Cardiomyocyte-specific disruption of Serca2 in adult mice causes sarco(endo)plasmic reticulum stress and apoptosis.

Reduced sarco(endo)plasmic reticulum (SR) Ca(2+) ATPase (SERCA2) contributes to the impaired cardiomyocyte Ca(2+) homeostasis observed in heart failure. We hypothesized that a reduction in SERCA2 also elicits myocardial ER/SR stress responses, including unfolded protein responses (UPR) and cardiomyocyte apoptosis, which may additionally contribute to the pathophysiology of this condition. Left ventricular myocardium from mice with cardiomyocyte-specific tamoxifen-inducible disruption of Serca2 (SERCA2 KO) was compared with aged-matched controls. In SERCA2 KO hearts, SERCA2 protein levels were markedly reduced to 2% of control values at 7 weeks following tamoxifen treatment. Serca2 disruption caused increased abundance of the ER stress-associated proteins CRT, GRP78, PERK, and eIF2α and increased phosphorylation of PERK and eIF2α, indicating UPR induction. Pro-apoptotic signaling was also activated in SERCA2 KO, as the abundance of CHOP, caspase 12, and Bax was increased. Indeed, TUNEL staining revealed an increased fraction of cardiomyocytes undergoing apoptosis in SERCA2 KO. ER-Tracker staining additionally revealed altered ER structure. These findings indicate that reduction in SERCA2 protein abundance is associated with marked ER/SR stress in cardiomyocytes, which induces UPR, apoptosis, and ER/SR structural alterations. This suggests that reduced SERCA2 abundance or function may contribute to the phenotype of heart failure also through induction of ER/SR stress responses.

Effects of bilirubin ditaurate overload on canalicular membrane function and ultrastructure of the pig liver.

AIM: Bilirubin overload caused by haemolysis of transfused blood and breakdown of extravasated blood constitutes an important causative factor of jaundice in trauma patients. Intravenous infusions of large amounts of unconjugated bilirubin (UCB) block biliary phospholipid secretion and produce canalicular membrane lesions in pigs, putatively because of enhanced cytotoxicity of bile. Severe intrahepatic cholestasis is the functional end result. The aim of the study was to investigate whether bilirubin ditaurate (BDT) overload, which also inhibits biliary phospholipid secretion, also induces intrahepatic cholestasis. METHODS: Six pigs were infused with 2.8 g of BDT for 150 min. Six control pigs were infused with albumin sham solution for 150 min. Bile samples were analysed for bile acid- and phospholipid-secretion rates. Bile and serum samples were analysed for bilirubin concentration. Liver biopsies were obtained for scanning electron microscopic studies (SEM). RESULTS: Biliary bile acid secretion fell by 7.7% and biliary phospholipid secretion rate fell by 88% after BDT infusion. Thus, infusion of BDT for 150 min did not cause intrahepatic cholestasis. SEM showed some variability in the size of canalicular membrane microvilli, but no evidence of gross destruction. CONCLUSION: BDT overload markedly lowers biliary phospholipid secretion. In contrast to UCB, BDT does not induce canalicular membrane damage nor cause intrahepatic cholestasis. Sustained, marked inhibition of phospholipid secretion does therefore not adequately explain UCB-induced cholestasis. Accumulation of UCB in the canalicular membrane may be the important factor in the pathogenesis of canalicular membrane lesions and intrahepatic cholestasis during UCB overload.

ABCB4 sequence variations in young adults with cholesterol gallstone disease.

BACKGROUND AND AIMS: Mutations in the gene encoding the ABCB4 [adenosine triphosphate (ATP)-binding cassette, sub-family B (MDR/TAP), member 4] transporter lower phosphatidylcholine output into bile and contribute to cholesterol gallstone formation by decreasing the solubility of cholesterol in bile. Mutations in ABCB4 have been identified in patients with low phospholipid-associated cholelithiasis. The aim of the present study was to determine the types and frequencies of ABCB4 mutations in cholecystectomized patients aged <40 years. PATIENTS AND METHODS: Hundred and four patients (mean age 30.6 years, range 12-39) were included in the study and the ABCB4 gene was sequenced. The frequency of missense mutations found in the patient material was measured in 95 healthy controls. The potential functional implications of the ABCB4 missense variations were assessed by computerized analysis (BLOSUM62 and Grantham substitution matrices, polymorphism phenotyping and sorting intolerant from tolerant). RESULTS: One patient was heterozygous for a frameshift mutation (c.1399_1400ins10/p.Y467F fsX25). Another patient was heterozygous for a nonsense mutation (c.3136C>T/p.R1046X). These two mutations are considered detrimental to ABCB4 protein function. In addition, six missense mutations were found in the ABCB4 gene, and three of these were only present in patients. CONCLUSION: In our study, <2% of young gallstone patients were found to be heterozygous for detrimental ABCB4 mutations. The functional implication of several missense mutations remains to be clarified. Thus, mutations in the ABCB4 gene are a rare cause of gallstone disease.

Gene expression profiles reflect sclerosing cholangitis activity in abcb4 (-/-) mice.

OBJECTIVE: Abcb4 (-/-) mice secrete phosphatidylcholine-deficient bile and develop sclerosing cholangitis (SC), a condition that involves differential hepatic transcription of genes governing inflammation, tissue remodelling and fibrosis. The objective of this study was to test the hypothesis that genes involved in the regulation of tissue inflammation and fibrosis display transcription rates that parallel differences in abcb4 (-/-) SC activity. The activity of abcb4 (-/-) SC can be altered through dietary intervention: abcb4 (-/-) mice fed cholic acid (CA) display high SC activity, whereas ursodeoxycholic acid (UDCA)-fed mice display low SC activity. MATERIAL AND METHODS: Differential hepatic transcription of genes was measured in abcb4 (-/-) mice maintained on CA- and UDCA-supplemented diets using cDNA microarrays. Abcb4 (+/+) mice served as controls. Differential transcription of selected genes was verified by real-time polymerase chain reaction. Liver tissue pathology was quantified by histopathology scoring. RESULT: Histopathology score, reflecting increased inflammation and fibrosis, was increased in CA-fed mice compared with UDCA-fed mice. cDNA microarray analysis showed up-regulation of 1582 genes in livers of CA-fed mice in contrast to 573 genes in UDCA-fed mice. Differential transcription of Ccl2, Ccl20, Cxcl10, Nfkappab1, Nfkappab2, Tgfbeta1, Tgfbeta2, Sparc, Ctgf, Lgals3, Elf3, Spp1, Pdgfa, Pdgfrb, Col1a1, Col1a2 and Col4a1 genes paralleled the unequal SC activities of CA- and UDCA-fed abcb4 (-/-) mice. CONCLUSIONS: The numbers of differentially transcribed genes and the transcriptional activity of genes relating to inflammation, tissue remodelling and fibrosis parallel disease activity in CA- and UDCA-fed abcb4 (-/-) mice harbouring SC. Data on their hepatic transcription can gauge SC disease activity.

Multiple inflammatory-, tissue remodelling- and fibrosis genes are differentially transcribed in the livers of Abcb4 (-/ - ) mice harbouring chronic cholangitis.

OBJECTIVE: Abcb4 (-/-) mice secrete phosphatidylcholine-free, cytotoxic bile and develop chronic cholangitis. The aim of this study was to identify differentially transcribed genes whose products contribute to the liver tissue pathology during this disease. MATERIAL AND METHODS: Hepatic gene transcription was measured in 3-, 6-, 9- and 20-week-old Abcb4 (-/-) mice (FVB.129P2-abcb4(tm1Bor)/J) using cDNA microarrays, with FVB/NJ Abcb4 (+/+) mice serving as controls. Focus was on inflammatory-, remodelling- and fibrosis genes. Marked differential transcription of inflammatory-, tissue remodelling- and fibrosis genes found by cDNA microarrays was verified by real-time polymerase chain reaction (PCR). Liver pathology was quantified by histopathology scoring. RESULTS: Transcription of clade A3 Serpin genes showed early, marked down-regulation. The chemokine genes Ccl2, Ccl20 and Cxcl10 were markedly up-regulated. Tissue remodelling- and fibrosis genes exhibiting markedly up-regulated transcription included: Ctgf, Elf3, Lgals3, Mmp12, Mmp15, Spp1, Loxl2, Pdgfa, Pdgfrb, Sparc, Tgfb1, Tgfb2, Tgfbi, Tgfbr2 and Col1a1, Col1a2, Col2a1, Col3a1, Col4a1 genes. Microarray-based recordings of differential gene transcription of the majority of these genes harmonized with the liver histopathology score. Thus, cDNA microarray-based analysis showed increasing differential transcription of several inflammatory-, tissue remodelling- and fibrosis genes during the first 9 weeks of disease and a tendency towards differential transcription to stabilize at an elevated level from 9 to 20 weeks of disease. CONCLUSIONS: Multiple genes regulating inflammation, tissue remodelling and fibrosis not previously linked to Abcb4 (-/-) cholangitis are identified as being differentially transcribed in Abcb4 (-/-) livers, where they contribute to the pathogenesis of liver tissue pathology.

Differential regulation of vacuolar H+ -ATPase and Na+/H+ exchanger 3 in rat cholangiocytes after bile duct ligation.

The cholangiocytes lining the intrahepatic bile ducts modify the primary secretion from the hepatocytes. The cholangiocytes secrete HCO (3)(-) into bile when stimulated with secretin in many species, including man. However, in rats, secretin stimulation neither affects biliary HCO (3)(-) concentration nor bile flow, whereas following bile duct ligation (BDL) it induces hypercholeresis with significant increase of NaHCO(3) concentration. We hypothesized that BDL might affect the expression of cholangiocyte H(+) transporters and thereby choleresis, and determined the expression and localization of the 31 kDa vacuolar type H(+)-ATPase (V-ATPase) subunit and of Na(+)/H(+) exchanger NHE3 in the livers of control and BDL rats by real-time PCR, in situ hybridization, immunoblotting, and immunohistochemistry. In controls, secretin had no effect on bile flow, whereas following BDL, secretin increased bile flow approximately threefold. V-ATPase and NHE3 were expressed in control cholangiocytes showing intracellular and apical distribution, respectively. BDL significantly up-regulated V-ATPase mRNA and protein expression and was associated with redistribution to the apical pole in approximately 60% of the cholangiocytes lining the small bile ductules. In contrast, NHE3 expression was significantly down-regulated by BDL at the mRNA and protein level. The data demonstrate expression of V-ATPase in rat cholangiocytes. BDL-induced down-regulation of NHE3 may contribute to a reduction of Na(+) and HCO (3)(-) reabsorption and thus to their net secretion into bile. Apical localization of V-ATPase in cholangiocytes may indicate its involvement in pH regulation and/or HCO (3)(-) salvage to compensate for NHE3 down-regulation in BDL.