Background-J Wave Syndromes have emerged conceptually to encompass the pleiotropic expression of J point abnormalities including Brugada syndrome (BrS) and early repolarization syndrome (ERS). Recently, KCNJ8, which encodes the cardiac K ATP Kir6.1 channel, has been implicated in ERS following the identification of a functionally uncharacterized missense mutation, S422L. Here, we sought to further explore KCNJ8 as a novel susceptibility gene for J wave syndromes.

Rationale: Cardioprotective pathways may involve a mitochondrial ATP-sensitive potassium (mitoK ATP ) channel but its composition is not fully understood. Objective: We hypothesized that the mitoK ATP channel contains a sulfonylurea receptor (SUR)2 regulatory subunit and aimed to identify the molecular structure. Methods and Results: Western blot analysis in cardiac mitochondria detected a 55-kDa mitochondrial SUR2 (mitoSUR2) short form, 2 additional short forms (28 and 68 kDa), and a 130-kDa long form. RACE (Rapid Amplification of cDNA Ends) identified a 1.5-Kb transcript, which was generated by a nonconventional intraexonic splicing ( Key Words: K ATP channel Ⅲ SUR2 Ⅲ ischemia Ⅲ intraexonic splicing Ⅲ mitochondria A lternative splicing generates multiple mRNAs from a single gene, which are subsequently translated into diverse proteins with different structures and functions. 1 Up to 60% of mammalian genes are alternatively spliced. 2 Eukaryotic ion channel genes are known to have multiple splice variants. The ATP-sensitive potassium (K ATP ) channels are ubiquitously distributed in many tissue types. Sarcolemmal K ATP (sarcK ATP ) channels consist of a potassium inward-rectifier pore-forming subunit (Kir6.0) and a sulfonylurea receptor (SUR) regulatory subunit. 3 Various isoforms and splice variants for the SUR genes have been reported. 4,5 The cardiac muscle splice variant (SUR2A) differs from the vascular smooth muscle splice variant (SUR2B) in the alternative use of the SUR2 C-terminal exon. 6,7 Subtypes of splice variants for SUR2A or SUR2B that lack exon 14 or exon 17 exist in mice 7,8 and humans. 9 Moreover, sarcolemmal SUR short variants are found in heart 10 and pancreatic ␤ cells. 11,12 The copresence of multiple splice variants increases the functional diversity and genetic complexity of K ATP channels.In addition to a sarcolemmal location, 13 the K ATP channel is present in the inner membrane of mitochondria (mitoK ATP ). 14 Both forms of channels are involved in cardioprotective pathways, 15 but earlier pharmacological evidence suggests that the mitoK ATP channel is more critical in conferring protection. 16,17 However, the molecular composition of the mitoK ATP channel is uncertain, hampering present efforts in elucidating its role in preconditioning signaling. 18 Putative mitoK ATP channel subunits in the sizes of 55 and 63

The methodology to create induced pluripotent stem cells (iPSCs) affords the opportunity to generate cells specific to the individual providing the host tissue. However, existing methods of reprogramming as well as the types of source tissue have significant limitations that preclude the ability to generate iPSCs in a scalable manner from a readily available tissue source. We present the first study whereby iPSCs are derived in parallel from multiple donors using episomal, non-integrating, oriP/EBNA1-based plasmids from freshly drawn blood. Specifically, successful reprogramming was demonstrated from a single vial of blood or less using cells expressing the early lineage marker CD34 as well as from unpurified peripheral blood mononuclear cells. From these experiments, we also show that proliferation and cell identity play a role in the number of iPSCs per input cell number. Resulting iPSCs were further characterized and deemed free of transfected DNA, integrated transgene DNA, and lack detectable gene rearrangements such as those within the immunoglobulin heavy chain and T cell receptor loci of more differentiated cell types. Furthermore, additional improvements were made to incorporate completely defined media and matrices in an effort to facilitate a scalable transition for the production of clinic-grade iPSCs.

Background-Sudden infant death syndrome (SIDS) is a leading cause of death during the first 6 months after birth. About 5% to 10% of SIDS may stem from cardiac channelopathies such as long-QT syndrome. We recently implicated mutations in ␣1-syntrophin (SNTA1) as a novel cause of long-QT syndrome, whereby mutant SNTA1 released inhibition of associated neuronal nitric oxide synthase by the plasma membrane Ca-ATPase PMCA4b, causing increased peak and late sodium current (I Na ) via S-nitrosylation of the cardiac sodium channel. This study determined the prevalence and functional properties of SIDS-associated SNTA1 mutations. Methods and Results-Using polymerase chain reaction, denaturing high-performance liquid chromatography, and DNA sequencing of SNTA1's open reading frame, 6 rare (absent in 800 reference alleles) missense mutations (G54R, P56S, T262P, S287R, T372M, and G460S) were identified in 8 (Ϸ3%) of 292 SIDS cases. These mutations were engineered using polymerase chain reaction-based overlap extension and were coexpressed heterologously with SCN5A, neuronal nitric oxide synthase, and PMCA4b in HEK293 cells. I Na was recorded using the whole-cell method. A significant 1.4-to 1.5-fold increase in peak I Na and 2.3-to 2.7-fold increase in late I Na compared with controls was evident for S287R-, T372M-, and G460S-SNTA1 and was reversed by a neuronal nitric oxide synthase inhibitor. These 3 mutations also caused a significant depolarizing shift in channel inactivation, thereby increasing the overlap of the activation and inactivation curves to increase window current. Conclusions-Abnormal biophysical phenotypes implicate mutations in SNTA1 as a novel pathogenic mechanism for the subset of channelopathic SIDS. Functional studies are essential to distinguish pathogenic perturbations in channel interacting proteins such as ␣1-syntrophin from similarly rare but innocuous ones. (Circ Arrhythm Electrophysiol. 2009;2:667-676.)