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Aims: To examine the role of the basic Helix-loop-Helix (bHLH) transcription factor HAND1 in embryonic and adult myocardium., Methods and Results: Hand1 is expressed within the cardiomyocytes of the left ventricle (LV) and myocardial cuff between embryonic days (E) 9.5-13.5. Hand gene dosage plays an important role in ventricular morphology and the contribution of Hand1 to congenital heart defects requires further interrogation. Conditional ablation of Hand1 was carried out using either Nkx2.5 knockin Cre (Nkx2.5Cre) or α-myosin heavy chain Cre (αMhc-Cre) driver. Interrogation of transcriptome data via ingenuity pathway analysis reveals several gene regulatory pathways disrupted including translation and cardiac hypertrophy-related pathways. Embryo and adult hearts were subjected to histological, functional, and molecular analyses. Myocardial deletion of Hand1 results in morphological defects that include cardiac conduction system defects, survivable interventricular septal defects, and abnormal LV papillary muscles (PMs). Resulting Hand1 conditional mutants are born at Mendelian frequencies; but the morphological alterations acquired during cardiac development result in, the mice developing diastolic heart failure., Conclusion: Collectively, these data reveal that HAND1 contributes to the morphogenic patterning and maturation of cardiomyocytes during embryogenesis and although survivable, indicates a role for Hand1 within the developing conduction system and PM development., (Published on behalf of the European Society of Cardiology. All rights reserved. © The Author(s) 2019. For permissions, please email: journals.permissions@oup.com.)

Purpose of Review: Many forms of heart disease result in the essentially irreversible loss of cardiomyocytes. The ability to promote cardiomyocyte renewal may be a promising approach to reverse injury in diseased hearts. The purpose of this review is to describe the impact of cardiomyocyte cell cycle activation on cardiac function and structure in several different models of myocardial disease., Recent Findings: Transgenic mice expressing cyclin D2 (D2 mice) exhibit sustained cardiomyocyte renewal in the adult heart. Earlier studies demonstrated that D2 mice exhibited progressive myocardial regeneration in experimental models of myocardial infarction, and that cardiac function was normalized to values seen in sham-operated litter mates by 180 days post-injury. D2 mice also exhibited markedly improved atrial structure in a genetic model of atrial fibrosis. More recent studies revealed that D2 mice were remarkably resistant to heart failure induced by chronic elevated afterload as compared with their wild type (WT siblings), with a 6-fold increase in median survival as well as retention of relatively normal cardiac function. Finally, D2 mice exhibited a progressive recovery in cardiac function to normal levels and a concomitant reduction in adverse myocardial remodeling in an anthracycline cardiotoxicity model. The studies reviewed here make a strong case for the potential utility of inducing cardiomyocyte renewal as a means to treat injured hearts. Several challenges which must be met to develop a viable therapeutic intervention based on these observations are discussed.

Correlative evidence suggests that polyploidization of heart muscle, which occurs naturally in post-natal mammals, creates a barrier to heart regeneration. Here, we move beyond a correlation by demonstrating that experimental polyploidization of zebrafish cardiomyocytes is sufficient to suppress their proliferative potential during regeneration. Initially, we determined that zebrafish myocardium becomes susceptible to polyploidization upon transient cytokinesis inhibition mediated by dominant-negative Ect2. Using a transgenic strategy, we generated adult animals containing mosaic hearts composed of differentially labeled diploid and polyploid-enriched cardiomyocyte populations. Diploid cardiomyocytes outcompeted their polyploid neighbors in producing regenerated heart muscle. Moreover, hearts composed of equivalent proportions of diploid and polyploid cardiomyocytes failed to regenerate altogether, demonstrating that a critical percentage of diploid cardiomyocytes is required to achieve heart regeneration. Our data identify cardiomyocyte polyploidization as a barrier to heart regeneration and suggest that mobilizing rare diploid cardiomyocytes in the human heart will improve its regenerative capacity., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Uniparental parthenotes are considered an unwanted byproduct of in vitro fertilization. In utero parthenote development is severely compromised by defective organogenesis and in particular by defective cardiogenesis. Although developmentally compromised, apparently pluripotent stem cells can be derived from parthenogenetic blastocysts. Here we hypothesized that nonembryonic parthenogenetic stem cells (PSCs) can be directed toward the cardiac lineage and applied to tissue-engineered heart repair. We first confirmed similar fundamental properties in murine PSCs and embryonic stem cells (ESCs), despite notable differences in genetic (allelic variability) and epigenetic (differential imprinting) characteristics. Haploidentity of major histocompatibility complexes (MHCs) in PSCs is particularly attractive for allogeneic cell-based therapies. Accordingly, we confirmed acceptance of PSCs in MHC-matched allotransplantation. Cardiomyocyte derivation from PSCs and ESCs was equally effective. The use of cardiomyocyte-restricted GFP enabled cell sorting and documentation of advanced structural and functional maturation in vitro and in vivo. This included seamless electrical integration of PSC-derived cardiomyocytes into recipient myocardium. Finally, we enriched cardiomyocytes to facilitate engineering of force-generating myocardium and demonstrated the utility of this technique in enhancing regional myocardial function after myocardial infarction. Collectively, our data demonstrate pluripotency, with unrestricted cardiogenicity in PSCs, and introduce this unique cell type as an attractive source for tissue-engineered heart repair.

Aims: Although pharmacological interventions that mobilize stem cells and enhance their homing to damaged tissue can limit adverse post-myocardial infarction (MI) remodelling, cardiomyocyte renewal with this approach is limited. While experimental cell cycle induction can promote cardiomyocyte renewal following MI, this process must compete with the more rapid processes of scar formation and adverse remodelling. The current study tested the hypothesis that the combination of enhanced stem cell mobilization/homing and cardiomyocyte cell cycle induction would result in increased myocardial renewal in injured hearts., Methods and Results: Myocardial infarction was induced by coronary artery ligation in adult MHC-cycD2 transgenic mice (which exhibit constitutive cardiomyocyte cell cycle activity) and their non-transgenic littermates. Mice were then treated with saline or with granulocyte colony-stimulating factor (G-CSF) plus the dipeptidylpeptidase-IV (DPP-IV) inhibitor Diprotin A (DipA) for 7 days. Infarct thickness and cardiomyocyte number/infarct/section were significantly improved in MHC-cycD2 mice with G-CSF plus DipA treatment when compared with MHC-cycD2 transgene expression or G-CSF plus DipA treatment alone. Echocardiographic analyses revealed that stem cell mobilization/homing and cardiomyocyte cell cycle activation had an additive effect on functional recovery., Conclusion: These data strongly suggest that G-CSF plus DPP-IV inhibition, combined with cardiomyocyte cell cycle activation, leads to enhanced myocardial regeneration following MI. The data are also consistent with the notion that altering adverse post-injury remodelling renders the myocardium more permissive for cardiomyocyte repopulation.

Rationale: Telethonin (also known as titin-cap or t-cap) is a 19-kDa Z-disk protein with a unique β-sheet structure, hypothesized to assemble in a palindromic way with the N-terminal portion of titin and to constitute a signalosome participating in the process of cardiomechanosensing. In addition, a variety of telethonin mutations are associated with the development of several different diseases; however, little is known about the underlying molecular mechanisms and telethonin's in vivo function., Objective: Here we aim to investigate the role of telethonin in vivo and to identify molecular mechanisms underlying disease as a result of its mutation., Methods and Results: By using a variety of different genetically altered animal models and biophysical experiments we show that contrary to previous views, telethonin is not an indispensable component of the titin-anchoring system, nor is deletion of the gene or cardiac specific overexpression associated with a spontaneous cardiac phenotype. Rather, additional titin-anchorage sites, such as actin-titin cross-links via α-actinin, are sufficient to maintain Z-disk stability despite the loss of telethonin. We demonstrate that a main novel function of telethonin is to modulate the turnover of the proapoptotic tumor suppressor p53 after biomechanical stress in the nuclear compartment, thus linking telethonin, a protein well known to be present at the Z-disk, directly to apoptosis ("mechanoptosis"). In addition, loss of telethonin mRNA and nuclear accumulation of this protein is associated with human heart failure, an effect that may contribute to enhanced rates of apoptosis found in these hearts., Conclusions: Telethonin knockout mice do not reveal defective heart development or heart function under basal conditions, but develop heart failure following biomechanical stress, owing at least in part to apoptosis of cardiomyocytes, an effect that may also play a role in human heart failure.

Cardiomyocytes exhibit robust proliferative activity during development. After birth, cardiomyocyte proliferation is markedly reduced. Consequently, regenerative growth in the postnatal heart via cardiomyocyte proliferation (and, by inference, proliferation of stem-cell-derived cardiomyocytes) is limited and often insufficient to affect repair following injury. Here, we review studies wherein cardiomyocyte cell cycle proliferation was induced via targeted expression of cyclin D2 in postnatal hearts. Cyclin D2 expression resulted in a greater than 500-fold increase in cell cycle activity in transgenic mice as compared to their nontransgenic siblings. Induced cell cycle activity resulted in infarct regression and concomitant improvement in cardiac hemodynamics following coronary artery occlusion. These studies support the notion that cell-cycle-based strategies can be exploited to drive myocardial repair following injury.

Transforming growth factor-beta (TGF-beta) family members, including TGF-betas, activins, and bone morphogenetic proteins, exert diverse biological activities in cell proliferation, differentiation, apoptosis, embryonic development, and many other processes. These effects are largely mediated by Smad proteins. Smad7 is a negative regulator for the signaling of TGF-beta family members. Dysregulation of Smad7 is associated with pathogenesis of a variety of human diseases. However, the in vivo physiological roles of Smad7 have not been elucidated due to the lack of a mouse model with significant loss of Smad7 function. Here we report generation and initial characterization of Smad7 mutant mice with targeted deletion of the indispensable MH2 domain. The majority of Smad7 mutant mice died in utero due to multiple defects in cardiovascular development, including ventricular septal defect and non-compaction, as well as outflow tract malformation. The surviving adult Smad7 mutant mice had impaired cardiac functions and severe arrhythmia. Further analyses suggest that Smad2/3 phosphorylation was elevated in atrioventricular cushion in the heart of Smad7 mutant mice, accompanied by increased apoptosis in this region. Taken together, these observations pinpoint an important role of Smad7 in the development and function of the mouse heart in vivo.

Aims: Cultured cardiac explants produce a heterogeneous population of cells including a distinctive population of refractile cells described here as small round cardiac explant derived cells (EDCs). The aim of this study was to explore the source, morphology and cardiogenic potential of EDCs., Methods: Transgenic MLC2v-Cre/ZEG, and actin-eGFP mice were used for lineage-tracing of EDCs in vitro and in vivo. C57B16 mice were used as cell transplant recipients of EDCs from transgenic hearts, as well as for the general characterisation of EDCs. The activation of cardiac-specific markers were analysed by: immunohistochemistry with bright field and immunofluorescent microscopy, electron microscopy, PCR and RT-PCR. Functional engraftment of transplanted cells was further investigated with calcium transient studies., Results: Production of EDCs was highly dependent on the retention of blood-derived cells or factors in the cultured explants. These cells shared some characteristics of cardiac myocytes in vitro and survived engraftment in the adult heart in vivo. However, EDCs failed to differentiate into functional cardiac myocytes in vivo as demonstrated by the absence of stimulation-evoked intracellular calcium transients following transplantation into the peri-infarct zone., Conclusions: This study highlights that positive identification based upon one parameter alone such as morphology or immunofluorescene is not adequate to identify the source, fate and function of adult cardiac explant derived cells.

Many forms of pediatric and adult heart disease result from a deficiency in cardiomyocyte number. Through repopulation of the heart with new cardiomyocytes (that is, induction of regenerative cardiac growth), cardiac disease potentially can be reversed, provided that the newly formed myocytes structurally and functionally integrate in the preexisting myocardium. A number of approaches have been utilized to effect regenerative growth of the myocardium in experimental animals. These include interventions aimed at enhancing the ability of cardiomyocytes to proliferate in response to cardiac injury, as well as transplantation of cardiomyocytes or myogenic stem cells into diseased hearts. Here we review efforts to induce myocardial regeneration. We also provide a critical review of techniques currently used to assess cardiac regeneration and functional integration of de novo cardiomyocytes.

Skeletal myoblast transplantation is a potential treatment for congestive heart failure. To study the functional activity of both donor and host myocytes following transplantation, skeletal myoblasts expressing an enhanced green fluorescent protein (EGFP) transgene were transplanted into hearts of nontransgenic recipients, and changes in intracellular calcium concentration ([Ca2+]i) were monitored in donor and host cells. While the vast majority of donor-derived myocytes were observed to be functionally isolated from the host myocardium, a small population of donor myocytes exhibited action potential-induced calcium transients in synchrony with adjacent host cardiomyocytes. In many cases, the durations of these [Ca2+]i transients were heterogeneous compared with those in neighboring host cardiomyocytes. In other studies, EGFP-expressing donor myoblasts were transplanted into the hearts of adult transgenic recipient mice expressing a cardiomyocyte-restricted beta-gal reporter gene. A small population of myocytes was observed to express both reporter transgenes, indicating that the transplanted myoblasts fused with host cardiomyocytes at a very low frequency. These cells also expressed connexin43, a component of gap junctions. Thus engraftment of skeletal myoblasts generated spatial heterogeneity of [Ca2+]i signaling at the myocardial/skeletal muscle interface, most likely as a consequence of fusion events between donor myoblasts and host cardiomyocytes.

The ability to image calcium signals at subcellular levels within the intact depolarizing heart could provide valuable information toward a more integrated understanding of cardiac function. Accordingly, a system combining two-photon excitation with laser-scanning microscopy was developed to monitor electrically evoked [Ca(2+)](i) transients in individual cardiomyocytes within noncontracting Langendorff-perfused mouse hearts. [Ca(2+)](i) transients were recorded at depths

Accurate nuclear identification is crucial for distinguishing the role of cardiac myocytes in intrinsic and experimentally induced regenerative growth of the myocardium. Conventional histologic analysis of myocyte nuclei relies on the optical sectioning capabilities of confocal microscopy in conjunction with immunofluorescent labeling of cytoplasmic proteins such as troponin T, and dyes that bind to double-strand DNA to identify nuclei. Using heart sections from transgenic mice in which the cardiomyocyte-restricted α-cardiac myosin heavy chain promoter targeted the expression of nuclear localized β-galactosidase reporter in >99% of myocytes, we systematically compared the fidelity of conventional myocyte nuclear identification using confocal microscopy, with and without the aid of a membrane marker. The values obtained with these assays were then compared with those obtained with anti-β-galactosidase immune reactivity in the same samples. In addition, we also studied the accuracy of anti-GATA4 immunoreactivity for myocyte nuclear identification. Our results demonstrate that, although these strategies are capable of identifying myocyte nuclei, the level of interobserver agreement and margin of error can compromise accurate identification of rare events, such as cardiomyocyte apoptosis and proliferation. Thus these data indicate that morphometric approaches based on segmentation are justified only if the margin of error for measuring the event in question has been predetermined and deemed to be small and uniform. We also illustrate the value of a transgene-based approach to overcome these intrinsic limitations of identifying myocyte nuclei. This latter approach should prove quite useful when measuring rare events. [ABSTRACT FROM AUTHOR]

Postnatal cardiac hypertrophies have traditionally been classified into physiological or pathological hypertrophies. Both of them are induced by hemodynamic load. Cardiac postnatal hypertrophic growth is regarded as a part of the cardiac maturation process that is independent of the cardiac working load. However, the functional significance of this biological event has not been determined, mainly because of the difficulty in creating an experimental condition for testing the growth potential of functioning heart in the absence of hemodynamic load. Recently, we generated a novel transgenic mouse model (αMHC-BMP10) in which the cardiac-specific growth factor bone morphogenetic protein 10 (BMP10) is overexpressed in postnatal myocardium. These αMHC-BMP10 mice appear to have normal cardiogenesis throughout embryogenesis, but develop to smaller hearts within 6 weeks after birth. xMHC-BMP10 hearts are about half the normal size with 100% penetrance. Detailed morphometric analysis of cardiomyocytes clearly indicated that the compromised cardiac growth in zMHC- BMP 10 mice was solely because of defect in cardiomyocyte postnatal hypertrophic growth. Physiological analysis further demonstrated that the responses of these hearts to both physiological (e.g. exercise-induced hypertrophy) and pathological hypertrophic stimuli remain normal. In addition, the αMHC-BMP10 mice develop subaortic narrowing and concentric myocardial thickening without obstruction by four weeks of age. Systematic analysis of potential intracellular pathways further suggested a novel genetic pathway regulating this previously undefined cardiac postnatal hypertrophic growth event. This is the first demonstration that cardiac postnatal hypertrophic growth can be specifically modified genetically and dissected out from physiological and pathological hypertrophies. [ABSTRACT FROM AUTHOR]