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.)

The cardiac sarcolemmal ATP-sensitive potassium channel (K ATP ) consists of a Kir6.2 pore and a SUR2 regulatory subunit, which is an ATP-binding cassette (ABC) transporter. K ATP channels have been proposed to play protective roles during ischemic preconditioning. A SUR2 mutant mouse was previously generated by disrupting the first nucleotide-binding domain (NBD1), where a glibenclamide action site was located. In the mutant ventricular myocytes, a non-conventional glibenclamide-insensitive (10 μM), ATP-sensitive current (I KATPn ) was detected in 33% of singlechannel recordings with an average amplitude of 12.3±5.4 pA per patch, an IC 50 to ATP inhibition at 10 μM, and a mean burst duration at 20.6±1.8 ms. Newly designed SUR2-isoform or variantspecific antibodies identified novel SUR2 short forms in the sizes of 28 and 68 kDa in addition to a 150-kDa long form in the sarcolemmal membrane of wild-type (WT) heart. We hypothesized that channels constituted by these short forms that lack NBD1, confer I KATPn . The absence of the long form in the mutant corresponded to loss of the conventional glibenclamide-sensitive K ATP currents (I KATP ) in isolated cardiomyocytes and vascular smooth muscle cells but the SUR2 short forms remained intact. Nested exonic RT-PCR in the mutant indicated that the short forms lacked NBD1 but contained NBD2. The SUR2 short forms co-immunoprecipitated with Kir6.1 or Kir6.2 suggesting that the short forms may function as hemi-transporters reported in other eukaryotic ABC transporter subgroups. Our results indicate that different K ATP compositions may co-exist in cardiac sarcolemmal membrane.

Based on autopsy data collected in Southern China from [2001][2002][2003][2004][2005][2006], 975 cases of sudden unexplained nocturnal death syndrome (SUNDS) were surveyed. Genetic screening of SCN5A, the gene encoding the voltage dependent cardiac Na channel, was performed in 74 available SUNDS cases. The annual occurrence rate of SUNDS in the area was estimated to be 1 per 100,000 people. 80.6% of deaths occurred between the ages of 21 to 40 years and the case number peaked at age 30 years. In 75.4% of cases where witnesses were present, victims died in their sleep between 11 PM and 4 AM and many showed abrupt respiratory distress shortly preceding death. The monthly distribution of emergency fever cases in the area during the same period was positively correlated to that of SUNDS cases (r s = 0.611, P = 0.0025). Four polymorphisms in SCN5A were identified in both SUNDS and control groups. Compared with controls, the allele frequency of C5457 and C3666+69 were significant higher in SUNDS (P<0.005) while the genotypes of both 5457CC (P=0.012, OR=2.0, 95% CI=1.3-3.2) and 3666+69CC (P=0.004, OR=2.1, 95% CI=1.3-3.3) in SUNDS cases were significantly higher. This is the first report of an epidemiological survey and SCN5A gene screening in SUNDS in the Han population of China. The genotypes of 5457CC and 3666+69CC in SCN5A gene may be Chinese SUNDS susceptible polymorphisms.

Targeting of ion channels to caveolae, a subset of lipid rafts, allow cells to respond efficiently to extracellular signals. Hyperpolarization-activated cyclic nucleotide-gated channel (HCN) 4 is a major subunit for the cardiac pacemaker. Caveolin-3 (Cav3), abundantly expressed in muscle cells, is responsible for forming caveolae. P104L, a Cav3 mutant, has a dominant negative effect on wild type (WT) Cav3 and associates with limb-girdle muscular dystrophy and cardiomyopathy. HCN4 was previously shown to localize to lipid rafts, but how caveolae regulate the function of HCN4 is unknown. We hypothesize that Cav3 associates with HCN4 and regulates the function of HCN4 channel. In this study, we applied whole-cell patch clamp analysis, immunostaining, biotinylation, and immunoprecipitation methods to investigate this hypothesis. The immunoprecipitation results indicated an association of HCN4 and Cav3 in the heart and in HEK293 cells. Our immunostaining results showed that HCN4 colocalized with Cav3 but only partially colocalized with P104L in HEK293 cells. Transient expression of Cav3, but not P104L, in HEK 293 cells stably expressing HCN4 caused a 45% increase in HCN4 current (IHCN4) density. Transient expression of P104L caused a two-fold increase in the activation time constant for IHCN4 and shifted the voltage of the steady-state inactivation to a more negative potential. We conclude that HCN4 associates with Cav3 to form a HCN4 macromolecular complex. Our results indicated that disruption of caveolae using P104L alters HCN4 function and could cause a reduction of cardiac pacemaker activity.

Aims Myocardial work is a novel echocardiographic algorithm that corrects speckle-tracking-derived global longitudinal strain (GLS) for afterload using non-invasive systolic blood pressure as a surrogate for left ventricular systolic pressure (LVSP). Yet, in patients with severe aortic stenosis, non-invasive systolic blood pressure does not equal LVSP. Methods and results We evaluated 35 patients with severe aortic stenosis who underwent transcatheter aortic valve replacement (TAVR). Transthoracic echocardiography, including myocardial mechanics, was performed pre- and post-TAVR. We performed simultaneous echocardiographic and cardiac catheterization measurements in 23 of the 35 patients at the time of TAVR. Peak and mean aortic gradients were calculated from echocardiographic and cardiac catheterization data. Peak-to-peak LV-aortic gradient correlated highly with mean LV-aortic gradient (r = 0.96); measured LVSP correlated highly with our novel method of non-invasively estimated LVSP (non-invasive systolic blood pressure cuff + Doppler-derived mean aortic gradient, r = 0.92). GLS improved from pre- to post-TAVR (−14.2% ± 4.3 vs. −15.1% ± 3.2), and myocardial work reduced from corrected pre-TAVR to post-TAVR (global work index: 1856.2 mmHg% ± 704.6 vs. 1534.8 ± 385.0). Conclusion We propose that non-invasive assessment of myocardial work can be reliably performed in aortic stenosis by the addition of mean aortic gradient to non-invasive systolic blood pressure. From this analysis, we note the novel and unique finding that GLS can improve as myocardial work reduces post-TAVR in patients with severe aortic stenosis. Both GLS and myocardial work post-TAVR remain below normal values, requiring further studies.