Metazoans like hydras achieve immortality through continuous stem cell-based whole-body turnover. It remains unclear whether mammals with extreme longevity have unrecognized regenerative potential. Using single-nucleus RNA-sequencing analysis, we show that long-lived mammal naked mole-rats may harbor regeneration-competent cardiomyocytes despite lacking cardiac stem cells. We validate that mole-rat cardiomyocytes exhibit higher renewal capacity than similar-sized mice. Cross-species comparisons uncover that the Bcl6 transcription factor, induced by neurohormonal activity, is upregulated in the adult hearts of mice and humans but not naked mole-rats, zebrafish, or leopard geckos. Murine cardiomyocyte-specific deletion of Bcl6 promotes cardiomyogenesis and heart repair. Bcl6 proteins form nuclear condensates and repress Akt1 expression, genetic normalization of which restores cardiomyocyte proliferation. This work identifies a critical brake of cardiomyocyte renewal downstream of nerve-endocrine-heart communication and unveils unusual cellular regenerative capacity in long-lived mammals that may provide insights into why most adult human organs cannot regenerate.

Adult human hearts have limited regenerative capacity. The loss of cardiomyocytes (CMs) following injury, therefore, can lead to myocardial dysfunction and heart failure. Although mammalian hearts do have the potential to regenerate in response to injury, this capacity sharply declined shortly after birth. It has been shown that cardiac tissue-resident macrophages (cTMs) are required for cardiac regeneration. The underlying mechanisms, however, remain poorly understood. Herein, we aimed to identify distinct macrophage populations that are required for cardiac regeneration and to determine the underlying mechanisms. Single-cell RNASeq identified distinct populations of cardiac macrophages that dominate in post-MI neonatal vs. adult mouse heart. One of the neonate-dominant cardiac macrophage clusters, CD36+ cTMs, exhibiting multiple pro-regenerative properties, including phagocytic, anti-inflammatory and pro-reparative transcriptional signatures. Myocardial infarction (MI) injury resulted in an acute (Day 1) depletion of CD36hi cTMs in both neonatal and adult mouse hearts. The population of CD36+ cTMs was quickly replenished in the neonatal mouse heart by Day 3 following MI, whereas the recovery of CD36+ cTMs population was markedly delayed (up to 21 days) in the adult mouse heart. Functional blockade of CD36 using a monocloncal Anti-CD36 antibody or inducing conditional deletion of CD36 in cTMs (using Cx3cr1-CreERT2;CD36fl/fl or CD36cKO mice) resulted in a significant reduction in CM proliferation and excessive scarring in the neonatal mouse heart following apical resection. Adoptive transfer of CD36+, but not CD36cKO, neonatal cTMs to adult mice increased CM self-renewal, reduced scar sizes and improved cardiac function following MI injury. Mechanistically, deficiency of CD36 resulted in impaired phagocytic activity of cTMs, leading to delayed clearance of CM debris and excessive myocardial inflammation, thereby hindering myocardial regeneration. Taken together, these results demonstrate an indispensable yet previously unrecognized role of CD36+ cTMs to promote cardiac repair and regeneration. Targeting CD36 in cTMs, therefore, could be a powerful new strategy to promote myocardial regeneration and functional recovery in MI and HF.

Balancing between regenerative processes and fibrosis is crucial for heart repair. However, strategies to regulate the balance between these two process are a barrier to the development of effective therapies for heart regeneration. While Interleukin 11 (IL11) is known as a fibrotic factor for the heart, its contribution to heart regeneration remains poorly understood. Here, we uncovered that il11a can initiate robust regenerative programs in the zebrafish heart, including cell cycle reentry of cardiomyocytes (CMs) and coronary expansion, even in the absence of injury. However, the prolonged il11a induction in uninjured hearts causes persistent fibroblast emergence, resulting in cardiac fibrosis. While deciphering the regenerative and fibrotic effects, we found that il11-dependent fibrosis, but not il11-dependent regeneration, is mediated through ERK activity, implying that the dual effects of il11a on regeneration and fibrosis can be uncoupled. To harness the regenerative ability of il11a for injured hearts, we devised a combinatorial treatment through il11a induction with ERK inhibition. Using this approach, we observed enhanced CM proliferation with mitigated fibrosis, achieving a balance between stimulating regenerative processes and curbing fibrotic outcomes. Thus, our findings unveil the mechanistic insights into regenerative roles of il11 signaling, offering the potential therapeutic avenues that utilizes a paracrine regenerative factor to foster cardiac repair without exacerbating the fibrotic responses.

In humans, new-born infants have the ability to regenerate their heart during early life. This is modelled in the mouse, where regenerative capacity is maintained for the first week after birth but lost thereafter. Reactivation of this process holds great therapeutic potential, however, the molecular pathways that might be targeted to extend neonatal regeneration remain elusive. Here, we explore a role for hypoxia and HIF signalling on the regulation of epicardial activity which is essential for heart development and modulates the response to injury. Hypoxic regions were found in the epicardium from mid-gestation, associated with HIF1α and HIF2α, and expression of the epicardial master regulator Wilms’ tumour 1 (WT1). Epicardial deletion of Hif1a reduced WT1 levels, leading to impaired coronary vasculature. Moreover, targeting of the HIF degradation enzyme PHD through pharmacological inhibition 35 with a clinically approved drug or epicardial-specific deletion stabilised HIF and promoted WT1 activity ex vivo. A combination of genetic and pharmacological stabilisation of HIF during neonatal heart injury led to prolonged epicardial activation, increased vascularisation, augmented infarct resolution and preserved function beyond the 7-day regenerative window. Pharmacological PHD inhibition also reduced scar size in adult infarcted hearts and improved function. Together, these findings suggest therapeutic modulation of HIF signalling may represent a viable strategy for treating ischaemic heart disease.

Future gene therapies for tissue regeneration will require precise spatiotemporal control of transgene expression to mitigate off-target effects, tissue overgrowth, and oncogenesis. Epigenetic profiling in regenerating tissues of zebrafish have revealed genetic enhancers that gain open chromatin marks after injury and during regeneration, thus termed tissue regeneration enhancer elements (TREEs). Paired with a minimal permissive promoter, zebrafish TREEs can direct gene expression in mammalian injury sites, using both transgenic lacZ reporter mice and recombinant adeno-associated viruses (AAVs). While promising, TREE-directed AAVs require further characterization of off-target expression and dynamics in individual recipients over time. Bioluminescence imaging can provide a useful tool for tracking TREE-directed expression in a whole-body manner with systemically delivered AAVs. Here, we characterize TREE-driven AAV transgene expression in a mouse myocardial injury model of ischemia-reperfusion to map spatial and temporal dynamics. Specifically, we administered AAV vectors harboring TREEs (REN or 2ankrd1aEN) upstream of an Hsp68 minimal promoter and a firefly luciferase reporter transgene, intravenously to adult mice pre- or post-injury. IVIS imaging revealed that REN and 2ankrd1aEN transiently direct luminescence in the heart after myocardial injury, with off-target expression in the liver. To limit off-target expression, we incorporated an engineered liver-detargeted AAV9 capsid variant cc84. Packaged with cc84, 2ankrd1aEN showed significantly higher luminescence in the heart after infarction compared to the Hsp68 promoter alone control, with minimal liver expression. We also used a capsid variant screening method in mice to discover new AAV capsids (IR41 and IR42) with enriched transduction at infarcted myocardial tissue vs healthy remote myocardium. IR41-packaged TREEs further enhanced transduction in infarcted myocardium of mice. Taken together, bioluminescence imaging in parallel with engineered AAV capsids provides valuable insights into on- and off-target aspects of the transient and injury-site specific nature of TREE-driven expression in mice.

The resolution of cardiac injury varies dramatically between species, ranging from complete functional and anatomical regeneration to scarring and reduction of contractility. In contrast to adult mammals, regenerating species such as the salamander Ambystoma mexicanum (axolotl), promote pro-healing processes such as cardiomyocytes dedifferentiation and proliferation and by that replenishing the damaged myocardium. Although several interventions have been identified to promote mammalian cardiac regeneration, the processes which govern dedifferentiation have not been thoroughly investigated. We hypothesised that axolotl cardiomyocytes at the injury border zone may express factors regulating regenerative processes. In particular, we were interested in factors that may cause cellular dedifferentiation that are not found in mammalian cardiomyocytes. To identify these, we generated single nucleus RNA and ATAC-seq analysis of the regenerating axolotl heart. Leveraging the large size of the heart compared to other regenerative species, we performed spatial transcriptomic analysis and obtained the first-of-its-kind, spatially resolved “atlas” of heart regeneration. Using trajectory analysis we identified a pro-regenerative CM population with unique transcriptomic signature which localizes to the border zone. We analysed which cell types are in proximity with border zone -CMs, allowing us to curate the “spatially relevant” ligand-receptor interactions. We identified conserved pro-regenerative signalling events such as NRG1-ERBB2 and AGRIN-DAG1, as well as signalling pairs which were not previously described in this context, such as Tyrosine-protein kinase receptor UFO (AXL), found solely in the injury-responsive CMs, and its ligand Growth arrest-specific protein 6 (Gas6) in endothelial cells. Over expression of AXL in mice and IPS derived human CMs promoted robust sarcomere disassembly but surprisingly not proliferation, showing for the first time, decoupling of dedifferentiation from proliferation. Our results provide a novel atlas of axolotl cardiac cells, a comprehensive analysis of changes occurring during cardiac regeneration, and identification of novel pro-regenerative pathways.