Physiological transitions during postnatal heart development
The mammalian heart undergoes profound adaptations during postnatal development to accommodate an increase in the contractile and metabolic demands of the postnatal environment. Almost every facet of cardiac physiology is altered during this maturational phase as the heart transitions from a fetal to an adult phenotype (Fig. 3). Physiological adaptations occurring during cardiac maturation have been extensively reviewed elsewhere and are only summarized briefly below.
The constellation of physiological changes occurring during cardiomyocyte maturation include the following: organization of the contractile apparatus through sarcomere expansion, alignment and myofibril switching; electrophysiological changes, including formation of a T tubule system, sarcoplasmic reticulum expansion and dyad formation; metabolic maturation, including an increase in mitochondrial content and a switch from glycolysis to fatty acid oxidation; and cardiomyocyte cell cycle withdrawal, polyploidization, multinucleation and hypertrophy.
Beyond the cardiomyocyte, cardiac maturation is associated with changes in cellular composition marked by an expansion in the number of fibroblasts, endothelial cells and immune cells in the heart. The transcriptional landscape of cardiac fibroblasts changes during maturation, which contributes to alterations in the composition of the extracellular matrix that allow the heart to withstand the increased mechanical loading on the ventricles after birth. In addition, postnatal angiogenesis increases the vascularization of the heart as it grows, which results in an increased metabolic demand. Interestingly, the developmental timing of immune system maturation and cardiac maturation are intimately linked. Postnatal maturation is associated with changes in the composition of immune cells in the heart and a shift from anti-inflammatory to pro-inflammatory states of multiple immune cell types (including macrophages, B cells and regulatory T cells). Collectively, these changes in the cellular microenvironment are thought to contribute to the postnatal loss of cardiac regenerative capacity in mammals, although the underlying mechanisms remain poorly understood.
Postnatal maturation of the heart is driven by extensive rewiring of the cardiac transcriptome and epigenome. Thousands of genes related to heart contraction, metabolism and proliferation are rewired in cardiomyocytes as they transition from a neonatal to an adult state, underpinning the major physiological changes occurring during cardiomyocyte maturation. Indeed, the transcriptional changes occurring during this maturational phase are so extensive that neonatal and adult cardiomyocytes could be considered as two distinct cell types. One of the most important transcriptional regulators controlling this maturational gene programme is SRF. SRF depletion affects almost every facet of the maturational gene programme in cardiomyocytes, including sarcomere isoform switching, sarcomere organization, mitochondrial biogenesis, lipid metabolism, oxidative respiration and T tubule formation. Similarly, deletion of SRF co-factors, including members of the myocardin-related transcription factor family, homeodomain-only protein homeobox (HOPX) and transcription factor GATA4 (refs. ), also result in cardiomyocyte maturation defects in mice. In addition to the SRF-related transcription factors, multiple ligand-activated transcription factors belonging to the nuclear hormone receptor superfamily have been implicated in cardiomyocyte maturation. Cardiac maturation is associated with a surge in serum levels of thyroid hormone T3 and glucocorticoids in the perinatal period. T3 and glucocorticoids exert broad effects on cardiomyocyte maturation in vitro, by increasing cardiomyocyte contractility, mitochondrial respiration, calcium handling, T tubule organization, cardiomyocyte hypertrophy and polyploidization. Cardiomyocyte-specific deletion of the major thyroid hormone receptor genes (Thra and Thrb) in mice has been found to impair cardiomyocyte maturation and prolong the proliferative and regenerative capacity of the neonatal heart. Similarly, cardiomyocyte-specific deletion of the glucocorticoid receptor in mice after birth delays cell cycle arrest, hypertrophy, and the structural and metabolic maturation of cardiomyocytes, and prolongs the regenerative capacity of the neonatal heart. In addition, numerous peroxisome proliferator-activated receptors and oestrogen-related receptors are required for postnatal mitochondrial biogenesis and the switch to an oxidative metabolic state. The progesterone receptor has also been identified as a regulator of sex-specific cardiomyocyte maturation programmes in humans via modulation of metabolic gene networks. Thus, multiple nuclear hormone receptors have essential roles in metabolic and contractile maturation of mammalian cardiomyocytes, as well as in cell cycle arrest and loss of regenerative capacity.
Transcriptional maturation of cardiomyocytes also involves coordinated control of alternative splicing on a genome-wide scale. Genome-wide analyses of alternative splicing have revealed that hundreds of genes involved in myofibril assembly, the cell cycle, vesicular trafficking and membrane organization undergo developmental splicing events during cardiac maturation. These genome-wide changes in alternative splicing are associated with changes in the expression of several RNA-binding proteins, including regulators of mRNA splicing. For example, CUGBP Elav-like family member (CELF) and muscleblind-like protein (MBNL) are reciprocally regulated during postnatal cardiac development. CELF proteins are downregulated whereas MBNL proteins are upregulated during cardiac maturation. Genetic gain-of-function and loss-of-function studies in mice suggest that CELF and MBNL proteins account for more than half of the developmentally regulated alternative splicing events occurring during postnatal heart maturation. The dramatic downregulation of CELF proteins after birth seems to be mediated by a microRNA regulatory mechanism dependent on miR-23a and miR-23b. Several other splicing factors also influence postnatal heart development, including RNA-binding protein 20, which regulates maturational splicing of TTN transcripts (which encodes titin), and the serine-arginine-rich family of splicing factors (SRSF1, SRSF2 and SRSF10), which regulate alternative splicing of genes encoding calcium handling proteins. In addition, RNA-binding protein FOX1 homologue 1 (RBFOX1) is dramatically induced during cardiac maturation from neonatal to adult stages. Ectopic expression of RBFOX1 enhances the maturational properties of neonatal rat cardiomyocytes and human pluripotent stem cell-derived cardiomyocytes (PSC-CMs), including improved sarcomere organization, increased contractility, electrophysiological maturation, hypertrophy and binucleation. These studies collectively suggest that post-transcriptional RNA splicing events substantially influence the transcriptional landscape of mature cardiomyocytes.
Silencing of the fetal gene programme and acquisition of the adult transcriptional programme involves remodelling of the epigenome through DNA methylation and covalent histone modifications. DNA methylation is highly dynamic during postnatal heart development. Site-specific DNA hypermethylation during cardiac maturation is associated with transcriptional silencing of fetal genes encoding sarcomeric proteins and several canonical developmental signalling networks, including the WNT, Notch, Hedgehog, transforming growth factor-β and fibroblast growth factor pathways. In addition, genome-wide analyses have identified large genomic regions (predominantly in gene bodies) that are demethylated during postnatal cardiomyocyte maturation, and which strongly correlate with a mature gene expression profile in adult cardiomyocytes.
As the heart matures, cardiomyocytes become progressively 'locked' into a terminally differentiated state by the acquisition of stable epigenetic marks. Repressive histone marks, such as histone 3 lysine 27 trimethylation (H3K27me3) and H3 lysine 9 dimethylation (H3K9me2), are acquired and maintained in inactivated genes in adult cardiomyocytes. By contrast, the presence of histone modifications such as H3K27 acetylation (H3K27ac), H3 lysine 4 methylation (H3K4me1) and H3K4me3 is associated with actively transcribed genes. The important regulatory role of these cis-regulatory elements in cardiac development and disease has been extensively reviewed previously. Genome-wide changes in histone methylation and acetylation during cardiomyocyte maturation are, at least in part, driven by postnatal changes in cellular metabolism. In mice, inhibition of fatty acid oxidation in postnatal cardiomyocytes via conditional cardiomyocyte-specific deletion of carnitine palmitoyltransferase 1b has been shown to result in an accumulation of α-ketoglutarate. α-Ketoglutarate directly activates the lysine demethylase KDM5, which broadly demethylates H3K4me3 domains in genes related to cardiomyocyte maturation, leading to partial reactivation of the fetal gene programme. Thus, the metabolic switch from glycolysis to fatty acid oxidation helps to shape the chromatin landscape and maintain the mature gene expression programme of adult cardiomyocytes. However, the implications of many other metabolite interactions with epigenetic proteins influencing cardiomyocyte cell states requires further investigation to completely define the epigenetic networks underpinning cardiac maturation.
Transcriptional changes during cardiomyocyte maturation also strongly correlate with changes in chromatin accessibility. Genome-wide analyses using the assay for transposase-accessible chromatin with sequencing (ATAC-seq) have detected changes in chromatin accessibility around thousands of genomic loci during cardiomyocyte maturation in mice and humans. Loss of chromatin accessibility around cell cycle genes is associated with transcriptional repression of the proliferative gene programme in postnatal cardiomyocytes, whereas activation of the mature metabolic and contractile gene programmes is associated with a gain of accessibility around these loci. How these changes in genome-wide chromatin accessibility are coordinated at the level of higher-order chromatin compartmentalization is poorly understood. Chromatin is highly ordered in topologically associated domains (TADs), which can be further spatially segregated into A and B compartments. One of the only studies investigating developmental changes in TADs during cardiac differentiation and maturation found that cardiomyocyte-specific A and B compartments are predominantly established during cardiac differentiation and remain relatively stable during maturation from the fetal to the adult stage. However, the proportion of low methylated regions, characteristic of cis-regulatory elements such as enhancers, increases during cardiomyocyte maturation and these regions are predominantly confined to A compartments.
Furthermore, in mice, deletion of transcriptional repressor CTCF (also known as CCCTC-binding factor), a crucial regulator of chromatin architecture, resulted in the aberrant activation of genes involved in mitochondrial metabolism and premature cardiomyocyte maturation. Of note, the regulation of the classic cardiac fetal gene locus of Nppa (encoding natriuretic peptide A) and Nppb (encoding natriuretic peptide B) was previously shown to be mediated by transcriptional repressor CTCF-dependent loops, with its expression perturbed when CTCF is depleted in cardiomyocytes. Further studies are required to dissect the mechanisms responsible for the developmental regulation of higher-order chromatin organization in cardiomyocytes. In this regard, a study published in 2021 indicated that cardiomyocytes establish distinct nuclear and chromatin organization during maturation (for example, H3K9me3 relocalization to the nuclear periphery) in response to mechanical cues from the environment (such as changes in extracellular matrix substrate stiffness). Given the widespread transcriptional changes that also occur in numerous cardiac non-myocyte populations during cardiac maturation, further investigation of the complex cell-cell interactions and the effect of the cellular microenvironment on the acquisition and maintenance of the adult epigenetic landscape is warranted.