Enzo R. Porrello

Transient Regenerative Potential of the Neonatal Mouse Heart

The heart in a newborn mouse can rebuild itself after injury, but this regenerative capacity is lost within a few days. Certain fish and amphibians retain a robust capacity for cardiac regeneration throughout life, but the same is not true of the adult mammalian heart. Whether the capacity for cardiac regeneration is absent in mammals or whether it exists and is switched off early after birth has been unclear. We found that the hearts of 1-day-old neonatal mice can regenerate after partial surgical resection, but this capacity is lost by 7 days of age. This regenerative response in 1-day-old mice was characterized by cardiomyocyte proliferation with minimal hypertrophy or fibrosis, thereby distinguishing it from repair processes. Genetic fate mapping indicated that the majority of cardiomyocytes within the regenerated tissue originated from preexisting cardiomyocytes. Echocardiography performed 2 months after surgery revealed that the regenerated ventricular apex had normal systolic function. Thus, for a brief period after birth, the mammalian heart appears to have the capacity to regenerate.

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.

Hippo pathway effector Yap promotes cardiac regeneration

The adult mammalian heart has limited potential for regeneration. Thus, after injury, cardiomyocytes are permanently lost, and contractility is diminished. In contrast, the neonatal heart can regenerate owing to sustained cardiomyocyte proliferation. Identification of critical regulators of cardiomyocyte proliferation and quiescence represents an important step toward potential regenerative therapies. Yes-associated protein (Yap), a transcriptional cofactor in the Hippo signaling pathway, promotes proliferation of embryonic cardiomyocytes by activating the insulin-like growth factor and Wnt signaling pathways. Here we report that mice bearing mutant alleles of Yap and its paralog WW domain containing transcription regulator 1 (Taz) exhibit gene dosage-dependent cardiac phenotypes, suggesting redundant roles of these Hippo pathway effectors in establishing proper myocyte number and maintaining cardiac function. Cardiac-specific deletion of Yap impedes neonatal heart regeneration, resulting in a default fibrotic response. Conversely, forced expression of a constitutively active form of Yap in the adult heart stimulates cardiac regeneration and improves contractility after myocardial infarction. The regenerative activity of Yap is correlated with its activation of embryonic and proliferative gene programs in cardiomyocytes. These findings identify Yap as an important regulator of cardiac regeneration and provide an experimental entry point to enhance this process.

miR-15 Family Regulates Postnatal Mitotic Arrest of Cardiomyocytes

Rationale: Mammalian cardiomyocytes withdraw from the cell cycle during early postnatal development, which significantly limits the capacity of the adult mammalian heart to regenerate after injury. The regulatory mechanisms that govern cardiomyocyte cell cycle withdrawal and binucleation are poorly understood. Objective: Given the potential of microRNAs (miRNAs) to influence large gene networks and modify complex developmental and disease phenotypes, we searched for miRNAs that were regulated during the postnatal switch to terminal differentiation. Methods and Results: Microarray analysis revealed subsets of miRNAs that were upregulated or downregulated in cardiac ventricles from mice at 1 and 10 days of age (P1 and P10). Interestingly, miR-195 (a member of the miR-15 family) was the most highly upregulated miRNA during this period, with expression levels almost 6-fold higher in P10 ventricles relative to P1. Precocious overexpression of miR-195 in the embryonic heart was associated with ventricular hypoplasia and ventricular septal defects in &bgr;-myosin heavy chain–miR-195 transgenic mice. Using global gene profiling and argonaute-2 immunoprecipitation approaches, we showed that miR-195 regulates the expression of a number of cell cycle genes, including checkpoint kinase 1 (Chek1), which we identified as a highly conserved direct target of miR-195. Finally, we demonstrated that knockdown of the miR-15 family in neonatal mice with locked nucleic acid–modified anti-miRNAs was associated with an increased number of mitotic cardiomyocytes and derepression of Chek1. Conclusions: These findings suggest that upregulation of the miR-15 family during the neonatal period may be an important regulatory mechanism governing cardiomyocyte cell cycle withdrawal and binucleation.

Macrophages are required for neonatal heart regeneration

Myocardial infarction (MI) leads to cardiomyocyte death, which triggers an immune response that clears debris and restores tissue integrity. In the adult heart, the immune system facilitates scar formation, which repairs the damaged myocardium but compromises cardiac function. In neonatal mice, the heart can regenerate fully without scarring following MI; however, this regenerative capacity is lost by P7. The signals that govern neonatal heart regeneration are unknown. By comparing the immune response to MI in mice at P1 and P14, we identified differences in the magnitude and kinetics of monocyte and macrophage responses to injury. Using a cell-depletion model, we determined that heart regeneration and neoangiogenesis following MI depends on neonatal macrophages. Neonates depleted of macrophages were unable to regenerate myocardia and formed fibrotic scars, resulting in reduced cardiac function and angiogenesis. Immunophenotyping and gene expression profiling of cardiac macrophages from regenerating and nonregenerating hearts indicated that regenerative macrophages have a unique polarization phenotype and secrete numerous soluble factors that may facilitate the formation of new myocardium. Our findings suggest that macrophages provide necessary signals to drive angiogenesis and regeneration of the neonatal mouse heart. Modulating inflammation may provide a key therapeutic strategy to support heart regeneration.

Meis1 regulates postnatal cardiomyocyte cell cycle arrest

The neonatal mammalian heart is capable of substantial regeneration following injury through cardiomyocyte proliferation. However, this regenerative capacity is lost by postnatal day 7 and the mechanisms of cardiomyocyte cell cycle arrest remain unclear. The homeodomain transcription factor Meis1 is required for normal cardiac development but its role in cardiomyocytes is unknown. Here we identify Meis1 as a critical regulator of the cardiomyocyte cell cycle. Meis1 deletion in mouse cardiomyocytes was sufficient for extension of the postnatal proliferative window of cardiomyocytes, and for re-activation of cardiomyocyte mitosis in the adult heart with no deleterious effect on cardiac function. In contrast, overexpression of Meis1 in cardiomyocytes decreased neonatal myocyte proliferation and inhibited neonatal heart regeneration. Finally, we show that Meis1 is required for transcriptional activation of the synergistic CDK inhibitors p15, p16 and p21. These results identify Meis1 as a critical transcriptional regulator of cardiomyocyte proliferation and a potential therapeutic target for heart regeneration.

Angiotensin II Type 2 Receptor Antagonizes Angiotensin II Type 1 Receptor–Mediated Cardiomyocyte Autophagy

Autophagy has emerged as an important process in the pathogenesis of cardiovascular diseases, but the proximal triggers for autophagy are unknown. Angiotensin II plays a central role in the pathogenesis of cardiac hypertrophy and heart failure. In this study, we used angiotensin II type 1 (AT1) and type 2 (AT2) receptor–expressing adenoviruses in cultured neonatal cardiomyocytes to provide the first demonstration that neonatal cardiomyocyte autophagic activity is differentially modulated by AT1 and AT2 receptor subtypes. Angiotensin II stimulation (48 hours) of neonatal cardiomyocytes expressing the AT1 receptor alone (Ad-AT1; 10 multiplicities of infection) induced a significant increase in the number of HcRed-LC3 autophagosomes per cell (17.3±1.6 versus 33.3±4.1 autophagosomes per cell; P<0.05). Coexpression of a high ratio of AT2:AT1 (Ad-AT2:Ad-AT1 multiplicity of infection ratio: 20:5) receptors completely abrogated the AT1-mediated increase in autophagy (9.3±1.4 versus 33.3±4.1 autophagosomes per cell; P<0.05). Treatment with the AT2 receptor antagonist PD123319 did not reverse the AT2-mediated antiautophagic effect. AT1- and AT2-mediated autophagic responses were also assessed in cardiomyocytes from a genetic model that exhibits neonatal myocardial growth suppression. In these neonate myocyte cultures, AT1 receptor activation induced a marked increase in the number of myocytes containing cytoplasmic vacuoles compared with the control (22.7±4.1% versus 1.1±0.6%; P<0.001) and was characterized by a nonapoptotic autophagic phenotype. The incidence of cardiomyocyte autophagic vacuolization in this myocyte population decreased dramatically to only 0.4±0.2% in myocytes infected with a high ratio of Ad-AT2:Ad-AT1. This study provides the first description of reciprocal regulation of cardiomyocyte autophagic induction by the AT1 and AT2 receptor subtypes.

The angiotensin II type 2 (AT2) receptor: an enigmatic seven transmembrane receptor.

Angiotensin II (AngII) interacts with two receptor subtypes, AT1 and AT2, belonging to the seven transmembrane receptor superfamily. Pharmacological investigations initially suggested that AT2 receptors antagonize AT1 effects. Data from AT2 receptor transgenic and knock-out mice have not been entirely consistent with this interpretation. At the cellular level, a clear mechanistic model of AT2 transduction and signalling has yet to emerge. The AT2 receptor displays the hallmark motifs and signature residues of a G protein-coupled receptor (GPCR), but fails to demonstrate most of the classic features of GPCR signalling. In recent years, unbiased screens for AT2-interacting proteins have identified novel partner proteins involved in AT2 signalling, providing new insight into the mechanisms of AT2 action. A growing body of evidence suggests that the AT2 receptor is constitutively active (i.e. signals without AngII). This review critically evaluates controversies surrounding physiological functions and signalling mechanisms of the AT2 receptor, primarily in a cardiovascular context. Recent advances in the field are highlighted and findings challenging the concept that the AT2 receptor is a conventional angiotensin receptor are considered.

A neonatal blueprint for cardiac regeneration

Adult mammals undergo minimal regeneration following cardiac injury, which severely compromises cardiac function and contributes to the ongoing burden of heart failure. In contrast, the mammalian heart retains a transient capacity for cardiac regeneration during fetal and early neonatal life. Recent studies have established the importance of several evolutionarily conserved mechanisms for heart regeneration in lower vertebrates and neonatal mammals including induction of cardiomyocyte proliferation, epicardial cell activation, angiogenesis, extracellular matrix deposition and immune cell infiltration. In this review, we provide an up-to-date account of the molecular and cellular basis for cardiac regeneration in lower vertebrates and neonatal mammals. The historical context for these recent findings and their ramifications for the future development of cardiac regenerative therapies are also discussed.

Expression, Regulation and Putative Nutrient-Sensing Function of Taste GPCRs in the Heart

G protein-coupled receptors (GPCRs) are critical for cardiovascular physiology. Cardiac cells express >100 nonchemosensory GPCRs, indicating that important physiological and potential therapeutic targets remain to be discovered. Moreover, there is a growing appreciation that members of the large, distinct taste and odorant GPCR families have specific functions in tissues beyond the oronasal cavity, including in the brain, gastrointestinal tract and respiratory system. To date, these chemosensory GPCRs have not been systematically studied in the heart. We performed RT-qPCR taste receptor screens in rodent and human heart tissues that revealed discrete subsets of type 2 taste receptors (TAS2/Tas2) as well as Tas1r1 and Tas1r3 (comprising the umami receptor) are expressed. These taste GPCRs are present in cultured cardiac myocytes and fibroblasts, and by in situ hybridization can be visualized across the myocardium in isolated cardiac cells. Tas1r1 gene-targeted mice (Tas1r1Cre/Rosa26tdRFP) strikingly recapitulated these data. In vivo taste receptor expression levels were developmentally regulated in the postnatal period. Intriguingly, several Tas2rs were upregulated in cultured rat myocytes and in mouse heart in vivo following starvation. The discovery of taste GPCRs in the heart opens an exciting new field of cardiac research. We predict that these taste receptors may function as nutrient sensors in the heart.