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.