Cotranslational Complexes Encoding Ion Channels In The Heart

The expression of ion channels underlying the heart’s rhythmic beating must be precisely coordinated to fulfill their physiological roles and protect the heart from arrhythmias. How cells determine the proper ratios of different ion channels mediating repolarization, the most vulnerable phase of the ventricular action potential, is poorly understood. Here, we tested the hypothesis that transcripts encoding ion channels necessary for repolarization are cotranslationally associated and regulated. Immunoprecipitation of hERG1a or Nav1.5 nascent proteins from human heart followed by RT-PCR revealed association of hERG1a, hERG1b, SCN5A, and CACNA1C mRNAs but not RYR2 or KCNJ2 transcripts. Specific silencing of hERG1b transcripts concomitantly reduced expression of the associated transcripts as determined by RT-qPCR in a manner not attributable off-target. Patch-clamp electrophysiology in cardiomyocytes derived from human iPSCs revealed a corresponding reduction in IKr, INa, INa,late, and ICa,L currents following hERG1b (or hERG1a) mRNA silencing, indicating that the mRNAs affected by knockdown were those undergoing translation. To observe mRNA complexes directly, we used single-molecule fluorescent in situ hybridization (smFISH) combined with immunofluorescence. We noted fractional colocalization of CACNA1C, KCNQ1, or SCN5A with hERG transcripts significantly greater than predicted by chance. Within translational complexes, the fraction associated was enriched 3-fold and diminished by puromycin, a translation inhibitor. We used multicolor smFISH experiments to test the hypothesis that the coregulated mRNA species associated in a supercomplex, but instead primarily found transcript pairs in translational complexes. These results rule out a single cotranslational target for coordinate regulation and indicate that hERG/IKr channel biogenesis arises from a heterogeneous population of cotranslational complexes with other mRNA species. Supported by T32 GM008688 (MBJ), AHA 20PRE35080125 (MBJ), and NIH R01HL131403 (GAR).