Ischaemia-induced autophagy leads to degradation of gap junction protein connexin43 in cardiomyocytes

GJIC (gap junction intercellular communication) between cardiomyocytes is essential for synchronous heart contraction and relies on Cx (connexin)-containing channels. Increased breakdown of Cx43 has been often associated with various cardiac diseases. However, the mechanisms whereby Cx43 is degraded in ischaemic heart remain unknown. The results obtained in the present study, using both HL-1 cells and organotypic heart cultures, show that simulated ischaemia induces degradation of Cx43 that can be prevented by chemical or genetic inhibitors of autophagy. Additionally, ischaemia-induced degradation of Cx43 results in GJIC impairment in HL-1 cells, which can be restored by autophagy inhibition. In cardiomyocytes, ubiquitin signals Cx43 for autophagic degradation, through the recruitment of the ubiquitin-binding proteins Eps15 (epidermal growth factor receptor substrate 15) and p62, that assist in Cx43 internalization and targeting to autophagic vesicles, via LC3 (light chain 3). Moreover, we establish that degradation of Cx43 in ischaemia or I/R (ischaemia/reperfusion) relies upon different molecular players. Indeed, degradation of Cx43 during early periods of ischaemia depends on AMPK (AMP-activated protein kinase), whereas in late periods of ischaemia and I/R Beclin 1 is required. In the Langendorff-perfused heart, Cx43 is dephosphorylated in ischaemia and degraded during I/R, where Cx43 degradation correlates with autophagy activation. In summary, the results of the present study provide new evidence regarding the molecular mechanisms whereby Cx43 is degraded in ischaemia, which may contribute to the development of new strategies that aim to preserve GJIC and cardiac function in ischaemic heart.

Tumor necrosis factor alpha protects heart cultures against hypoxic damage via activation of PKA and phospholamban to prevent calcium overload

This study aims to elucidate the mechanisms by which tumor necrosis factor alpha (TNFα) provides protection from hypoxic damage to neonatal rat cardiomyocyte cultures. We show that when intracellular Ca2+ ([Ca2+]i) levels are elevated by extracellular Ca2+ ([Ca2+]o) or by hypoxia, then TNFα decreased [Ca2+]i in individual cardiomyocytes. However, TNFα did not reduce [Ca2+]i after its increase by thapsigargin, (a SERCA2a inhibitor), indicating that TNFα attenuates Ca2+ overload through Ca2+ uptake by SERCA2a. TNFα did not reduce [Ca2+]i, following its elevation when [Ca2+]o levels were elevated in TNFα receptor knock-out mice. H-89, a protein kinase A (PKA) inhibitor, attenuated the protective effect of TNFα when the cardiomyoctyes were subjected to hypoxia, as determined by lactate dehydrogenase (LDH) and creatine kinase (CK) released and from the cardiomyocytes. Moreover, when the levels of [Ca2+]i were increased by hypoxia, H-89, but not KN93, (a calmodulin kinase II inhibitor), prevented the reduction in [Ca2+]i by TNFα. TNFα increased the phosphorylation of PKA in normoxic and hypoxic cardiomyoctes, indicating that the cardioprotective effect of TNFα against hypoxic damage was via PKA activation. Hypoxia decreased phosphorylated phospholamban levels; however, TNFα attenuated this decrease following hypoxia. It is suggested that TNFα activates phospholamban phosphorylation in hypoxic heart cultures via PKA to stimulate SERCA2a activity to limit Ca2+ overload.

282 CHARACTERIZATION OF THE DEVELOPMENTAL POTENTIAL OF FETAL SOMATIC STEM CELLS ISOLATED FROM DIFFERENT TISSUES

Recently we discovered a novel type of stem cells which can be derived from primary cultures of fetal connective tissue using a high-density culture (HD) system (Kues et al. 2005 Biol. Reprod. 70, 1020–1028). This cell type shows most of the characteristics of true embryonic stem cells and is a promising alternative for developing therapeutically useful cells. The goal of the present study was to analyze and characterize stem cell populations obtained from five different tissues, i.e., brain, heart, lung, liver, and adrenal gland, using this HD culture system. Explant cultures of the double transgenic strain OG2/Rosa26 were established from mouse fetuses at Day 13.5 post-conception. In this strain, cells with pluripotent properties are characterized by the green fluorescence of the green fluorescent protein (GFP) which is under the transcriptional control of the Oct-4 promoter. In a total of five replicates, cells were subcultivated and reseeded at a concentration of 25 000 cells/cm–2. Cell culture conditions were as recently reported for fetal somatic stem cell derivation (Kues et al. 2005 Biol. Reprod. 72, 1020–1028). After colony formation and/or observation of green fluorescence under UV illumination, mRNA was isolated for RT-PCR analysis of the expression of genes known to be involved in pluripotency, i.e., Oct-4, Nanog, Stat3, TNAP, Rex1, and Sox2. Under these culture conditions, 12.5% of the liver and 80% of the brain explants showed senescence and could not be seeded at the required concentration. No colony formation was observed in brain and heart cultures. Cells from heart explants showed a three-dimensional growth over the whole surface of the attached cells and were difficult to separate. On average, colony formation was observed after 11 days � 8 (mean � SD) (fetal fibroblasts), 10 days � 7 (lung), 18 days � 11 (liver), and 4 days � 1 (adrenal gland). Green fluorescence was detected after 17 days � 9 (fetal fibroblasts), 17 days � 9 (lung), 35 days � 13 (heart, randomly distributed), 26 days � 10 (brain), 26 days � 8 (liver), and 8 days � 5 (adrenal gland). A single large clump of stem-like cells formed in 43% of the fetal fibroblast, 35% of the lung, and 41% of the liver derivatives. Oct-4 expression was not observed in any of the cultures. Nanog and Stat3 were expressed in all cell cultures, and TNAP was expressed in all cultures from heart, brain, lung, and adrenal gland. Rex1 was expressed in all cultures from brain and adrenal gland, and in 25% of the heart cultures and in 16.6% of the fetal fibroblasts. Sox2 was expressed in all cultures from brain and in 16.6% of fetal fibroblast cultures. These preliminary results show that cells with some of the typical features of stem cells can be derived from various tissues of the body, but that the efficiency is different among tissues. Only cells derived from the adrenal gland exhibited better colony formation than the original fibroblasts. This work was funded by DFG grant Ni 256/28-1.

Electron Microscope observations on Mitotic Chromosomes

A method is described by which single mitotic cells growing in tissue culture can be selected under the light microscope and then sectioned for electron-microscopic study. The method has been applied to mitotic cells in newt heart cultures and to monolayer cultures of human fibroblasts. The structure of the chromosomes and of the mitotic apparatus at various phases of mitosis are described and discussed. The effects of pH and of divalent cations on the fixation of chromosomes by buffered osmic mixtures are also considered.

CARBOHYDRATE UTILIZATION BY CHICK EMBRYONIC HEART CULTURES

Freshly explanted chick embryonic heart fragments were cultivated in completely synthetic media. Survival of such cultures in the complete medium, containing glucose, was established at approximately 35 to 40 days, while in the absence of carbohydrate the cultures died within 3 to 5 days. Survival was considered to be a more physiological measurement than rapid cell multiplication for normal tissues and was adopted as the criterion for all experiments reported. Fifty-two compounds were tested for their ability to replace glucose, as the sole carbohydrate, in this system. Of these, seven (mannose, fructose, galactose, β-glucose, maltose, glucose-1-phosphate, and glucose-6-phosphate) replaced glucose completely. Five others (sorbitol, alpha-methyl-D-glucoside, turanose, dextrin, and fructose-6-phosphate) were partially active. The remainder were negative. Comparison is made of the present results with those obtained by other workers using malignant cells in the presence of serum–enzymes. The present results suggest that the ability to replace glucose decreases progressively as compounds down the Embden–Meyerhof pathway are tested.

CARBOHYDRATE UTILIZATION BY CHICK EMBRYONIC HEART CULTURES

Freshly explanted chick embryonic heart fragments were cultivated in completely synthetic media. Survival of such cultures in the complete medium, containing glucose, was established at approximately 35 to 40 days, while in the absence of carbohydrate the cultures died within 3 to 5 days. Survival was considered to be a more physiological measurement than rapid cell multiplication for normal tissues and was adopted as the criterion for all experiments reported. Fifty-two compounds were tested for their ability to replace glucose, as the sole carbohydrate, in this system. Of these, seven (mannose, fructose, galactose, β-glucose, maltose, glucose-1-phosphate, and glucose-6-phosphate) replaced glucose completely. Five others (sorbitol, alpha-methyl-D-glucoside, turanose, dextrin, and fructose-6-phosphate) were partially active. The remainder were negative. Comparison is made of the present results with those obtained by other workers using malignant cells in the presence of serum–enzymes. The present results suggest that the ability to replace glucose decreases progressively as compounds down the Embden–Meyerhof pathway are tested.

TRANSAMINATION IN THE METABOLISM OF ß-2-THIENYL-DL-ALANINE IN NORMAL AND NEOPLASTIC CELLS IN VITRO

In tissue cultures of C-57 black mouse heart and sarcoma T-241, ß-2-thienyl-DL-alanine acts specifically as a phenylalanine antagonist. Heart cultures can transaminate between ß-2-thienyl-DL-alanine and phenylpyruvate to form L-phenylalanine and thus block the toxic action of the remaining ß-2-thienyl-DL-alanine, whereas sarcoma T-241 cultures cannot. Of eleven mouse tumors and four rat tumors tested for their ability to perform this reaction, nine tumors had little or no activity. The ß-2-thienylpyruvic acid resulting from transamination further reacts to form a red compound the exact structure of which is not yet known.