Primary Mouse Cardiomyocytes Research Articles

Mouse and Human Resistins Impair Glucose Transport in Primary Mouse Cardiomyocytes, and Oligomerization Is Required for This Biological Action

The adipocytokine resistin impairs glucose tolerance and insulin sensitivity in rodents. Here, we examined the effect of resistin on glucose uptake in isolated adult mouse cardiomyocytes. Murine resistin reduced insulin-stimulated glucose uptake, establishing the heart as a resistin target tissue. Notably, human resistin also impaired insulin action in mouse cardiomyocytes, providing the first evidence that human and mouse resistin homologs have similar functions. Resistin is a cysteine-rich molecule that circulates as a multimer of a dimeric form dependent upon a single intermolecular disulfide bond, which, in the mouse, involves Cys26; mutation of this residue to alanine (C26A) produces a monomeric molecule that appears to be bioactive in the liver. Remarkably, unlike native resistin, monomeric C26A resistin had no effect on basal or insulin-stimulated glucose uptake in mouse cardiomyocytes. Resistin impairs glucose uptake in cardiomyocytes by mechanisms that involve altered vesicle trafficking. Thus, in cardiomyocytes, both mouse and human resistins directly impair glucose transport; and in contrast to effects on the liver, these actions of resistin require oligomerization.

The Adenosine Transporter, mENT1, Is a Target for Adenosine Receptor Signaling and Protein Kinase Cϵ in Hypoxic and Pharmacological Preconditioning in the Mouse Cardiomyocyte Cell Line, HL-1

Brief exposure of the heart to hypoxia results in less cellular damage after subsequent hypoxia, an effect known as preconditioning (PC). PC has been widely studied but is still not fully understood. Adenosine (Ado), adenosine receptors, and protein kinase C (PKC) have been implicated as integral components of PC. Adenosine (nucleoside) transporters (NTs) facilitate flux of Ado across cell membranes, but their role in PC is unknown. Therefore, we used the murine cardiomyocyte cell line, HL-1, and asked if there was feedback regulation of NTs by Ado, Ado receptors, and PKC following either hypoxic or pharmacological PC. Activation (by specific agonists) of A1 or A3 Ado receptors or PKC resulted in PC in HL-1. The A1 (but not A3) receptor is coupled to PKCepsilon, and activation of PKCepsilon (by specific peptide agonist) resulted in PC. Moreover, PKCepsilon stimulates Ado uptake via the predominant NT in HL-1, mouse equilibrative nucleoside transporter 1 (mENT1). Studies in primary neonatal mouse cardiomyocytes confirmed our observations in HL-1 cells. Hypoxic challenge led to a rapid increase in, and efflux of, intracellular Ado from cells, which was blocked by NT inhibitors (dipyridamole/nitrobenzylthioinosine). Moreover, NT inhibition during hypoxia or PC was highly protective, suggesting that Ado loss contributes to decreased cell viability. Our data suggest that hypoxic challenge causes an efflux of Ado via ENTs, activation of A1 and/or A3 receptors, signaling through PKCepsilon, and activation of ENT1. Since Ado is required for ATP synthesis on reperfusion, this feedback regulation of mENT1 would promote reuptake of Ado.

Design of Artificial Myocardial Microtissues

Cultivation technologies promoting organization of mammalian cells in three dimensions are essential for gene-function analyses as well as drug testing and represent the first step toward the design of tissue replacements and bioartificial organs. Embedded in a three-dimensional environment, cells are expected to develop tissue-like higher order intercellular structures (cell-cell contacts, extracellular matrix) that orchestrate cellular functions including proliferation, differentiation, apoptosis, and angiogenesis with unmatched quality. We have refined the hanging drop cultivation technology to pioneer beating heart microtissues derived from pure primary rat and mouse cardiomyocyte cultures as well as mixed populations reflecting the cell type composition of rodent hearts. Phenotypic characterization combined with detailed analysis of muscle-specific cell traits, extracellular matrix components, as well as endogenous vascular endothelial growth factor (VEGF) expression profiles of heart microtissues revealed (1). a linear cell number-microtissue size correlation, (2). intermicrotissue superstructures, (3). retention of key cardiomyocyte-specific cell qualities, (4). a sophisticated extracellular matrix, and (5). a high degree of self-organization exemplified by the tendency of muscle structures to assemble at the periphery of these myocardial spheroids. Furthermore (6). myocardial spheroids support endogenous VEGF expression in a size-dependent manner that will likely promote vascularization of heart microtissues produced from defined cell mixtures as well as support connection to the host vascular system after implantation. As cardiomyocytes are known to be refractory to current transfection technologies we have designed lentivirus-based transduction strategies to lead the way for genetic engineering of myocardial microtissues in a clinical setting.

Ca 2+-dependent interaction of S100A1 with the sarcoplasmic reticulum Ca 2+-ATPase2a and phospholamban in the human heart

The Ca 2+-binding S100A1 protein displays a specific and high expression level in the human myocardium and is considered to be an important regulator of heart contractility. Diminished protein levels detected in dilated cardiomyopathy possibly contribute to impaired Ca 2+ handling and contractility in heart failure. To elucidate the S100A1 signaling pathway in the human heart, we searched for S100A1 target proteins by applying S100A1-specific affinity chromatography and immunoprecipitation techniques. We detected the formation of a Ca 2+-dependent complex of S100A1 with SERCA2a and PLB in the human myocardium. Using confocal laser scanning microscopy, we showed that all three proteins co-localize at the level of the SR in primary mouse cardiomyocytes and confirmed these results by immunoelectron microscopy in human biopsies. Our results support a regulatory role of S100A1 in the contraction–relaxation cycle in the human heart.

Transcriptional regulation of S100A1 and expression during mouse heart development

S100A1, a member of the large EF-hand family of Ca 2+-binding proteins, is mainly expressed in the mammalian heart. To assess the underlying mechanisms for cell- and tissue-specific expression we isolated and characterized the mouse S100A1 gene. The gene displays a high degree of homology to the human and rat genes, especially in the exonic sequences. In its promoter region and the first intron, we identified regulatory elements characteristic for cardiac and slow skeletal muscle restricted genes. Transfection assays with luciferase constructs containing different parts of the S100A1 gene demonstrated the active expression in primary mouse cardiomyocytes and that its 5′-upstream region containing a putative cardiac enhancer showed a greatly increased activity. Furthermore, we investigated the expression of the S100A1 mRNA during embryonic mouse development, using in situ hybridization. S100A1 transcripts were first detected in the primitive heart at embryonic day (E) 8, with equal levels in the atrium and ventricle. During development up to E17.5 we detected a shift in the S100A1 expression pattern with lower levels in atrial and high levels in ventricular myocardium. The regulatory elements identified in the mouse S100A1 promoter correspond well with the observed expression pattern and suggest that S100A1 has an important function during heart muscle development.

Morin hydrate: A potential antioxidant in minimizing the free-radicals-mediated damage to cardiovascular cells by anti-tumor drugs

The co-incubation of morin hydrate with either doxorubicin or mitomycin C could minimize the toxicity of these anti-tumor drugs on cardiovascular cells, such as red blood cells, human umbilical vein endothelial cells (ECV304) and primary mouse cardiomyocytes, whereas morin hydrate did not lower the cytotoxicity of the drugs on human hepatocellular carcinoma cells (HepG2). Morin hydrate may not exert its antioxidant effect by enhancing the antioxidant enzymatic activity because it did not cause any induction on the mRNA levels of manganese superoxide dismutase expression in ECV304 cells and HepG2 cells.

Metallothionein-overexpressing neonatal mouse cardiomyocytes are resistant to H2O2 toxicity.

To study cellular and molecular events of cardiac protection by metallothionein (MT) from oxidative injury, a primary neonatal cardiomyocyte culture was established from a specific cardiac MT-overexpressing transgenic mouse model. Ventricular cardiomyocytes were isolated from 1- to 3-day-old neonatal mice and cultured in an Eagle's minimum essential medium supplemented with 20% fetal bovine serum under an atmosphere of 5% CO2-95% air at 37 degreesC. Forty-eight hours after plating was completed, the purity of such cultures was 95% myocytes, assessed by an immunocytochemical assay. Over 80% of the cardiomyocytes beat spontaneously on the first day of culture and synchronously in a confluent monolayer after the sixth day of culture. Cellular MT concentrations in the transgenic cardiomyocytes before culturing and on the sixth day postculturing were about seven- and twofold higher than nontransgenic controls, respectively. However, there were no significant differences in cell morphology, glutathione content, and antioxidant enzymatic activities between these two types of cardiomyocytes. When these cells were challenged by H2O2, the transgenic cardiomyocytes displayed a significant resistance to the toxic effect of this oxidant, as measured by cell viability, lactate dehydrogenase leakage, and morphological alterations. In addition, the transgenic cells were highly protected from H2O2-induced lipid peroxidation. These observations demonstrate that MT protects the cultured cardiomyocytes from H2O2 toxicity by preventing its interaction with macromolecules such as lipids, and this cultured primary neonatal mouse cardiomyocyte system provides a valuable tool to directly study cellular and molecular events of MT in cardiac protection against oxidative injury.