Dissecting the Physiological and Functional Consequences of Alternative Splicing

To better understand the underlying disease mechanisms in DM, research has been linking splicing alterations triggered by MBNL depletion to functional defects measurable in animal models and patients. While the association between myotonia and mis-splicing of the skeletal muscle chloride channel, CLCN1, is the most prominent example, identification of such protein isoform expression/phenotype links is essential for a variety of efforts including understanding basic disease mechanisms, developing biomarker panels, and identifying putative therapeutic targets.

Mis-Splicing and Cardiac Function in DM

The cardiac consequences of DM1 have been well-characterized from a physiological viewpoint and are important contributors to disease morbidity and mortality. Yet only recently has information come to light about potential linkages between mis-splicing and the characteristic pattern of heart abnormalities in DM1 (Freyermuth et al., 2016). The authors identified mis-splicing of SCN5A in a transcriptome screen of DM1 patient heart samples—leading to expression of a fetal isoform of this key cardiac sodium channel accompanied by altered electrophysiological properties. Furthermore, altering Scn5a splicing in the mouse reproduced the cardiac conduction system delays and arrhythmia.

A recent publication (Pang et al., 2018) by Dr. Tom Cooper and colleagues at Baylor College of Medicine has extended understanding of the link between Scn5a mis-splicing and cardiac defects in a mouse model. The normal developmental switch in Scn5a is driven by replacement of exon 6A in fetal heart by exon 6B inclusion in the adult. To achieve the mis-splicing pattern associated with cardiac conduction defects, the research team deleted exon 6B using CRISPR/Cas9 technology.

Deletion of exon 6B resulted in redirection of Scn5a splicing to include the fetal exon 6A. Assessment of mice using surface ECG showed decreased basal heart rate (in mice homozygous for the deletion), prolonged PR interval (homozygous mice), and prolonged QRS interval (both homozygous and heterozygous mice). In parallel to DM1 patients, deleted mice did not show alterations of ejection fraction, fractional shortening, or heart structure. In an in vivo intracardiac pacing paradigm, mice showed increased susceptibility to arrhythmias, including delays in sinoatrial node function (homozygotes only) and prolonged atrioventricular effective refractory period (both homozygotes and hets). Finally, optical mapping of isolated, perfused hearts confirmed significant delays in activation and propagation of conduction (both homozygotes and hets).

Taken together, the electrophysiological consequences in mice expressing the fetal isoform of Scn5a mirror conduction defects and sensitivity to arrhythmias in DM1 patients. Although this CRISPR/Cas9-generated mouse model has higher expression of the embryonic isoform than seen in DM1 patients, the research team’s data suggests that severity of cardiac electrophysiological function defects are closely related to the ratio of embryonic and adult isoforms of Scn5a.

References:

Splicing misregulation of SCN5A contributes to cardiac-conduction delay and heart arrhythmia in myotonic dystrophy.
Freyermuth F, Rau F, Kokunai Y, Linke T, Sellier C, Nakamori M, Kino Y, Arandel L, Jollet A, Thibault C, Philipps M, Vicaire S, Jost B, Udd B, Day JW, Duboc D, Wahbi K, Matsumura T, Fujimura H, Mochizuki H, Deryckere F, Kimura T, Nukina N, Ishiura S, Lacroix V, Campan-Fournier A, Navratil V, Chautard E, Auboeuf D, Horie M, Imoto K, Lee KY, Swanson MS, Lopez de Munain A, Inada S, Itoh H, Nakazawa K, Ashihara T, Wang E, Zimmer T, Furling D, Takahashi MP, Charlet-Berguerand N.
Nat Commun. 2016 Apr 11;7:11067. doi: 10.1038/ncomms11067.

CRISPR-Mediated Expression of the Fetal Scn5a Isoform in Adult Mice Causes Conduction Defects and Arrhythmias
Pang PD, Alsina KM, Cao S, Koushik AB, Wehrens XHT, Cooper TA
Am Heart Assoc. 2018;7:e010393.