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.

Existing cardiomyocytes generate cardiomyocytes at a low rate after birth in mice.

The mammalian heart has long been considered a postmitotic organ, implying that the total number of cardiomyocytes is set at birth. Analysis of cell division in the mammalian heart is complicated by cardiomyocyte binucleation shortly after birth, which makes it challenging to interpret traditional assays of cell turnover [Laflamme MA, Murray CE (2011) Nature 473(7347):326-335; Bergmann O, et al. (2009) Science 324(5923):98-102]. An elegant multi-isotope imaging-mass spectrometry technique recently calculated the low, discrete rate of cardiomyocyte generation in mice [Senyo SE, et al. (2013) Nature 493(7432):433-436], yet our cellular-level understanding of postnatal cardiomyogenesis remains limited. Herein, we provide a new line of evidence for the differentiated α-myosin heavy chain-expressing cardiomyocyte as the cell of origin of postnatal cardiomyogenesis using the "mosaic analysis with double markers" mouse model. We show limited, life-long, symmetric division of cardiomyocytes as a rare event that is evident in utero but significantly diminishes after the first month of life in mice; daughter cardiomyocytes divide very seldom, which this study is the first to demonstrate, to our knowledge. Furthermore, ligation of the left anterior descending coronary artery, which causes a myocardial infarction in the mosaic analysis with double-marker mice, did not increase the rate of cardiomyocyte division above the basal level for up to 4 wk after the injury. The clonal analysis described here provides direct evidence of postnatal mammalian cardiomyogenesis.

Developmental heterogeneity of cardiac fibroblasts does not predict pathological proliferation and activation.

RATIONALE: Fibrosis is mediated partly by extracellular matrix-depositing fibroblasts in the heart. Although these mesenchymal cells are reported to have multiple embryonic origins, the functional consequence of this heterogeneity is unknown. OBJECTIVE: We sought to validate a panel of surface markers to prospectively identify cardiac fibroblasts. We elucidated the developmental origins of cardiac fibroblasts and characterized their corresponding phenotypes. We also determined proliferation rates of each developmental subset of fibroblasts after pressure overload injury. METHODS AND RESULTS: We showed that Thy1(+)CD45(-)CD31(-)CD11b(-)Ter119(-) cells constitute the majority of cardiac fibroblasts. We characterized these cells using flow cytometry, epifluorescence and confocal microscopy, and transcriptional profiling (using reverse transcription polymerase chain reaction and RNA-seq). We used lineage tracing, transplantation studies, and parabiosis to show that most adult cardiac fibroblasts derive from the epicardium, a minority arises from endothelial cells, and a small fraction from Pax3-expressing cells. We did not detect generation of cardiac fibroblasts by bone marrow or circulating cells. Interestingly, proliferation rates of fibroblast subsets on injury were identical, and the relative abundance of each lineage remained the same after injury. The anatomic distribution of fibroblast lineages also remained unchanged after pressure overload. Furthermore, RNA-seq analysis demonstrated that Tie2-derived and Tbx18-derived fibroblasts within each operation group exhibit similar gene expression profiles. CONCLUSIONS: The cellular expansion of cardiac fibroblasts after transaortic constriction surgery was not restricted to any single developmental subset. The parallel proliferation and activation of a heterogeneous population of fibroblasts on pressure overload could suggest that common signaling mechanisms stimulate their pathological response.

Prospective isolation of human embryonic stem cell-derived cardiovascular progenitors that integrate into human fetal heart tissue.

A goal of regenerative medicine is to identify cardiovascular progenitors from human ES cells (hESCs) that can functionally integrate into the human heart. Previous studies to evaluate the developmental potential of candidate hESC-derived progenitors have delivered these cells into murine and porcine cardiac tissue, with inconclusive evidence regarding the capacity of these human cells to physiologically engraft in xenotransplantation assays. Further, the potential of hESC-derived cardiovascular lineage cells to functionally couple to human myocardium remains untested and unknown. Here, we have prospectively identified a population of hESC-derived ROR2(+)/CD13(+)/KDR(+)/PDGFRα(+) cells that give rise to cardiomyocytes, endothelial cells, and vascular smooth muscle cells in vitro at a clonal level. We observed rare clusters of ROR2(+) cells and diffuse expression of KDR and PDGFRα in first-trimester human fetal hearts. We then developed an in vivo transplantation model by transplanting second-trimester human fetal heart tissues s.c. into the ear pinna of a SCID mouse. ROR2(+)/CD13(+)/KDR(+)/PDGFRα(+) cells were delivered into these functioning fetal heart tissues: in contrast to traditional murine heart models for cell transplantation, we show structural and functional integration of hESC-derived cardiovascular progenitors into human heart.

Overexpression of BCL2 enhances survival of human embryonic stem cells during stress and obviates the requirement for serum factors.

The promise of pluripotent stem cells as a research and therapeutic tool is partly undermined by the technical challenges of generating and maintaining these cells in culture. Human embryonic stem cells (hESCs) are exquisitely sensitive to culture conditions, and require constant signaling by growth factors and cell-cell and cell-matrix interactions to prevent apoptosis, senescence, and differentiation. Previous work from our laboratory demonstrated that overexpression of the prosurvival gene BCL2 in mouse embryonic stem cells overrode the requirement of serum factors and feeder cells to maintain mESCs in culture. To determine whether this prosurvival gene could similarly protect hESCs, we generated hESC lines that constitutively or inducibly express BCL2. We find that BCL2 overexpression significantly decreases dissociation-induced apoptosis, resulting in enhanced colony formation from sorted single cells, and enhanced embryoid body formation. In addition, BCL2-hESCs exhibit normal growth in the absence of serum, but require basic fibroblast growth factor to remain undifferentiated. Furthermore, they maintain their pluripotency markers, form teratomas in vivo, and differentiate into all three germ layers. Our data suggest that the BCL2 signaling pathway plays an important role in inhibiting hESC apoptosis, such that its overexpression in hESCs offers both a survival benefit in conditions of stress by resisting apoptosis and obviates the requirement for serum or a feeder layer for maintenance.

Actin-organizing protein palladin modulates C2C12 cell fate determination.

Background: Cell confluency and serum deprivation promote the transition of C2C12 myoblasts into myocytes and subsequence fusion into myotubes. However, despite all myoblasts undergoing the same serum deprivation trigger, their responses vary: whether they become founder myocytes, remain proliferative, or evolve into fusion-competent myocytes remains unclear. We have previously shown that depletion of the scaffolding protein palladin in myoblasts inhibits cell migration and promotes premature muscle differentiation, pointing to its potential significance in muscle development and the necessity for a more in-depth examination of its function in cellular heterogeneity. Methods and results: Here, we showed that the subcellular localization of palladin might contribute to founder-fate cell decision in the early differentiation process. Depleting palladin in C2C12 myoblasts depleted integrin-ß3 plasma membrane localization of and focal adhesion formation at the early stage of myogenesis, decreased kindlin-2 and metavinculin expression during the myotube maturation process, leading to the inability of myocytes to fuse into preexisting mature myotubes. This aligns with previous findings where early differentiation into nascent myotubes occurred but compromised maturation. In contrast, wildtype C2C12 overexpressing the 140-kDa palladin isoform developed a polarized morphology with star-like structures toward other myoblasts. However, this behaviour was not observed in palladin-depleted cells, where the 140-kDa palladin overexpression could not recover cell migration capacity, suggesting other palladin isoforms are also needed to establish cell polarity. Conclusion: Our study identifies a counter-intuitive role for palladin in regulating myoblast-to-myocyte cell fate decisions and impacting their ability to form mature multinucleated myotubes by influencing cell signalling pathways and cytoskeletal organization, necessary for skeletal muscle regeneration and repair studies.

Takotsubo cardiomyopathy as the primary manifestation of decompensated Graves' disease.

Takotsubo cardiomyopathy (TCM) is characterized by transient left ventricular dysfunction with apical ballooning, usually observed in postmenopausal women after a stressful event. We discuss a rare presentation of TCM induced by thyrotoxicosis secondary to Graves' disease. This case raises interesting questions about the pathogenesis, diagnosis, and management of TCM. Learning objectives: 1. To recognize hyperthyroidism as a possible etiology of takutsubo cardiomyopathy.2. To identify the effect of radioiodine contrast on diagnosis of some types of takutsubo cardiomyopathy.

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.

Rad regulation of CaV1.2 channels controls cardiac fight-or-flight response.

Fight-or-flight responses involve ß-adrenergic-induced increases in heart rate and contractile force. In the present study, we uncover the primary mechanism underlying the heart's innate contractile reserve. We show that four protein kinase A (PKA)-phosphorylated residues in Rad, a calcium channel inhibitor, are crucial for controlling basal calcium current and essential for ß-adrenergic augmentation of calcium influx in cardiomyocytes. Even with intact PKA signaling to other proteins modulating calcium handling, preventing adrenergic activation of calcium channels in Rad-phosphosite-mutant mice (4SA-Rad) has profound physiological effects: reduced heart rate with increased pauses, reduced basal contractility, near-complete attenuation of ß-adrenergic contractile response and diminished exercise capacity. Conversely, expression of mutant calcium-channel ß-subunits that cannot bind 4SA-Rad is sufficient to enhance basal calcium influx and contractility to adrenergically augmented levels of wild-type mice, rescuing the failing heart phenotype of 4SA-Rad mice. Hence, disruption of interactions between Rad and calcium channels constitutes the foundation toward next-generation therapeutics specifically enhancing cardiac contractility.

Characterization of active site residues of nitroalkane oxidase.

The flavoenzyme nitroalkane oxidase catalyzes the oxidation of primary and secondary nitroalkanes to the corresponding aldehydes and ketones plus nitrite. The structure of the enzyme shows that Ser171 forms a hydrogen bond to the flavin N5, suggesting that it plays a role in catalysis. Cys397 and Tyr398 were previously identified by chemical modification as potential active site residues. To more directly probe the roles of these residues, the S171A, S171V, S171T, C397S, and Y398F enzymes have been characterized with nitroethane as substrate. The C397S and Y398 enzymes were less stable than the wild-type enzyme, and the C397S enzyme routinely contained a substoichiometric amount of FAD. Analysis of the steady-state kinetic parameters for the mutant enzymes, including deuterium isotope effects, establishes that all of the mutations result in decreases in the rate constants for removal of the substrate proton by approximately 5-fold and decreases in the rate constant for product release of approximately 2-fold. Only the S171V and S171T mutations alter the rate constant for flavin oxidation. These results establish that these residues are not involved in catalysis, but rather are required for maintaining the protein structure.

The electronic role of DNA-functionalized carbon nanotubes: efficacy for in situ polymerization of conducting polymer nanocomposites.

We have found that the polymerization process was 4,500 times faster when a self-doped polyaniline nanocomposite was fabricated using in situ polymerization in the presence of single-stranded DNA-dispersed and -functionalized single-walled carbon nanotubes (ssDNA-SWNTs). More importantly, the quality of the composite was significantly improved: fewer short oligomers were produced, and the self-doped polyaniline backbone had a longer conjugation length and existed in the more stable and conductive emeraldine state. The functionality of the boronic acid group in the composite and the highly improved electronic performance may lead to broad applications of the composite in flexible electronic devices. Blending of preformed polymer with carbon nanotubes is straightforward and widely used to fabricate nanocomposites. We demonstrate that this simple mixing approach might not fully and synergistically combine the merits of each individual component. Surprisingly, these advantages also cannot be obtained using in situ polymerization with preoxidized ssDNA-SWNTs, which is renowned as the "seed" method for production of conducting-polymer nanowires. The electronic structures of the carbon nanotubes and the monomer-nanotube interaction during polymerization greatly impact the kinetics of nanocomposite fabrication and the electronic performance of the resulting composites.

A Nonoxidative Electrochemical Sensor Based on a Self-Doped Polyaniline/Carbon Nanotube Composite for Sensitive and Selective Detection of the Neurotransmitter Dopamine: A Review.

Most of the current techniques for in vivo detection of dopamine exploit the ease of oxidation of this compound. The major problem during the detection is the presence of a high concentration of ascorbic acid that is oxidized at nearly the same potential as dopamine on bare electrodes. Furthermore, the oxidation product of dopamine reacts with ascorbic acid present in samples and regenerates dopamine again, which severely limits the accuracy of the detection. Meanwhile, the product could also form a melanin-like insulating film on the electrode surface, which decreases the sensitivity of the electrode. Various surface modifications on the electrode, new materials for making the electrodes, and new electrochemical techniques have been exploited to solve these problems. Recently we developed a new electrochemical detection method that did not rely on direct oxidation of dopamine on electrodes, which may naturally solve these problems. This approach takes advantage of the high performance of our newly developed poly(anilineboronic acid)/carbon nanotube composite and the excellent permselectivity of the ion-exchange polymer Nafion. The high affinity binding of dopamine to the boronic acid groups of the polymer affects the electrochemical properties of the polyaniline backbone, which act as the basis for the transduction mechanism of this non-oxidative dopamine sensor. The unique reduction capability and high conductivity of single-stranded DNA functionalized single-walled carbon nanotubes greatly improved the electrochemical activity of the polymer in a physiologically-relevant buffer, and the large surface area of the carbon nanotubes increased the density of the boronic acid receptors. The high sensitivity and selectivity of the sensor show excellent promise toward molecular diagnosis of Parkinson's disease. In this review, we will focus on the discussion of this novel detection approach, the new interferences in this detection approach, and how to eliminate these interferences toward in vivo and in vitro detection of the neurotransmitter dopamine.

Interference of ascorbic acid in the sensitive detection of dopamine by a nonoxidative sensing approach.

The electrochemistry of a poly(anilineboronic acid)/carbon nanotube composite was studied in the presence of dopamine and ascorbic acid. To understand the binding affinity of dopamine and ascorbic acid to the boronic acid functional groups in the composite, the association constants between the diol groups in dopamine and ascorbic acid and the boronic acid were experimentally determined using a fluorescence-based binding assay. The results demonstrate that ascorbic acid could severely interfere with the detection of dopamine in nonoxidative boronic acid-binding approaches: Ascorbic acid was able to electrocatalytically reduce the fully oxidized polyaniline backbone during the electrochemical oxidation process; similarly to dopamine, ascorbic acid was also able to bind to the boronic acid groups through its planar diol group even though the binding affinity is much lower. The examination of the dopamine transduction mechanism and ascorbic acid interference mechanism in this nonoxidative approach will benefit the design of future boronic acid-based sensors.

Enhanced sensitivity for biosensors: multiple functions of DNA-wrapped single-walled carbon nanotubes in self-doped polyaniline nanocomposites.

A nanocomposite of poly(anilineboronic acid), a self-doped polyaniline, with ss-DNA-wrapped single-walled carbon nanotubes (ss-DNA/SWNTs) was fabricated on a gold electrode by in situ electrochemical polymerization of 3-aminophenylboronic acid monomers in the presence of ssDNA/SWNTs. We used this nanocomposite to detect nanomolar concentrations of dopamine and found that the sensitivity increased 4 orders of magnitude compared to the detection at an electrode modified with only poly(anilineboronic acid). For the first time, this work reports the multiple functions of the ss-DNA/SWNTs in the fabrication and biosensor application of a self-doped polyaniline/ss-DNA/SWNT nanocomposite. First, the ss-DNA/SWNTs acted as effective molecular templates during polymerization of self-doped polyaniline so that not only was the polymerization speed increased but also the quality of the polymer was greatly improved. Second, they functioned as novel active stabilizers after the polymerization, significantly enhancing the stability of the film. Furthermore, the ss-DNA/SWNTs also acted as conductive polyanionic doping agents in the resulting polyaniline film, which showed enhanced conductivity and redox activity. Finally, the large surface area of carbon nanotubes greatly increased the density of the functional groups available for sensitive detection of the target analyte. We envision that polyaniline with other functional groups as well as other conducting polymers may be produced for different targeted applications by this approach.