The human adult cardiomyocyte phenotype.

AIM: Determination of the phenotype of adult human atrial and ventricular myocytes based on gene expression and morphology. METHODS: Atrial and ventricular cardiomyocytes were obtained from patients undergoing cardiac surgery using a modified isolation procedure. Myocytes were isolated and cultured with or without serum. The relative cell attachment promoting efficiency of several reagents was evaluated and compared. Morphological changes during long-term culture were assessed with phase contrast microscopy, morphometric analysis and immunocytochemistry or RT-PCR of sarcomeric markers including alpha-actinin, myosin light chain-2 (MLC-2) and the adhesion molecule, cadherin. RESULTS: The isolation method produced viable rod-shaped atrial (16.6+/-6.0%, mean+/-S.E.; n=5) and ventricular cells (22.4+/-8.0%, mean+/-S.E.; n=5) in addition to significant numbers of apoptotic and necrotic cells. Cell dedifferentiation was characterized by the loss of sarcomeric structure, condensation and extrusion of sarcomeric proteins. Cells cultured with low serum recovered and assumed a flattened, spread form with two distinct morphologies apparent. Type I cells were large, had extensive sarcolemmal spreading, with stress fibers and nascent myofibrils, whilst type II cells appeared smaller, with more mature myofibril organisation and focal adhesions. CONCLUSION: Characterization of the redifferentiation capabilities of cultured adult cardiac myocytes in culture, provides an important system for comparing cardiomyocytes differentiating from human stem cells and provides the basis for an in vitro transplantation model to study interaction and communication between primary adult and stem cell-derived cardiomyocytes.

Cardiomyocyte differentiation of mouse and human embryonic stem cells.

Ischaemic heart disease is the leading cause of morbidity and mortality in the western world. Cardiac ischaemia caused by oxygen deprivation and subsequent oxygen reperfusion initiates irreversible cell damage, eventually leading to widespread cell death and loss of function. Strategies to regenerate damaged cardiac tissue by cardiomyocyte transplantation may prevent or limit post-infarction cardiac failure. We are searching for methods for inducing pluripotent stem cells to differentiate into transplantable cardiomyocytes. We have already shown that an endoderm-like cell line induced the differentiation of embryonal carcinoma cells into immature cardiomyocytes. Preliminary results show that human and mouse embryonic stem cells respond in a similar manner. This study presents initial characterization of these cardiomyocytes and the mouse myocardial infarction model in which we will test their ability to restore cardiac function.

Platelet derived growth factor-BB induced calcium transients in cultured human peritoneal mesothelial cells.

The peritoneal membrane is used as an artificial dialysis organ for patients with end-stage renal failure during continuous ambulatory peritoneal dialysis (CAPD). Resident peritoneal mast cells may influence the course of the inflammatory process during acute infection or prolonged exposure to dialysate in a paracrine fashion, with the release of preformed mediators, including platelet derived growth factor (PDGF), which may activate human peritoneal mesothelial cells (HPMC) and other peritoneal cells. PDGF receptor interactions have multiple intracellular effects, typified by cell proliferation, protein synthesis, cytoskeletal organization, extracellular matrix turnover, and angiogenesis. PDGF may mediate HPMC inflammatory response by stimulating cytokine release. The presence of transcripts for both PDGF-B polypeptide and PDGF-beta receptor in cultured HPMC was confirmed using a reverse transcriptase-polymerase chain reaction. To investigate the activity of PDGF-beta receptors in HPMC, the authors examined intracellular calcium (Ca2+(i)) mobilization in HPMC in response to PDGF-BB (100 ng/ml), histamine (1.0 mmol/L), and 4-brA23187 (1.0 micromol/L) using the calcium indicator, fura-2. HPMC calcium mobilization in response to PDGF was assessed in Krebs-Ringer saline, with and without added external calcium (Ca2+(ext)). HPMC responded to PDGF with a transient rise in Ca2+(i) (approximately 1.6-fold) that returned to an elevated resting value. In the absence of Ca2+(ext), PDGF produced a Ca2+(i) transient indicative of Ca2+ release from intracellular stores. These results suggest that HPMC have functional PDGF-beta receptors that may bind and respond to PDGF in both an autocrine and paracrine

Mast cell histamine-induced calcium transients in cultured human peritoneal mesothelial cells.

OBJECTIVE: Peritoneal inflammation results from a complex interplay of events initiated by macrophage activity in response to infection, with the stimulation of mesothelial cell cytokine release amplifying the recruitment of blood-borne defense cells to the site of injury. Resident peritoneal mast cells may add to this complexity with mast cell derived cytokines released during this cascade. This study examined the influence of histamine, a mast cell-derived inflammatory mediator, on the initial activation of human peritoneal mesothelial cells (HPMC) by intracellular free calcium (Ca2+(i)) mobilization, and changes to the actin cytoskeleton. DESIGN: HPMC signal transduction was examined in response to histamine (1.0 mmol/L) compared to fetal bovine serum (FBS) (0.1%) and 4-br-A23187 (1.0 micromol/L). Intracellular free calcium was measured in fura-2 loaded cells with and without external calcium (Ca2+(ext)), or Ca2+(ext) with verapamil (100 micromol/L). Following treatment with agonists, HPMC actin cytoskeleton was stained using direct immunocytochemistry. RESULTS: HPMC responded to histamine with a twofold transient rise in Ca2+(i) which returned to the baseline, in contrast with FBS- and A23187-induced Ca2+(i) transients, which returned to elevated resting values. In the absence of Ca2+(ext), all agents produced a calcium transient indicative of calcium release from intracellular stores. Histamine induced calcium-dependent changes to the cytoskeleton and cellular organization, including increased actin stress fibers. CONCLUSION: Histamine produced large specific receptor-mediated calcium transients in HPMC, which included components of calcium release from intracellular stores and receptor-mediated calcium influx processes. The observed response to histamine raises the possibility that histamine derived from resident mast cells may modulate mesothelial cell function, in part by calcium-dependent pathways, and influence the performance of the peritoneal membrane during peritoneal dialysis.

L-cysteine improves growth of human peritoneal mesothelial cells in vitro.

OBJECTIVE: To improve the growth characteristics of human peritoneal mesothelial cells (HPMC). DESIGN: The effect of commonly used agents, L-cysteine and epidermal growth factor (EGF), added individually ("single") or mixed with hydrocortisone and apo-transferrin ("admixture") in the culture medium (M199) on cultured HPMC, was investigated. METHODS: Growth agents were added to M199 medium along with 2% fetal bovine serum and L-glutamine. Growth was determined by the analysis of thymidine ([methyl-3H] thymidine) incorporation into deoxyribonucleic acid, total cell protein, and by cell counts. Morphology was assessed by phase contrast light microscopy and scanning electron microscopy. RESULTS: HPMC exposed to L-cysteine 0.25 x 10(-3) mol/L (30 micrograms/mL) exhibited significantly improved attachment and growth. Attached cells appeared flat and well spread out shortly after seeding, and produced a tight polygonal monolayer in 14 days, in contrast to the growth of HPMC in control M199 medium, which failed to reach confluence. After an initial lag period in cell growth, EGF (0.01 microgram/mL) produced a greater increase in cell growth than L-cysteine did; however, this was associated with changes in HPMC morphology. During the growth period (14 days), EGF-stimulated HPMC appeared distorted and irregular compared to L-cysteine-treated cells, which had the characteristic tight "cobblestone" appearance. CONCLUSION: L-cysteine improved cell attachment with preservation of the characteristic morphology of HPMC. Epidermal growth factor improved cell growth but produced changes in morphology. The addition of L-cysteine to the culture medium has an important cell growth enhancement role due to the improved cell attachment and cell viability.

Altered cellular biosynthesis in human peritoneal mesothelial cells exposed to dialysis solutions.

Peritoneal dialysis (PD) solutions can produce changes in the peritoneal membrane and structure which are related to the high glucose concentrations and low pH of these solutions. This study was designed to examine the effects of high glucose (76-214 mM) and low pH (5.3) solutions on human peritoneal mesothelial cells in culture. Changes in mesothelial cell biosynthesis and cell numbers were evident within 2 hours of exposure to unmodified dialysis solutions. In the development of new dialysis solutions, human peritoneal mesothelial cells in culture provide a good in vitro model to determine potential toxicity.

Altered free calcium transients in pig kidney cells (LLC-PK1) cultured with penicillin/streptomycin.

The effect of a conventional antibiotic (penicillin/streptomycin) mixture on the widely used kidney epithelial cell line, LLC-PK1, was investigated by measuring growth and intracellular free calcium. Free calcium concentration was the same in cells cultured for 3 to 7 wk with ("plus") and without ("minus") antibiotics both at rest and when challenged with high (14 mM) external calcium. When exposed to vasopressin, minus cells exhibited significantly smaller calcium transients than plus cells. A similar difference existed for transients elicited by a calcium ionophore, 4-br-A23187. After longer periods of culture (> 20 wk), minus cells grew slower than plus cells but on reaching confluence (minus cells took 1 day longer) the morphologies and viabilities were indistinguishable. The finding that culture with penicillin/streptomycin reversibly modified some properties of LLC-PK1 cells, at least partly through altered calcium homeostasis, is of importance for workers using this cell model to study drug effects and raises the general possibility of similar effects on other cultured cells.