Postnatal Cardiomyocyte Proliferation Research Articles

Dissecting signal transduction pathways that regulate cardiomyocyte proliferation

Abstract Funding Acknowledgements None. Introduction During cardiac development, the Notch, Hippo, and Wnt/β-catenin signalling pathways play a crucial role in the tuning of cardiomyocyte proliferation. The individual engagement of each of these pathways during the early postnatal life, however, is still unclear. Purpose We wanted to develop a method to visualize over time, both in vitro and in vivo, the topography of activation of the Notch, Wnt/β-catenin, and Hippo pathways and their crosstalk upon cardiomyocyte treatment with a series of pro-proliferative stimuli. Methods We obtained Adeno-Associated Virus (AAV) constructs expressing distinct fluorescent proteins (ECFP, GFP, mCherry, DsRed) under the control of promoters that are specifically activated downstream of the signalling cascades of interest (a synthetic promoter consisting of 12 CSL binding sites for Notch1, a synthetic promoter consisting of 8 TEAD binding sites for YAP, and a synthetic promoter containing 7 TCF/LEF1 binding sites for β-catenin). Results First, by high content microscopy we followed the time of the activation of the AAV vectors coding for fluorescent reporters, either alone or in combination. We observed that a crosstalk exists between Yap and Notch signalling in proliferating cardiomyocytes, as detected by the activation of their respective fluorescent reporters. We confirmed our data by gene expression analysis and co-immunoprecipitation using specific antibodies in fractionated cardiomyocyte extracts. Next, we observe that miR-1825, miR-199a-3p, and miR-590-3p – three pro-regenerative miRNAs in cardiomyocytes– specifically activate both Yap and Notch signalling. Finally we characterized the reporter systems in vivo, to dissect the intracellular signalling pathways that are active in the first week of postnatal life. Beta-catenin signalling was quite undetectable in any of the conditions tested. In contrast, both Notch and Yap showed significant activation, consistent with their role in regulating early postnatal cardiomyocyte proliferation. Conclusions The developed AAV-fluorescent reporters allow to efficiently and specifically visualize the pro-proliferative activation of Notch and Yap signalling both in vitro and in vivo. The results obtained are supportive of a crosstalk existing between Notch and Yap pathways in proliferating cardiomyocytes.

Hydrogen Sulfide Promotes Postnatal Cardiomyocyte Proliferation by Upregulating SIRT1 Signaling Pathway.

Hydrogen sulfide (H2S) has been identified as a novel gasotransmitter and a substantial antioxidant that can activate various cellular targets to regulate physiological and pathological processes in mammals. However, under physiological conditions, it remains unclear whether it is involved in regulating cardiomyocyte (CM) proliferation during postnatal development in mice. This study mainly aimed to evaluate the role of H2S in postnatal CM proliferation and its regulating molecular mechanisms. We found that sodium hydrosulfide (NaHS, the most widely used H2S donor, 50-200 μM) increased neonatal mouse primary CM proliferation in a dose-dependent manner in vitro. Consistently, exogenous administration of H2S also promoted CM proliferation and increased the total number of CMs at postnatal 7 and 14 days in vivo. Moreover, we observed that the protein expression of SIRT1 was significantly upregulated after NaHS treatment. Inhibition of SIRT1 with EX-527 or si-SIRT1 decreased CM proliferation, while enhancement of the activation of SIRT1 with SRT1720 promoted CM proliferation. Meanwhile, pharmacological and genetic blocking of SIRT1 repressed the effect of NaHS on CM proliferation. Taken together, these results reveal that H2S plays a promotional role in proliferation of CMs in vivo and in vitro and SIRT1 is required for H2S-mediated CM proliferation, which indicates that H2S may be a potential modulator for heart development in postnatal time window.

Cardiomyocyte proliferation is suppressed by ARID1A-mediated YAP inhibition during cardiac maturation

The inability of adult human cardiomyocytes to proliferate is an obstacle to efficient cardiac regeneration after injury. Understanding the mechanisms that drive postnatal cardiomyocytes to switch to a non-regenerative state is therefore of great significance. Here we show that Arid1a, a subunit of the switching defective/sucrose non-fermenting (SWI/SNF) chromatin remodeling complex, suppresses postnatal cardiomyocyte proliferation while enhancing maturation. Genome-wide transcriptome and epigenome analyses revealed that Arid1a is required for the activation of a cardiomyocyte maturation gene program by promoting DNA access to transcription factors that drive cardiomyocyte maturation. Furthermore, we show that ARID1A directly binds and inhibits the proliferation-promoting transcriptional coactivators YAP and TAZ, indicating ARID1A sequesters YAP/TAZ from their DNA-binding partner TEAD. In ischemic heart disease, Arid1a expression is enhanced in cardiomyocytes of the border zone region. Inactivation of Arid1a after ischemic injury enhanced proliferation of border zone cardiomyocytes. Our study illuminates the pivotal role of Arid1a in cardiomyocyte maturation, and uncovers Arid1a as a crucial suppressor of cardiomyocyte proliferation.

Effects of maternal hypothyroidism on postnatal cardiomyocyte proliferation and cardiac disease responses of the progeny.

Maternal hypothyroidism (MH) could adversely affect the cardiac disease responses of the progeny. This study tested the hypothesis that MH reduces early postnatal cardiomyocyte (CM) proliferation so that the adult heart of MH progeny has a smaller number of larger cardiac myocytes, which imparts adverse cardiac disease responses following injury. Thyroidectomy (TX) was used to establish MH. The progeny from mice that underwent sham or TX surgery were termed Ctrl (control) or MH (maternal hypothyroidism) progeny, respectively. MH progeny had similar heart weight (HW) to body weight (BW) ratios and larger CM size consistent with fewer CMs at postnatal day 60 (P60) compared with Ctrl (control) progeny. MH progeny had lower numbers of EdU+, Ki67+, and phosphorylated histone H3 (PH3)+ CMs, which suggests they had a decreased CM proliferation in the postnatal timeframe. RNA-seq data showed that genes related to DNA replication were downregulated in P5 MH hearts, including bone morphogenetic protein 10 (Bmp10). Both in vivo and in vitro studies showed Bmp10 treatment increased CM proliferation. After transverse aortic constriction (TAC), the MH progeny had more severe cardiac pathological remodeling compared with the Ctrl progeny. Thyroid hormone (T4) treatment for MH mothers preserved their progeny's postnatal CM proliferation capacity and prevented excessive pathological remodeling after TAC. Our results suggest that CM proliferation during early postnatal development was significantly reduced in MH progeny, resulting in fewer CMs with hypertrophy in adulthood. These changes were associated with more severe cardiac disease responses after pressure overload.NEW & NOTEWORTHY Our study shows that compared with Ctrl (control) progeny, the adult progeny of mothers who have MH (MH progeny) had fewer CMs. This reduction of CM numbers was associated with decreased postnatal CM proliferation. Gene expression studies showed a reduced expression of Bmp10 in MH progeny. Bmp10 has been linked to myocyte proliferation. In vivo and in vitro studies showed that Bmp10 treatment of MH progeny and their myocytes could increase CM proliferation. Differences in CM number and size in adult hearts of MH progeny were linked to more severe cardiac structural and functional remodeling after pressure overload. T4 (synthetic thyroxine) treatment of MH mothers during their pregnancy, prevented the reduction in CM number in their progeny and the adverse response to disease stress.

Inhibition of β1-AR/Gαs signaling promotes cardiomyocyte proliferation in juvenile mice through activation of RhoA-YAP axis.

The regeneration potential of the mammalian heart is incredibly limited, as cardiomyocyte proliferation ceases shortly after birth. β-adrenergic receptor (β-AR) blockade has been shown to improve heart functions in response to injury; however, the underlying mechanisms remain poorly understood. Here, we inhibited β-AR signaling in the heart using metoprolol, a cardio-selective β blocker for β1-adrenergic receptor (β1-AR) to examine its role in heart maturation and regeneration in postnatal mice. We found that metoprolol enhanced cardiomyocyte proliferation and promoted cardiac regeneration post myocardial infarction, resulting in reduced scar formation and improved cardiac function. Moreover, the increased cardiomyocyte proliferation was also induced by the genetic deletion of Gnas, the gene encoding G protein alpha subunit (Gαs), a downstream effector of β-AR. Genome wide transcriptome analysis revealed that the Hippo-effector YAP, which is associated with immature cardiomyocyte proliferation, was upregulated in the cardiomyocytes of β-blocker treated and Gnas cKO hearts. Moreover, the increased YAP activity is modulated by RhoA signaling. Our pharmacological and genetic studies reveal that β1-AR-Gαs-YAP signaling axis is involved in regulating postnatal cardiomyocyte proliferation. These results suggest that inhibiting β-AR-Gαs signaling promotes the regenerative capacity and extends the cardiac regenerative window in juvenile mice by activating YAP-mediated transcriptional programs.

Wnt11 in regulation of physiological and pathological cardiac growth.

Wnt11 regulates early cardiac development and left ventricular compaction in the heart, but it is not known how Wnt11 regulates postnatal cardiac maturation and response to cardiac stress in the adult heart. We studied cell proliferation/maturation in postnatal and adolescent Wnt11 deficient (Wnt11-/-) heart and subjected adult mice with partial (Wnt11+/-) and complete Wnt11 (Wnt11-/-) deficiency to cardiac pressure overload. In addition, we subjected primary cardiomyocytes to recombinant Wnt proteins to study their effect on cardiomyocyte growth. Wnt11 deficiency did not affect cardiomyocyte proliferation or maturation in the postnatal or adolescent heart. However, Wnt11 deficiency led to enlarged heart phenotype that was not accompanied by significant hypertrophy of individual cardiomyocytes. Analysis of stressed adult hearts from wild-type mice showed a progressive decrease in Wnt11 expression in response to pressure overload. When studied in experimental cardiac pressure overload, Wnt11 deficiency did not exacerbate cardiac hypertrophy or remodeling and cardiac function remained identical between the genotypes. When subjecting cardiomyocytes to hypertrophic stimulus, the presence of recombinant Wnt11 together with Wnt5a reduced protein synthesis. In conclusion, Wnt11 deficiency does not affect postnatal cardiomyocyte proliferation but leads to cardiac growth. Interestingly, Wnt11 deficiency alone does not substantially modulate hypertrophic response to pressure overload in vivo. Wnt11 may require cooperation with other noncanonical Wnt proteins to regulate hypertrophic response under stress.

Prolonged Myocardial Regenerative Capacity in Neonatal Opossum.

Early neonates of both large and small mammals are able to regenerate the myocardium through cardiomyocyte proliferation for only a short period after birth. This myocardial regenerative capacity declines in parallel with withdrawal of cardiomyocytes from the cell cycle in the first few postnatal days. No mammalian species examined to date has been found capable of a meaningful regenerative response to myocardial injury later than 1 week after birth. We examined cardiomyocyte proliferation in neonates of the marsupial opossum (Monodelphis domestica) by immunostaining at various times after birth. The regenerative capacity of the postnatal opossum myocardium was assessed after either apex resection or induction of myocardial infarction at postnatal day 14 or 29, whereas that of the postnatal mouse myocardium was assessed after myocardial infarction at postnatal day 7. Bioinformatics data analysis, immunofluorescence staining, and pharmacological and genetic intervention were applied to determine the role of AMPK (5'-AMP-activated protein kinase) signaling in regulation of the mammalian cardiomyocyte cell cycle. Opossum neonates were found to manifest cardiomyocyte proliferation for at least 2 weeks after birth at a frequency similar to that apparent in early neonatal mice. Moreover, the opossum heart at postnatal day 14 showed substantial regenerative capacity both after apex resection and after myocardial infarction injury, whereas this capacity had diminished by postnatal day 29. Transcriptomic and immunofluorescence analyses indicated that AMPK signaling is activated in postnatal cardiomyocytes of both opossum and mouse. Pharmacological or genetic inhibition of AMPK signaling was sufficient to extend the postnatal window of cardiomyocyte proliferation in both mouse and opossum neonates as well as of cardiac regeneration in neonatal mice. The marsupial opossum maintains cardiomyocyte proliferation and a capacity for myocardial regeneration for at least 2 weeks after birth. As far as we are aware, this is the longest postnatal duration of such a capacity among mammals examined to date. AMPK signaling was implicated as an evolutionarily conserved regulator of mammalian postnatal cardiomyocyte proliferation.

The euchromatic histone-lysine N-methyltransferases EHMT1/2 suppress post-natal cardiomyocyte proliferation via histone 3 lysine 9 (H3K9) dimethylation

Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – EU funding. Main funding source(s): KULeuven C14/17/088 Flemish Research Foundation (FWO) G0C6419N Cardiomyocytes proliferate rapidly during developmental growth in utero, but this proliferative capacity dramatically declines postnatally, rendering the heart unable to repair itself subsequent to injury or during ageing. The mechanisms governing this postnatal loss of cell cycle activity are not fully understood. During maturation, epigenomic remodelling drives wholesale changes in the cardiomyocyte transcriptome from the neonatal to the adult state. This is associated with silencing of foetally-expressed genes and activation of expression of genes required for adulthood. Here, we tested the hypothesis that epigenomic remodelling silences cell cycle-associated genes and hence cell cycle activity postnatally. In this regard, we focused on the role of the repressive H3K9me2 mark. Using immunostaining of Ki-67 and Aurora Kinase B as well as analysis of EdU incorporation to identify cell cycle activity in primary cardiomyocytes culture and in cryosections from rat hearts spanning from embryonic day 18 to adulthood, the abundance of H3K9me2 was found to be inversely correlated with cell proliferation. In line with these data, the expression of a gene panel associated with cell cycle activity decreased postnatally and was inversely related with H3K9me2 abundance in cardiomyocyte nuclei, purified by flow cytometry based on H3K9me2 abundance. Inhibition of the euchromatic histone lysine methyltransferases (Ehmt1/2 aka GLP/G9a), with the highly selective inhibitor A-366, enhanced proliferation of neonatal rat cardiomyocytes in vitro, as well as in vivo, in neonatal mice, via concomitant reduction of the repressive histone mark, H3K9me2. Similar effects were observed in vitro by overexpression of the histone di/tri-methyl demethylase KDM4B. Notably, forced induction of cell cycle activity by overexpression of cyclin/cyclin-dependent kinase significantly reduced H3K9me2 levels, in vitro. Together, these data point to a key role of H3K9me2 and its deposition by Ehmts in post-natal suppression of proliferation. Targeting this repressive mark could provide a strategy to enhance cardiac repair for therapeutic benefit.

Early Postnatal Cardiomyocyte Proliferation Requires High Oxidative Energy Metabolism

Cardiac energy metabolism must cope with early postnatal changes in tissue oxygen tensions, hemodynamics, and cell proliferation to sustain development. Here, we tested the hypothesis that proliferating neonatal cardiomyocytes are dependent on high oxidative energy metabolism. We show that energy-related gene expression does not correlate with functional oxidative measurements in the developing heart. Gene expression analysis suggests a gradual overall upregulation of oxidative-related genes and pathways, whereas functional assessment in both cardiac tissue and cultured cardiomyocytes indicated that oxidative metabolism decreases between the first and seventh days after birth. Cardiomyocyte extracellular flux analysis indicated that the decrease in oxidative metabolism between the first and seventh days after birth was mostly related to lower rates of ATP-linked mitochondrial respiration, suggesting that overall energetic demands decrease during this period. In parallel, the proliferation rate was higher for early cardiomyocytes. Furthermore, in vitro nonlethal chemical inhibition of mitochondrial respiration reduced the proliferative capacity of early cardiomyocytes, indicating a high energy demand to sustain cardiomyocyte proliferation. Altogether, we provide evidence that early postnatal cardiomyocyte proliferative capacity correlates with high oxidative energy metabolism. The energy requirement decreases as the proliferation ceases in the following days, and both oxidative-dependent metabolism and anaerobic glycolysis subside.

MiR-31a-5p promotes postnatal cardiomyocyte proliferation by targeting RhoBTB1

A limited number of microRNAs (miRNAs, miRs) have been reported to control postnatal cardiomyocyte proliferation, but their strong regulatory effects suggest a possible therapeutic approach to stimulate regenerative capacity in the diseased myocardium. This study aimed to investigate the miRNAs responsible for postnatal cardiomyocyte proliferation and their downstream targets. Here, we compared miRNA profiles in cardiomyocytes between postnatal day 0 (P0) and day 10 (P10) using miRNA arrays, and found that 21 miRNAs were upregulated at P10, whereas 11 were downregulated. Among them, miR-31a-5p was identified as being able to promote cardiomyocyte proliferation as determined by proliferating cell nuclear antigen (PCNA) expression, double immunofluorescent labeling for α-actinin and 5-ethynyl-2-deoxyuridine (EdU) or Ki-67, and cell number counting, whereas miR-31a-5p inhibition could reduce their levels. RhoBTB1 was identified as a target gene of miR-31a-5p, mediating the regulatory effect of miR-31a-5p in cardiomyocyte proliferation. Importantly, neonatal rats injected with a miR-31a-5p antagomir at day 0 for three consecutive days exhibited reduced expression of markers of cardiomyocyte proliferation including PCNA expression and double immunofluorescent labeling for α-actinin and EdU, Ki-67 or phospho-histone-H3. In conclusion, miR-31a-5p controls postnatal cardiomyocyte proliferation by targeting RhoBTB1, and increasing miR-31a-5p level might be a novel therapeutic strategy for enhancing cardiac reparative processes.

Live cell screening platform identifies PPAR\u03b4 as a regulator of cardiomyocyte proliferation and cardiac repair

Zebrafish can efficiently regenerate their heart through cardiomyocyte proliferation. In contrast, mammalian cardiomyocytes stop proliferating shortly after birth, limiting the regenerative capacity of the postnatal mammalian heart. Therefore, if the endogenous potential of postnatal cardiomyocyte proliferation could be enhanced, it could offer a promising future therapy for heart failure patients. Here, we set out to systematically identify small molecules triggering postnatal cardiomyocyte proliferation. By screening chemical compound libraries utilizing a Fucci-based system for assessing cell cycle stages, we identified carbacyclin as an inducer of postnatal cardiomyocyte proliferation. In vitro, carbacyclin induced proliferation of neonatal and adult mononuclear rat cardiomyocytes via a peroxisome proliferator-activated receptor δ (PPARδ)/PDK1/p308Akt/GSK3β/β-catenin pathway. Inhibition of PPARδ reduced cardiomyocyte proliferation during zebrafish heart regeneration. Notably, inducible cardiomyocyte-specific overexpression of constitutively active PPARδ as well as treatment with PPARδ agonist after myocardial infarction in mice induced cell cycle progression in cardiomyocytes, reduced scarring, and improved cardiac function. Collectively, we established a cardiomyocyte proliferation screening system and present a new drugable target with promise for the treatment of cardiac pathologies caused by cardiomyocyte loss.

Cardiac resident macrophages are involved in hypoxia‑induced postnatal cardiomyocyte proliferation.

Induction of cardiomyocyte proliferation, the most promising approach to reverse myocardial attrition, has been gaining importance as a therapy for cardiovascular disease. Hypoxia and macrophages were previously independently reported to promote cardiomyocyte proliferation in mice. However, whether hypoxia promotes cardiomyocyte proliferation in humans, and the association between hypoxia and macrophages in cardiomyocyte proliferation, have not to the best of our knowledge been previously investigated. The present study investigated the cardiomyocyte proliferation in 22 acyanotic and 29 cyanotic patients. Cardiomyocyte proliferation in a hypoxic mouse model (15% O2) was subsequently performed and the macrophage subsets were analyzed. A C-C chemokine receptor type 2 (CCR2) inhibitor was used to increase the number of resident macrophages in order to investigate the effect of macrophages on cardiomyocyte proliferation. The results demonstrated that cardiomyocyte proliferation in the cyanotic infant group was significantly increased compared with the acyanotic infant group and the hypoxia-treated C57BL/6J neonates confirmed the hypoxia-induced cardiomyocyte proliferation. However, hypoxia did not induce the proliferation of isolated cardiomyocytes. Notably, hypoxia treatment increased the number of cardiac resident macrophages in neonate hearts. Furthermore, increasing the number of resident macrophages significantly enhanced cardiomyocyte proliferation. In conclusion, postnatal hypoxia promoted cardiomyocyte proliferation in humans and animals, and cardiac resident macrophages may be involved in this process. Therefore, this novel mechanism may provide a promising strategy for cardiovascular disease treatment.

A p53-based genetic tracing system to follow postnatal cardiomyocyte expansion in heart regeneration.

In the field of heart regeneration, the proliferative potential of cardiomyocytes in postnatal mice is under intense investigation. However, solely relying on immunostaining of proliferation markers, the long-term proliferation dynamics and potential of the cardiomyocytes cannot be readily addressed. Previously, we found that a p53 promoter-driving reporter predominantly marked the proliferating lineages in mice. Here, we established a p53-based genetic tracing system to investigate postnatal cardiomyocyte proliferation and heart regeneration. By selectively tracing proliferative cardiomyocytes, a differential pattern of clonal expansion in p53+ cardiac myocytes was revealed in neonatal, adolescent and adult stages. In addition, the percentage of p53+ lineage cardiomyocytes increased continuously in the first month. Furthermore, these cells rapidly responded to heart injury and greatly contributed to the replenished myocardium. Therefore, this study reveals complex proliferating dynamics in postnatal cardiomyocytes and heart repair, and provides a novel genetic tracing strategy for studying postnatal cardiac turnover and regeneration.

Genetics of Cardiac Developmental Disorders: Cardiomyocyte Proliferation and Growth and Relevance to Heart Failure.

Cardiac developmental disorders represent the most common of human birth defects, and anomalies in cardiomyocyte proliferation drive many of these disorders. This review highlights the molecular mechanisms of prenatal cardiac growth. Trabeculation represents the initial ventricular growth phase and is necessary for embryonic survival. Later in development, the bulk of the ventricular wall derives from the compaction process, yet the arrest of this process can still be compatible with life. Cardiomyocyte proliferation and growth form the basis of both trabeculation and compaction, and mouse models indicate that cardiomyocyte interactions with the surrounding environment are critical for these proliferative processes. The human genetics of left ventricular noncompaction cardiomyopathy suggest that cardiomyocyte cell-autonomous mechanisms contribute to the compaction process. Understanding the determinants of prenatal or early postnatal cardiomyocyte proliferation and growth provides critical information that identifies risk factors for cardiovascular disease, including heart failure and its associated complications of arrhythmias and thromboembolic events.

Extra- and intracellular factors regulating cardiomyocyte proliferation in postnatal life

One of the striking differences that distinguish the adult from the embryonic heart in mammals and set it apart from the heart in urodeles and teleosts is the incapacity of cardiomyocytes to respond to damage by proliferation. While the molecular reasons underlying these characteristics still await elucidation, mounting evidence collected over the last several years indicates that cardiomyocyte proliferation can be modulated by different extracellular molecules. The exogenous administration of selected growth factors is capable of inducing neonatal and, in some instances, also adult cardiomyocyte proliferation. Other diffusible factors can regulate the proliferation and cardiac commitment of endogenous or implanted stem cells. While the individual role of these factors in the paracrine control of normal heart homeostasis still needs to be defined, this information is relevant for the development of novel therapeutic strategies for cardiac regeneration. In addition, recent evidence indicates that postnatal cardiomyocyte proliferation is controlled by genetically defined pathways, such as the Hippo pathway, and can be modulated by perturbing the endogenous cardiomyocyte microRNA network; the identification of the cytokines that activate these molecular circuits holds great potential for clinical translation.

Mutations in Alström protein impair terminal differentiation of cardiomyocytes.

Cardiomyocyte cell division and replication in mammals proceed through embryonic development and abruptly decline soon after birth. The process governing cardiomyocyte cell cycle arrest is poorly understood. Here we carry out whole exome sequencing in an infant with evidence of persistent postnatal cardiomyocyte replication to determine the genetic risk factors. We identify compound heterozygous ALMS1 mutations in the proband, and confirm their presence in her affected sibling, one copy inherited from each heterozygous parent. Next, we recognise homozygous or compound heterozygous truncating mutations in ALMS1 in four other children with high levels of postnatal cardiomyocyte proliferation. Alms1 mRNA knockdown increases multiple markers of proliferation in cardiomyocytes, the percentage of cardiomyocytes in G2/M phases, and the number of cardiomyocytes by 10% in cultured cells. Homozygous Alms1-mutant mice have increased cardiomyocyte proliferation at two weeks postnatal compared to wild-type littermates. We conclude that deficiency of Alström protein impairs postnatal cardiomyocyte cell cycle arrest.

Targeted MicroRNA Interference Promotes Postnatal Cardiac Cell Cycle Re-Entry.

Mammalian heart cells undergo a marked reduction in proliferative activity shortly after birth, and thereafter grow predominantly by hypertrophy. Our understanding of the molecular mechanisms underlying cardiac maturation and senescence is based largely on studies at the whole-heart level. Here, we investigate the molecular basis of the acquired quiescence of purified neonatal and adult cardiomyocytes, and use microRNA interference as a novel strategy to promote cardiomyocyte cell cycle re-entry. Expression of cyclins and cyclin-dependent kinases (CDKs) and positive modulators were down-regulated, while CDK inhibitors and negative cell cycle modulators were up-regulated during postnatal maturation of cardiomyocytes. The expression pattern of microRNAs also changed dramatically, including increases in miR-29a, miR-30a and miR-141. Treatment of neonatal cardiomyocytes with miRNA inhibitors anti-miR-29a, anti-miR-30a, and antimiR-141 resulted in more cycling cells and enhanced expression of Cyclin A2 (CCNA2). Thus, targeted microRNA interference can reactivate postnatal cardiomyocyte proliferation.

Regulation of neonatal and adult mammalian heart regeneration by the miR-15 family.

We recently identified a brief time period during postnatal development when the mammalian heart retains significant regenerative potential after amputation of the ventricular apex. However, one major unresolved question is whether the neonatal mouse heart can also regenerate in response to myocardial ischemia, the most common antecedent of heart failure in humans. Here, we induced ischemic myocardial infarction (MI) in 1-d-old mice and found that this results in extensive myocardial necrosis and systolic dysfunction. Remarkably, the neonatal heart mounted a robust regenerative response, through proliferation of preexisting cardiomyocytes, resulting in full functional recovery within 21 d. Moreover, we show that the miR-15 family of microRNAs modulates neonatal heart regeneration through inhibition of postnatal cardiomyocyte proliferation. Finally, we demonstrate that inhibition of the miR-15 family from an early postnatal age until adulthood increases myocyte proliferation in the adult heart and improves left ventricular systolic function after adult MI. We conclude that the neonatal mammalian heart can regenerate after myocardial infarction through proliferation of preexisting cardiomyocytes and that the miR-15 family contributes to postnatal loss of cardiac regenerative capacity.

Lack of endothelial nitric oxide synthase decreases cardiomyocyte proliferation and delays cardiac maturation

We recently demonstrated that deficiency in endothelial nitric oxide synthase (eNOS) results in congenital septal defects and postnatal heart failure. The aim of this study was to investigate the role of eNOS in cardiomyocyte proliferation and maturation during postnatal development. Cultured eNOS knockout (eNOS(-/-)) cardiomyocytes displayed fewer cells and lower bromodeoxyuridine (BrdU) incorporation in vitro compared with wild-type (WT) cardiomyocytes (P < 0.05). Treatment with the nitric oxide (NO) donor diethylenetriamine NONOate increased BrdU incorporation and cell counts in eNOS(-/-) cardiomyocytes (P < 0.05). Inhibition of nitric oxide synthase activity using N(G)-nitro-L-arginine methyl ester decreased the level of BrdU incorporation and cell counts in WT cardiomyocytes (P < 0.05). Vascular endothelial growth factor (VEGF) increased the level of BrdU incorporation in cultured WT cardiomyocytes in a dose- and time-dependent manner (P < 0.05). Conversely, VEGF did not alter BrdU incorporation in eNOS(-/-) cardiomyocytes (P = not significant). Furthermore, deficiency in eNOS significantly decreased BrdU labeling indexes in neonatal hearts in vivo. Although WT hearts displayed a rapid decrease in atrial natriuretic peptide (ANP) expression in the first week of neonatal life, ANP expression in eNOS(-/-) hearts remain elevated. Our study demonstrated that NO production from eNOS is necessary for postnatal cardiomyocyte proliferation and maturation, suggesting that eNOS plays an important role during postnatal heart development.