Background
Pitx2 is the homeobox gene located in proximity to the human 4q25 familial atrial fibrillation locus. When deleted in the mouse germline, Pitx2 haploinsufficiency predisposes to pacing induced atrial fibrillation indicating that reduced Pitx2 promotes an arrhythmogenic substrate. Previous work focused on Pitx2 developmental functions that predispose to atrial fibrillation. Although Pitx2 is expressed in postnatal left atrium, it is unknown whether Pitx2 has distinct postnatal and developmental functions.
Methods and Results
To investigate Pitx2 postnatal function, we conditionally inactivated Pitx2 in the postnatal atrium while leaving its developmental function intact. Unstressed adult Pitx2 homozygous mutant mice display variable R-R interval with diminished P-wave amplitude characteristic of sinus node dysfunction, an atrial fibrillation risk factor in human patients. An integrated genomics approach in the adult heart revealed Pitx2 target genes encoding cell junction proteins, ion channels, and critical transcriptional regulators. Importantly, many Pitx2 target genes have been implicated in human atrial fibrillation by genome wide association studies. Immunofluorescence and transmission electron microscopy studies in adult Pitx2 mutant mice revealed structural remodeling of the intercalated disc characteristic of human atrial fibrillation patients.
Conclusions
Our findings, revealing that Pitx2 has genetically separable postnatal and developmental functions, unveil direct Pitx2 target genes that include channel and calcium handling genes as well as genes that stabilize the intercalated disc in postnatal atrium.
Mutations in the laminin b2 gene (LAMB2) cause Pierson syndrome, a severe congenital nephrotic syndrome with ocular and neurologic defects. LAMB2 is a component of the laminin-521 (a5b2g1) trimer, an important constituent of the glomerular basement membrane (GBM). The C321R-LAMB2 missense mutation leads to congenital nephrotic syndrome but only mild extrarenal symptoms; the mechanisms underlying the development of proteinuria with this mutation are unclear. We generated three transgenic mouse lines, in which rat C321R-LAMB2 replaced mouse LAMB2 in the GBM. During the first postnatal month, expression of C321R-LAMB2 attenuated the severe proteinuria exhibited by Lamb2 2/2 mice in a dose-dependent fashion; proteinuria eventually increased, however, leading to renal failure. The C321R mutation caused defective secretion of laminin-521 from podocytes to the GBM accompanied by podocyte endoplasmic reticulum (ER) stress, likely resulting from protein misfolding. Moreover, ER stress preceded the onset of significant proteinuria and was manifested by induction of the ER-initiated apoptotic signal C/EBP homologous protein (CHOP), ER distention, and podocyte injury. Treatment of cells expressing C321R-LAMB2 with the chemical chaperone taurodeoxycholic acid (TUDCA), which can facilitate protein folding and trafficking, greatly increased the secretion of the mutant LAMB2. Taken together, these results suggest that the mild variant of Pierson syndrome caused by the C321R-LAMB2 mutation may be a prototypical ER storage disease, which may benefit from treatment approaches that target the handling of misfolded proteins.
Both FS-LASIK and SMILE procedures achieved good visual outcomes in the correction of myopia and myopic astigmatism. SMILE had a lower induction rate of spherical aberration at 6 months postoperatively in the analysis of 6 mm diameter than that of FS-LASIK.
MyHC isoforms have complex expression patterns, exhibiting not only longitudinal and cross-sectional variation of each isoform, but also of coexpression in single fibers. The highly heterogeneous MyHC expression reflects the complex contractile profiles of EOMs, which in turn are a function of the requirements of eye movements, which range from extremely fast saccades to sustained position, each with a need for precise coordination of each eye.
Myasthenia gravis (MG) is a neuromuscular transmission disorder in which damage to acetylcholine receptors (AChR) on motor endplates by autoantibody-induced complement attack causes muscle weakness. To determine whether and, if so, to what extent, blockade of complement cascade at the C5 step ameliorates disease, we evaluated the effect of administering a functionally blocking anti-C5 mAb in passive experimental MG in Lewis rats induced with AChR Ab McAb-3. In contrast to uniform severe weakness at 24 h requiring euthanasia in untreated animals, anti-C5 mAb-pretreated rats showed no weakness at 48 h. Anti-C5 mAb treatment 24 h after disease induction restored strength in two-thirds of the rats. Immunofluorescence staining of endplates from the treated animals showed that C9 deposition at AChR was reduced and ultrastructural analyses showed that endplates were intact. The results argue that targeting C5 may warrant testing in MG patients and that this approach may be particularly valuable for myasthenic crisis.
Nicotinic acetylcholine receptors (nAChRs) mediate ganglionic transmission in the peripheral autonomic nervous system in mammals. Functional neuronal nAChRs have been shown to assemble from a combination of alpha and beta subunits, including alpha3, alpha5, alpha7, beta2, and beta4 in RNA-injected oocytes, but the subunit composition of functional neuronal nAChRs in vivo in mammals remains unknown. We examined the subunit composition of functional nAChRs in the intracardiac parasympathetic ganglion in a physiologically intact system in vivo. We report here that localized perfusion of the canine intracardiac ganglion in situ with an antagonist specific for nAChRs containing an alpha3/beta2 subunit interface (alpha-conotoxin MII 100-200 nm) resulted in reversible attenuation of the sinus cycle length (SCL) response by approximately 70% to electrical stimulation of the preganglionic vagus nerve. Perfusion with antagonist specific for receptors containing an alpha3/beta4 subunit interface (alpha-conotoxin AuIB 1 micrometer) resulted in attenuation in SCL responses (approximately 20%) compared with baseline when applied by itself, but not in animals pretreated with alpha-conotoxin MII. Perfusion of the ganglion with alpha-bungarotoxin (1 micrometer, which blocks alpha7 receptors) caused a reduction in SCL response by approximately 30% compared with baseline when perfused on its own and when added after blockade with MII and AuIB. Perfusion with hexamethonium bromide resulted in complete blockade of ganglionic transmission, confirming total perfusion of the ganglion and the nicotinic nature of ganglionic transmission at this synapse. Immunohistochemistry using monoclonal antibodies against specific nicotinic subunits confirmed the presence of alpha3, alpha7, beta2, and beta4 subunits. We conclude that functional ganglionic transmission in the canine intracardiac ganglion is mediated primarily by receptors containing an alpha3/beta2 subunit interface, with a smaller contribution by receptors containing alpha7 nAChRs. Despite the presence of beta4 subunits in functional channels, a contribution of a distinct alpha3/beta4 receptor population that does not include an alpha3/beta2 subunit interface was less clear.
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