We generated patient-specific pluripotent stem cells from members of a family affected by long-QT syndrome type 1 and induced them to differentiate into functional cardiac myocytes. The patient-derived cells recapitulated the electrophysiological features of the disorder. (Funded by the European Research Council and others.)

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Patient-specific induced pluripotent stem cells (iPSCs) will assist research on genetic cardiac maladies if the disease phenotype is recapitulated in vitro. However, genetic background variations may confound disease traits, especially for disorders with incomplete penetrance, such as long-QT syndromes (LQTS). To study the LQT2-associated c.A2987T (N996I) KCNH2 mutation under genetically defined conditions, we derived iPSCs from a patient carrying this mutation and corrected it. Furthermore, we introduced the same point mutation in human embryonic stem cells (hESCs), generating two genetically distinct isogenic pairs of LQTS and control lines. Correction of the mutation normalized the current (IKr) conducted by the HERG channel and the action potential (AP) duration in iPSC-derived cardiomyocytes (CMs). Introduction of the same mutation reduced IKr and prolonged the AP duration in hESC-derived CMs. Further characterization of N996I-HERG pathogenesis revealed a trafficking defect. Our results demonstrated that the c.A2987T KCNH2 mutation is the primary cause of the LQTS phenotype. Precise genetic modification of pluripotent stem cells provided a physiologically and functionally relevant human cellular context to reveal the pathogenic mechanism underlying this specific disease phenotype.

Dihydropyridines (DHPs) block the vascular smooth muscle L-type Ca2+ channel at lower concentrations than the cardiac Ca2+ channel, although their alpha 1 subunit, which binds the DHPs, is derived from the same gene. This alpha 1C gene gives rise to several splice variants, among which the alpha 1C-b variant is affected by lower concentrations of nisoldipine than the alpha 1C-a variant. Functional expression of chimeras of alpha 1C-a and alpha 1C-b subunits demonstrated that the transmembrane segment IS6 is responsible for the different dihydropyridine sensitivity. Northern blot analysis showed that transcripts coding for the IS6 segment of the alpha 1C-a subunit were expressed in heart but not in aorta, whereas the IS6 segment of the alpha 1C-b subunit was expressed predominantly in vascular smooth muscle. In situ hybridization of rat heart sections confirmed this expression pattern of IS6 alpha 1C-a and IS6 alpha 1C-b in ventricular and smooth muscle myocytes, respectively. These results suggest that the different dihydropyridine sensitivities of cardiac and vascular L-type Ca2+ channels are caused at least partially by the tissue-specific expression of alternatively spliced IS6 segments of the alpha 1C gene.

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Mouse pancreatic β- and α-cells are equipped with voltage-gated Na+ currents that inactivate over widely different membrane potentials (half-maximal inactivation (V0.5) at −100 mV and −50 mV in β- and α-cells, respectively). Single-cell PCR analyses show that both α- and β-cells have Nav1.3 (Scn3) and Nav1.7 (Scn9a) α subunits, but their relative proportions differ: β-cells principally express Nav1.7 and α-cells Nav1.3. In α-cells, genetically ablating Scn3a reduces the Na+ current by 80%. In β-cells, knockout of Scn9a lowers the Na+ current by >85%, unveiling a small Scn3a-dependent component. Glucagon and insulin secretion are inhibited in Scn3a−/− islets but unaffected in Scn9a-deficient islets. Thus, Nav1.3 is the functionally important Na+ channel α subunit in both α- and β-cells because Nav1.7 is largely inactive at physiological membrane potentials due to its unusually negative voltage dependence of inactivation. Interestingly, the Nav1.7 sequence in brain and islets is identical and yet the V0.5 for inactivation is >30 mV more negative in β-cells. This may indicate the presence of an intracellular factor that modulates the voltage dependence of inactivation.

In arterial vascular smooth muscle cells (VSMCs),

There is a need for in vivo monitoring of cell engraftment and survival after cardiac cell transplantation therapy. This study assessed the feasibility and usefulness of combined PET and MRI for monitoring cell engraftment and survival after cell transplantation. Methods: Human endothelial progenitor cells (HEPCs), derived from CD341 mononuclear cells of umbilical cord blood, were retrovirally transduced with the sodium iodide symporter (NIS) gene for reporter gene imaging by 124 I-PET and labeled with iron oxides for visualization by MRI. Imaging and histologic analysis were performed on 3 groups of nude rats on days 1, 3, and 7 after intramyocardial injection of 4 million HEPCs. Results: In vitro studies demonstrated stable expression of functional NIS protein and normal viability of HEPCs after transduction. On day 1, after intramyocardial transplantation, iron-and NIS-labeled HEPCs were visualized successfully on MRI as a regional signal void in the healthy myocardium and on PET as 124 I accumulation. The 124 I uptake decreased on day 3 and was undetectable on day 7, and the MRI signal remained unchanged throughout the follow-up period. Histologic analysis with CD31 and CD68 antibodies confirmed the presence of either labeled or nonlabeled control transplanted HEPCs at the site of injection on day 1 but not on day 7, when only iron-loaded macrophages were seen. Furthermore, deoxyuride-59-triphosphate biotin nick end labeling showed extensive apoptosis at the site of transplantation. Conclusion: The combination of MRI and PET allows imaging of localization and survival of transplanted HEPCs together with morphologic information about the heart. Although iron labeling rapidly loses specificity for cell viability because of phagocytosis of iron particles released from dead cells, reporter gene expression provided specific information on the number of surviving cells. This multimodality approach allows complementary analysis of cell localization and viability.