Immunolocalization of basic fibroblast growth factor during chicken cardiac development

Basic fibroblast growth factor (bFGF) has been identified in cultured cardiac myocytes as well as in myocardial tissue of both embryonic and adult organisms; bFGF has also been demonstrated to regulate proliferation and differentiation of these cells in culture. Such studies suggest a possible role for bFGF in cardiac myogenesis. In vitro studies using cultured endothelial and neuronal cells also suggest that myocyte-derived bFGF may be involved in the regulation of vascularization and/or innervation of the developing heart. We have generated a spatial and temporal map for bFGF in the developing chick heart using immunohistochemical techniques and our monospecific polyclonal rabbit antihuman bFGF IgG. A progressive decrease in bFGF expression was seen in the highly trabeculated region of the ventricular myocardium, relative to the myocardium directly underlying the epicardial tissue, with increasing developmental age. bFGF expression was limited to the cytoplasm of cardiac myocytes; neither vascular endothelium nor smooth muscle contained anti-bFGF immunoreactive material. A correlation between the temporal and spatial pattern of bFGF expression seen here, with the pattern of myocyte proliferation and differentiation reported by others, suggests a role for bFGF in the autocrine regulation of myocyte proliferation and differentiation.     

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.     

Blebbistatin extends culture life of adult mouse cardiac myocytes and allows efficient and stable transgene expression

The characterization of cellular phenotypes of heart disorders can be achieved by isolating cardiac myocytes from mouse models or genetically modifying wild-type cells in culture. However, adult mouse cardiac myocytes show extremely low tolerance to isolation and primary culture conditions. Previous studies indicate that 2,3-butanedione monoximine (BDM), a nonspecific excitation-contraction coupling inhibitor, can improve the viability of isolated adult mouse cardiac myocytes. The mechanisms of the beneficial and unwanted nonspecific actions of BDM on cardiac myocytes are not understood. To understand what contributes to murine adult cardiac myocyte stability in primary culture and improve this model system for experimental use, the specific myosin II inhibitor blebbistatin was explored as a media supplement to inhibit mouse myocyte contraction. Enzymatically isolated adult mouse cardiac myocytes were cultured with blebbistatin or BDM as a media supplement. Micromolar concentrations of blebbistatin significantly increased the viability, membrane integrity, and morphology of adult cardiac myocytes compared with cells treated with previously described 10 mM BDM. Cells treated with blebbistatin also showed efficient adenovirus gene transfer and stable transgene expression, and unlike BDM, blebbistatin does not appear to interfere with cell adhesion. Higher concentrations of BDM actually worsened myocyte membrane integrity and transgene expression. Therefore, the specific inhibition of myosin II activity by blebbistatin has significant beneficial effects on the long-term viability of adult mouse cardiac myocytes. Furthermore, the unwanted effects of BDM on adult mouse cardiac myocytes, perhaps due to its nonspecific activities or action as a chemical phosphatase, can be avoided by using blebbistatin.     

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.     

Myocyte enlargement, differentiation, and proliferation kinetics in the fetal sheep heart.

The generation of new myocytes is an essential process of in utero heart growth. Most, or all, cardiac myocytes lose their capacity for proliferation during the perinatal period through the process of terminal differentiation. An increasing number of studies focus on how experimental interventions affect cardiac myocyte growth in the fetal sheep. Nevertheless, fundamental questions about normal growth of the fetal heart remain unanswered. In this study, we determined that during the last third of gestation the hearts of fetal sheep grew primarily by four processes. 1) Myocyte proliferation contributed substantially to daily cardiac mass gain, and the number of cardiac myocytes continued to increase to term. 2) The (hitherto unrecognized) contribution to cardiac growth by the increase in myocyte size associated with the transition from mononucleation to binucleation (terminal differentiation) became considerable from approximately 115 days of gestational age (dGA) until term (145dGA). Because binucleation became the more frequent outcome of myocyte cell cycle activity after approximately 115dGA, the number of binucleated myocytes increased at the expense of the number of mononucleated myocytes. Both the interval between nuclear divisions and the duration of cell cycle activity in myocytes decreased substantially during this same period. Finally, cardiac growth was in part due to enlargement of 3) mononucleated and 4) binucleated myocytes, which grew in cross-sectional diameter but not length during the last third of gestation. These data on normal cardiac growth may enable a more detailed understanding of the consequences of experimental and pathological interventions in prenatal life.     

A method to collect isolated myocytes and whole tissue from the same heart

A technique for isolation of cardiac myocytes and collection of whole heart tissue from individual hearts of adult rats is described in this study. After excision of the apical half of the left ventricle (LV) and cauterization of the cut edge, aortas were cannulated and high-quality isolated cardiac myocytes were collected after collagenase perfusion of the basal portion. Myocyte dimensions from the basal portion of cauterized and noncauterized hearts from matching rats were identical. Additionally, myocyte dimensions from the basal and apical halves of the LV were compared with the use of whole heart-isolated myocyte preps. No regional differences between basal and apical LV myocyte size were found. Therefore, this cauterization method can be used to collect isolated myocytes from the basal half and whole heart tissue from the apical half, with each half being representative of the other with respect to myocyte dimensions.     

The capacity for reactive DNA synthesis of the myocytes in the heart conduction system in experimental and clinical myocardial pathology]

The DNA synthesis has been studied in the conductive system (CS) myocytes, compared to that in atrial and ventricular myocytes: 1) in the left ventricular myocardial infarction induced in two- and three-week-old and adult rats, 2) after isoproterenol injections to adult rats and mice, and 3) in the hypertrophied human heart. The extent of DNA synthesis reactivation was evaluated by the cumulative labeling indices in experiments with multiple 3HTdR injections to rats and mice. In the human cardiac myocyte nuclei, the DNA content was determined by the Feulgen-cytophotometry. The difference between the control and experimental mean values of the labeling indices for CS myocyte nuclei was statistically significant only for atrioventricular part of the CS in the infarcted hearts of adult rats. In the human heart CS the ability of myocytes to polyploidization varies from one cell type to another, the lowest being in nodal cells.     

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.     

Trk C receptor signaling regulates cardiac myocyte proliferation during early heart development in vivo

Neurotrophin-3 (NT-3) is a member of the neurotrophin family of growth factors, best characterized by its survival- and differentiation-inducing effects on developing neurons bearing the trk C receptor tyrosine kinase. Through analysis of NT-3 and trk C gene-targeted mice we have identified NT-3 as critically regulating cardiac septation, valvulogenesis, and conotruncal formation. Although these defects could reflect cardiac neural crest dysfunction, the expression of NT-3 and trk C by cardiac myocytes prior to neural crest migration prompted analysis of cell-autonomous actions of NT-3 on cardiac myocytes. Retroviral-mediated overexpression of truncated trk C receptor lacking kinase activity was used to inhibit activation of trk C by endogenous NT-3, during early heart development in ovo. During the first week of chicken development, expression of truncated trk C reduced myocyte clone size by more than 60% of control clones. Direct mitogenic actions of NT-3 on embryonic cardiac myocytes were demonstrated by analysis of BrdU incorporation or PCNA immunoreactivity in control and truncated trk C-expressing clones. Inhibition of trk C signaling reduced cardiac myocyte proliferation during the first week of development, but had no effect at later times. These studies demonstrate that endogenous NT-3:trk C signaling regulates cardiac myocyte proliferation during cardiac looping and the establishment of ventricular trabeculation but that myocyte proliferation becomes NT-3 independent during the second week of embryogenesis.     

Sequential growth of fetal sheep cardiac myocytes in response to simultaneous arterial and venous hypertension

While the fetal heart grows by myocyte enlargement and proliferation, myocytes lose their capacity for proliferation in the perinatal period after terminal differentiation. The relationship between myocyte enlargement, proliferation, and terminal differentiation has not been studied under conditions of combined arterial and venous hypertension, as occurs in some clinical conditions. We hypothesize that fetal arterial and venous hypertension initially leads to cardiomyocyte proliferation, followed by myocyte enlargement. Two groups of fetal sheep received intravascular plasma infusions for 4 or 8 days (from 130 days gestation) to increase vascular pressures. Fetal hearts were arrested in diastole and dissociated. Myocyte size, terminal differentiation (%binucleation), and cell cycle activity (Ki-67[+] cells as a % of mononucleated myocytes) were measured. We found that chronic plasma infusion greatly increased venous and arterial pressures. Heart (but not body) weights were approximately 30% greater in hypertensive fetuses than controls. The incidence of cell cycle activity doubled in hypertensive fetuses compared with controls. After 4 days of hypertension, myocytes were (approximately 11%) longer, but only after 8 days were they wider (approximately 12%). After 8 days, %binucleation was approximately 50% greater in hypertensive fetuses. We observed two phases of cardiomyocyte growth and maturation in response to fetal arterial and venous hypertension. In the early phase, the incidence of cell cycle activity increased and myocytes elongated. In the later phase, the incidence of cell cycle activity remained elevated, %binucleation increased, and cross sections were greater. This study highlights unique fetal adaptations of the myocardium and the importance of experimental duration when interpreting fetal cardiac growth data.     

Erythropoietin and retinoic acid,secreted from the epicardium,are required for cardiac myocyte proliferation

We have established a heart slice primary culture, which allows us to mechanically separate distinct cardiac cell populations and assay their relative mitogenic and trophic effects on cardiac myocyte proliferation and survival. Using this system, we have found that a signal(s) from the epicardium, but not the trabeculae and endocardium, is required in embryonic day 10 (E10) chick heart slices for continued cardiac myocyte proliferation and survival. An examination of potential epicardial growth or trophic factors has revealed that blockade of either retinoic acid (RA) or erythopoietin (epo) signaling from the epicardium inhibits cardiac myocyte proliferation and survival. The blockade of cardiac myocyte proliferation following administration of an RA antagonist can be rescued by exogenous epo. Conversely, the blockade of cardiac myocyte proliferation following administration of an anti-epo receptor antisera can be rescued by exogenous RA. Thus, our findings suggest that RA and epo signals work in parallel to support myocardial cell proliferation. In addition, we have found that these factors do not act directly on myocardial cells. Rather, they induce another soluble factor(s) in the epicardium that directly regulates proliferation of cardiac myocytes. We therefore postulate that the epicardium controls normal heart growth in ventricular segments of the embryonic chick heart by secreting a cardiac myocyte mitogen whose expression (or activity) is regulated by both RA and erythropoietin signaling.     

Power output is linearly related to MyHC content in rat skinned myocytes and isolated working hearts

The amount of work the heart can perform during ejection is governed by the inherent contractile properties of individual myocytes. One way to alter contractile properties is to alter contractile proteins such as myosin heavy chain (MyHC), which is known to demonstrate isoform plasticity in response to disease states. The purpose of this study was to examine myocyte functionality over the complete range of MyHC expression in heart, from 100% alpha-MyHC to 100% beta-MyHC, using euthyroid and hypothyroid rats. Peak power output in skinned cardiac myocytes decreased as a nearly linear function of beta-MyHC expression during maximal (r2 = 0.85, n = 44 myocyte preparations) and submaximal (r2 = 0.82, n = 31 myocyte preparations) Ca2+ activation. To determine whether single myocyte function translated to the level of the whole heart, power output was measured in working heart preparations expressing varied ratios of MyHC. Left ventricular power output of isolated working heart preparations also decreased as a linear function of increasing beta-MyHC expression (r2 = 0.82, n = 34 myocyte preparations). These results demonstrate that power output is highly dependent on MyHC expression in single myocytes, and this translates to the performance of working left ventricles.     

A primary culture system for sustained expression of a calcium sensor in preserved adult rat ventricular myocytes

For studying heart pathologies on the cellular level, cultured adult cardiac myocytes represent an important approach. We aimed to explore a novel adult rat ventricular myocyte culture system with minimised dedifferentiation allowing extended experimental manipulation of the cells such as expression of exogenous proteins. Various culture conditions were investigated including medium supplement, substrate coating and electrical pacing for one week. Adult myocytes were probed for (i) viability, (ii) morphology, (iii) frequency dependence of contractions, (iv) Ca(2+) transients, and (v) their tolerance towards adenovirus-mediated expression of the Ca(2+) sensor "inverse pericam". Conventionally, in either serum supplemented or serum-free medium, myocytes dedifferentiated into flat cells within 3 days or cell physiology and morphology were impaired, respectively. In contrast, myocytes cultured in medium supplemented with an insulin-transferrin-selenite mixture on substrates coated with extracellular matrix proteins showed an increased cell attachment and a conserved cross-striation. Moreover, these myocytes displayed optimised preservation of their contractile behaviour and Ca(2+) signalling even under conditions of continuous electrical pacing. Sustained expression of inverse pericam did not alter myocyte function and allowed long lasting high speed Ca(2+) imaging of electrically driven adult myocytes. Our single-cell model thus provides a new advance for high-content screening of these highly specialised cells.     

Lymph heart musculature in birds

Development and innervation of the lymph heart musculature of chicken, emu, rhea, and duck was studied by electron microscopy at post-hatch ages from 3 days to adulthood. Development of innervation was monitored by acetylcholinesterase staining. Horseradish peroxidase was used to determine the extent of the transverse tubule network. Chickens were unusual among these birds in that lymph heart myocytes had already undergone a definitive differentiation and degeneration by 3 days. In ducks and ratite birds, lymph heart myocytes more slowly but progressively differentiate a cytomorphology that does not conform in all characteristics to cardiac or skeletal muscle and even resembles in some aspects, smooth muscle. Myofibrils become the dominant cytoplasmic structure, transverse tubules form "internal couplings" with agranular reticulum cisternae, and "external couplings" are formed between myocytes at myomyal junctions. The myomyal junctions also contain AChE-positive reaction product and some subplasmalemmal vesicles that lack a dense core. The lymph heart myocardium of ducks of 2 weeks demonstrated mitotic figures. In adult ducks the myosatellite cell numbers diminish and a characteristic pattern of myocyte degeneration appears. In juvenile ducks and ratites some myocytes differentiate to conductile cells, much as the conductile myocytes and myofibers of the blood heart. The lymph heart innervation is described, and the role of nerve in differentiation and maintenance of myocyte morphology in the lymph heart is discussed.     

Measurement of cardiac mechanical function in isolated ventricular myocytes from rats and mice by computerized video-based imaging

Isolated adult cardiac ventricular myocytes have been a useful model for cardiovascular research for more than 20 years. With the recent advances in cellular physiology and transgenic techniques, direct measurement of isolated ventricular myocyte mechanics is becoming an increasingly important technique in cardiac physiology that provides fundamental information on excitation-contraction coupling of the heart, either in drug intervention or pathological states. The goal of this article is to describe the isolation of ventricular myocytes from both rats and mice, and the use of real-time beat-to-beat simultaneous recording of both myocyte contraction and intracellular Ca²⁺ transients. Published: December 11, 2001     

Fetal myocardium in the kidney capsule: an in vivo model of repopulation of myocytes by bone marrow cells

Debate surrounds the question of whether the heart is a post-mitotic organ in part due to the lack of an in vivo model in which myocytes are able to actively regenerate. The current study describes the first such mouse model--a fetal myocardial environment grafted into the adult kidney capsule. Here it is used to test whether cells descended from bone marrow can regenerate cardiac myocytes. One week after receiving the fetal heart grafts, recipients were lethally irradiated and transplanted with marrow from green fluorescent protein (GFP)-expressing C57Bl/6J (B6) donors using normal B6 recipients and fetal donors. Levels of myocyte regeneration from GFP marrow within both fetal myocardium and adult hearts of recipients were evaluated histologically. Fetal myocardium transplants had rich neovascularization and beat regularly after 2 weeks, continuing at checkpoints of 1, 2, 4, 6, 8 and12 months after transplantation. At each time point, GFP-expressing rod-shaped myocytes were found in the fetal myocardium, but only a few were found in the adult hearts. The average count of repopulated myocardium with green rod-shaped myocytes was 996.8 cells per gram of fetal myocardial tissue, and 28.7 cells per adult heart tissue, representing a thirty-five fold increase in fetal myocardium compared to the adult heart at 12 months (when numbers of green rod-shaped myocytes were normalized to per gram of myocardial tissue). Thus, bone marrow cells can differentiate to myocytes in the fetal myocardial environment. The novel in vivo model of fetal myocardium in the kidney capsule appears to be valuable for testing repopulating abilities of potential cardiac progenitors.     

Role of troponin I phosphorylation in protein kinase C-mediated enhanced contractile performance of rat myocytes

Our goal was to define the role of phosphorylated cardiac troponin-I in the adult myocyte contractile performance response to activated protein kinase C. In agreement with earlier work, endothelin enhanced both adult rat myocyte contractile performance and cardiac troponin-I phosphorylation. Protein kinase C participated in both responses. The role of cardiac troponin-I phosphorylation in the contractile function response to protein kinase C was further investigated using gene transfer into myocytes of troponin-I isoforms/mutants lacking one or more phosphorylation sites previously identified in purified cardiac troponin-I. Sarcomeric replacement with slow skeletal troponin-I-abrogated protein kinase C-mediated troponin-I phosphorylation. In functional studies, endothelin slowed relaxation in myocytes expressing slow skeletal troponin-I, while the relaxation rate increased in myocytes expressing cardiac troponin-I. Based on these results, acceleration of myocyte relaxation during protein kinase C activation largely depended on cardiac troponin-I phosphorylation. Experiments with troponin-I isoform chimeras provided evidence that phosphorylation sites in the amino portion of cardiac troponin I-mediated the protein kinase C acceleration of relaxation. The cardiac troponin-I Thr-144 phosphorylation site identified in earlier biochemical studies was not significantly phosphorylated during the acute contractile response. Thus, amino-terminal protein kinase C-dependent phosphorylation sites in cardiac troponin-I are likely responsible for the accelerated relaxation observed in adult myocytes.     

Cytoskeletal modulation of electrical and mechanical activity in cardiac myocytes

The cardiac myocyte has an intracellular scaffold, the cytoskeleton, which has been implicated in several cardiac pathologies including hypertrophy and failure. In this review we describe the role that the cytoskeleton plays in modulating both the electrical activity (through ion channels and exchangers) and mechanical (or contractile) activity of the adult heart. We focus on the 3 components of the cytoskeleton, actin microfilaments, microtubules, and desmin filaments. The limited visual data available suggest that the subsarcolemmal actin cytoskeleton is sparse in the adult myocyte. Selective disruption of cytoskeletal actin by pharmacological tools has yet to be verified in the adult cell, yet evidence exists for modulation of several ionic currents, including I(CaL), I(Na), I(KATP), I(SAC) by actin microfilaments. Microtubules exist as a dense network throughout the adult cardiac cell, and their structure, architecture, kinetics and pharmacological manipulation are well described. Both polymerised and free tubulin are functionally significant. Microtubule proliferation reduces contraction by impeding sarcomeric motion; modulation of sarcoplasmic reticulum Ca(2+) release may also be involved in this effect. The lack of effect of microtubule disruption on cardiac contractility in adult myocytes, and the concentration-dependent modulation of the rate of contraction by the disruptor nocodazole in neonatal myocytes, support the existence of functionally distinct microtubule populations. We address the controversy regarding the stimulation of the beta-adrenergic signalling pathway by free tubulin. Work with mice lacking desmin has demonstrated the importance of intermediate filaments to normal cardiac function, but the precise role that desmin plays in the electrical and mechanical activity of cardiac muscle has yet to be determined.     

Hepatocyte growth factor protects cardiac myocytes against oxidative stress-induced apoptosis

Hepatocyte growth factor (HGF) has been proposed as an endogenous cardioprotective agent against oxidative stress. The mechanism of HGF action in the heart, however, has not yet been elucidated. The present study demonstrates that HGF protects adult cardiac myocytes against oxidative stress-induced apoptosis. HGF, at the concentrations which can be detected in the plasma of humans subsequent to myocardial infarction, effectively attenuated death of isolated adult rat cardiac myocytes and cultured HL-1 cardiac muscle cells induced by apoptosis-inducing oxidative stress stimuli such as daunorubicin, serum deprivation, and hydrogen peroxide. We identified expression of c-Met HGF receptor in adult cardiac myocytes, which can be rapidly tyrosine phosphorylated in response to HGF treatment. HGF also activated MEK, p44/42 MAPK, and p90RSK. To determine if MEK-MAPK pathway may be involved in the mechanism of HGF-mediated cardiac myocyte protection, effects of a specific MEK inhibitor, PD98059, were studied. Pretreatment of cells with PD98059 partially blocked HGF signaling for protection against hydrogen peroxide-induced cell death. Thus, HGF protects cardiac myocytes against oxidative stress, in part, via activating MEK-MAPK pathway.