Regulation of cardiac melusin gene expression by hypertrophic stimuli in the rat.

AIM: Melusin is an integrin ß1-interacting protein proposed to act as a biomechanical sensor in the heart. We characterized mechanisms and signalling pathways regulating cardiac melusin expression. METHODS: Infusion of arginine(8) -vasopressin (AVP) in Sprague-Dawley (SD) rats, spontaneously hypertensive rats (SHR) and double transgenic rats (dTGR) harbouring both human angiotensinogen and renin genes as well as infusion of angiotensin II (Ang II) in SD rats were used. The effect of direct left ventricular (LV) wall stretch was analysed by using isolated perfused rat heart preparation. For the cell culture studies, mouse atrial HL-1 cell line and neonatal rat ventricular myocytes (NRVMs) were used. RESULTS: Left atrial melusin mRNA levels increased already after 30 min of AVP infusion. Ang II caused significant upregulation of left atrial melusin mRNA (2.1-fold at 6 h, P < 0.05) and protein (1.9-fold at 72 h, P < 0.05) levels. In contrast, LV melusin mRNA levels remained unchanged in response to both infusions, as well as to aortic banding-induced pressure overload. Direct LV wall stress or late-stage hypertensive heart disease did not modify LV melusin gene expression either. Interestingly, in atrial HL-1 cells, cyclic stretching increased melusin mRNA levels. Stretching and treatments with hypertrophic agonists increased melusin mRNA and protein levels in NRVMs, endothelin-1 being the most potent. PD98059, an extracellular signal-regulated protein kinase 1/2 inhibitor, markedly attenuated the endothelin-1-induced upregulation of melusin gene expression in NRVMs. CONCLUSION: Multiple hypertrophic stimuli regulate melusin expression predominately in the atria, which may represent a necessary initial step in early adaptive remodelling processes.

Sequence variations and two levels of MCT1 and CD147 expression in red blood cells and gluteus muscle of horses.

MCT1-CD147 complex is the prime lactate transporter in mammalian plasma membranes. In equine red blood cells (RBCs), activity of the complex and expression of MCT1 and CD147 is bimodal; high in 70% and low in 30%. We studied whether sequence variations contribute to the bimodal expression of MCT1 and CD147. Samples of blood and cremaster muscle were collected in connection of castration from 24 horses. Additional gluteus muscle samples were collected from 15 Standardbreds of which seven were known to express low amounts of CD147 in RBCs. The cDNA of MCT1 and CD147 together with a promoter region of CD147 was sequenced. The amounts of MCT1 and CD147 expressed in RBC and muscle membranes were measured by Western blot and mRNA levels in muscles by qPCR. MCT1 and CD147 were expressed in 20 castrates, and in four only were traces found. Sequence variations found in MCT1 were not linked to MCT1 expression. In CD147 linked heterozygous single nucleotide polymorphisms (SNPs) 389A>G (Met(125)Val) and 990C>T (3'-UTR) were associated to low expression of CD147. Also a mutation 168A>G (Ile(51)Val) in CD147 was associated to low MCT1 and CD147 expression. Low MCT1 and CD147 mRNA levels in gluteus were found in Standardbreds with low CD147 expression in RBCs. The results suggest that sequence variations affect the expression level of CD147, but do not explain its bimodality. The levels of MCT1 and CD147 mRNA correlated with the expression of CD147 and suggest that bimodality of their expression is regulated at transcriptional level.

Increased expression of glucocorticoid receptor beta in lymphocytes of patients with severe atopic dermatitis unresponsive to topical corticosteroid.

BACKGROUND: Variable response to topical glucocorticoid therapy occurs in the treatment of severe atopic dermatitis (AD). Glucocorticoid receptor (GR)-beta does not bind glucocorticoids but antagonizes the activity of the classic GRalpha, and could thus account for glucocorticoid insensitivity. OBJECTIVES: To investigate GRalpha and GRbeta mRNA and protein expression in lymphocytes of patients with AD before and after treatment with topical corticosteroids. METHODS: Blood was collected from 11 healthy donors, 10 patients with mild AD and 13 patients with severe AD. mRNA was isolated from peripheral blood mononuclear cells. Expression of GRalpha and GRbeta mRNA was determined by reverse transcriptase-polymerase chain reaction and quantitated. Expression of the GRs was confirmed by Western blot analysis. RESULT: The expression of GRalpha mRNA was detected in all subjects. GRbeta mRNA was detected in four out of 11 healthy volunteers, five out of 10 patients with mild AD and 11 out of 13 patients with severe AD. The incidence of GRbeta mRNA expression was higher in patients with severe AD (85%) than in patients with mild AD (50%), and significantly higher than in healthy volunteers (36%, P = 0.033). Four of the 13 patients with severe AD showed a 3.3-13.2-fold increase in the expression of GRbeta mRNA during a 2-week treatment with topical corticosteroids. In these patients the response to topical corticosteroids was poor. CONCLUSIONS: Expression of GRbeta is increased during topical corticosteroid treatment in the lymphocytes of patients with AD and, in particular, glucocorticoid-insensitive AD is associated with increased expression of GRbeta.

The effect of a short-term hypocaloric diet on liver gene expression and metabolic risk factors in obese women.

BACKGROUND AND AIMS: Most gene expression studies examining the effect of obesity and weight loss have been performed using adipose tissue. However, the liver also plays a central role in maintaining energy balance. We wanted to study the effects of a hypocaloric diet on overall hepatic gene expression and metabolic risk factors. METHODS AND RESULTS: The study subjects were middle-aged, obese women. The diet intervention subjects (n=12) were on a hypocaloric, low-fat diet for 8 weeks with a daily energy intake of 5.0 MJ (1200 kcal), while the control subjects (n=19) maintained their weight. Liver biopsies were taken at the end of the diet period during a gallbladder operation. Hepatic gene expression was analyzed using microarrays by comparing the gene expression profiles from four subjects per group. A global decrease in gene expression was observed with 142 down-regulated genes and only one up-regulated gene in the diet intervention group. The diet resulted in a mean weight loss of 5% of body weight. Triglyceride and fasting insulin concentrations decreased significantly after the diet. CONCLUSIONS: The global decrease in hepatic gene expression was unexpected but the results are interesting, since they included several genes not previously linked to weight reduction. However, since the comparison was made only after the weight reduction, other factors in addition to weight loss may also have been involved in the differences in gene expression between the groups. The decrease in triglyceride and fasting plasma insulin concentrations is in accordance with results from previous weight-loss studies.

Changes in natriuretic peptides, apelin and adrenomedullin after off-pump and on-pump coronary artery bypass surgery.

AIM: The authors have evaluated the postoperative changes of natriuretic peptides, apelin and adrenomedullin after off-pump (OPCAB) and on-pump coronary artery bypass surgery (CCAB) to assess the impact of these techniques on the myocardium. METHODS: Twenty-two patients underwent OPCAB and 24 patients underwent CCAB. Plasma levels of NT-proANP, NT-proBNP, apelin and adrenomedullin were measured preoperatively, and on the 1st, 3rd, and 5th postoperative day. RESULTS: Natriuretic peptides, apelin and adrenomedullin increased significantly postoperatively. Natriuretic peptides were markedly elevated on the fifth postoperative day. Apelin was still increasing, but adrenomedullin, although elevated, clearly decreased toward baseline levels on the fifth postoperative day. CCAB was associated with significantly higher postoperative cTnI, but levels of natriuretic peptides, adrenomedullin and apelin did not differ significantly after CCAB and OPCAB. cTnI, echocardiographic parameters, cardiac index, and degree of postoperative pericardial effusion did not correlate with levels of natriuretic peptides, apelin and adrenomedullin. Postoperative levels of natriuretic peptides were significantly associated with parameters of renal function, age, and extracardiac arteriopathy. The correlation between preoperative estimated glomerular filtration rate and natriuretic peptides increased along the study intervals (NT-proANP rho: -0.181, -0.350, -0.364, and -0.442; NT-proBNP rho: -0.112, -0.420, -0.405 and -0.550). Also adrenomedullin correlated with parameters of renal function. The postoperative levels of apelin were not associated with any variable. CONCLUSION: A marked, sustained and similar increase in these five markers of cardiac adaptation was detected after OPCAB and CCAB. The upregulation of these peptides should be further investigated to evaluate their potential beneficial/harmful impact on the outcome after coronary surgery.

Distinct expression of type XIII collagen in neuronal structures and other tissues during mouse development.

Type XIII collagen is a type II transmembrane protein found in adhesive structures of mature tissues. We describe here its expression and spatio-temporal localization during mouse fetal development. Type XIII collagen mRNAs were expressed at a constant rate during development, with an increase of expression towards birth. Strong type XIII collagen expression was detected in the central and peripheral nervous systems of the developing mouse fetus in mid-gestation. Cultured primary neurons also expressed this collagen, and it was found to enhance neurite outgrowth. The results suggest that type XIII collagen is a new member among the proteins involved in nervous system development. Strong expression during early development was also detected in the heart, with localization to cell-cell contacts and accentuation in the intercalated discs perinatally. During late fetal development, type XIII collagen was observed in many tissues, including cartilage, bone, skeletal muscle, lung, intestine and skin. Clear developmental shifts in expression suggest a role in endochondral ossification of bone and the branching morphogenesis in the lung. Notable structures lacking type XIII collagen were the endothelia of most blood vessels and the endocardium. Its initially unique staining pattern began to concentrate in the same adhesive structures where it exists in adult tissues, and started to resemble that of the beta1 integrin subunit and vinculin during late intrauterine development and in the perinatal period.

Lack of type XV collagen causes a skeletal myopathy and cardiovascular defects in mice.

Type XV collagen occurs widely in the basement membrane zones of tissues, but its function is unknown. To understand the biological role of this protein, a null mutation in the Col15a1 gene was introduced into the germ line of mice. Despite the complete lack of type XV collagen, the mutant mice developed and reproduced normally, and they were indistinguishable from their wild-type littermates. However, Col15a1-deficient mice showed progressive histological changes characteristic for muscular diseases after 3 months of age, and they were more vulnerable than controls to exercise-induced muscle injury. Despite the antiangiogenic role of type XV collagen-derived endostatin, the development of the vasculature appeared normal in the null mice. Nevertheless, ultrastructural analyses revealed collapsed capillaries and endothelial cell degeneration in the heart and skeletal muscle. Furthermore, perfused hearts showed a diminished inotropic response, and exercise resulted in cardiac injury, changes that mimic early or mild heart disease. Thus, type XV collagen appears to function as a structural component needed to stabilize skeletal muscle cells and microvessels.

Gene structure of a new cardiac peptide hormone: a model for heart-specific gene expression.

Volume excess and mechanical load lead to the induction of the endocrine activity of the heart. The increased production and secretion of A- and B-type natriuretic peptides (ANP and BNP), in turn, unload the heart due to their physiological effects. To find out the mechanisms of cardiac-specific expression and sensitivity to mechanical stimuli of the natriuretic peptide genes, we have used salmon (Salmo salar) as our model organism, because osmoregulating fish have a particularly well developed defense mechanism against volume excess. We have previously cloned a complementary DNA from salmon heart encoding a novel vasorelaxant cardiac hormone, salmon cardiac peptide (sCP). Its production is restricted to the heart, and its release is very sensitive to mechanical load. We have now cloned the gene encoding sCP. The structure of the gene suggests that sCP may represent an ancestral form of the mammalian natriuretic peptides. Remarkably, despite the large phylogenetic distance, the sCP promoter is as effective as mammalian ANP promoters in cultured neonatal rat atrial cardiomyocytes. Therefore, structural and functional comparisons of the promoters of sCP and ANP provide an excellent means of identifying the elements and transcription factors required for atrial-specific gene expression and the regulation of the endocrine function of the heart. Isolation of the protein product of sCP gene from salmon atrium demonstrated that the storage form of sCP is the prohormone of 126 amino acids. The final processing of the prohormone appears to take place during exocytosis of the secretory granules, as the released and circulating form is the biologically active 29-amino acid sCP.