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Among the 15 types of congenital long QT syndrome, the LQTS 2 caused by hERG mutate is the most common type in China. Nowadays, more and more researches have proved LQTS has not only abnormal cardioelectrical activities, but also cardiovascular structural deficiency and remodeling: LQTS is associated with contraction inability, left atrial and ventricular dilated and heart deformity; ARVC, DCM, LVNC are also accompanied with LQTS; fetuses and infants heart deformity or sudden death are associated with LQTS as well. The mechanisms of LQTS structural deficiency are still under exploring. By biopsy, LQTS patients cardiomyocytes characterized by increased apoptosis; hERG mutation mice's hearts are developmental defected and increased apoptosis. These proofs above pointed apoptosis of cardiomyocyte might be the potential mechanism of LQTS induced heart deformity. L539fs/47-hERG is a complicated mutation in a Chinese family consists of 19 bps deletion at 1619-1637 accompanied with a 1692 A→G nonsense mutation, which causes hERG protein shorten and channel function defected. It can also prolong the phase 2 repolarization which slow down the L-type Ca2+ channel deactivation, make Ca2+ accumulate in cytoplasmic. High Ca2+ concentration can cause cell apoptosis via activation of Calpain. Calpain is a proteinase widely distributed in mammal cells. It is activated by Ca2+ and then induce CASPASE and None-CASPASE cell apoptosis pathways. According to these above evidences, we make the hypothesis that hERG mutation could induce cell apoptosis via activating Calpain pathway. In order to prove this theory and explore the potential mechanism of hERG mutation induced apoptosis, we decide to use L539fs/47-hERG cell model to detect the level of apoptosis and the expression of Calpain simultaneously.
We have constructed L539fs/47-hERG-pcDNA3.0 and WT-hERG-pcDNA3.0 plasmids, each of them was transiently transfected to HEK293 cell respectively to mimic L539fs/47-hERG mutation or WT-hERG cell models. After 24 hours incubation, the laser confocal scan microscope was used to detect hERG protein distribution. Hoechst33342 cell apoptosis stain was conducted and observed under fluorescence microscope. The whole cell protein extract Calpain1 expression level was detected through Western Blot technic.
At 24 hours after transfection, it can be seen that abnormal hERG protein accumulated in endoplasmic reticulum and failed to rivet into membrane. Using inverted phase contrast microscope, L539fs/47-hERG homozygous cell typing has more floated and bright cell in the medium compared with the heterozygosis typing or WT-hERG cell. After Hoechst33342 stain it can be seen that the number of apoptosis cells are significantly higher in L539fs/47-hERG cell than WT-hERG (P<0.05). Comparing with the WT-hERG, the Calpain1 is elevated in L539fs/47-hERG mutation cell (P=0.010).
The results indicated that L539fs/47-hERG mutation can induce cell apoptosis, and the elevation pf Calpain1 expression may play a regulatory role in this pathophysiology process. It's the first time to prove that LQTS type 2 mutation L539fs/47-hERG can cause cell apoptosis, which gives researchers a new sight to comprehension the potential mechanism of LQTS patients' structural deficiency and systole abnormal. Calpain play a considerable role in regulating L539fs/47-hERG induced apoptosis, which provide clinicians a new potential medication target in LQTS type 2 patients management.