A calcineurin-Hoxb13 axis regulates growth mode of mammalian cardiomyocytes.

A major factor in the progression to heart failure in humans is the inability of the adult heart to repair itself after injury. We recently demonstrated that the early postnatal mammalian heart is capable of regeneration following injury through proliferation of preexisting cardiomyocytes1,2 and that Meis1, a three amino acid loop extension (TALE) family homeodomain transcription factor, translocates to cardiomyocyte nuclei shortly after birth and mediates postnatal cell cycle arrest3. Here we report that Hoxb13 acts as a cofactor of Meis1 in postnatal cardiomyocytes. Cardiomyocyte-specific deletion of Hoxb13 can extend the postnatal window of cardiomyocyte proliferation and reactivate the cardiomyocyte cell cycle in the adult heart. Moreover, adult Meis1-Hoxb13 double-knockout hearts display widespread cardiomyocyte mitosis, sarcomere disassembly and improved left ventricular systolic function following myocardial infarction, as demonstrated by echocardiography and magnetic resonance imaging. Chromatin immunoprecipitation with sequencing demonstrates that Meis1 and Hoxb13 act cooperatively to regulate cardiomyocyte maturation and cell cycle. Finally, we show that the calcium-activated protein phosphatase calcineurin dephosphorylates Hoxb13 at serine-204, resulting in its nuclear localization and cell cycle arrest. These results demonstrate that Meis1 and Hoxb13 act cooperatively to regulate cardiomyocyte maturation and proliferation and provide mechanistic insights into the link between hyperplastic and hypertrophic growth of cardiomyocytes.

Adducin Regulates Sarcomere Disassembly During Cardiomyocyte Mitosis.

BACKGROUND: Recent interest in understanding cardiomyocyte cell cycle has been driven by potential therapeutic applications in cardiomyopathy. However, despite recent advances, cardiomyocyte mitosis remains a poorly understood process. For example, it is unclear how sarcomeres are disassembled during mitosis to allow the abscission of daughter cardiomyocytes. METHODS: Here, we use a proteomics screen to identify adducin, an actin capping protein previously not studied in cardiomyocytes, as a regulator of sarcomere disassembly. We generated many adeno-associated viruses and cardiomyocyte-specific genetic gain-of-function models to examine the role of adducin in neonatal and adult cardiomyocytes in vitro and in vivo. RESULTS: We identify adducin as a regulator of sarcomere disassembly during mammalian cardiomyocyte mitosis. α/γ-adducins are selectively expressed in neonatal mitotic cardiomyocytes, and their levels decline precipitously thereafter. Cardiomyocyte-specific overexpression of various splice isoforms and phospho-isoforms of α-adducin in identified Thr445/Thr480 phosphorylation of a short isoform of α-adducin as a potent inducer of neonatal cardiomyocyte sarcomere disassembly. Concomitant overexpression of this α-adducin variant along with γ-adducin resulted in stabilization of the adducin complex and persistent sarcomere disassembly in adult mice, which is mediated by interaction with α-actinin. CONCLUSIONS: These results highlight an important mechanism for coordinating cytoskeletal morphological changes during cardiomyocyte mitosis.

Mitochondrial fatty acid utilization increases chromatin oxidative stress in cardiomyocytes.

The inability of adult mammalian cardiomyocytes to proliferate underpins the development of heart failure following myocardial injury. Although the newborn mammalian heart can spontaneously regenerate for a short period of time after birth, this ability is lost within the first week after birth in mice, partly due to increased mitochondrial reactive oxygen species (ROS) production which results in oxidative DNA damage and activation of DNA damage response. This increase in ROS levels coincides with a postnatal switch from anaerobic glycolysis to fatty acid (FA) oxidation by cardiac mitochondria. However, to date, a direct link between mitochondrial substrate utilization and oxidative DNA damage is lacking. Here, we generated ROS-sensitive fluorescent sensors targeted to different subnuclear compartments (chromatin, heterochromatin, telomeres, and nuclear lamin) in neonatal rat ventricular cardiomyocytes, which allowed us to determine the spatial localization of ROS in cardiomyocyte nuclei upon manipulation of mitochondrial respiration. Our results demonstrate that FA utilization by the mitochondria induces a significant increase in ROS detection at the chromatin level compared to other nuclear compartments. These results indicate that mitochondrial metabolic perturbations directly alter the nuclear redox status and that the chromatin appears to be particularly sensitive to the prooxidant effect of FA utilization by the mitochondria.

Mechanism of Eccentric Cardiomyocyte Hypertrophy Secondary to Severe Mitral Regurgitation.

BACKGROUND: Primary valvular heart disease is a prevalent cause of morbidity and mortality in both industrialized and developing countries. Although the primary consequence of valvular heart disease is myocardial dysfunction, treatment of valvular heart diseases centers around valve repair or replacement rather than prevention or reversal of myocardial dysfunction. This is particularly evident in primary mitral regurgitation (MR), which invariably results in eccentric hypertrophy and left ventricular (LV) failure in the absence of timely valve repair or replacement. The mechanism of LV dysfunction in primary severe MR is entirely unknown. METHODS: Here, we developed the first mouse model of severe MR. Valvular damage was achieved by severing the mitral valve leaflets and chords with iridectomy scissors, and MR was confirmed by echocardiography. Serial echocardiography was performed to follow up LV morphology and systolic function. Analysis of cardiac tissues was subsequently performed to evaluate valve deformation, cardiomyocyte morphology, LV fibrosis, and cell death. Finally, dysregulated pathways were assessed by RNA-sequencing analysis and immunofluorescence. RESULTS: In the ensuing 15 weeks after the induction of MR, gradual LV dilatation and dysfunction occurred, resulting in severe systolic dysfunction. Further analysis revealed that severe MR resulted in a marked increase in cardiac mass and increased cardiomyocyte length but not width, with electron microscopic evidence of sarcomere disarray and the development of sarcomere disruption. From a mechanistic standpoint, severe MR resulted in activation of multiple components of both the mammalian target of rapamycin and calcineurin pathways. Inhibition of mammalian target of rapamycin signaling preserved sarcomeric structure and prevented LV remodeling and systolic dysfunction. Immunohistochemical analysis uncovered a differential pattern of expression of the cell polarity regulator Crb2 (crumbs homolog 2) along the longitudinal axis of cardiomyocytes and close to the intercalated disks in the MR hearts. Electron microscopy images demonstrated a significant increase in polysome localization in close proximity to the intercalated disks and some areas along the longitudinal axis in the MR hearts. CONCLUSIONS: These results indicate that LV dysfunction in response to severe MR is a form of maladaptive eccentric cardiomyocyte hypertrophy and outline the link between cell polarity regulation and spatial localization protein synthesis as a pathway for directional cardiomyocyte growth.

Neonatal Heart Regeneration: Comprehensive Literature Review.

BACKGROUND: The adult mammalian heart is incapable of meaningful functional recovery after injury, and thus promoting heart regeneration is 1 of the most important therapeutic targets in cardiovascular medicine. In contrast to the adult mammalian heart, the neonatal mammalian heart is capable of regeneration after various types of injury. Since the first report in 2011, a number of groups have reported their findings on neonatal heart regeneration. The current review provides a comprehensive analysis of heart regeneration studies in neonatal mammals conducted to date, outlines lessons learned, and poses unanswered questions. METHODS: We performed a PubMed search using the keywords "neonatal" and "heart" and "regeneration." In addition, we assessed all publications that cited the first neonatal heart regeneration reports: Porrello et al, Science, Feb 2011 for apical resection injury; Porrello et al, PNAS, Dec 2012 for coronary ligation injury; and Mahmoud et al, Nature Methods, Jan 2014 for surgical methodology. Publications were examined for surgical models used, timing of surgery, and postinjury assessment including anatomic, histological, and functional assessment, as well as conclusions drawn. RESULTS: We found 30 publications that performed neonatal apical resection, 19 publications that performed neonatal myocardial infarction by coronary artery ligation, and 6 publications that performed cryoinjury using liquid nitrogen-cooled metal probes. Both apical resection and ischemic infarction injury in neonatal mice result in a robust regenerative response, mediated by cardiomyocyte proliferation. On the other hand, several reports have demonstrated that cryoinjury is associated with incomplete heart regeneration in neonatal mice. Not surprisingly, several studies suggest that injury size, as well as surgical and histological techniques, can strongly influence the observed regenerative response and final conclusions. Studies have utilized these neonatal cardiac injury models to identify factors that either inhibit or stimulate heart regeneration. CONCLUSIONS: Overall, there is consensus that both apical resection and coronary ligation injuries during the first 2 days of life result in heart regeneration in neonatal mammals, whereas cryoinjury was not associated with a similar regenerative response. This regenerative response is mediated by proliferation of preexisting cardiomyocytes, and is modifiable by injury size and surgical technique, as well as metabolic, immunologic, genetic, and environmental factors.

Cardiogenic genes expressed in cardiac fibroblasts contribute to heart development and repair.

RATIONALE: Cardiac fibroblasts are critical to proper heart function through multiple interactions with the myocardial compartment, but appreciation of their contribution has suffered from incomplete characterization and lack of cell-specific markers. OBJECTIVE: To generate an unbiased comparative gene expression profile of the cardiac fibroblast pool, identify and characterize the role of key genes in cardiac fibroblast function, and determine their contribution to myocardial development and regeneration. METHODS AND RESULTS: High-throughput cell surface and intracellular profiling of cardiac and tail fibroblasts identified canonical mesenchymal stem cell and a surprising number of cardiogenic genes, some expressed at higher levels than in whole heart. While genetically marked fibroblasts contributed heterogeneously to interstitial but not cardiomyocyte compartments in infarcted hearts, fibroblast-restricted depletion of one highly expressed cardiogenic marker, T-box 20, caused marked myocardial dysmorphology and perturbations in scar formation on myocardial infarction. CONCLUSIONS: The surprising transcriptional identity of cardiac fibroblasts, the adoption of cardiogenic gene programs, and direct contribution to cardiac development and repair provoke alternative interpretations for studies on more specialized cardiac progenitors, offering a novel perspective for reinterpreting cardiac regenerative therapies.

Hypoxia-induced stabilization of HIF2A promotes cardiomyocyte proliferation by attenuating DNA damage.

Introduction: Gradual exposure to a chronic hypoxic environment leads to cardiomyocyte proliferation and improved cardiac function in mouse models through a reduction in oxidative DNA damage. However, the upstream transcriptional events that link chronic hypoxia to DNA damage have remained obscure. Aim: We sought to determine whether hypoxia signaling mediated by the hypoxia-inducible factor 1 or 2 (HIF1A or HIF2A) underlies the proliferation phenotype that is induced by chronic hypoxia. Methods and Results: We used genetic loss-of-function models using cardiomyocyte-specific HIF1A and HIF2A gene deletions in chronic hypoxia. We additionally characterized a cardiomyocyte-specific HIF2A overexpression mouse model in normoxia during aging and upon injury. We performed transcriptional profiling with RNA-sequencing on cardiac tissue, from which we verified candidates at the protein level. We find that HIF2A - rather than HIF1A - mediates hypoxia-induced cardiomyocyte proliferation. Ectopic, oxygen-insensitive HIF2A expression in cardiomyocytes reveals the cell-autonomous role of HIF2A in cardiomyocyte proliferation. HIF2A overexpression in cardiomyocytes elicits cardiac regeneration and improvement in systolic function after myocardial infarction in adult mice. RNA-sequencing reveals that ectopic HIF2A expression attenuates DNA damage pathways, which was confirmed with immunoblot and immunofluorescence. Conclusion: Our study provides mechanistic insights about a new approach to induce cardiomyocyte renewal and mitigate cardiac injury in the adult mammalian heart. In light of evidence that DNA damage accrues in cardiomyocytes with aging, these findings may help to usher in a new therapeutic approach to overcome such age-related changes and achieve regeneration.

Mitochondrial Substrate Utilization Regulates Cardiomyocyte Cell Cycle Progression.

The neonatal mammalian heart is capable of regeneration for a brief window of time after birth. However, this regenerative capacity is lost within the first week of life, which coincides with a postnatal shift from anaerobic glycolysis to mitochondrial oxidative phosphorylation, particularly towards fatty-acid utilization. Despite the energy advantage of fatty-acid beta-oxidation, cardiac mitochondria produce elevated rates of reactive oxygen species when utilizing fatty acids, which is thought to play a role in cardiomyocyte cell-cycle arrest through induction of DNA damage and activation of DNA-damage response (DDR) pathway. Here we show that inhibiting fatty-acid utilization promotes cardiomyocyte proliferation in the postnatatal heart. First, neonatal mice fed fatty-acid deficient milk showed prolongation of the postnatal cardiomyocyte proliferative window, however cell cycle arrest eventually ensued. Next, we generated a tamoxifen-inducible cardiomyocyte-specific, pyruvate dehydrogenase kinase 4 (PDK4) knockout mouse model to selectively enhance oxidation of glycolytically derived pyruvate in cardiomyocytes. Conditional PDK4 deletion resulted in an increase in pyruvate dehydrogenase activity and consequently an increase in glucose relative to fatty-acid oxidation. Loss of PDK4 also resulted in decreased cardiomyocyte size, decreased DNA damage and expression of DDR markers and an increase in cardiomyocyte proliferation. Following myocardial infarction, inducible deletion of PDK4 improved left ventricular function and decreased remodelling. Collectively, inhibition of fatty-acid utilization in cardiomyocytes promotes proliferation, and may be a viable target for cardiac regenerative therapies.

Redox Regulation of Heart Regeneration: An Evolutionary Tradeoff.

Heart failure is a costly and deadly disease, affecting over 23 million patients worldwide, half of which die within 5 years of diagnosis. The pathophysiological basis of heart failure is the inability of the adult heart to regenerate lost or damaged myocardium. Although limited myocyte turnover does occur in the adult heart, it is insufficient for restoration of contractile function (Nadal-Ginard, 2001; Laflamme et al., 2002; Quaini et al., 2002; Hsieh et al., 2007; Bergmann et al., 2009, 2012). In contrast to lower vertebrates (Poss et al., 2002; Poss, 2007; Jopling et al., 2010; Kikuchi et al., 2010; Chablais et al., 2011; González-Rosa et al., 2011; Heallen et al., 2011), adult mammalian heart cardiomyogenesis following injury is very limited (Nadal-Ginard, 2001; Laflamme et al., 2002; Quaini et al., 2002; Bergmann et al., 2009, 2012) and is insufficient to restore normal cardiac function. Studies in the late 90s elegantly mapped the DNA synthesis and cell cycle dynamics of the mammalian heart during development and following birth (Soonpaa et al., 1996; Soonpaa and Field, 1997, 1998), where they showed that DNA synthesis drops significantly around birth with low-level DNA synthesis few days after birth. Around P5 to P7, cardiomyocytes undergo a final round of DNA synthesis without cytokinesis, and the majority become binucleated and exit the cell cycle permanently. Therefore, due to the similarities between the immature mammalian heart and lower vertebrates (Poss, 2007; Walsh et al., 2010), it became important to determine whether they have similar regenerative abilities. Recently, we demonstrated that removal of up to 15% of the apex of the left ventricle of postnatal day 1 (P1) mice results in complete regeneration within 3 weeks without any measurable fibrosis and cardiac dysfunction (Porrello et al., 2011). This response is characterized by robust cardiomyocyte proliferation with gradual restoration of normal cardiac morphology. In addition to the histological evidence of proliferating myocytes, genetic fate-mapping studies confirmed that the majority of newly formed cardiomyocytes are derived from proliferation of preexisting cardiomyocytes (Porrello et al., 2011). More recently, we established an ischemic injury model where the left anterior descending coronary artery was ligated in P1 neonates (Porrello et al., 2013). The injury response was similar to the resection model, with robust cardiomyocyte proliferation throughout the myocardium, as well as restoration of normal morphology by 21 days. However, this regenerative capacity is lost by P7, after which injury results in the typical cardiomyocyte hypertrophy and scar-formation characteristic of the adult mammalian heart. Not surprisingly, the loss of this regenerative capacity coincides with binucleation and cell cycle exit of cardiomyocytes (Soonpaa et al., 1996; Walsh et al., 2010). An important approach toward a deeper understanding the loss of cardiac regenerative capacity in mammals is to first consider why , and not only how , this happens. Regeneration of the early postnatal heart following resection or ischemic infarction involves replacement of lost myocardium and vasculature with restoration of normal myocardial thickness and architecture, with long-term normalization of systolic function. Why would the heart permanently forego such a remarkable regenerative program shortly after birth? The answer may lie in within the fundamental principal of evolutionary tradeoff.

Abnormal mitochondrial L-arginine transport contributes to the pathogenesis of heart failure and rexoygenation injury.

BACKGROUND: Impaired mitochondrial function is fundamental feature of heart failure (HF) and myocardial ischemia. In addition to the effects of heightened oxidative stress, altered nitric oxide (NO) metabolism, generated by a mitochondrial NO synthase, has also been proposed to impact upon mitochondrial function. However, the mechanism responsible for arginine transport into mitochondria and the effect of HF on such a process is unknown. We therefore aimed to characterize mitochondrial L-arginine transport and to investigate the hypothesis that impaired mitochondrial L-arginine transport plays a key role in the pathogenesis of heart failure and myocardial injury. METHODS AND RESULTS: In mitochondria isolated from failing hearts (sheep rapid pacing model and mouse Mst1 transgenic model) we demonstrated a marked reduction in L-arginine uptake (p<0.05 and p<0.01 respectively) and expression of the principal L-arginine transporter, CAT-1 (p<0.001, p<0.01) compared to controls. This was accompanied by significantly lower NO production and higher 3-nitrotyrosine levels (both p<0.05). The role of mitochondrial L-arginine transport in modulating cardiac stress responses was examined in cardiomyocytes with mitochondrial specific overexpression of CAT-1 (mtCAT1) exposed to hypoxia-reoxygenation stress. mtCAT1 cardiomyocytes had significantly improved mitochondrial membrane potential, respiration and ATP turnover together with significantly decreased reactive oxygen species production and cell death following mitochondrial stress. CONCLUSION: These data provide new insights into the role of L-arginine transport in mitochondrial biology and cardiovascular disease. Augmentation of mitochondrial L-arginine availability may be a novel therapeutic strategy for myocardial disorders involving mitochondrial stress such as heart failure and reperfusion injury.

Effect of oxygen on cardiac differentiation in mouse iPS cells: role of hypoxia inducible factor-1 and Wnt/beta-catenin signaling.

BACKGROUND: Disturbances in oxygen levels have been found to impair cardiac organogenesis. It is known that stem cells and differentiating cells may respond variably to hypoxic conditions, whereby hypoxia may enhance stem cell pluripotency, while differentiation of multiple cell types can be restricted or enhanced under hypoxia. Here we examined whether HIF-1alpha modulated Wnt signaling affected differentiation of iPS cells into beating cardiomyocytes. OBJECTIVE: We investigated whether transient and sustained hypoxia affects differentiation of cardiomyocytes derived from murine induced pluripotent stem (iPS) cells, assessed the involvement of HIF-1alpha (hypoxia-inducible factor-1alpha) and the canonical Wnt pathway in this process. METHODS: Embryoid bodies (EBs) derived from iPS cells were differentiated into cardiomyocytes and were exposed either to 24 h normoxia or transient hypoxia followed by a further 13 days of normoxic culture. RESULTS: At 14 days of differentiation, 59 ± 2% of normoxic EBs were beating, whilst transient hypoxia abolished beating at 14 days and EBs appeared immature. Hypoxia induced a significant increase in Brachyury and islet-1 mRNA expression, together with reduced troponin C expression. Collectively, these data suggest that transient and sustained hypoxia inhibits maturation of differentiating cardiomyocytes. Compared to normoxia, hypoxia increased HIF-1alpha, Wnt target and ligand genes in EBs, as well as accumulation of HIF-1alpha and beta-catenin in nuclear protein extracts, suggesting involvement of the Wnt/beta-catenin pathway. CONCLUSION: Hypoxia impairs cardiomyocyte differentiation and activates Wnt signaling in undifferentiated iPS cells. Taken together the study suggests that oxygenation levels play a critical role in cardiomyocyte differentiation and suggest that hypoxia may play a role in early cardiogenesis.

Nerve growth factor stimulates cardiac regeneration via cardiomyocyte proliferation in experimental heart failure.

Although the adult heart likely retains some regenerative capacity, heart failure (HF) typically remains a progressive disorder. We hypothesise that alterations in the local environment contribute to the failure of regeneration in HF. Previously we showed that nerve growth factor (NGF) is deficient in the failing heart and here we hypothesise that diminished NGF limits the cardiac regenerative response in HF. The capacity of NGF to augment cardiac regeneration was tested in a zebrafish model of HF. Cardiac injury with a HF phenotype was induced in zebrafish larvae at 72 hours post fertilization (hpf) by exposure to aristolochic acid (AA, 2.5 µM, 72-75 hpf). By 168 hpf, AA induced HF and death in 37.5% and 20.8% of larvae respectively (p<0.001). NGF mRNA expression was reduced by 42% (p<0.05). The addition of NGF (50 ng/ml) after exposure to AA reduced the incidence of HF by 50% (p<0.01) and death by 65% (p<0.01). Mechanistically, AA mediated HF was characterised by reduced cardiomyocyte proliferation as reflected by a 6.4 fold decrease in BrdU+ cardiomyocytes (p<0.01) together with features of apoptosis and loss of cardiomyocytes. Following AA exposure, NGF increased the abundance of BrdU+ cardiomyocytes in the heart by 4.8 fold (p<0.05), and this was accompanied by a concomitant significant increase in cardiomyocyte numbers. The proliferative effect of NGF on cardiomyocytes was not associated with an anti-apoptotic effect. Taken together the study suggests that NGF stimulates a regenerative response in the failing zebrafish heart, mediated by stimulation of cardiomyocyte proliferation.