Expression of replication factor C 40-kDa subunit is down-regulated during neonatal development in rat ventricular myocardium

During neonatal development, cardiac myocytes undergo a transition from hyperplastic to hypertrophic growth. Whether these cells are terminally differentiated and permanently withdrawn from the cell cycle shortly after birth is controversial. Nevertheless, the clinical observation that functionally significant myocardial regeneration has not been documented in cardiovascular disease or injury during adulthood seems to support the notion that the vast majority of cardiac myocytes do not proliferate once they differentiate. Regardless of the controversy, the elucidation on how mitosis is blocked in cardiac myocytes may facilitate development of new cardiovascular therapies, based on the regeneration of the adult myocardium. To better understand postnatal myocardial development, we performed suppression subtractive hybridization to isolate genes that are differentially expressed in day one or day seven postnatal rat ventricular myocardium. Here we report the down-regulated mRNA expression of the 40-kDa subunit of replication factor C (RFC p40 or RFC2), which is an essential processive factor for proliferating cellular nuclear antigen-dependent DNA replication during neonatal myocardial development.     

The c-myc proto-oncogene regulates cardiac development in transgenic mice.

During the maturation of the cardiac myocyte, a transition occurs from hyperplastic to hypertrophic growth. The factors that control this transition in the developing heart are unknown. Proto-oncogenes such as c-myc have been implicated in the regulation of cellular proliferation and differentiation, and in the heart the switch from myocyte proliferation to terminal differentiation is synchronous with a decrease in c-myc mRNA abundance. To determine whether c-myc can influence myocyte proliferation or differentiation, we examined the in vivo effect of increasing c-myc expression during embryogenesis and of preventing the decrease in c-myc mRNA expression that normally occurs during cardiac development. The model system used was a strain of transgenic mice exhibiting constitutive expression of c-myc mRNA in cardiac myocytes throughout development. In these transgenic mice, increased c-myc mRNA expression was found to be associated with both atrial and ventricular enlargement. This increase in cardiac mass was secondary to myocyte hyperplasia, with the transgenic hearts containing more than twice as many myocytes as did nontransgenic hearts. The results suggest that in the transgenic animals there is additional hyperplastic growth during fetal development. However, this additional proliferative growth is not reflected in abnormal myocyte maturation, as assessed by the expression of the cardiac and skeletal isoforms of alpha-actin. The results of this study indicate that constitutive expression of c-myc mRNA in the heart during development results in enhanced hyperplastic growth and suggest a regulatory role for this proto-oncogene in cardiac myogenesis.     

Over Expression of Plk1 Does Not Induce Cell Division in Rat Cardiac Myocytes In Vitro

Background

Mammalian cardiac myocytes withdraw from the cell cycle during post-natal development, resulting in a non-proliferating, fully differentiated adult phenotype that is unable to repair damage to the myocardium, such as occurs following a myocardial infarction. We and others previously have shown that forced expression of certain cell cycle molecules in adult cardiac myocytes can promote cell cycle progression and division in these cells. The mitotic serine/threonine kinase, Polo-like kinase-1 (Plk1), is known to phosphorylate and activate a number of mitotic targets, including Cdc2/Cyclin B1, and to promote cell division.

Principal Findings

The mammalian Plk family are all differentially regulated during the development of rat cardiac myocytes, with Plk1 showing the most dramatic decrease in both mRNA, protein and activity in the adult. We determined the potential of Plk1 to induce cell cycle progression and division in cultured rat cardiac myocytes. A persistent and progressive loss of Plk1 expression was observed during myocyte development that correlated with the withdrawal of adult rat cardiac myocytes from the cell cycle. Interestingly, when Plk1 was over-expressed in cardiac myocytes by adenovirus infection, it was not able to promote cell cycle progression, as determined by cell number and percent binucleation.

Conclusions

We conclude that, in contrast to Cdc2/Cyclin B1 over-expression, the forced expression of Plk1 in adult cardiac myocytes is not sufficient to induce cell division and myocardial repair.     

Transgenic animals as a tool for studying the effect of the c-myc proto-oncogene on cardiac development

Transgenic animals provide a model system to elucidate the role of specific proteins in development. This model is now being used increasingly in the cardiovascular system to study cardiac growth and differentiation. During cardiac myocyte development a transition occurs from hyperplastic to hypertrophic growth. In the heart the switch from myocyte proliferation to terminal differentiation is synchronous with a decrease in c-myc mRNA abundance. To determine whether c-myc functions to regulate myocyte proliferation and/or differentiation, we examined the in vivo effect of increasing c-myc expression during fetal development and of preventing the decrease in c-myc mRNA expression that normally occurs during myocyte development. The model system used was a strain of transgenic mice exhibiting constitutive expression of c-myc mRNA in cardiac myocytes throughout development. Increased c-myc mRNA expression is associated with both atrial and ventricular enlargement in the transgenic mice. This increase in cardiac mass is secondary to myocyte hyperplasia, with the transgenic hearts containing greater than twice as many myocytes as nontransgenic hearts. The results of this study indicate that constitutive expression of c-myc mRNA in the heart during development results in enhanced hyperplastic growth, and suggest a regulatory role for the c-myc protooncogene in cardiac myogenesis.     

MLP参与心肌肥大发生过程与Calcineurin/NFAT信号通路有关

心肌肥大是心肌细胞面对多种病理刺激时的共同反应,以心肌细胞体积增大和胚胎期基因的重新表达为标志.心肌发育调控基因肌肉LIM蛋白(muscle LIM protein,MLP)的表达异常与心肌肥大有关.为研究MLP参与心肌肥大发生的分子机制,采用去氧肾上腺素（phenylephrine, PE）刺激大鼠原代培养心肌细胞,建立心肌细胞肥大模型,采用RNAi技术敲减MLP的表达,分析MLP与肥大信号通路钙调神经磷酸酶（calcineurin）/活化T细胞核因子（nuclear factor of activated T-cells, NFAT）的关系.结果显示, 原代培养的心肌细胞经一定浓度的PE刺激后细胞表面积增加,肥大标志蛋白ANP、BNP表达增高,并伴有MLP表达上调. RNAi方法敲减MLP的表达则明显抑制PE诱导的心肌细胞表面积增加和BNP表达增高,并且直接 影响NFAT的转录激活活性,提示MLP与心肌肥大的发生密切相关,并且可能是通过calcineurin/NFAT信号通路而参与心肌肥大的发生.     

Western array analysis of cell cycle protein changes during the hyperplastic to hypertrophic transition in heart development

Cardiac myocytes proliferate most rapidly during the hyperplastic phase of heart development; however, the level of cell cycle activity is drastically down regulated after birth. Further growth of the heart is achieved by hypertrophic growth of cardiac myocytes. The mechanism that controls the switch from hyperplastic proliferation to hypertrophic growth in cardiac myocytes is unknown. Understanding this fundamental mechanism of cardiac myocyte biology would be most beneficial for studies directed towards myocardial regeneration. In this study, we identified changes in the expression of proteins involved in cell cycle regulation during the hyperplastic to hypertrophic transition of cardiac myocytes. Using a high-throughput immunoblotting technique, we examined 200+ proteins in primary cultures of cardiac myocytes at different developmental time points to determine the important regulators of this transition. In addition, we also analyzed samples from an immortalized cardiac myocyte cell line to compare expression levels of cell cycle regulatory proteins to our primary cultures. Our findings by this uncovered proteomic screen identified several potential key regulatory proteins and provide insight into the important components of cardiac myocyte cell cycle regulation.     

Complete cDNA sequence of rat atrial myosin light chain 1: patterns of expression during development and with hypertension.

Distinct atrial and ventricular isoforms of myosin light chain 1 (LC1) exist in mammals. The atrial LC1 is also expressed in fetal ventricular and skeletal muscle. Here we present a full length cDNA encoding a rat atrial LC1, based upon homology with previously reported LC1 sequences and its atrial-specific pattern of RNA hybridization in adult cardiac muscle. Atrial and ventricular RNA expression were studied during rat development and with chronic hypertension. Atrial LC1 mRNA was expressed in rat atria throughout development, and was coexpressed with ventricular LC1 mRNA in the hearts of 12-day and 16-day embryos, and in the ventricles of newborn rats (less than 24 hours). In 9 day-old neonates, atrial LC1 mRNA expression was restricted to rat atrium. In adult rats exhibiting renovascular hypertension, the expression of the atrial and ventricular LC1 mRNAs was unchanged from that seen in normal control animals.     

Atrial chamber-specific expression of sarcolipin is regulated during development and hypertrophic remodeling

Intracellular Ca2+ regulation is critical in the normal cardiac function and development of pathologic hearts. Phospholamban, an endogenous inhibitor of sarcoplasmic reticulum Ca2+ ATPase in the sarcoplasmic reticulum, plays an important role in Ca2+ cycling in heart. Recently, sarcolipin has been identified as having a similar function as phospholamban in skeletal muscle. Because phospholamban is differentially expressed in atrial and ventricular myocardia and its expression is often altered in diseased hearts, we investigated the cardiac chamber specificity of sarcolipin expression and its regulation during development and hypertrophic remodeling. Northern blot analysis revealed that the expression of mouse sarcolipin mRNA was most abundant in the atria and was undetectable in the ventricles, indicating an atrial chamber-specific expression pattern. Atrial chamber-specific expression of sarcolipin mRNA was increased during development. These findings were confirmed by in situ hybridization studies. In addition, sarcolipin expression was down-regulated in the atria of hypertrophic heart when induced by ventricular specific overexpression of the activated H-ras gene. In humans, sarcolipin mRNA was also expressed in the atria but not detected in the ventricles, although sarcolipin expression was most abundant in skeletal muscle. Taken together, sarcolipin is likely to be an atrial chamber-specific regulator of Ca2+ cycling in heart.     

Striated muscle-specific beta(1D)-integrin and FAK are involved in cardiac myocyte hypertrophic response pathway

Alterations in the extracellular matrix occur during the cardiac hypertrophic process. Because integrins mediate cell-matrix adhesion and beta(1D)-integrin (beta1D) is expressed exclusively in cardiac and skeletal muscle, we hypothesized that beta1D and focal adhesion kinase (FAK), a proximal integrin-signaling molecule, are involved in cardiac growth. With the use of cultured ventricular myocytes and myocardial tissue, we found the following: 1) beta1D protein expression was upregulated perinatally; 2) alpha(1)-adrenergic stimulation of cardiac myocytes increased beta1D protein levels 350% and altered its cellular distribution; 3) adenovirally mediated overexpression of beta1D stimulated cellular reorganization, increased cell size by 250%, and induced molecular markers of the hypertrophic response; and 4) overexpression of free beta1D cytoplasmic domains inhibited alpha(1)-adrenergic cellular organization and atrial natriuretic factor (ANF) expression. Additionally, FAK was linked to the hypertrophic response as follows: 1) coimmunoprecipitation of beta1D and FAK was detected; 2) FAK overexpression induced ANF-luciferase; 3) rapid and sustained phosphorylation of FAK was induced by alpha(1)-adrenergic stimulation; and 4) blunting of the alpha(1)-adrenergically modulated hypertrophic response was caused by FAK mutants, which alter Grb2 or Src binding, as well as by FAK-related nonkinase, a dominant interfering FAK mutant. We conclude that beta1D and FAK are both components of the hypertrophic response pathway of cardiac myocytes.     

Skeletal muscle satellite cells appear during late chicken embryogenesis.

The emergence of avian satellite cells during development has been studied using markers that distinguish adult from fetal cells. Previous studies by us have shown that myogenic cultures from fetal (Embryonic Day 10) and adult 12-16 weeks) chicken pectoralis muscle (PM) each regulate expression of the embryonic isoform of fast myosin heavy chain (MHC) differently. In fetal cultures, embryonic MHC is coexpressed with a ventricular MHC in both myocytes (differentiated myoblasts) and myotubes. In contrast, myocytes and newly formed myotubes in adult cultures express ventricular but not embryonic MHC. In the current study, the appearance of myocytes and myotubes which express ventricular but not embryonic MHC was used to determine when adult myoblasts first emerge during avian development. By examining patterns of MHC expression in mass and clonal cultures prepared from embryonic and posthatch chicken skeletal muscle using double-label immunofluorescence with isoform-specific monoclonal antibodies, we show that a significant number of myocytes and myotubes which stain for ventricular but not embryonic MHC are first seen in cultures derived from PM during fetal development (Embryonic Day 18) and comprise the majority, if not all, of the myoblasts present at hatching and beyond. These results suggest that adult type myoblasts become dominant in late embryogenesis. We also show that satellite cell cultures derived from adult slow muscle give results similar to those of cultures derived from adult fast muscle. Cultures derived from Embryonic Day 10 hindlimb form myocytes and myotubes that coexpress ventricular and embryonic MHCs in a manner similar to cells of the Embryonic Day 10 PM. Thus, adult and fetal expression patterns of ventricular and embryonic MHCs are correlated with developmental age but not muscle fiber type.     

Muscle ring finger protein-1 inhibits PKC{epsilon} activation and prevents cardiomyocyte hypertrophy

Much effort has focused on characterizing the signal transduction cascades that are associated with cardiac hypertrophy. In spite of this, we still know little about the mechanisms that inhibit hypertrophic growth. We define a novel anti-hypertrophic signaling pathway regulated by muscle ring finger protein-1 (MURF1) that inhibits the agonist-stimulated PKC-mediated signaling response in neonatal rat ventricular myocytes. MURF1 interacts with receptor for activated protein kinase C (RACK1) and colocalizes with RACK1 after activation with phenylephrine or PMA. Coincident with this agonist-stimulated interaction, MURF1 blocks PKCepsilon translocation to focal adhesions, which is a critical event in the hypertrophic signaling cascade. MURF1 inhibits focal adhesion formation, and the activity of downstream effector ERK1/2 is also inhibited in the presence of MURF1. MURF1 inhibits phenylephrine-induced (but not IGF-1-induced) increases in cell size. These findings establish that MURF1 is a key regulator of the PKC-dependent hypertrophic response and can blunt cardiomyocyte hypertrophy, which may have important implications in the pathophysiology of clinical cardiac hypertrophy.