Alterations in myocardial creatinine kinase (CK) and lactate dehydrogenase (LDH) isoenzyme-distribution in a model of left ventricular dysfunction.

The purpose of the current study was to evaluate myocardial creatinine kinase (CK) and lactate dehydrogenase (LDH) systems in a model of epinephrine-induced cardiomyopathy in rabbits. Eight rabbits received four repetitive epinephrine infusions (300 mg/kg/60 min, i.v.) in 12-day intervals and eight untreated rabbits served as controls (CTRL). Echocardiography demonstrated a significant deterioration of LV function as well as increased LV-diameter and -mass index in catecholamine-induced cardiomyopathy. Histological examination revealed that repetitive catecholamine infusion resulted in LV fibrous areas with collagenous content and an increase in myocyte width (16.9+/-0.8 microm vs. CTRL 12.9+/-0.9; P<0.05). LV dysfunction was associated with a decreased total LV lactate dehydrogenase activity (LDH; 0.43+/-0.03 IU/mg protein vs. CTRL 0.52+/-0.04; P<0.05) whereas total creatinine kinase activity was unchanged (CK; 7.30+/-0.63 IU/mg protein vs. CTRL 9.20+/-0.49, n.s.). Furthermore, myocardial LDH isoenzymes were shifted with a decrease in LDH(1) and an increase in LDH2 and LDH3 (LDH(1): 84.90+/-2.60% vs. CTRL 94.50+/-0.40; LDH2: 7.30+/-1.20% vs. 1.50+/-0.13; LDH3: 5.40+/-0.90% vs. 3.20+/-0.25; all P<0.05). Foetal B-CK isoenzymes were significantly increased (CK-MB 5.30+/-0.66 vs. 2.20+/-0.35%; P<0.05). The current study demonstrates changes in cardiac energy metabolism including an impaired LDH activity with a shift towards anaerobic isoenzymes as well as a more efficient CK system in a model of catecholamine-induced LV dysfunction

Sarco(endo)plasmic reticulum ATPase is a molecular partner of Wolfram syndrome 1 protein, which negatively regulates its expression.

This is a pre-copyedited, author-produced PDF of an article accepted for publication in Human Molecular Genetics following peer review. The version of record Zatyka, M., Xavier, G. D. S., Bellomo, E. A., Leadbeater, W., Astuti, D., Smith, J., Michelangeli, F., Rutter, G. A., Barrett, T. G. (2015). Sarco(endo)plasmic reticulum ATPase is a molecular partner of Wolfram syndrome 1 protein, which negatively regulates its expression. Human Molecular Genetics, 24(3), 814-27 is available online at: http://dx.doi.org/10.1093/hmg/ddu499Wolfram syndrome is an autosomal recessive disorder characterized by neurodegeneration and diabetes mellitus. The gene responsible for the syndrome (WFS1) encodes an endoplasmic reticulum (ER)-resident transmembrane protein that is involved in the regulation of the unfolded protein response (UPR), intracellular ion homeostasis, cyclic adenosine monophosphate production and regulation of insulin biosynthesis and secretion. In this study, single cell Ca(2+) imaging with fura-2 and direct measurements of free cytosolic ATP concentration ([ATP]CYT) with adenovirally expressed luciferase confirmed a reduced and delayed rise in cytosolic free Ca(2+) concentration ([Ca(2+)]CYT), and additionally, diminished [ATP]CYT rises in response to elevated glucose concentrations in WFS1-depleted MIN6 cells. We also observed that sarco(endo)plasmic reticulum ATPase (SERCA) expression was elevated in several WFS1-depleted cell models and primary islets. We demonstrated a novel interaction between WFS1 and SERCA by co-immunoprecipitation in Cos7 cells and with endogenous proteins in human neuroblastoma cells. This interaction was reduced when cells were treated with the ER stress inducer dithiothreitol. Treatment of WFS1-depleted neuroblastoma cells with the proteasome inhibitor MG132 resulted in reduced accumulation of SERCA levels compared with wild-type cells. Together these results reveal a role for WFS1 in the negative regulation of SERCA and provide further insights into the function of WFS1 in calcium homeostasis

The MLL fusion gene, MLL-AF4, regulates cyclin-dependent kinase inhibitor CDKN1B (p27(kip1)) expression

MLL, involved in many chromosomal translocations associated with acute myeloid and lymphoid leukemia, has >50 known partner genes with which it is able to form in-frame fusions. Characterizing important downstream target genes of MLL and of MLL fusion proteins may provide rational therapeutic strategies for the treatment of MLL-associated leukemia. We explored downstream target genes of the most prevalent MLL fusion protein, MLL-AF4. To this end, we developed inducible MLL-AF4 fusion cell lines in different backgrounds. Overexpression of MLL-AF4 does not lead to increased proliferation in either cell line, but rather, cell growth was slowed compared with similar cell lines inducibly expressing truncated MLL. We found that in the MLL-AF4-induced cell lines, the expression of the cyclin-dependent kinase inhibitor gene CDKN1B was dramatically changed at both the RNA and protein (p27(kip1)) levels. In contrast, the expression levels of CDKN1A (p21) and CDKN2A (p16) were unchanged. To explore whether CDKN1B might be a direct target of MLL and of MLL-AF4, we used chromatin immunoprecipitation (ChIP) assays and luciferase reporter gene assays. MLL-AF4 binds to the CDKN1B promoter in vivo and regulates CDKN1B promoter activity. Further, we confirmed CDKN1B promoter binding by ChIP in MLL-AF4 as well as in MLL-AF9 leukemia cell lines. Our results suggest that CDKN1B is a downstream target of MLL and of MLL-AF4, and that, depending on the background cell type, MLL-AF4 inhibits or activates CDKN1B expression. This finding may have implications in terms of leukemia stem cell resistance to chemotherapy in MLL-AF4 leukemias