The epithelial sodium channel (ENaC) is essential for sodium homoeostasis in many epithelia. ENaC activity is required for lung fluid clearance in newborn animals and for maintenance of blood volume and blood pressure in adults. In vitro studies show that the ubiquitin ligase Nedd4-2 ubiquitinates ENaC to regulate its cell surface expression. Here we show that knockout of Nedd4-2 in mice leads to increased ENaC expression and activity in embryonic lung. This increased ENaC activity is the likely reason for premature fetal lung fluid clearance in Nedd4-2−/− animals, resulting in a failure to inflate lungs and perinatal lethality. A small percentage of Nedd4-2−/− animals survive up to 22 days, and these animals also show increased ENaC expression and develop lethal sterile inflammation of the lung. Thus, we provide critical in vivo evidence that Nedd4-2 is essential for correct regulation of ENaC expression, fetal and postnatal lung function and animal survival.

Natural killer T cells are an immunoregulatory population of lymphocytes that plays a critical role in controlling the adaptive immune system and contributes to the regulation of autoimmune responses. We have previously reported deficiencies in the numbers and function of NKT cells in the nonobese diabetic (NOD) mouse strain, a well-validated model of type 1 diabetes and systemic lupus erythematosus. In this study, we report the results of a genetic linkage analysis of the genes controlling NKT cell numbers in a first backcross (BC1) from C57BL/6 to NOD.Nkrp1b mice. The numbers of thymic NKT cells of 320 BC1 mice were determined by fluorescence-activated cell analysis using anti-TCR Ab and CD1/α-galactosylceramide tetramer. Tail DNA of 138 female BC1 mice was analyzed for PCR product length polymorphisms at 181 simple sequence repeats, providing greater than 90% coverage of the autosomal genome with an average marker separation of 8 cM. Two loci exhibiting significant linkage to NKT cell numbers were identified; the most significant (Nkt1) was on distal chromosome 1, in the same region as the NOD mouse lupus susceptibility gene Babs2/Bana3. The second most significant locus (Nkt2) mapped to the same region as Idd13, a NOD-derived diabetes susceptibility gene on chromosome 2.

MicroRNAs (miRNAs) regulate post-transcriptional gene expression during development and disease. We have determined the miRNA expression levels of early- and end-stage hypertrophic cardiomyopathy (HCM) in a severe, transgenic mouse model of the disease. Five miRNAs were differentially expressed at an early stage of HCM development. Time-course analysis revealed that decreased expression of miR-1 and miR-133a commences at a pre-disease stage, and precedes upregulation of target genes causal of cardiac hypertrophy and extracellular matrix remodelling, suggesting a role for miR-1 and miR-133a in early disease development. At end-stage HCM, 16 miRNA are dysregulated to form an expression profile resembling that of other forms of cardiac hypertrophy, suggesting common responses. Analysis of the mRNA transcriptome revealed that miRNAs potentially target 15.7% upregulated and 4.8% downregulated mRNAs at end-stage HCM, and regulate mRNAs associated with cardiac hypertrophy and electrophysiology, calcium signalling, fibrosis, and the TGF-β signalling pathway. Collectively, these results define the miRNA expression signatures during development and progression of severe HCM and highlight critical miRNA regulated gene networks that are involved in disease pathogenesis.

Background-Familial hypertrophic cardiomyopathy (FHC) is characterized by genetic and clinical heterogeneity. Five percent of FHC families have 2 FHC-causing mutations, which results in earlier disease onset, increased cardiac dysfunction, and a higher incidence of sudden death events. These observations suggest a relationship between the number of gene mutations and phenotype severity in FHC. Methods and Results-We sought to develop, characterize, and investigate the pathogenic mechanisms in a double-mutant murine model of FHC. This model (designated TnI-203/MHC-403) was generated by crossbreeding mice with the Gly203Ser cardiac troponin I (TnI-203) and Arg403Gln ␣-myosin heavy chain (MHC-403) FHC-causing mutations.The mortality rate in TnI-203/MHC-403 mice was 100% by age 21 days. At age 14 days, TnI-203/MHC-403 mice developed a significantly increased ratio of heart weight to body weight, marked interstitial myocardial fibrosis, and increased expression of atrial natriuretic factor and brain natriuretic peptide compared with nontransgenic, TnI-203, and MHC-403 littermates. By age 16 to 18 days, TnI-203/MHC-403 mice rapidly developed a severe dilated cardiomyopathy and heart failure, with inducibility of ventricular arrhythmias, which led to death by 21 days. Downregulation of mRNA levels of key regulators of Ca 2ϩ homeostasis in TnI-203/MHC-403 mice was observed. Increased levels of phosphorylated STAT3 were observed in TnI-203/MHC-403 mice and corresponded with the onset of disease, which suggests a possible cardioprotective response. Conclusions-TnI-203/MHC-403 double-mutant mice develop a severe cardiac phenotype characterized by heart failure and early death. The presence of 2 disease-causing mutations may predispose individuals to a greater risk of developing severe heart failure than human FHC caused by a single gene mutation.

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Key pointsr Genetic mutations in cardiac troponin I (cTnI) are associated with development of hypertrophic cardiomyopathy characterized by myocyte remodelling, disorganization of cytoskeletal proteins and altered energy metabolism.r The L-type Ca 2+ channel is the main route for calcium influx and is crucial to cardiac excitation and contraction. The channel also regulates mitochondrial function in the heart by a functional communication between the channel and mitochondria via the cytoskeletal network.r We find that L-type Ca 2+ channel kinetics are altered in cTnI-G203S cardiac myocytes and that activation of the channel causes a significantly greater increase in mitochondrial membrane potential and metabolic activity in cTnI-G203S cardiac myocytes. r We propose that L-type Ca 2+ channel antagonists, such as diltiazem, might be effective in reducing the cardiomyopathy by normalizing mitochondrial metabolic activity.Abstract Genetic mutations in cardiac troponin I (cTnI) account for 5% of families with hypertrophic cardiomyopathy. Hypertrophic cardiomyopathy is associated with disorganization of cytoskeletal proteins and altered energy metabolism. The L-type Ca 2+ channel (I Ca-L ) plays an important role in regulating mitochondrial function. This involves a functional communication between the channel and mitochondria via the cytoskeletal network. We investigate the role of I Ca-L in regulating mitochondrial function in 25-to 30-week-old cardiomyopathic mice expressing the human disease-causing mutation Gly203Ser in cTnI (cTnI-G203S). The inactivation rate of I Ca-L is significantly faster in cTnI-G203S myocytes [cTnI-G203S: τ 1 = 40.68 ± 3.22, n = 10 vs. wild-type (wt): τ 1 = 59.05 ± 6.40, n = 6, P < 0.05]. Activation of I Ca-L caused a greater increase in mitochondrial membrane potential ( m , 29.19 ± 1.85%, n = 15 vs. wt: 18.84 ± 2.01%, n = 10, P < 0.05) and metabolic activity (24.40 ± 6.46%, n = 8 vs. wt: 9.98 ± 1.57%, n = 9, P < 0.05). The responses occurred because of impaired communication between I Ca-L and F-actin, involving lack of dynamic movement of actin-myosin and block of the mitochondrial voltage-dependent anion channel. Similar responses were observed in precardiomyopathic mice. I Ca-L antagonists nisoldipine and diltiazem decreased m to basal levels. We conclude that the Gly203Ser mutation

Wang X, McLennan SV, Allen TJ, Tsoutsman T, Semsarian C, Twigg SM. Adverse effects of high glucose and free fatty acid on cardiomyocytes are mediated by connective tissue growth factor.

Major advances have been made over the last decade in our understanding of the molecular basis of several cardiac conditions. Hypertrophic cardiomyopathy (HCM) was the first cardiac disorder in which a genetic basis was identified and as such, has acted as a paradigm for the study of an inherited cardiac disorder. HCM can result in clinical symptoms ranging from no symptoms to severe heart failure and premature sudden death. HCM is the commonest cause of sudden death in those aged less than 35 years, including competitive athletes. At least ten genes have now been identified, defects in which cause HCM. All of these genes encode proteins which comprise the basic contractile unit of the heart, i.e. the sarcomere. While much is now known about which genes cause disease and the various clinical presentations, very little is known about how these gene defects cause disease, and what factors modify the expression of the mutant genes. Studies in both cell culture and animal models of HCM are now beginning to shed light on the signalling pathways involved in HCM, and the role of both environmental and genetic modifying factors. Understanding these mechanisms will ultimately improve our knowledge of the basic biology of heart muscle function, and will therefore provide new avenues for treating cardiovascular disease in man.