Non-technical summary Long QT syndrome (LQTS) is a genetic disorder characterized by recurrent syncope and sudden cardiac death (SCD). Type 1 (LQT1) and Type 2 (LQT2) LQTS account for 90% of the genotyped mutations in patients with this disorder. These syndromes have been associated with different sympathetic modes for initiation of cardiac arrest. Using isolated cardiomyocytes and Langendorff-perfused hearts from transgenic rabbit models of LQT1 and LQT2, we have identified differential conditions and cellular mechanisms for the generation of early afterdepolarizations (EADs), abnormal depolarizations during the plateau and repolarization phase of action potentials and the hallmark of the arrhythmias in LQTS. These differences explain why different types of increased autonomic nervous system activity, i.e. sympathetic surge vs. high sympathetic tone, are associated with the initiation of polymorphic ventricular tachycardia in LQTS patients with different genetic background.Abstract Early after-depolarization (EAD), or abnormal depolarization during the plateau phase of action potentials, is a hallmark of long-QT syndrome (LQTS). More than 13 genes have been identified as responsible for LQTS, and elevated risks for EADs may depend on genotypes, such as exercise in LQT1 vs. sudden arousal in LQT2 patients. We investigated mechanisms underlying different high-risk conditions that trigger EADs using transgenic rabbit models of LQT1 and LQT2, which lack I Ks and I Kr (slow and fast components of delayed rectifying K + current), respectively. Single-cell patch-clamp studies show that prolongation of action potential duration (APD) can be further enhanced by lowering extracellular potassium concentration ([K + ] o ) from 5.4 to 3.6 mM. However, only LQT2 myocytes developed spontaneous EADs following perfusion with lower [K + ] o , while there was no EAD formation in littermate control (LMC) or LQT1 myocytes, although APDs were also prolonged in LMC myocytes and LQT1 myocytes. Isoprenaline (ISO) prolonged APDs and triggered EADs in LQT1 myocytes in the presence of lower [K + ] o . In contrast, continuous ISO perfusion diminished APD prolongation and reduced the incidence of EADs in LQT2 myocytes. These different effects of ISO on LQT1 and LQT2 were verified by optical mapping of the whole heart, suggesting that ISO-induced EADs are genotype specific. Further voltage-clamp studies revealed that ISO increases L-type calcium current (I Ca ) faster than I Ks (time constant 9.2 s for I Ca and 43.6 s for I Ks ), and computer simulation demonstrated a high-risk window of EADs in LQT2 during ISO perfusion owing to mismatch in the time courses of I Ca and I Ks , which may explain why a sympathetic surge rather than high sympathetic tone can be an effective trigger of EADs in LQT2 perfused hearts. In summary, EAD formation is genotype specific, such that EADs can be elicited in LQT2 myocytes simply by lowering [K + ] o , while LQT1 myocytes require sympathetic stimulation. Slower activation of I Ks than of I Ca by ISO may expla...

Myotubularins (MTMs) belong to a large subfamily of phosphatases that dephosphorylate the 3 position of phosphatidylinositol 3-phosphate [PI(3)P] and PI(3,5)P 2 . MTM1 is mutated in X-linked myotubular myopathy, and MTMR2 and MTMR13 are mutated in Charcot-Marie-Tooth syndrome. However, little is known about the general mechanism(s) whereby MTMs are regulated or the specific biological processes regulated by the different MTMs. We identified a Ca 2؉ -activated K channel, K Ca 3.1 (also known as KCa4, IKCa1, hIK1, or SK4), that specifically interacts with the MTMR6 subfamily of MTMs via coiled coil (CC) domains on both proteins. Overexpression of MTMR6 inhibited K Ca 3.1 channel activity, and this inhibition required MTMR6's CC and phosphatase domains. This inhibition is specific; MTM1, a closely related MTM, did not inhibit K Ca 3.1. However, a chimeric MTM1 in which the MTM1 CC domain was swapped for the MTMR6 CC domain inhibited K Ca 3.1, indicating that MTM CC domains are sufficient to confer target specificity. K Ca 3.1 was also inhibited by the PI(3) kinase inhibitors LY294002 and wortmannin, and this inhibition was rescued by the addition of PI(3)P, but not other phosphoinositides, to the patch pipette solution. PI(3)P also rescued the inhibition of K Ca 3.1 by MTMR6 overexpression. These data, when taken together, indicate that K Ca 3.1 is regulated by PI(3)P and that MTMR6 inhibits K Ca 3.1 by dephosphorylating the 3 position of PI(3)P, possibly leading to decreased PI(3)P in lipid microdomains adjacent to K Ca 3.1. K Ca 3.1 plays important roles in controlling proliferation by T cells, vascular smooth muscle cells, and some cancer cell lines. Thus, our findings not only provide unique insights into the regulation of K Ca 3.1 channel activity but also raise the possibility that MTMs play important roles in the negative regulation of T cells and in conditions associated with pathological cell proliferation, such as cancer and atherosclerosis.Myotubularins (MTM) are a large family of evolutionarily conserved lipid phosphatases (PT) that specifically dephosphorylate the 3Ј position of phosphatidylinositol 3-phosphate [PI(3)P] and PI(3,5)P 2 (28, 39). Fourteen MTMs in mammalian cells have been identified, and they can be divided into six subgroups based on sequence alignment and phylogenetic comparison. Members of one of these subgroups lack phosphatase activity due to a mutation in a critical residue within the phosphatase domain. MTM1, the founding member of this gene family, is mutated in X-linked myotubular myopathy, and MTMR2 and MTMR13 are mutated in Charcot-Marie-Tooth (CMT) syndrome type 4B (3, 30, 37). In addition to containing a phosphatase domain, most MTMs are composed of a GRAM domain which may mediate association of MTMs with membranes, a Rac-induced localization domain which mediates the association with Rac-induced membrane ruffles, and a C-terminal coiled coil (CC) domain (10,28,29,39). Recent data have indicated that the MTM CC domains mediate specific heterodimerization between a MTM (PT ac...

To bridge the gap between two-dimensional cell culture and tissue, various three-dimensional (3-D) cell culture approaches have been developed for the investigation of cardiac myocytes (CMs) and cardiac fibroblasts (CFs). However, several limitations still exist. This study was designed to develop a cardiac 3-D culture model with a scaffold-free technology that can easily and inexpensively generate large numbers of microtissues with cellular distribution and functional behavior similar to cardiac tissue. Using micromolded nonadhesive agarose hydrogels containing 822 concave recesses (800 μm deep × 400 μm wide), we demonstrated that neonatal rat ventricular CMs and CFs alone or in combination self-assembled into viable (Live/Dead stain) spherical-shaped microtissues. Importantly, when seeded simultaneously or sequentially, CMs and CFs self-sorted to be interspersed, reminiscent of their myocardial distribution, as shown by cell type-specific CellTracker or antibody labeling. Microelectrode recordings and optical mapping revealed characteristic triangular action potentials (APs) with a resting membrane potential of -66 ± 7 mV (n = 4) in spontaneously contracting CM microtissues. Under pacing, optically mapped AP duration at 90% repolarization and conduction velocity were 100 ± 30 ms and 18.0 ± 1.9 cm/s, respectively (n = 5 each). The presence of CFs led to a twofold AP prolongation in heterogenous microtissues (CM-to-CF ratio of 1:1). Importantly, Ba(2+)-sensitive inward rectifier K(+) currents and Ca(2+)-handling proteins, including sarco(endo)plasmic reticulum Ca(2+)-ATPase 2a, were detected in CM-containing microtissues. Furthermore, cell type-specific adenoviral gene transfer was achieved, with no impact on microtissue formation or cell viability. In conclusion, we developed a novel scaffold-free cardiac 3-D culture model with several advancements for the investigation of CM and CF function and cross-regulation.

Coetzee. Consequences of cardiac myocyte-specific ablation of KATP channels in transgenic mice expressing dominant negative Kir6 subunits.

KCa3.1 is an intermediate conductance Ca2؉ -activated K ؉ channel that is expressed predominantly in hematopoietic cells, smooth muscle cells, and epithelia where it functions to regulate membrane potential, Ca 2؉ influx, cell volume, and chloride secretion. We recently found that the KCa3.1 channel also specifically requires phosphatidylinositol-3 phosphate [PI(3)P] for channel activity and is inhibited by myotubularin-related protein 6 (MTMR6), a PI(3)P phosphatase. We now show that PI(3)P indirectly activates KCa3.1. Unlike KCa3.1 channels, the related KCa2.1, KCa2.2, or KCa2.3 channels do not require PI(3)P for activity, suggesting that the KCa3.1 channel has evolved a unique means of regulation that is critical for its biological function. By making chimeric channels between KCa3.1 and KCa2.3, we identified a stretch of 14 amino acids in the carboxy-terminal calmodulin binding domain of KCa3.1 that is sufficient to confer regulation of KCa2.3 by PI(3)P. However, mutation of a single potential phosphorylation site in these 14 amino acids did not affect channel activity. These data together suggest that PI(3)P and these 14 amino acids regulate KCa3.1 channel activity by recruiting an as yet to be defined regulatory subunit that is required for Ca 2؉ gating of KCa3.1. INTRODUCTIONKCa3.1 subunits (also known as IKCa and KCNN4) are components of an intermediate conductance Ca 2ϩ -activated K ϩ channel that is found predominantly in peripheral tissues such as blood cells, epithelia, and smooth muscle cells where it functions to couple alterations in cytosolic Ca 2ϩ to K ϩ flux Jensen et al., 2001;Wulff et al., 2003). KCa3.1 channels thereby play an important physiological role to set the membrane potential at negative values close to the K ϩ equilibrium potential Jensen et al., 2001;Wulff et al., 2003;Stocker, 2004). This effect on membrane potential can have diverse physiological responses in a variety of cell types, including water movement and volume regulation in red blood cells, mitogen activation of T-lymphocytes, Cl Ϫ secretion of exocrine epithelial cells, and control of proliferation by a variety of cells such as Tand B-lymphocytes, vascular smooth muscle cells, keratinocytes, and some cancer cell lines (Khanna et al., 1999;Ghanshani et al., 2000;Fanger et al., 2001;Koegel and Alzheimer, 2001;Kohler et al., 2003;Maher and Kuchel, 2003; OuadidAhidouch et al., 2004;Wulff et al., 2004). Based on these activities, pharmacological modulation of KCa3.1 channels has been proposed to treat proliferative diseases such as restenosis after angioplasty and cancer, transplant rejection as well as secretory diarrheas, sickle cell anemia, and cystic fibrosis (Rufo et al., 1997;Jensen et al., 2001;Maher and Kuchel, 2003;Wulff et al., 2003).Over the past several years, the mechanism of activation of KCa3.1 and the three related small conductance Ca 2ϩ -activated K ϩ channels, KCa2.1, KCa2.2, and KCa2.3 channels (also known as SK1, SK2, and SK3), has been determined. These channels are all structurally similar and possess...

The targeting of ion channels to particular membrane microdomains and their organization in macromolecular complexes allow excitable cells to respond efficiently to extracellular signals. In this study, we describe the formation of a complex that contains two scaffolding proteins: caveolin-3 (Cav-3) and a membrane-associated guanylate kinase (MAGUK), SAP97. Complex formation involves the association of Cav-3 with a segment of SAP97 localized between its PDZ2 and PDZ3 domains. In heterologous expression systems, this scaffolding complex can recruit Kv1.5 to form a tripartite complex in which each of the three components interacts with the other two. These interactions regulate the expression of currents encoded by a glycosylation-deficient mutant of Kv1.5. We conclude that the association of Cav-3 with SAP97 may constitute the nucleation site for the assembly of macromolecular complexes containing potassium channels.

BackgroundRemodeling of cardiac repolarizing currents, such as the downregulation of slowly activating K+ channels (IKs), could underlie ventricular fibrillation (VF) in heart failure (HF). We evaluated the role of I ks remodeling in VF susceptibility using a tachypacing HF model of transgenic rabbits with Long QT Type 1 (LQT1) syndrome.Methods and ResultsLQT1 and littermate control (LMC) rabbits underwent three weeks of tachypacing to induce cardiac myopathy (TICM). In vivo telemetry demonstrated steepening of the QT/RR slope in LQT1 with TICM (LQT1-TICM; pre: 0.26±0.04, post: 0.52±0.01, P<0.05). In vivo electrophysiology showed that LQT1-TICM had higher incidence of VF than LMC-TICM (6 of 11 vs. 3 of 11, respectively). Optical mapping revealed larger APD dispersion (16±4 vs. 38±6 ms, p<0.05) and steep APD restitution in LQT1-TICM compared to LQT1-sham (0.53±0.12 vs. 1.17±0.13, p<0.05). LQT1-TICM developed spatially discordant alternans (DA), which caused conduction block and higher-frequency VF (15±1 Hz in LQT1-TICM vs. 13±1 Hz in LMC-TICM, p<0.05). Ca2+ DA was highly dynamic and preceded voltage DA in LQT1-TICM. Ryanodine abolished DA in 5 out of 8 LQT1-TICM rabbits, demonstrating the importance of Ca2+ in complex DA formation. Computer simulations suggested that HF remodeling caused Ca2+-driven alternans, which was further potentiated in LQT1-TICM due to the lack of IKs.ConclusionsCompared with LMC-TICM, LQT1-TICM rabbits exhibit steepened APD restitution and complex DA modulated by Ca2+. Our results strongly support the contention that the downregulation of IKs in HF increases Ca2+ dependent alternans and thereby the risk of VF.

In contrast to the other heterotrimeric GTP-binding proteins (G proteins) G s and Gi, the functional role of Go is still poorly defined. To investigate the role of G␣o in the heart, we generated transgenic mice with cardiac-specific expression of a constitutively active form of G␣ o1 ‫ء‬ (G␣ o ‫,)ء‬ the predominant G␣ o isoform in the heart. G␣ o expression was increased 3-to 15-fold in mice from 5 independent lines, all of which had a normal life span and no gross cardiac morphological abnormalities. We demonstrate enhanced contractile function in G␣ o‫ء‬ transgenic mice in vivo, along with increased L-type Ca 2ϩ channel current density, calcium transients, and cell shortening in ventricular G␣ o‫-ء‬expressing myocytes compared with wild-type controls. These changes were evident at baseline and maintained after isoproterenol stimulation. Expression levels of all major Ca 2ϩ handling proteins were largely unchanged, except for a modest reduction in Na ϩ /Ca 2ϩ exchanger in transgenic ventricles. In contrast, phosphorylation of the ryanodine receptor and phospholamban at known PKA sites was increased 1.6-and 1.9-fold, respectively, in G␣ o‫ء‬ ventricles. Density and affinity of ␤-adrenoceptors, cAMP levels, and PKA activity were comparable in G␣ o‫ء‬ and wild-type myocytes, but protein phosphatase 1 activity was reduced upon G␣ o‫ء‬ expression, particularly in the vicinity of the ryanodine receptor. We conclude that G␣ o‫ء‬ exerts a positive effect on Ca 2ϩ cycling and contractile function. Alterations in protein phosphatase 1 activity rather than PKA-mediated phosphorylation might be involved in hyperphosphorylation of key Ca 2ϩ handling proteins in hearts with constitutive G␣ o activation. G proteins; signal transduction; calcium; contraction; transgenic mice CARDIAC CONTRACTILE FUNCTION is determined by the intrinsic contractile properties of the heart and is subject to neurohumoral regulation. The main receptors involved in regulating contraction are prototypical G protein-coupled receptors (42). They activate heterotrimeric G proteins that are comprised of ␣-, ␤-, and ␥-subunits (40). G proteins are classified according to their ␣-subunits, because they primarily determine downstream signaling specificity. Several different G proteins are expressed in the heart (53). Members of the G s and G i/o subfamilies play a key role in transmitting extracellular signals that regulate myocyte cell shortening (42): 1) activation of G s protein-coupled receptors (such as ␤-adrenergic receptors) leads to increased adenylyate cyclase activity and cAMP and PKA activation. PKA then phosphorylates several Ca 2ϩ handling and contractile proteins, resulting in increased contraction and relaxation. 2) Activation of cardiac receptors that are coupled to members of the pertussis toxin-sensitive G i/o subfamily (such as A 1 adenosine and M 2 muscarinic receptors) negatively regulate contractile function in mammalian ventricles in the presence of elevated cAMP ("accentuated antagonism"). While G␣ i has the capacity to directly inhib...