Understanding how cell identity transitions occur and whether there are multiple paths between the same beginning and end states are questions of wide interest. Here we show that acquisition of naive pluripotency can follow transcriptionally and mechanistically distinct routes. Starting from post-implantation epiblast stem cells (EpiSCs), one route advances through a mesodermal state prior to naive pluripotency induction, whereas another transiently resembles the early inner cell mass and correspondingly gains greater developmental potency. These routes utilize distinct signaling networks and transcription factors but subsequently converge on the same naive endpoint, showing surprising flexibility in mechanisms underlying identity transitions and suggesting that naive pluripotency is a multidimensional attractor state. These route differences are reconciled by precise expression of Oct4 as a unifying, essential, and sufficient feature. We propose that fine-tuned regulation of this "transition factor" underpins multidimensional access to naive pluripotency, offering a conceptual framework for understanding cell identity transitions.

Fluorescence recovery after photobleaching (FRAP) has been useful in delineating cardiac myofilament biology, and innovations in fluorophore chemistry have expanded the array of microscopic assays used. However, one assumption in FRAP is the irreversible photobleaching of fluorescent proteins after laser excitation. Here we demonstrate reversible photobleaching regarding the photoconvertible fluorescent protein mEos3.2. We used CRISPR/Cas9 genome editing in human induced pluripotent stem cells (hiPSCs) to knock-in mEos3.2 into the COOH terminus of titin to visualize sarcomeric titin incorporation and turnover. Upon cardiac induction, the titin-mEos3.2 fusion protein is expressed and integrated in the sarcomeres of hiPSC-derived cardiomyocytes (CMs). STORM imaging shows M-band clustered regions of bound titin-mEos3.2 with few soluble titin-mEos3.2 molecules. FRAP revealed a baseline titin-mEos3.2 fluorescence recovery of 68% and half-life of ~1.2 h, suggesting a rapid exchange of sarcomeric titin with soluble titin. However, paraformaldehyde-fixed and permeabilized titin-mEos3.2 hiPSC-CMs surprisingly revealed a 55% fluorescence recovery. Whole cell FRAP analysis in paraformaldehyde-fixed, cycloheximide-treated, and untreated titin-mEos3.2 hiPSC-CMs displayed no significant differences in fluorescence recovery. FRAP in fixed HEK 293T expressing cytosolic mEos3.2 demonstrates a 58% fluorescence recovery. These data suggest that titin-mEos3.2 is subject to reversible photobleaching following FRAP. Using a mouse titin-eGFP model, we demonstrate that no reversible photobleaching occurs. Our results reveal that reversible photobleaching accounts for the majority of titin recovery in the titin-mEos3.2 hiPSC-CM model and should warrant as a caution in the extrapolation of reliable FRAP data from specific fluorescent proteins in long-term cell imaging.

Engineered 3D cardiac tissue constructs (ECTCs) can replicate complex cardiac physiology under normal and pathological conditions. Currently, most measurements of ECTC contractility are either made isometrically, with fixed length and without control of the applied force, or auxotonically against a variable force, with the length changing during the contraction. The "I-Wire" platform addresses the unmet need to control the force applied to ECTCs while interrogating their passive and active mechanical and electrical characteristics. A six-well plate with inserted PDMS casting molds containing neonatal rat cardiomyocytes cultured with fibrin for 13-15 days is mounted on the motorized mechanical stage of an inverted microscope equipped with a fast sCMOS camera. A calibrated flexible probe provides strain load of the ECTC via lateral displacement, and the microscope detects the deflections of both the probe and the ECTC. The ECTCs exhibited longitudinally aligned cardiomyocytes with well-developed sarcomeric structure, recapitulated the Frank-Starling force-tension relationship, and demonstrated expected transmembrane action potentials, electrical and mechanical restitutions, and responses to both beta-adrenergic stimulation and blebbistatin. The I-Wire platform enables creation and mechanical and electrical characterization of ECTCs, and hence can be valuable in the study of cardiac diseases, drug screening, drug development, and the qualification of cells for tissue-engineered regenerative medicine.Statement of SignificanceThere is a growing interest in creating engineered heart tissue constructs for basic cardiac research, applied research in cardiac pharmacology, and repair of damaged hearts. We address an unmet need to characterize fully the performance of these tissues with our simple "I-Wire" assay that allows application of controlled forces to three-dimensional cardiac fiber constructs and measurement of both the electrical and mechanical properties of the construct. The advantage of I-Wire over other approaches is that the constructs being measured are truly three-dimensional, rather than a single layer of cells grown within a microfluidic device. We anticipate that the I-Wire will be extremely useful for the evaluation of myocardial constructs created using cardiomyocytes derived from human induced pluripotent stem cells. (C) 2016 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Introduction: The giant myofilament protein titin is a critical determinant of myofibril elasticity and sarcomere structure in striated muscle. Although significant advances have been made in our understanding of the biophysical and mechanical properties of titin, the mechanisms governing sarcomeric titin turnover are poorly understood. Our goal is to establish a cardiomyocyte cell culture model that would enable visualization of sarcomeric titin turnover, in real-time. Methods and Results: To directly visualize titin incorporation and turnover in cardiac sarcomeres, we used CRISPR/Cas9 genome editing in human induced pluripotent stem cells (hiPSC) to knock-in a photoconvertible fluorescent protein, mEos3.2, into the C-terminus of titin to produce hiPSC-derived cardiomyocytes (hiPSC-CM) that express endogenous sarcomeric titin-mEos3.2 fusion proteins. Cardiomyocyte induction of the titin-mEos3.2 hiPSC line demonstrated that the titin-mEos3.2 fusion protein is expressed in the sarcomeres in the expected st...

Induced pluripotent stem cells (iPSCs) from patients' somatic tissues provide a viable source to create autologous cardiomyocytes (CMs) for potential cardiac-related cell therapies. However, a gap between the generation of iPSC-derived cardiomyocytes (iPSC–CMs) and the successful intra-cardiac engraftment of the cells to restore heart function remains to be bridged. Clinical data reporting engraftment of cells within human heart tissue has not been without its challenges, with significant cell loss from the site of delivery due to the physical stress of the cardiac cycle and the hostile inflammatory response within the infarct zone [ 1 Hofmann M. Wollert K.C. Meyer G.P. Menke A. Arseniev L. Hertenstein B. et al. Monitoring of bone marrow cell homing into the infarcted human myocardium. Circulation. 2005; 111: 2198-2202 Crossref PubMed Scopus (833) Google Scholar , 2 Schierling W. Kunz-Schughart L.A. Muders F. Riegger G.A. Griese D.P. Fates of genetically engineered haematopoietic and mesenchymal stem cell grafts in normal and injured rat hearts. J. Tissue Eng. Regen. Med. 2008; 2: 354-364 Crossref PubMed Scopus (7) Google Scholar , 3 Segers V.F. Lee R.T. Stem-cell therapy for cardiac disease. Nature. 2008; 451: 937-942 Crossref PubMed Scopus (996) Google Scholar ]. Hydrogels have been proven to support the survival of multiple cell types and have served as a platform for cell transplantation [ 4 Kutschka I. Chen I.Y. Kofidis T. Arai T. von Degenfeld G. Sheikh A.Y. et al. Collagen matrices enhance survival of transplanted cardiomyoblasts and contribute to functional improvement of ischemic rat hearts. Circulation. 2006; 114: I167-I173 PubMed Google Scholar , 5 Suuronen E.J. Veinot J.P. Wong S. Kapila V. Price J. Griffith M. et al. Tissue-engineered injectable collagen-based matrices for improved cell delivery and vascularization of ischemic tissue using CD133+ progenitors expanded from the peripheral blood. Circulation. 2006; 114: I138-I144 Crossref PubMed Scopus (128) Google Scholar ]. Yet the use of tissue-adhesive, temperature-sensitive hydrogels to deliver iPSC-derived cardiomyocytes to infarcted heart remains to be explored. Therefore, we developed a polymer hydrogel to encapsulate, deliver, and integrate iPSC–CMs into infarcted myocardium to restore heart function.

Little is known about the right ventricular (RV) proteome in human heart failure (HF), including possible differences compared to the left ventricular (LV) proteome. We used 2-dimensional differential in-gel electrophoresis (pH: 4-7, 10-150 kDa), followed by liquid chromatography tandem mass spectrometry, to compare the RV and LV proteomes in 12 explanted human hearts. We used Western blotting and multiple-reaction monitoring for protein verification and RNA sequencing for messenger RNA and protein expression correlation. In all 12 hearts, the right ventricles (RVs) demonstrated differential expression of 11 proteins relative to the left ventricles (LVs), including lesser expression of CRYM, TPM1, CLU, TXNL1, and COQ9 and greater expression of TNNI3, SAAI, ERP29, ACTN2, HSPB2, and NDUFS3. Principal-components analysis did not suggest RV-versus-LV proteome partitioning. In the nonischemic RVs (n = 6), 7 proteins were differentially expressed relative to the ischemic RVs (n = 6), including increased expression of CRYM, B7Z964, desmin, ANXA5, and MIME and decreased expression of SERPINA1 and ANT3. Principal-components analysis demonstrated partitioning of the nonischemic and ischemic RV proteomes, and gene ontology analysis identified differences in hemostasis and atherosclerosis-associated networks. There were no proteomic differences between RVs with echocardiographic dysfunction (n = 8) and those with normal function (n = 4). Messenger RNA and protein expression did not correlate consistently, suggesting a major role for RV posttranscriptional protein expression regulation. Differences in contractile, cytoskeletal, metabolic, signaling, and survival pathways exist between the RV and the LV in HF and may be related to the underlying HF etiology and differential posttranscriptional regulation.

Rationale: The lack of measurable single-cell contractility of human-induced pluripotent stem cell-derived cardiac myocytes (hiPSC-CMs) currently limits the utility of hiPSC-CMs for evaluating contractile performance for both basic research and drug discovery.Objective: To develop a culture method that rapidly generates contracting single hiPSC-CMs and allows quantification of cell shortening with standard equipment used for studying adult CMs.Methods and Results: Single hiPSC-CMs were cultured for 5 to 7 days on a 0.4- to 0.8-mm thick mattress of undiluted Matrigel (mattress hiPSC-CMs) and compared with hiPSC-CMs maintained on a control substrate (<0.1-mm thick 1: 60 diluted Matrigel, control hiPSC-CMs). Compared with control hiPSC-CMs, mattress hiPSC-CMs had more rod-shape morphology and significantly increased sarcomere length. Contractile parameters of mattress hiPSC-CMs measured with video-based edge detection were comparable with those of freshly isolated adult rabbit ventricular CMs. Morphological and contractile properties of mattress hiPSC-CMs were consistent across cryopreserved hiPSC-CMs generated independently at another institution. Unlike control hiPSC-CMs, mattress hiPSC-CMs display robust contractile responses to positive inotropic agents, such as myofilament calcium sensitizers. Mattress hiPSC-CMs exhibit molecular changes that include increased expression of the maturation marker cardiac troponin I and significantly increased action potential upstroke velocity because of a 2-fold increase in sodium current (I-Na).Conclusions: The Matrigel mattress method enables the rapid generation of robustly contracting hiPSC-CMs and enhances maturation. This new method allows quantification of contractile performance at the single-cell level, which should be valuable to disease modeling, drug discovery, and preclinical cardiotoxicity testing.

Human induced pluripotent stem cell derived cardiomyocytes (hiPSC-CMs) have great potential as tools for human heart disease modeling and drug discovery. However, their contractile properties have not been routinely evaluated; as current methods are not accessible for most laboratories. We sought to develop a more efficient method to evaluate hiPSC-CM mechanical properties, at the single cell level. Individual hiPSC-CMs were cultured on a hydrogel based platform, termed the “hydrogel mattress,” and their cellular contractile properties evaluated using video-based edge detection. We found that hiPSC-CMs maintained on the mattress reproducibly exhibited robust cell shortening, in dramatic contrast to hiPSC-CMs maintained in a standard manner. We further found that contraction and peak cell shortening amplitude of hiPSC-CMs on mattress was comparable to that of freshly isolated adult ventricular mouse CM. Importantly, hiPSC-CMs maintained on the mattress exhibited several characteristics of a native CM, in terms of myocyte elongation, calcium handling and pharmacological response. Finally, using this platform, we could calculate the traction force generated by individual CMs. In summary, the Hydrogel mattress platform is a simple and reliable in vitro platform that not only enables the quantification of contractile performance of isolated hiPSC-CMs, but also enhances CM maturation. This flexible platform can be extended to in vitro disease modeling, drug discovery and cardiotoxicity testing.

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) have great potential as tools for cell based therapy, disease modeling and drug discovery. However, their contractile properties have not been routinely evaluated; as methods to do so are cumbersome and not easily translated to standard laboratories. We sought to develop a more efficient protocol to evaluate hiPSC-CM mechanical properties, at the single cell level. Individual hiPSC-CMs were cultured on a hydrogel based platform, termed the hydrogel mattress, and their cellular contractile properties were evaluated using video based edge detection (Figure A). We found individual hiPSC-CMs maintained for 5 days on the mattress assumed an elongated shape (Figure B) and exhibited robust, reproducible cell shortening (~10% versus <1% for standard culture). We further found hiPSC-CM cellular contraction and peak cell shortening amplitude was comparable to that of freshly isolated adult rabbit and mouse ventricular CMs (Figure C). In addition, hiPSC-CMs maintained on the mattress exhibited characteristics of mature CMs including action potential waveform, calcium handling and pharmacological response to myofilament calcium sensitizers. We further show a positive correlation with the established traction force microscopy method. We demonstrate the hydrogel mattress platform enables routine characterization and quantification of contractile performance of isolated hiPSC-CMs. This platform can be extended to in vitro disease modeling, drug discovery and drug induced cardiotoxicity testing. Further, the mattress can be easily adapted to virtually any laboratory, thus streamlining cellular mechanical evaluation of individual hiPSC-CMs in vitro.

AIMSAccumulating evidence suggest that sarcomere signalling complexes play a pivotal role in cardiomyocyte hypertrophy by communicating stress signals to the nucleus to induce gene expression. Ankyrin repeat domain 1 (ANKRD1) is a transcriptional regulatory protein that also associates with sarcomeric titin; however, the exact role of ANKRD1 in the heart remains to be elucidated. We therefore aimed to examine the role of ANKRD1 in cardiomyocyte hypertrophic signalling.METHODS AND RESULTSIn neonatal rat ventricular myocytes, we found that ANKRD1 is part of a sarcomeric signalling complex that includes ERK1/2 and cardiac transcription factor GATA4. Treatment with hypertrophic agonist phenylephrine (PE) resulted in phosphorylation of ERK1/2 and GATA4 followed by nuclear translocation of the ANKRD1/ERK/GATA4 complex. Knockdown of Ankrd1 attenuated PE-induced phosphorylation of ERK1/2 and GATA4, inhibited nuclear translocation of the ANKRD1 complex, and prevented cardiomyocyte growth. Mice lacking Ankrd1 are viable with normal cardiac function. Chronic PE infusion in wild-type mice induced significant cardiac hypertrophy with reactivation of the cardiac fetal gene program which was completely abrogated in Ankrd1 null mice. In contrast, ANKRD1 does not play a role in haemodynamic overload as Ankrd1 null mice subjected to transverse aortic constriction developed cardiac hypertrophy comparable to wild-type mice.CONCLUSIONOur study reveals a novel role for ANKRD1 as a selective regulator of PE-induced signalling whereby ANKRD1 recruits and localizes GATA4 and ERK1/2 in a sarcomeric macro-molecular complex to enhance GATA4 phosphorylation with subsequent nuclear translocation of the ANKRD1 complex to induce hypertrophic gene expression.

Organ development is a highly regulated process involving the coordinated proliferation and differentiation of diverse cellular populations. The pathways regulating cell proliferation and their effects on organ growth are complex and for many organs incompletely understood. In all vertebrate species, the cardiac natriuretic peptides (ANP and BNP) are produced by cardiomyocytes in the developing heart. However, their role during cardiogenesis is not defined. Using the embryonic zebrafish and neonatal mammalian cardiomyocytes we explored the natriuretic peptide signaling network during myocardial development. We observed that the cardiac natriuretic peptides ANP and BNP and the guanylate cyclase-linked natriuretic peptide receptors Npr1 and Npr2 are functionally redundant during early cardiovascular development. In addition, we demonstrate that low levels of the natriuretic peptides preferentially activate Npr3, a receptor with Gi activator sequences, and increase cardiomyocyte proliferation through inhibition of adenylate cyclase. Conversely, high concentrations of natriuretic peptides reduce cardiomyocyte proliferation through activation of the particulate guanylate cyclase-linked natriuretic peptide receptors Npr1 and Npr2, and activation of protein kinase G. These data link the cardiac natriuretic peptides in a complex hierarchy modulating cardiomyocyte numbers during development through opposing effects on cardiomyocyte proliferation mediated through distinct cyclic nucleotide signaling pathways.

Titin is the largest known protein and a critical determinant of myofibril elasticity and sarcomere structure in striated muscle. Accumulating evidence that mRNA transcripts are post-transcriptionally regulated by specific motifs located in the flanking untranslated regions (UTRs) led us to consider the role of titin 5'-UTR in regulating its translational efficiency. Titin 5'-UTR is highly homologous between human, mouse, and rat, and sequence analysis revealed the presence of a stem-loop and two upstream AUG codons (uAUGs) converging on a shared in frame stop codon. We generated a mouse titin 5'-UTR luciferase reporter construct and targeted the stem-loop and each uAUG for mutation. The wild-type and mutated constructs were transfected into the cardiac HL-1 cell line and primary neonatal rat ventricular myocytes (NRVM). SV40 driven 5'-UTR luciferase activity was significantly suppressed by wild-type titin 5'-UTR (similar to 70% in HL-1 cells and similar to 60% in NRVM). Mutating both uAUGs was found to alleviate titin 5'-UTR suppression, while eliminating the stem-loop had no effect. Treatment with various growth stimuli: pacing, PMA or neuregulin had no effect on titin 5'-UTR luciferase activity. Doxorubicin stress stimuli reduced titin 5'-UTR suppression, while H2O2 had no effect. A reported single nucleotide polymorphism (SNP) rs13422986 at position -4 of the uAUG2 was introduced and found to further repress titin 5'-UTR luciferase activity. We conclude that the uAUG motifs in titin 5'-UTR serve as translational repressors in the control of titin gene expression, and that mutations/SNPs of the uAUGs or doxorubicin stress could alter titin translational efficiency. (C) 2014 Elsevier Inc. All rights reserved.

Aim: To evaluate the efficacy of electrically conductive, biocompatible composite scaffolds in modulating the cardiomyogenic differentiation of human mesenchymal stem cells (hMSCs). Materials & methods: Electrospun scaffolds of poly(epsilon-caprolactone) with or without carbon nanotubes were developed to promote the in vitro cardiac differentiation of hMSCs. Results: Results indicate that hMSC differentiation can be enhanced by either culturing in electrically conductive, carbon nanotube-containing composite scaffolds without electrical stimulation in the presence of 5-azacytidine, or extrinsic electrical stimulation in nonconductive poly(epsilon-caprolactone) scaffolds without carbon nanotube and azacytidine. Conclusion: This study suggests a first step towards improving hMSC cardiomyogenic differentiation for local delivery into the infarcted myocardium. Original submitted 23 July 2012; Revised submitted 31 October 2012; Published online 27 March 2013

Ankyrin repeat domain 1 protein (Ankrd1), also known as cardiac ankyrin repeat protein (CARP), increases dramatically after tissue injury, and its overexpression improves aspects of wound healing. Reports that Ankrd1/CARP protein stability may affect cardiovascular organization, together with our findings that the protein is crucial to stability of the cardiomyocyte sarcomere and increased in wound healing, led us to compare the contribution of Ankrd1/CARP stability to its abundance. We found that the 26S proteasome is the dominant regulator of Ankrd1/CARP degradation, and that Ankrd1/CARP half-life is significantly longer in cardiomyocytes (h) than endothelial cells (min). In addition, higher endothelial cell density decreased the abundance of the protein without affecting steady state mRNA levels. Taken together, our data and that of others indicate that Ankrd1/CARP is highly regulated at multiple levels of its expression. The striking difference in protein half-life between a muscle and a non-muscle cell type suggests that post-translational proteolysis is correlated with the predominantly structural versus regulatory role of the protein in the two cell types.

Doxorubicin (Adriamycin) is an effective anti-cancer drug, but its clinical usage is limited by a dose-dependent cardiotoxicity characterized by widespread sarcomere disarray and loss of myofilaments. Cardiac ankyrin repeat protein (CARP, ANKRD1) is a transcriptional regulatory protein that is extremely susceptible to doxorubicin; however, the mechanism(s) of doxorubicin-induced CARP depletion and its specific role in cardiomyocytes have not been completely defined. We report that doxorubicin treatment in cardiomyocytes resulted in inhibition of CARP transcription, depletion of CARP protein levels, inhibition of myofilament gene transcription, and marked sarcomere disarray. Knockdown of CARP with small interfering RNA (siRNA) similarly inhibited myofilament gene transcription and disrupted cardiomyocyte sarcomere structure. Adenoviral overexpression of CARP, however, was unable to rescue the doxorubicin-induced sarcomere disarray phenotype. Doxorubicin also induced depletion of the cardiac transcription factor GATA4 in cardiomyocytes. CARP expression is regulated in part by GATA4, prompting us to examine the relationship between GATA4 and CARP in cardiomyocytes. We show in co-transfection experiments that GATA4 operates upstream of CARP by activating the proximal CARP promoter. GATA4-siRNA knockdown in cardiomyocytes inhibited CARP expression and myofilament gene transcription, and induced extensive sarcomere disarray. Adenoviral overexpression of GATA4 (AdV-GATA4) in cardiomyocytes prior to doxorubicin exposure maintained GATA4 levels, modestly restored CARP levels, and attenuated sarcomere disarray. Interestingly, siRNA-mediated depletion of CARP completely abolished the Adv-GATA4 rescue of the doxorubicin-induced sarcomere phenotype. These data demonstrate co-dependent roles for GATA4 and CARP in regulating sarcomere gene expression and maintaining sarcomeric organization in cardiomyocytes in culture. The data further suggests that concurrent depletion of GATA4 and CARP in cardiomyocytes by doxorubicin contributes in large part to myofibrillar disarray and the overall pathophysiology of anthracycline cardiomyopathy.

1 New Advances in Cardiac Phenotyping 2 3 4 Ronglih Liao, Bruno K. Podesser, and Chee Chew Lim 5 6 Cardiac Muscle Research Laboratory, Cardiovascular Division, Department of 7 Medicine, Brigham and Women’s Hospital and Harvard Medical School, Boston 8 Massachusetts, 2 Ludwig Boltzmann Cluster for Cardiovascular Research, Core Unit for 9 Biomedical Research, Allgemeines Krankenhaus Wien, Medical University of Vienna, 10 Austria, Division of Cardiovascular Medicine, Department of Medicine, Vanderbilt 11 University School of Medicine, Nashville, Tennessee 12 13 14 15 Address for correspondence: 16 Chee Chew Lim, PhD Ronglih Liao, PhD 17 Vanderbilt University Medical Center Cardiac Muscle Research Laboratory 18 2220 Pierce Ave, PRB 332 Brigham and Women’s Hospital 19 Nashville, TN 37232 Harvard Medical School 20 email: chee.lim@vanderbilt.edu 77 Avenue Louis Pasteur, NRB 431 21 22 Boston, MA 02115 23 24 email: rliao@rics.bwh.harvard.edu 25 26 Articles in PresS. Am J Physiol Heart Circ Physiol (May 25, 2012). doi:10.1152/ajpheart.00333.2012

The isolated retrograde-perfused Langendorff heart and the isolated ejecting heart have, over many decades, resulted in fundamental discoveries that form the underpinnings of our current understanding of the biology and physiology of the heart. These two experimental methodologies have proven invaluable in studying pharmacological effects on myocardial function, metabolism, and vascular reactivity and in the investigation of clinically relevant disease states such as ischemia-reperfusion injury, diabetes, obesity, and heart failure. With the advent of the genomics era, the isolated mouse heart preparation has gained prominence as an ex vivo research tool for investigators studying the impact of gene modification in the intact heart. This review summarizes the historical development of the isolated heart and provides a practical guide for the establishment of the Langendorff and ejecting heart preparations with a particular emphasis on the murine heart. In addition, current applications and novel methods of recording cardiovascular parameters in the isolated heart preparation will be discussed. With continued advances in methodological recordings, the isolated mouse heart preparation will remain physiologically relevant for the foreseeable future, serving as an integral bridge between in vitro assays and in vivo approaches.

Endothelial progenitor cells (EPCs) are mobilized into the vascular space and home to damaged tissues, where they promote repair in part through a process of angiogenesis. Neuregulins (NRGs) are ligands in the epidermal growth factor family that signal through type I receptor tyrosine kinases in the erbB family (erbB2, erbB3, and erbB4) and regulate endothelial cell biology, promoting angiogenesis. Stimuli such as ischemia and exercise that promote EPC mobilization also induce cleavage and release of transmembrane NRG from cardiac microvascular endothelial cells (CMECs). We hypothesized that NRG/erbB signaling may regulate EPC biology. Using an embryonic (e)EPC cell line that homes to and repairs injured myocardium, we were able to detect erbB2 and erbB3 transcripts. Identical receptor expression was found in EPCs isolated from rat bone marrow and human whole blood. NRG treatment of eEPCs induces phosphorylation of kinases including Akt, GSK-3β, and Erk1/2 and the nuclear accumulation and transcriptional activation of β-catenin. NRG does not induce eEPC proliferation or migration but does protect eEPCs against serum deprivation-induced apoptosis. These results suggest a role for tissue-derived NRG in the regulation of EPC survival.

Introduction: ErbB2 plays a critical role in developing and adult heart. Neuregulin-1beta (Nrg) modulates PI3Kinase, MAPK, and Src/FAK signalling pathways in cardiac myocytes and thereby modulates cell survival and other phenotypes. Nrg is activated in the heart by ischemia/reperfusion injury, leading us to hypothesize that Nrg/ErbB modulates myocyte-matrix and myocyte-myocyte coupling and the response of the heart to ischemic injury at the intercalated disk. Methods: Co-immunoprecipitation assays were performed to assess interactions between ErbB receptors and components of the FAC. Isolated mouse hearts were perfused according to the Langerdorff method, and subjected 15 min ischemia followed by 30 min of reperfusion (I/R). Hearts were fixed and immunostaining, histological and electron microscopy analysis was performed. Results: Nrg induced ErbB2/ErbB4 co-immunoprecipitation with Src, FAK, p130CAS, and Paxilin in isolated myocytes. In isolated hearts subjected to I/R injury, phosphorylation of erbB2, FA...