Manuela Mura1, Ashish Mehta2, Chrishan J Ramachandra2, Rita Zappatore3, Federica Pisano1, Maria Chiara Ciuffreda1, Vincenzo Barbaccia4, Lia Crotti5, Peter J Schwartz6, Winston Shim7, Massimiliano Gnecchi8. 1. Laboratory of Experimental Cardiology for Cell and Molecular Therapy, Fondazione IRCCS Policlinico San Matteo, Pavia, Italy; Coronary Care Unit and Laboratory of Clinical and Experimental Cardiology, Fondazione IRCCS Policlinico San Matteo, Pavia, Italy. 2. National Heart Research Institute Singapore, National Heart Centre Singapore, Singapore; Cardiovascular Academic Clinical Program, DUKE-NUS Medical School, Singapore. 3. Laboratory of Oncohaematological Cytogenetics and Molecular Diagnostics, Division of Haematology, Fondazione IRCCS Policlinico San Matteo, Pavia, Italy. 4. Department of Clinical-Surgical, Diagnostic and Pediatric Science, Institute of Dermatology, University of Pavia, Fondazione IRCCS Policlinico San Matteo, Pavia, Italy. 5. IRCCS Istituto Auxologico Italiano, Center for Cardiac Arrhythmias of Genetic Origin and Laboratory of Cardiovascular Genetics, Milan, Italy; Department of Cardiovascular, Neural and Metabolic Sciences, San Luca Hospital IRCCS Istituto Auxologico Italiano, Milan, Italy; Department of Molecular Medicine, Unit of Cardiology, University of Pavia, Pavia, Italy. 6. IRCCS Istituto Auxologico Italiano, Center for Cardiac Arrhythmias of Genetic Origin and Laboratory of Cardiovascular Genetics, Milan, Italy. 7. National Heart Research Institute Singapore, National Heart Centre Singapore, Singapore; Cardiovascular & Metabolic Disorders Program, DUKE-NUS Medical School, Singapore. Electronic address: winston.shim.s.n@nhcs.com.sg. 8. Laboratory of Experimental Cardiology for Cell and Molecular Therapy, Fondazione IRCCS Policlinico San Matteo, Pavia, Italy; Coronary Care Unit and Laboratory of Clinical and Experimental Cardiology, Fondazione IRCCS Policlinico San Matteo, Pavia, Italy; Department of Molecular Medicine, Unit of Cardiology, University of Pavia, Pavia, Italy; Department of Medicine, University of Cape Town, Cape Town, South Africa. Electronic address: m.gnecchi@unipv.it.
Abstract
BACKGROUND: Long QT Syndrome type 2 (LQT2) is caused by mutations in the KCNH2 gene that encodes for the α-subunit (hERG) of the ion channel conducting the rapid delayed rectifier potassium current (IKr). We have previously identified a disease causing mutation (IVS9-28A/G) in the branch point of the splicing of KCNH2 intron 9. However, the mechanism through which this mutation causes the disease is unknown. METHODS AND RESULTS: We generated human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) from fibroblasts of two IVS9-28A/G mutation carriers. IVS9-28A/G iPSC-CMs showed prolonged repolarization time, mimicking what observed at the ECG level in the same patients. The expression of the full-length ERG1a isoform resulted reduced, whereas the C-terminally truncated ERG1aUSO isoform was upregulated in mutant iPSC-CMs, with consequent alteration of the physiological ERG1aUSO/ERG1a ratio. Importantly, we observed an impairment of hERG trafficking to the cell membrane. The severity of the alterations in hERG expression and trafficking correlated with the clinical severity of the disease in the two patients under study. Finally, we were able to revert the trafficking defect and reduce the repolarization duration in LQT2 iPSC-CMs using the proteasome inhibitor ALLN. CONCLUSION: Our results highlight the key role of the KCNH2 intron 9 branch point in the regulation of KCNH2 isoform expression and hERG channel function, and allow to categorize the IVS9-28A/G mutation as LQT2 class 2 mutation. These findings may result in a more personalized clinical management of IVS9-28A/G mutation carriers.
BACKGROUND:Long QT Syndrome type 2 (LQT2) is caused by mutations in the KCNH2 gene that encodes for the α-subunit (hERG) of the ion channel conducting the rapid delayed rectifier potassium current (IKr). We have previously identified a disease causing mutation (IVS9-28A/G) in the branch point of the splicing of KCNH2 intron 9. However, the mechanism through which this mutation causes the disease is unknown. METHODS AND RESULTS: We generated human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) from fibroblasts of two IVS9-28A/G mutation carriers. IVS9-28A/G iPSC-CMs showed prolonged repolarization time, mimicking what observed at the ECG level in the same patients. The expression of the full-length ERG1a isoform resulted reduced, whereas the C-terminally truncated ERG1aUSO isoform was upregulated in mutant iPSC-CMs, with consequent alteration of the physiological ERG1aUSO/ERG1a ratio. Importantly, we observed an impairment of hERG trafficking to the cell membrane. The severity of the alterations in hERG expression and trafficking correlated with the clinical severity of the disease in the two patients under study. Finally, we were able to revert the trafficking defect and reduce the repolarization duration in LQT2 iPSC-CMs using the proteasome inhibitor ALLN. CONCLUSION: Our results highlight the key role of the KCNH2 intron 9 branch point in the regulation of KCNH2 isoform expression and hERG channel function, and allow to categorize the IVS9-28A/G mutation as LQT2 class 2 mutation. These findings may result in a more personalized clinical management of IVS9-28A/G mutation carriers.
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