Pore architecture of the ORAI1 store-operated calcium channel.

ORAI1 is the pore-forming subunit of the calcium release-activated calcium (CRAC) channel, a store-operated channel that is central to Ca(2+) signaling in mammalian cells. Electrophysiological data have shown that the acidic residues E106 in transmembrane helix 1 (TM1) and E190 in TM3 contribute to the high selectivity of ORAI1 channels for Ca(2+). We have examined the pore architecture of the ORAI1 channel using ORAI1 proteins engineered to contain either one or two cysteine residues. Disulfide cross-linking shows that ORAI1 assembles as a tetramer or a higher oligomer with TM1 centrally located. Cysteine side chains projecting from TM1 at position 88, 95, 102, or 106 cross-link efficiently to the corresponding side chain in a second ORAI1 monomer. Cysteine residues at position 190 or at surrounding positions in TM3 do not cross-link. We conclude that E106 residues in wild-type ORAI1 are positioned to form a Ca(2+) binding site in the channel pore and that E190 interacts less directly with ions traversing the pore. The cross-linking data further identify a relatively rigid segment of TM1 adjacent to E106 that is likely to contribute to the selectivity filter.

Plasma microvesicle analysis identifies microRNA 129-5p as a biomarker of heart failure in univentricular heart disease.

Biomarkers of heart failure in adults have been extensively studied. However, biomarkers to monitor the progression of heart failure in children with univentricular physiology are less well understood. We proposed that as mediators of diverse pathophysiology, miRNAs contained within circulating microvesicles could serve as biomarkers for the presence and progression of heart failure in univentricular patients. To test this, we studied the association of heart failure with elevations in specific miRNAs isolated from circulating microvesicles in a cohort of children with univentricular heart disease and heart failure. We conducted a single site cross-sectional observational study of 71 children aged 1 month-7 years with univentricular heart disease and heart failure. We demonstrated that levels of miR129-5p isolated from plasma microvesicles were inversely related to the degree of clinical heart failure as assessed by Ross score. We then showed that miR129-5p levels are downregulated in HL1 cells and human embryonic stem cell-derived cardiomyocytes exposed to oxidative stress. We demonstrated that bone morphogenetic protein receptor 2, which has been implicated in the development of pulmonary vascular disease, is a target of miR129-5p, and conversely regulated in response to oxidative stress in cell culture. Levels of miR129-5p were inversely related to the degree of clinical heart failure in patients with univentricular heart disease. This study demonstrates that miR129-5p is a sensitive and specific biomarker for heart failure in univentricular heart disease independent of ventricular morphology or stage of palliation. Further study is warranted to understand the targets affected by miR129-5p with the development of heart failure in patients with univentricular physiology.

miR-125b promotes early germ layer specification through Lin28/let-7d and preferential differentiation of mesoderm in human embryonic stem cells.

Unlike other essential organs, the heart does not undergo tissue repair following injury. Human embryonic stem cells (hESCs) grow indefinitely in culture while maintaining the ability to differentiate into many tissues of the body. As such, they provide a unique opportunity to explore the mechanisms that control human tissue development, as well as treat diseases characterized by tissue loss, including heart failure. MicroRNAs are small, non-coding RNAs that are known to play critical roles in the regulation of gene expression. We profiled the expression of microRNAs during hESC differentiation into myocardial precursors and cardiomyocytes (CMs), and determined clusters of human microRNAs that are specifically regulated during this process. We determined that miR-125b overexpression results in upregulation of the early cardiac transcription factors, GATA4 and Nkx2-5, and accelerated progression of hESC-derived myocardial precursors to an embryonic CM phenotype. We used an in silico approach to identify Lin28 as a target of miR-125b, and validated this interaction using miR-125b knockdown. Anti-miR-125b inhibitor experiments also showed that miR-125b controls the expression of miRNA let-7d, likely through the negative regulatory effects of Lin28 on let-7. We then determined that miR-125b overexpression inhibits the expression of Nanog and Oct4 and promotes the onset of Brachyury expression, suggesting that miR-125b controls the early events of human CM differentiation by inhibiting hESC pluripotency and promoting mesodermal differentiation. These studies identified miR-125b as an important regulator of hESC differentiation in general, and the development of hESC-derived mesoderm and cardiac muscle in particular. Manipulation of miR-125b-mediated pathways may provide a novel approach to directing the differentiation of hESC-derived CMs for cell therapy applications.

Age- and gender-related differences in the geometric properties and biomechanical significance of intracortical porosity in the distal radius and tibia.

Cortical bone contributes the majority of overall bone mass and bears the bulk of axial loads in the peripheral skeleton. Bone metabolic disorders often are manifested by cortical microstructural changes via osteonal remodeling and endocortical trabecularization. The goal of this study was to characterize intracortical porosity in a cross-sectional patient cohort using novel quantitative computational methods applied to high-resolution peripheral quantitative computed tomography (HR-pQCT) images of the distal radius and tibia. The distal radius and tibia of 151 subjects (57 male, 94 female; 47 +/- 16 years of age, range 20 to 78 years) were imaged using HR-pQCT. Intracortical porosity (Ct.Po) was calculated as the pore volume normalized by the sum of the pore and cortical bone volume. Micro-finite element analysis (microFE) was used to simulate 1% uniaxial compression for two scenarios per data set: (1) the original structure and (2) the structure with intracortical porosity artificially occluded. Differential biomechanical indices for stiffness (Delta K), modulus (Delta E), failure load (Delta F), and cortical load fraction (Delta Ct.LF) were calculated as the difference between original and occluded values. Regression analysis revealed that cortical porosity, as depicted by HR-pQCT, exhibited moderate but significant age-related dependence for both male and female cohorts (radius rho = 0.7; tibia rho = 0.5; p < .001). In contrast, standard cortical metrics (Ct.Th, Ct.Ar, and Ct.vBMD) were more weakly correlated or not significantly correlated with age in this population. Furthermore, differential microFE analysis revealed that the biomechanical deficit (Delta K) associated with cortical porosity was significantly higher for postmenopausal women than for premenopausal women (p < .001). Finally, porosity-related measures provided the only significant decade-wise discrimination in the radius for females in their fifties versus females in their sixties (p < .01). Several important conclusions can be drawn from these results. Age-related differences in cortical porosity, as detected by HR-pQCT, are more pronounced than differences in standard cortical metrics. The biomechanical significance of these structural differences increases with age for men and women and provides discriminatory information for menopause-related bone quality effects.