expresión Génica/inmunología , Corazón/crecimiento & desarrollo , Humanos , Masculino , Ratones , Infarto del Miocardio/tratamiento farmacológico , Infarto del Miocardio/patología , Daño por Reperfusión Miocárdica/patología , Daño por Reperfusión Miocárdica/prevención & control , Miocardio/citología , Miocardio/inmunología , Miocardio/patología , Miocitos Cardíacos/fisiología , Cultivo Primario de Células , RNA-Seq , Regeneración/efectos de los fármacos , Análisis de la Célula Individual , Subgrupos de Linfocitos T/efectos de los fármacosRESUMEN
BACKGROUND: Rodent cardiomyocytes (CM) undergo mitotic arrest and decline of mononucleated-diploid population post-birth, which are implicated in neonatal loss of heart regenerative potential. However, the dynamics of postnatal CM maturation are largely unknown in swine, despite a similar neonatal cardiac regenerative capacity as rodents. Here, we provide a comprehensive analysis of postnatal cardiac maturation in swine, including CM cell cycling, multinucleation and hypertrophic growth, as well as non-CM cardiac factors such as extracellular matrix (ECM), immune cells, capillaries, and neurons. Our study reveals discordance in postnatal pig heart maturational events compared to rodents. METHODS AND RESULTS: Left-ventricular myocardium from White Yorkshire-Landrace pigs at postnatal day (P)0 to 6 months (6mo) was analyzed. Mature cardiac sarcomeric characteristics, such as fetal TNNI1 repression and Cx43 co-localization to cell junctions, were not evident until P30 in pigs. In CMs, appreciable binucleation is observed by P7, with extensive multinucleation (4-16 nuclei per CM) beyond P15. Individual CM nuclei remain predominantly diploid at all ages. CM mononucleation at ~50% incidence is observed at P7-P15, and CM mitotic activity is measurable up to 2mo. CM cross-sectional area does not increase until 2mo-6mo in pigs, though longitudinal CM growth proportional to multinucleation occurs after P15. RNAseq analysis of neonatal pig left ventricles showed increased expression of ECM maturation, immune signaling, neuronal remodeling, and reactive oxygen species response genes, highlighting significance of the non-CM milieu in postnatal mammalian heart maturation. CONCLUSIONS: CM maturational events such as decline of mononucleation and cell cycle arrest occur over a 2-month postnatal period in pigs, despite reported loss of heart regenerative potential by P3. Moreover, CMs grow primarily by multinucleation and longitudinal hypertrophy in older pig CMs, distinct from mice and humans. These differences are important to consider for preclinical testing of cardiovascular therapies using swine, and may offer opportunities to study aspects of heart regeneration unavailable in other models.