The cardiomyocyte cell cycle

Many forms of cardiac disease are characterized by cardiomyocyte death due to necrosis, apoptosis and/or oncosis. Recently, the notion of promoting cardiac regeneration as a means to replace damaged heart tissue has engendered considerable interest. One approach to accomplish heart muscle regeneration entails promoting cardiomyocyte cell cycle activity in the surviving myocardium. Genetically modified mice have provided useful model systems to test the efficacy of specific pathways to promote cardiomyocyte proliferation in normal and diseased hearts. For example, expression of a heart-restricted dominant interfering version of p193 (an E3 ubiquitin ligase also known as Cul7) resulted in an induction of cardiomyocyte cell cycle activity at the infarct border zone and ventricular septum 4 weeks after permanent coronary artery occlusion. A concomitant reduction in hypertrophic cardiomyocyte growth was also observed in this model, suggesting that cell cycle activation partially counteracted the adverse ventricular remodeling that occurs postinfarction. In other studies, targeted expression of cyclin D2 promoted cardiomyocyte cell cycle activity in adult hearts. The level of cardiomyocyte cell cycle activity increased after myocardial infarction, ultimately resulting in a marked increase in cardiomyocyte number and a concomitant regression of infarct size. Collectively, these data suggest that modulation of cardiomyocyte cell cycle activity can be exploited to promote regenerative growth in injured hearts

The CUL7 E3 Ubiquitin Ligase Targets Insulin Receptor Substrate 1 for Ubiquitin-Dependent Degradation

Recent genetic studies have documented a pivotal growth regulatory role played by the Cullin 7 (CUL7) E3 ubiquitin ligase complex containing the Fbw8-substrate-targeting subunit, Skp1, and the ROC1 RING finger protein. In this report, we identified insulin receptor substrate 1 (IRS-1), a critical mediator of the insulin/insulin-like growth factor 1 signaling, as a proteolytic target of the CUL7 E3 ligase in a manner that depends on mammalian target of rapamycin and the p70 S6 kinase activities. Interestingly, while embryonic fibroblasts of Cul7-/- mice were found to accumulate IRS-1 and exhibit increased activation of IRS-1’s downstream Akt and MEK/ERK pathways, these null cells grew poorly and displayed phenotypes reminiscent of those associated with oncogene-induced senescence. Taken together, our findings demonstrate a key role for the CUL7 E3 in targeting IRS-1 for degradation, a process that may contribute to the regulation of cellular senescence

Cardiac Myocyte Diversity and a Fibroblast Network in the Junctional Region of the Zebrafish Heart Revealed by Transmission and Serial Block-Face Scanning Electron Microscopy

The zebrafish has emerged as an important model of heart development and regeneration. While the structural characteristics of the developing and adult zebrafish ventricle have been previously studied, little attention has been paid to the nature of the interface between the compact and spongy myocardium. Here we describe how these two distinct layers are structurally and functionally integrated. We demonstrate by transmission electron microscopy that this interface is complex and composed primarily of a junctional region occupied by collagen, as well as a population of fibroblasts that form a highly complex network. We also describe a continuum of uniquely flattened transitional cardiac myocytes that form a circumferential plate upon which the radially-oriented luminal trabeculae are anchored. In addition, we have uncovered within the transitional ring a subpopulation of markedly electron dense cardiac myocytes. At discrete intervals the transitional cardiac myocytes form contact bridges across the junctional space that are stabilized through localized desmosomes and fascia adherentes junctions with adjacent compact cardiac myocytes. Finally using serial block-face scanning electron microscopy, segmentation and volume reconstruction, we confirm the three-dimensional nature of the junctional region as well as the presence of the sheet-like fibroblast network. These ultrastructural studies demonstrate the previously unrecognized complexity with which the compact and spongy layers are structurally integrated, and provide a new basis for understanding development and regeneration in the zebrafish heart

Oncostatin M differentially regulates CXC chemokines in mouse cardiac fibroblasts

Ischemia-reperfusion injury in the heart is characterized by marked infiltration of neutrophils in the myocardial interstitial space. Studies in human, canine, and murine models have revealed oncostatin M (OSM) expression in infiltrating leukocytes. In an effort to assess possible roles of OSM in the myocardium, we used cardiac fibroblasts (mCFs) isolated from adult mouse heart to determine whether recombinant murine OSM regulates the synthesis and release of MIP2/ CXCL2, KC/CXCL1, and LIX/CXCL5, which are three potent neutrophil chemoattractants in the mouse. Our results demonstrate that mCFs express OSM receptors and that, within the IL-6 cytokine family, OSM uniquely induces significant release of KC and LIX in mCFs. In addition, although OSM activates the JAK-signal transducers and activators of transcription and MAPK pathways, we demonstrate that the OSM-mediated CXC chemokine release in mCFs is also dependent on the activation of the phosphatidylinositol 3-kinase pathway

The Giant Danio (D. aequipinnatus) as a Model of Cardiac Remodeling and Regeneration

The paucity of mammalian adult cardiac myocytes (CM) proliferation following myocardial infarction (MI) and the remodeling of the necrotic tissue that ensues, result in non-regenerative repair. In contrast, zebrafish (ZF) can regenerate after an apical resection or cryoinjury of the heart. There is considerable interest in models where regeneration proceeds in the presence of necrotic tissue. We have developed and characterized a cautery injury model in the giant danio (GD), a species closely related to ZF, where necrotic tissue remains part of the ventricle, yet regeneration occurs. By light and transmission electron microscopy (TEM), we have documented four temporally overlapping processes: 1) a robust inflammatory response analogous to that observed in MI, 2) concomitant proliferation of epicardial cells leading to wound closure, 3) resorption of necrotic tissue and its replacement by granulation tissue, 4) regeneration of the myocardial tissue driven by 5-EDU and [3H]thymidine incorporating CMs. In conclusion, our data suggest that the GD possesses robust repair mechanisms in the ventricle, and can serve as an important model of cardiac inflammation, remodeling and regeneration

Heart Development, Coronary Vascularization and Ventricular Maturation in a Giant Danio (Devario malabaricus)

Giant danios (genus Devario), like zebrafish, are teleosts belonging to the danioninae subfamily of cyprinids. Adult giant danios are used in a variety of investigations aimed at understanding cellular and physiological processes, including heart regeneration. Despite their importance, little is known about development and growth in giant danios, or their cardiac and coronary vessels development. To address this scarcity of knowledge, we performed a systematic study of a giant danio (Devario malabaricus), focusing on its cardiac development, from the segmentation period to ten months post-fertilization. Using light and scanning electron microscopy, we documented that its cardiovascular development and maturation proceed along well defined dynamic and conserved morphogenic patterns. The overall size and cardiovascular expansion of this species was significantly impacted by environmental parameters such as rearing densities. The coronary vasculature began to emerge in the late larval stage. More importantly, we documented two possible loci of initiation of the coronary vasculature in this species, and compared the emergence of the coronaries to that of zebrafish and gourami. This is the first comprehensive study of the cardiac growth in a Devario species, and our findings serve as an important reference for further investigations of cardiac biology using this species

Cardiac Repair and Regenerative Potential in the Goldfish (Carassius auratus) Heart

The remarkable ability of the heart to regenerate has been demonstrated in the zebrafish and giant danio, two fish members of the cyprinid family. Here we use light and electron microscopy to examine the repair response in the heart of another cyprinid, the goldfish (Carassius auratus), following cautery injury to a small portion of its ventricularmyocardium. We observed a robust inflammatory response in the first two weeks consisting primarily of infiltrating macrophages, heterophils, and melanomacrophages. These inflammatory cells were identified in the lumen of the spongy heart, within the site of the wound, and attached to endocardial cells adjacent to the site of injury. Marked accumulation of collagen fibers and increased connective tissue were also observed during the first and second weeks in a transition zone between healthy and injured myocardium as well as in adjacent sub-epicardial regions. The accumulation of collagen and connective tissue however did not persist. The presence of capillaries was also noted in the injured area during repair. The replacement of the cauterized region of the ventricle by myocardial tissue was achieved in 6 weeks. The presence of ethynyl deoxyuridinepositive cardiac myocytes and partially differentiated cardiac myocytes during repair suggest effective cardiac myocyte driven regeneration mechanisms also operate in the injured goldfish heart, and are similar to those observed in zebrafish and giant danio. Our data suggest the ability for cardiac regeneration may be widely conserved among cyprinids

Absence of cardiomyocyte differentiation following transplantation of adult cardiac-resident Sca-1+ cells into infarcted mouse hearts

Although several lines of evidence suggest that the glycosyl phosphatidylinositol-anchored cell surface protein Sca-1 marks cardiac-resident stem cells, a critical analysis of the literature raises some concerns regarding their cardiomyogenic potential.1 Here, isolated adult cardiac-resident Sca-1+ cells were engrafted into infarcted hearts and monitored for cardiomyogenic differentiation. Donor cells were prepared from ACT-EGFP; MHC-nLAC double-transgenic mice ([C57/Bl6J x DBA/2J]F1 genetic background; all procedures followed were in accordance with Institutional Guidelines). The ACT-EGFP transgene targets ubiquitous expression of an enhanced green fluorescent protein reporter, and the MHC-nLAC transgene targets cardiomyocyte-restricted expression of a nuclear-localized β-galactosidase reporter. Donor cell survival was monitored via EGFP fluorescence, while cardiomyogenic differentiation was monitored by reacting with the chromogenic β-galactosidase substrate 5-bromo-4-chloro-3-indolyl-β-D-galactoside (X-GAL), which gives rise to a blue product.2 Double-transgenic hearts were dispersed with Blendzyme and the resulting cells reacted with an APC-conjugated anti-Sca-1 antibody and a PE-conjugated cocktail of antibodies recognizing hematopoietic lineage markers.3 Sca-1+, EGFP+, lineage- cells were then isolated via fluorescence-activated cell sorting (FACS; characterization of the donor cells is provided in Figure 1A), and 100,000 cells were injected into the infarct border zone of non-transgenic [C57/Bl6J x DBA/2J]F1 mice immediately following permanent coronary artery occlusion

Cardiac Myocyte Diversity and a Fibroblast Network in the Junctional Region of the Zebrafish Heart Revealed by Transmission and Serial Block-Face Scanning Electron Microscopy

<div><p>The zebrafish has emerged as an important model of heart development and regeneration. While the structural characteristics of the developing and adult zebrafish ventricle have been previously studied, little attention has been paid to the nature of the interface between the compact and spongy myocardium. Here we describe how these two distinct layers are structurally and functionally integrated. We demonstrate by transmission electron microscopy that this interface is complex and composed primarily of a junctional region occupied by collagen, as well as a population of fibroblasts that form a highly complex network. We also describe a continuum of uniquely flattened transitional cardiac myocytes that form a circumferential plate upon which the radially-oriented luminal trabeculae are anchored. In addition, we have uncovered within the transitional ring a subpopulation of markedly electron dense cardiac myocytes. At discrete intervals the transitional cardiac myocytes form contact bridges across the junctional space that are stabilized through localized desmosomes and fascia adherentes junctions with adjacent compact cardiac myocytes. Finally using serial block-face scanning electron microscopy, segmentation and volume reconstruction, we confirm the three-dimensional nature of the junctional region as well as the presence of the sheet-like fibroblast network. These ultrastructural studies demonstrate the previously unrecognized complexity with which the compact and spongy layers are structurally integrated, and provide a new basis for understanding development and regeneration in the zebrafish heart.</p></div