Interactions between cardiac cells enhance cardiomyocyte hypertrophy and increase fibroblast proliferation

In cardiac hypertrophy, both excessive enlargement of cardiac myocytes (CMs) and progressive fibrosis are known to occur simultaneously. To investigate the nature of interactions between ventricular CMs and cardiac fibroblasts (CFs) in these conditions, we have established a "dedifferentiated model" of adult murine CMs in coculture with CFs. In such a model, which is recognized to study cardiac cell hypertrophy in vitro, dedifferentiated CMs in culture and in coculture were characterized by immunopositive staining to ANP (atrial natriuretic peptide) and beta-myosin heavy chain (beta-MHC). The results confirm that ANP secretion by CMs was significantly increased during the cultures. The increase size of cultured CMs was significantly higher in CM/CF cocultures than in CM cultures which was also observed when CMs were cultured with fibroblast conditioned medium (FCM). In addition, fibroblast proliferation studies showed that CMs favored fibroblast adhesion and/or growth at the beginning of the coculture and fibroblast proliferation throughout the time course of the coculture. Furthermore, a significant level of interleukin-6 (IL-6) production was detected by ELISA in CM/CF cocultures. A similar higher increase was observed when CMs were cultured in the presence of FCM. These results demonstrate that CFs enhance myocyte hypertrophy and that CMs regulate fibroblast adhesion and/or proliferation, suggesting a paracrine interaction between CMs and CFs which could involve IL-6.     

Network architecture of signaling from uncoupled helicase-polymerase to cell cycle checkpoints and trans-lesion DNA synthesis

Potentially, adult stem cell-based therapy provides a new therapeutic option for myocardial regeneration. However, to date, with regard to the benefits seen, the mechanisms involved in stem cell-based therapy are not well understood. Suggested pathways proposed so far include fusion of stem cells with cardiomyocytes, transdifferentiation into cardiac and vascular cells and secretion of paracrine factors. In a recent study, our group examined the fate of human adipose tissue-derived stem cells (hASCs) fused with rat cardiomyocytes after treatment with fusion-inducing hemagglutinating virus of Japan (HVJ). In this study, we demonstrated that cells of fused hASC cardiomyocytes display a cardiomyocyte phenotype and spontaneous rhythmic contraction and generate an action potential in vitro. As part of the work underlying this paper, we co-cultured rat neonatal cardiomyocytes with hASCs or pig bone marrow-derived mesenchymal stem cells (MSCs), where ASCs or MSCs had previously been transduced with a lentivirus encoding eGFP. Our data evidence early cardiac contractile proteins, such as Titin and MF20, identified in eGFP-positive cells, suggesting a cardiomyogenic phenotype. Recent work by others has shown that the myogenic conversion increased when BMSCs were cultured with apoptotic cells. In this Extra View article, we review the current understanding of stem cell-derived factors, fusion/partial fusion and the manner in which the exchange of cellular contents between stem cells and cardiomyocytes might contribute to the reprogramming of fully differentiated cardiomyocytes based on recently published literature.     

Cell transplantation for treatment of acute myocardial infarction: unique capacity for repair by skeletal muscle satellite cells

An adult heart injured by an ischemic episode has a limited capacity to regenerate. We administered three types of adult guinea pig cells [cardiomyocytes (CMs), cardiac fibroblasts (CFs), and skeletal myoblasts (Mbs)] to compare their suitability for repair of acute myocardial infarction. We used confocal fluorescent microscopy and a variety of specific immunomarkers and echocardiography to provide anatomic evidence for the viability of such cells and their possible functional beneficial effects. All cells were transfected with adenovirus-containing beta-galactosidase gene so that migration from the injection sites could be traced. Both freshly isolated CMs as well as CFs were found concentrated in the infarcted zone; these cells survived for at least 2 wk posttransplantation. Transplanted CMs were regularly striated and grew long projections that could form gap junctions with native CMs, which was evidenced by connexin43 labeling. In addition, CM transplantation resulted in increased angiogenesis in the infarcted areas. In contrast, transplanted CFs did not appear to make any gap junctional contacts with native CMs nor did they enhance local angiogenesis. Mbs cultured for 7 days and transfected Mbs were identified 7 days posttransplantation in the infarcted area. During that time and thereafter, Mbs proliferated and differentiated into myotubes that formed new, regularly striated myofibers that occupied most (50-70%) of the infarcted area by 2-3 wk. These newly formed myofibers maintained their Mb skeletal muscle origin as evidenced by their capacity to express myogenin and fast skeletal myosin. This skeletal phenotype appeared to downregulate with time, and Mbs partially transdifferentiated into a cardiac phenotype as indicated by labeling for cardiac-specific troponin T and cardiac myosin heavy chain. By the third week posttransplantation, new myofibers formed apparent contacts with the native CMs via putative gap junctions that expressed connexin43. Myocardial performance of animals that were successfully transplanted with Mbs was improved.     

Role of interleukin-6 in cardiomyocyte/cardiac fibroblast interactions during myocyte hypertrophy and fibroblast proliferation

The process of cardiac hypertrophy is considered to involve two components: that of cardiac myocyte (CM) enlargement and cardiac fibroblast (CF) proliferation. The interleukin-6 (IL-6) family cytokines have been implicated in a variety of cellular and molecular interactions between myocytes and non-myocytes (NCMs), which in turn have important roles in the development of cardiac hypertrophy. In the study of these interactions, we previously detected very high levels of IL-6 in supernatants of a "dedifferentiated model" of adult ventricular CMs cultured with CFs. In the present study, we have used this in vitro coculture system to examine how IL-6 is involved in the interactions between CMs and CFs during CM hypertrophy and CF proliferation. IL-6 and its signal transducer, 130-kDa glycoprotein (gp130), were detected by immunostaining cultured CMs and CFs with anti-IL-6 or anti-gp130 antibodies. Addition of anti-IL-6 or anti-gp130 antagonist antibodies into CM/CF cocultures induced a significant decrease in expression of atrial natriuretic peptide (ANP) and beta-myosin heavy chain (beta-MHC) in CMs. The presence of IL-6 antagonist also resulted in a decrease in the surface area of 12-day-old CMs cultured with CFs or in the presence of fibroblast conditioned medium (FCM), and decreased fibroblast proliferation in CM/CF cocultures, particularly in the presence of a gp130 antagonist. The results also show that angiotensin II (AngII) is mainly secreted by CFs and induces IL-6 secretion in CMs cultured with CFs or with FCM. In addition, the effects of IL-6 on cardiomyocyte hypertrophy and fibroblast proliferation were inhibited by addition of the AT-1 receptor antagonist, losartan. These results suggest that IL-6 contributes significantly to CM hypertrophy by an autocrine pathway and to fibroblast proliferation by a paracrine pathway and that these effects could be mediated by AngII.     

Dermatopontin promotes adhesion,spreading and migration of cardiac fibroblasts in vitro

Dermatopontin (DPT), an extracellular matrix (ECM) protein, has been previously shown to be upregulated in the infarct zone of experimentally induced myocardial infarction (MI) rats. However, the accurate role that DPT exerts in the ventricular remodeling process after MI remains poorly understood. In this study, we evaluated the expression pattern of DPT mRNA and protein as well as its secretion in cultured neonatal rat cardiomyocytes (CMs) and cardiac fibroblasts (CFs) under conditions of hypoxia and serum deprivation (hypoxia/SD). Further, we tested the possible roles of DPT in CFs adhesion, spreading, migration and proliferation, which greatly promote the ventricular remodeling process after MI. Results showed that hypoxia/SD stimulated DPT expression and secretion in CMs and CFs and that DPT promoted adhesion, spreading and migration of CFs whereas had no effect on CFs proliferation. In addition, functional blocking antibodies specific for integrin α3 and β1 significantly reduced CFs adhesion and migration that DPT induced, suggesting that integrin α3β1 is at least one receptor for CFs adhesion and migration to DPT. These results implicated that DPT participates in the ventricular remodeling process after MI and may act as a potential therapeutic target for ventricular remodeling.     

Cardiomyocytes fuse with surrounding noncardiomyocytes and reenter the cell cycle

The concept of the plasticity or transdifferentiation of adult stem cells has been challenged by the phenomenon of cell fusion. In this work, we examined whether neonatal cardiomyocytes fuse with various somatic cells including endothelial cells, cardiac fibroblasts, bone marrow cells, and endothelial progenitor cells spontaneously in vitro. When cardiomyocytes were cocultured with endothelial cells or cardiac fibroblasts, they fused and showed phenotypes of cardiomyocytes. Furthermore, cardiomyocytes reentered the G2-M phase in the cell cycle after fusing with proliferative noncardiomyocytes. Transplanted endothelial cells or skeletal muscle-derived cells fused with adult cardiomyocytes in vivo. In the cryoinjured heart, there were Ki67-positive cells that expressed both cardiac and endothelial lineage marker proteins. These results suggest that cardiomyocytes fuse with other cells and enter the cell cycle by maintaining their phenotypes.     

Characterization of heterokaryons between skeletal myoblasts and somatic cells formed by fusion with HVJ (Sendai virus); effects on myogenic differentiation

In skeletal myogenic differentiation, myoblasts fuse with myogenic cells spontaneously, but do not fuse with non-myogenic cells either in vivo or in vitro, suggesting that the fusion of myoblasts with non-myogenic cells is unsuitable for differentiation. To understand the inevitability of the fusion among myoblasts, we prepared heterokaryons in crosses between quail myoblasts transformed with a temperature-sensitive mutant of Rous sarcoma virus (QM-RSV cells) and rodent non-myogenic cells, such as tumor cells, fibroblasts, or neurogenic cells by HVJ (Sendai virus) and examined how myogenic differentiation was influenced in the prepared heterokaryons, focusing on myogenin expression and myofibril formation as markers of differentiation. When presumptive QM-RSV cells were fused with non-myogenic cells by HVJ and induced to differentiate, both myogenin expression and myofibril formation were suppressed. When myotubes of QM-RSV cells that had already expressed myogenin and formed myofibrils were fused with non-myogenic cells, both myogenin and myofibrils disappeared. Especially, fibrous structures of myofibrils were significantly lost and dots or aggregations of F-actin were formed within 24 hr after formation of heterokaryons. However, the fusion of presumptive or differentiated QM-RSV cells with rodent myoblasts did not disturb myogenin expression or myofibril formation. These results suggest that mutual fusion of myoblasts is indispensable for normal myogenic differentiation irrespective of the species, and that some factors inhibiting myogenic differentiation exist in the cytoplasm of non-myogenic cells, but not in myoblasts.     

Rhythmic contraction and intracellular Ca2 + oscillatory rhythm in spontaneously beating cultured cardiac myocytes

Cultured cardiac myocytes from neonatal rats show spontaneous and rhythmic contractions. The intracellular concentration of free Ca^2 + also changes rhythmically, associated with the rhythmic contraction of myocytes (Ca^2 + oscillation). This study aims to elucidate whether spontaneous rhythmic contraction affects the dynamics of intracellular Ca^2 + oscillation in cultured cardiac myocytes. In cultures at four days in vitro (4 DIV), spontaneous Ca^2 + oscillation was synchronized among myocytes. Treatment of cultures with an uncoupler of E - C coupling resulted in a cessation of the spontaneous contraction of cardiac myocytes, but did not abolish the Ca^2 + oscillation. The intercellular synchronization of intracellular Ca^2 + oscillation persisted, and both the intervals and the fluctuation of the oscillation tended to increase after the termination of rhythmic contraction. The present study demonstrated that mechanical factors associated with rhythmic contraction did not affect the intercellular synchronization of intracellular Ca^2 + oscillation, but possibly contributed to the stability of the oscillatory rhythm.     

Introduction of macromolecules into mammalian cells by cell fusion

Proteins with molecular weights of up to 500K can be enclosed in erythrocyte ghosts by exposing the ghosts to hypotonic solution containing these proteins. The proteins can then be introduced into recipient cells by fusing the ghosts with the cells using HVJ, PEG, or influenza virus. Some applications of this method are described. By an improved method, 15 kbp DNA and IgM (900 kDa) can be entrapped in erythrocyte membranes and these are then treated with liposomes containing gangliosides and HVJ. These treated membranes containing large macromolecules fuse with almost 100% of the recipient cells used. Naked liposomes infrequently fuse with cultured cells, so introduction of their contents into cells is very inefficient. However, liposomes constituted from lipid and glycoproteins (HN and F) of HVJ (Sendai virus), by removing a nonionic detergent, fuse with cells about 200 times more efficiently than naked liposomes. Naked liposomes can fuse with specific cells, such as cells infected with subacute sclerosing panencephalitis virus or with human immunodeficiency virus. Plasmid DNA and mRNA of up to about 40 kbp can be entrapped efficiently in liposomes associated with gangliosides formed by reverse-phase evaporation, and then reacted with HVJ. The contents of the resulting liposomes with HVJ can be introduced efficiently into cultured cells in a suspended or plated state, and nearly all the cells then express the gene transiently. This procedure is also effective for obtaining stable transformants of many kinds of cultured cells.     

Functional scaffold-free 3-D cardiac microtissues: a novel model for the investigation of heart cells

To bridge the gap between two-dimensional cell culture and tissue, various three-dimensional (3-D) cell culture approaches have been developed for the investigation of cardiac myocytes (CMs) and cardiac fibroblasts (CFs). However, several limitations still exist. This study was designed to develop a cardiac 3-D culture model with a scaffold-free technology that can easily and inexpensively generate large numbers of microtissues with cellular distribution and functional behavior similar to cardiac tissue. Using micromolded nonadhesive agarose hydrogels containing 822 concave recesses (800 μm deep × 400 μm wide), we demonstrated that neonatal rat ventricular CMs and CFs alone or in combination self-assembled into viable (Live/Dead stain) spherical-shaped microtissues. Importantly, when seeded simultaneously or sequentially, CMs and CFs self-sorted to be interspersed, reminiscent of their myocardial distribution, as shown by cell type-specific CellTracker or antibody labeling. Microelectrode recordings and optical mapping revealed characteristic triangular action potentials (APs) with a resting membrane potential of -66 ± 7 mV (n = 4) in spontaneously contracting CM microtissues. Under pacing, optically mapped AP duration at 90% repolarization and conduction velocity were 100 ± 30 ms and 18.0 ± 1.9 cm/s, respectively (n = 5 each). The presence of CFs led to a twofold AP prolongation in heterogenous microtissues (CM-to-CF ratio of 1:1). Importantly, Ba(2+)-sensitive inward rectifier K(+) currents and Ca(2+)-handling proteins, including sarco(endo)plasmic reticulum Ca(2+)-ATPase 2a, were detected in CM-containing microtissues. Furthermore, cell type-specific adenoviral gene transfer was achieved, with no impact on microtissue formation or cell viability. In conclusion, we developed a novel scaffold-free cardiac 3-D culture model with several advancements for the investigation of CM and CF function and cross-regulation.     

N-乙酰半胱氨酸对大鼠心脏成纤维细胞增殖和胶原合成的影响

目的:通过观察N-乙酰半胱氨酸(NAC)对大鼠心脏成纤维细胞(CFs)增殖和胶原合成的影响,探讨NAC对心脏重构的作用。方法:以培养的新生SD大鼠CFs为实验对象,给予不同浓度的NAC进行干预,48小时后用MTT比色法检测CFs增殖水平,用3H脯氨酸掺入法测定总胶原合成。结果:与对照组相比,不同浓度NAC作用下的CFs增殖水平和3H脯氨酸掺入量均比对照组低,且具有浓度依赖性(p<0.05)。结论:NAC能够抑制SD大鼠CFs增殖,并降低其胶原合成,因此NAC对心脏的病理性重构可能具有保护作用。     

N-乙酰半胱氨酸对大鼠心脏成纤维细胞增殖和胶原合成的影响

目的：通过观察N-乙酰半胱氨酸（NAC）对大鼠心脏成纤维细胞（CFs）增殖和胶原合成的影响,探讨NAC对心脏重构的作用。方法：以培养的新生SD大鼠CFs为实验对象,给予不同浓度的NAC进行干预,48小时后用MTT比色法检测CFs增殖水平,用3H脯氨酸掺入法测定总胶原合成。结果：与对照组相比,不同浓度NAC作用下的CFs增殖水平和3H脯氨酸掺入量均比对照组低,且具有浓度依赖性（p〈0.05）。结论：NAC能够抑制SD大鼠CFs增殖,并降低其胶原合成,因此NAC对心脏的病理性重构可能具有保护作用。     

Changes in the fluctuation of the contraction rhythm of spontaneously beating cardiac myocytes in cultures with and without cardiac fibroblasts

The heart functions as a syncytium of cardiac myocytes and surrounding supportive non-myocytes such as fibroblasts. There is a possibility that a variety of non-myocyte-derived factors affect the maturation of cardiac myocytes in the development of the heart. Cultured neonatal cardiac myocytes contract spontaneously and cyclically. The fluctuation of beating rhythm varies depending on the strength of coupling through gap junctions among cardiac myocytes, indicating that the development of intercellular communication via gap junctions is crucial to the stability of contraction rhythm in cardiac myocytes. In this study, we aimed at elucidating whether and how cardiac fibroblasts affect the development of cardiac myocytes from the point of view of the changes in the fluctuation of the contraction rhythm of cardiac myocytes in cardiac myocyte–fibroblast co-cultures. The present study suggested that cardiac fibroblasts co-cultured with cardiac myocytes enhanced the intercellular communication among myocytes via gap junctions, thereby stabilizing the spontaneous contraction rhythm of cultured cardiac myocytes.     

Characterization of myogenic cell membrane: spontaneous formation of heterokaryotic myotubes between two different kinds of myoblasts

In a previous study, it has been shown that presumptive mouse C2 myoblast cells are strongly resistant to HVJ (hemaglutinating virus of Japan, Sendai virus)-mediated cell fusion, but do become capable of fusion upon differentiation. Quail myoblasts transformed with a temperature-sensitive mutant of Rous sarcoma virus (QM-RSV cells) also become more sensitive to HVJ-mediated cell fusion during differentiation. Investigations were undertaken to see whether heterokaryotic myotubes were formed spontaneously by co-culture of two different kinds of myogenic cells, QM-RSV cells and C2 cells. When both cells were committed to myotube formation, they spontaneously fused without HVJ on co-culture. On the other hand, when both or one of the cells were in the presumptive state, heterokaryons were not formed by co-culturing. Furthermore, committed QM-RSV cells did not fuse with non-myogenic cells. These results indicate that the membranes of myogenic cells change to become capable of fusion for myotube formation during differentiation.     

Beating of multinucleated giant myocardial cells in culture

Cultured mouse myocardial cells grown as cell sheets in Petri dishes fused together and formed multinucleated giant cells on treatment with HVJ (Sendai virus). The giant cells had well organized myofibrils and beat spontaneously and rhythmically. The spontaneous beating activity of the giant cells changed in response to changes of the external potassium and calcium concentrations and on addition of ouabain in the same way as the beating of cultured myocardial cells not treated with HVJ. When a microelectrode was inserted into giant cells that exhibited spontaneous beating, action potentials were easily recorded.     

以单细胞免疫荧光技术观察烟草细胞融合过程中微管骨架的变化

本文建立了单细胞免疫荧光标记技术并以此结合单对细胞融合技术对细胞融合过程中微管骨架组织形式的动态变化进行了追踪观察。发现在聚乙二醇(PEG)诱导条件下,一旦细胞开始粘连,细胞内微管骨架便开始解聚。在细胞融合的整个过程中一直维持着这种解聚的状态,直到融合完成,在后续的培养中微管骨架才重新出现。在微管骨架呈解聚状态时融合产物不能完成与另外的细胞融合。实验揭示了细胞的再融合能力可能受细胞本身微管骨架状态的影响。该结果为解释高等植物如何避免多精入卵提供了新的可能性。     

Characterisation of myogenic cell membrane: II. Dynamic changes in membrane lipids during the differentiation of mouse C2 myoblast cells

Proliferating mouse C2 myoblast cells resist haemagglutinating virus of Japan, Sendai virus (HVJ) mediated cell fusion. However, differentiating C2 cells can be induced to fuse by HVJ, suggesting that the rigid membrane of C2 cells changes during the differentiation. To investigate this phenomenon, changes in membrane lipids which affect fluidity were examined. Membrane cholesterol gradually decreased with the differentiation of C2 cells. However, spontaneous fusion to form myotubes and artificial fusion induced by HVJ were both inhibited when the level of cholesterol was prevented from falling in the cell membrane. The membranes of differentiating C2 cells contained more unsaturated fatty acids than those of proliferating cells. Thus, when differentiating C2 cells were treated with stearate (a saturated fatty acid), they failed to form myotubes and were insensitive to HVJ-mediated fusion. Whereas, if proliferating C2 cells were given linolenate (an unsaturated fatty acid), they became capable of HVJ-induced fusion. These results indicate that differentiating C2 cells change their fusion sensitivity by decreasing cholesterol, probably at the same time as they increase the unsaturated fatty acid content of the cell membrane.     

Morphological changes in Ehrlich ascites tumor cells during the cell fusion reaction with HVJ (Sendai virus). III. Morphological characterization of HVJ glycoproteins integrated into the plasma membrane and their internalization by coated vesicles

On cell-cell fusion of Ehrlich ascites tumor (EAT) cells with HVJ, HVJ envelopes also fuse with the cell membrane, resulting in integration of the viral envelope glycoproteins into the fused cell membranes. Morphological characterization of the glycoproteins in the plasma membrane and the mode of their internalization were investigated in detail. In the fusion reaction, the glycoproteins were rapidly integrated into the cell membrane within 2 or 3 min on incubation at 37 °C and they remained at the fusion sites, not dispersing widely, during further incubation. Thus they were still present in clusters in the plasma membrane at the end of the fusion reaction. On culture of fused cells in culture medium, internalization of the viral glycoproteins was initiated by formation of coated vesicles and most of the integrated glycoproteins were endocytosed into the cytoplasm within 30 min. Soon after internalization, the coated vesicles fused with each other, losing their coat materials. The intact virions that remained unfused on the cell surface were also internalized, but coat materials did not appear on the inside surface of the cell membrane, unlike in the case of integrated glycoproteins.     

Inhibition of cardiac fibroblast proliferation and myofibroblast differentiation by resveratrol

Cardiac fibroblasts (CFs) regulate myocardial remodeling by proliferating, differentiating, and secreting extracellular matrix proteins. Prolonged activation of CFs leads to cardiac fibrosis and reduced myocardial contractile function. Resveratrol (RES) exhibits a number of cardioprotective properties; however, the possibility that this compound affects CF function has not been considered. The current study tests whether RES directly influences the growth and proliferation of CFs and differentiation to the hypersecretory myofibroblast phenotype. Pretreatment of CFs with RES (5-25 microM) inhibited basal and ANG II-induced extracellular signal-regulated kinase (ERK) 1/2 and ERK kinase activation. This inhibition by RES reduced basal proliferation and blocked ANG II-induced growth and proliferation of CFs in a concentration-dependent manner, as measured by [(3)H]leucine and [(3)H]thymidine incorporation, respectively. RES pretreatment attenuated ERK phosphorylation when CFs were stimulated with 0.2 nM epidermal growth factor (EGF), a concentration at which EGF-induced ERK activation over basal was similar to the phosphorylation induced by 100 nM ANG II. Akt phosphorylation in CFs was unaffected by treatment with either 100 nM ANG II or 25 microM RES. Pretreatment of CFs with RES also reduced both ANG II- and transforming growth factor-beta-induced CF differentiation to the myofibroblast phenotype, indicated by a reduction in alpha-smooth muscle actin expression and stress fiber organization in CFs. This study identifies RES as an anti-fibrotic agent in the myocardium by limiting CF proliferation and differentiation, two critical steps in the pathogenesis of cardiac fibrosis.