Young Jae Nam

Heart repair by reprogramming non-myocytes with cardiac transcription factors

The adult mammalian heart possesses little regenerative potential following injury. Fibrosis due to activation of cardiac fibroblasts impedes cardiac regeneration and contributes to loss of contractile function, pathological remodelling and susceptibility to arrhythmias. Cardiac fibroblasts account for a majority of cells in the heart and represent a potential cellular source for restoration of cardiac function following injury through phenotypic reprogramming to a myocardial cell fate. Here we show that four transcription factors, GATA4, HAND2, MEF2C and TBX5, can cooperatively reprogram adult mouse tail-tip and cardiac fibroblasts into beating cardiac-like myocytes in vitro. Forced expression of these factors in dividing non-cardiomyocytes in mice reprograms these cells into functional cardiac-like myocytes, improves cardiac function and reduces adverse ventricular remodelling following myocardial infarction. Our results suggest a strategy for cardiac repair through reprogramming fibroblasts resident in the heart with cardiogenic transcription factors or other molecules.

Reprogramming of human fibroblasts toward a cardiac fate

Reprogramming of mouse fibroblasts toward a myocardial cell fate by forced expression of cardiac transcription factors or microRNAs has recently been demonstrated. The potential clinical applicability of these findings is based on the minimal regenerative potential of the adult human heart and the limited availability of human heart tissue. An initial but mandatory step toward clinical application of this approach is to establish conditions for conversion of adult human fibroblasts to a cardiac phenotype. Toward this goal, we sought to determine the optimal combination of factors necessary and sufficient for direct myocardial reprogramming of human fibroblasts. Here we show that four human cardiac transcription factors, including GATA binding protein 4, Hand2, T-box5, and myocardin, and two microRNAs, miR-1 and miR-133, activated cardiac marker expression in neonatal and adult human fibroblasts. After maintenance in culture for 4–11 wk, human fibroblasts reprogrammed with these proteins and microRNAs displayed sarcomere-like structures and calcium transients, and a small subset of such cells exhibited spontaneous contractility. These phenotypic changes were accompanied by expression of a broad range of cardiac genes and suppression of nonmyocyte genes. These findings indicate that human fibroblasts can be reprogrammed to cardiac-like myocytes by forced expression of cardiac transcription factors with muscle-specific microRNAs and represent a step toward possible therapeutic application of this reprogramming approach.

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.

Regulation of p53 tetramerization and nuclear export by ARC

Inactivation of the transcription factor p53 is central to carcinogenesis. Yet only approximately one-half of cancers have p53 loss-of-function mutations. Here, we demonstrate a mechanism for p53 inactivation by apoptosis repressor with caspase recruitment domain (ARC), a protein induced in multiple cancer cells. The direct binding in the nucleus of ARC to the p53 tetramerization domain inhibits p53 tetramerization. This exposes a nuclear export signal in p53, triggering Crm1-dependent relocation of p53 to the cytoplasm. Knockdown of endogenous ARC in breast cancer cells results in spontaneous tetramerization of endogenous p53, accumulation of p53 in the nucleus, and activation of endogenous p53 target genes. In primary human breast cancers with nuclear ARC, p53 is almost always WT. Conversely, nearly all breast cancers with mutant p53 lack nuclear ARC. We conclude that nuclear ARC is induced in cancer cells and negatively regulates p53.

Induction of diverse cardiac cell types by reprogramming fibroblasts with cardiac transcription factors

Various combinations of cardiogenic transcription factors, including Gata4 (G), Hand2 (H), Mef2c (M) and Tbx5 (T), can reprogram fibroblasts into induced cardiac-like myocytes (iCLMs) in vitro and in vivo. Given that optimal cardiac function relies on distinct yet functionally interconnected atrial, ventricular and pacemaker (PM) cardiomyocytes (CMs), it remains to be seen which subtypes are generated by direct reprogramming and whether this process can be harnessed to produce a specific CM of interest. Here, we employ a PM-specific Hcn4-GFP reporter mouse and a spectrum of CM subtype-specific markers to investigate the range of cellular phenotypes generated by reprogramming of primary fibroblasts. Unexpectedly, we find that a combination of four transcription factors (4F) optimized for Hcn4-GFP expression does not generate beating PM cells due to inadequate sarcomeric protein expression and organization. However, applying strict single-cell criteria to GHMT-reprogrammed cells, we observe induction of diverse cellular phenotypes, including those resembling immature forms of all three major cardiac subtypes (i.e. atrial, ventricular and pacemaker). In addition, we demonstrate that cells induced by GHMT are directly reprogrammed and do not arise from an Nxk2.5+ progenitor cell intermediate. Taken together, our results suggest a remarkable degree of plasticity inherent to GHMT reprogramming and provide a starting point for optimization of CM subtype-specific reprogramming protocols.

The apoptosis inhibitor ARC undergoes ubiquitin-proteasomal-mediated degradation in response to death stimuli: identification of a degradation-resistant mutant.

Efficient induction of apoptosis requires not only the activation of death-promoting proteins but also the inactivation of inhibitors of cell death. ARC (apoptosis repressor with caspase recruitment domain) is an endogenous inhibitor of apoptosis that antagonizes both central apoptosis pathways. Despite its potent inhibition of cell death, cells that express abundant ARC eventually succumb. A possible explanation is that ARC protein levels decrease dramatically in response to death stimuli. The mechanisms that mediate decreases in ARC protein levels during apoptosis and whether these decreases initiate the subsequent cell death are not known. Here we show that endogenous ARC protein levels decrease in response to death stimuli in a variety of cell contexts as well as in a model of myocardial ischemia-reperfusion in intact mice. Decreases in ARC protein levels are not explained by alterations in the abundance of ARC transcripts. Rather, pulse-chase experiments show that decreases in steady state ARC protein levels during apoptosis result from marked destabilization of ARC protein. ARC protein destabilization, in turn, is mediated by the ubiquitin-proteasomal pathway, as mutation of ARC ubiquitin acceptor residues stabilizes ARC protein and preserves its steady state levels during apoptosis. In addition, this degradation-resistant ARC mutant exhibits improved cytoprotection. We conclude that decreases in ARC protein levels in response to death stimuli are mediated by increased ARC protein degradation via the ubiquitin-proteasomal pathway. Moreover, these data demonstrate that decreases in ARC protein levels are a trigger, and not merely a consequence, of the ensuing cell death.

Heart repair by cardiac reprogramming

Nearly one million Americans suffer a myocardial infarction each year, many of whom progress to heart failure, the single most common hospital discharge diagnosis in those over age 65 (ref. 1). The adult human heart has a limited regenerative response to injury such that the loss or dysfunction of cardiomyocytes results in reduced pump function, often culminating in heart failure, life-threatening arrhythmias and sudden death. Numerous clinical trials over the past decade have introduced a variety of autologous stem and progenitor cell types into failing human hearts as a strategy for regenerating new myocardium, but exogenous stem cells seem to give rise to few if any new muscle cells, bringing into question the biological basis for the limited functional improvement. Thus, there is still a dire need for innovative strategies for heart regeneration and repair. A series of recent studies in rodents has reported the ability of exogenous transcription factors and miRNAs to reprogram cardiac fibroblasts into cardiomyocytes2–6, resulting in dramatic improvement of cardiac contractility after myocardial infarction2,3,6. Much work remains to optimize such reprogramming methods and to define the mechanistic basis for functional improvement in this setting, but this initial evidence suggests a potentially transformative new approach for heart repair. Whereas skeletal and smooth muscle cells can be generated from fibroblasts by ectopic expression of single transcription factors7,8, the cardiac muscle phenotype has proven more elusive, as no single factor has been shown to be capable of generating cardiomyocytes from fibroblasts. An important step toward possible therapeutic generation of cardiomyocytes was provided by Ieda et al.9, who showed that three transcription factors—Gata4, Mef2c and Tbx5 (together referred to as GMT)—could activate cardiac gene expression in cultured mouse fibroblasts with a low efficiency of between 5 and 15%. Activation of cardiac genes by these factors seems to require precise levels of expression of the factors. Inclusion of a fourth factor, Hand2, in the GMT cocktail substantially increases reprogramming efficiency2. Several cardiac miRNAs have also been reported to activate cardiac gene expression in fibroblasts with low efficiency4. Because cardiac transcription factors and miRNAs function within complex regulatory networks involving feed-forward and autoregulatory interactions, it is likely that multiple combinations of these cardiac regulators may initiate the cardiac phenotype. Reprogramming by cardiac transcription factors and miRNAs seems to involve direct conversion of fibroblasts toward a cardiomyocyte-like fate without transition through a stem cell intermediate. This approach therefore differs from reprogramming methods that involve the generation of induced pluripotent stem cells and subsequent commitment to the cardiac lineage. Direct cardiac reprogramming of fibroblasts also circumvents potential teratogenicity and immunogenicity of induced pluripotent stem cells. Induced cardiac-like myocytes (iCLMs) seem to be relatively immature, and only a very low fraction show action potentials and strong contractility, well-developed sarcomeres, and binucleation, characteristics of adult cardiomyocytes. Thus, maturation to adult cardiac phenotypes may require prolonged periods in culture or additional factors not yet identified. Initial efforts to reprogram human fibroblasts to a cardiac fate have recently found a set of at least five factors different from the factor combination in mouse fibroblasts that can activate cardiac gene expression in adult human cardiac and dermal fibroblasts and in neonatal human foreskin fibroblasts10. Cardiac reprogramming of human fibroblasts is slower and less efficient than in mouse fibroblasts, perhaps reflecting stable epigenetic events that need to be overcome. Nearly half of the cells in the heart are fibroblasts, and their activation during heart disease leads to fibrosis, which impedes contractility and contributes to conduction abnormalities. Thus, targeting activated cardiac fibroblasts after injury to induce heart repair is particularly attractive (Fig. 1). Retroviruses, which infect only proliferating cells, were used to introduce GMT and GHMT into fibroblasts in the infarct zone of mice after myocardial infarction2,3,5. Lineage-tracing studies with fibroblast markers indicated that newly generated iCLMs were derived from fibroblasts. Figure 1 Heart repair by in vivo reprogramming of nonmyocytes into iCLMs. After myocardial infarction, a viral cocktail of cardiac transcription factors or miRNAs is directly injected into the border zone adjacent to the infarcted myocardium. The forced expression ... There are a few aspects of these studies that warrant consideration. First, the reprogramming efficiency in vivo seems to be higher compared to in vitro, suggesting that the milieu of the intact heart may favor reprogramming in ways that cannot be reproduced in culture. Second, introduction of reprogramming factors results in dramatic functional improvement after myocardial infarction2,3, indicating that, at least in mice, the impact of cardiac reprogramming exceeds the relatively modest and transient effects observed with autologous stem cell transplantation. Finally, the extent of functional improvement after in vivo reprogramming is greater than expected, given the relatively modest number of mature cardiomyocytes generated. This may suggest that reprogramming factors enhance cardiac function through mechanisms beyond simply reprogramming of fibroblasts toward a cardiomyocyte cell fate, perhaps also promoting neoangiogenesis, preventing cardiomyocyte death and/or inhibiting fibroblast proliferation. Although these initial studies point to a potentially promising new approach for heart repair, numerous technical and biological hurdles remain to be overcome. The efficiency of the reprogramming process remains relatively low, and reprogrammed cells show a spectrum of intermediate phenotypes, reflecting incomplete conversion to a mature cardiac phenotype. The latter issue is of concern, given the propensity of arrhythmias to arise from zones of cardiomyocyte heterogeneity11. The long-term stability and integration of reprogrammed cardiomyocytes with native cardiomyocytes also remains to be shown. Further optimization of reprogramming of human fibroblasts and demonstration of the therapeutic efficacy and safety of this approach in large animals is needed. Cells from the cardiac conduction system and vasculature are also lost after cardiac injury, and full restoration of cardiac function after injury will therefore require recreation of multiple cell types. Smooth muscle, endothelial and angioblast-like progenitor cells have been efficiently generated by reprogramming8,12,13 and inclusion of a vascular endothelial growth factor–expressing virus with GMT enhances functional recovery of mice after myocardial infarction, possibly through neovascularization of the injured myocardium6. In addition, forced expression of Tbx3, activated Notch or Tbx18 in working cardiomyocytes is sufficient to generate conduction system cells in vitro14–16 and in vivo16. Reprogramming experiments in rodents requires open chest surgery to directly inject viruses into the infarct zone; in humans, direct delivery of reprogramming factors during coronary artery bypass graft surgery could be a starting point. Given the potential for teratogenic viral insertions in the genome, as well as other complications associated with viral delivery, it will be important to develop nonintegrative methods for safe clinical application. Replacing cardiogenic transcription factors with small molecules or synthetic oligonucleotides with cardiogenic activity has long-term therapeutic possibilities; their combination with catheter-based delivery during a percutaneous coronary artery intervention after myocardial infarction could also reach widespread and effective use for intervention after heart attack. Whereas studies thus far have been limited to the reprogramming of fibroblasts to cardiomyocytes within the infarct zone of hearts after myocardial infarction, it will be of interest to determine whether this approach can also be applied to other forms of acquired and inherited forms of heart disease associated with loss or dysfunction of cardiomyocytes. As most heart diseases are associated with an increase in cardiac fibrosis, this approach may extend beyond post–myocardial infarction therapy. Given our desperate need for entirely new heart repair strategies, further studies are warranted to resolve the current challenges facing in vivo reprogramming approaches. Cellular reprogramming, perhaps in combination with biological scaffolds or other bioengineering strategies, has the potential to provide an alternative or complementary heart repair strategy to cell transplantation–based approaches, which have been in clinical trials for nearly a decade.

Induction of the apoptosis inhibitor ARC by Ras in human cancers

Inhibition of apoptosis is critical for carcinogenesis. ARC (apoptosis repressor with caspase recruitment domain) is an endogenous inhibitor of apoptosis that antagonizes both intrinsic and extrinsic apoptosis pathways. Although normally expressed in striated myocytes and neurons, ARC is markedly induced in a variety of primary human epithelial cancers and renders cancer cells resistant to killing. The mechanisms that mediate the induction of ARC in cancer are unknown. Herein we demonstrate that increases in ARC abundance are stimulated by Ras through effects on transcription and protein stability. Overexpression of activated N-Ras or H-Ras in normal cells is sufficient to increase ARC mRNA and protein levels. Similarly, transgenic expression of activated H-Ras induces ARC in both the normal mammary epithelium and resulting tumors of intact mice. Conversely, knockdown of endogenous N-Ras in breast and colon cancer cells significantly reduces ARC mRNA and protein levels. The promoter of the Nol3 locus, encoding ARC, is activated by N-Ras and H-Ras in a MEK/ERK-dependent manner. Ras also stabilizes ARC protein by suppressing its polyubiquitination and subsequent proteasomal degradation. In addition to the effects of Ras on ARC abundance, ARC mediates Ras-induced cell survival and cell cycle progression. Thus, Ras induces ARC in epithelial cancers, and ARC plays a role in the oncogenic actions of Ras.

The miR-15 Family Regulates Post-natal 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.

The Promise of Cardiac Regeneration by In Situ Lineage Conversion

Although remarkable advances in acute coronary care have significantly improved outcomes following myocardial infarction (MI), survivors often experience progressive heart failure, a devastating condition with limited curative options. Given that the adult heart cannot regenerate heart muscle cells to restore contractile function, there is great demand to develop therapeutic strategies to build new heart muscle (Figure). Thus, many strategies have been conceived to induce de novo generation of cardiomyocytes for heart repair. Figure. Generation of new cardiomyocytes by direct reprogramming of cardiac fibroblasts in situ. Following a myocardial infarction (MI), a fundamental issue remains the irreversible loss of cardiomyocytes with subsequent replacement by fibroblast-derived scar tissue ( left ). Although current pharmacological treatments can slow the progression of heart failure, the ultimate therapeutic goal would be to completely restore normal cardiac function ( right ). Recent work has shown that activated fibroblasts in the MI border zone can be directly converted into functional cardiomyocytes by the addition of transcription factors (cardiogenic genes). In work described in this issue of Circulation , Mohamed et al4 show that specific small molecules can further augment the cardiac reprogramming process in situ. By far the most well-studied approach …