Constitutive deletion of the obscurin-Ig58/59 domains induces atrial remodeling and Ca2+-based arrhythmogenesis
Alyssa Grogan, Annie Brong, Humberto C. Joca, Liron Boyman, Aaron D. Kaplan, Christopher W. Ward, Maura Greiser, Aikaterini Kontrogianni-Konstantopoulos
Alyssa Grogan, Annie Brong, Humberto C. Joca, Liron Boyman, Aaron D. Kaplan, Christopher W. Ward, Maura Greiser, Aikaterini Kontrogianni-Konstantopoulos
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Research Article Muscle biology

Constitutive deletion of the obscurin-Ig58/59 domains induces atrial remodeling and Ca2+-based arrhythmogenesis

Abstract

Obscurin is a giant protein that coordinates diverse aspects of striated muscle physiology. Obscurin immunoglobulin domains 58/59 (Ig58/59) associate with essential sarcomeric and Ca2+ cycling proteins. To explore the pathophysiological significance of Ig58/59, we generated the Obscn-ΔIg58/59 mouse model, expressing obscurin constitutively lacking Ig58/59. Males in this line develop atrial fibrillation by 6 months, with atrial and ventricular dilation by 12 months. As Obscn-ΔIg58/59 left ventricles at 6 months exhibit no deficits in sarcomeric ultrastructure or Ca2+ signaling, we hypothesized that susceptibility to arrhythmia may emanate from the atria. Ultrastructural evaluation of male Obscn-ΔIg58/59 atria uncovered prominent Z-disk streaming by 6 months and further misalignment by 12 months. Relatedly, isolated Obscn-ΔIg58/59 atrial cardiomyocytes exhibited increased Ca2+ spark frequency and age-specific alterations in Ca2+ cycling dynamics, coinciding with arrhythmia onset and progression. Quantitative analysis of the transverse-axial tubule (TAT) network using super-resolution microscopy demonstrated significant TAT depletion in Obscn-ΔIg58/59 atria. These structural and Ca2+ signaling deficits were accompanied by age-specific alterations in the expression or phosphorylation of T-cap protein, which links transverse tubules to Z-disks, and junctophilin 2, which connects transverse tubules to the sarcoplasmic reticulum. Collectively, our work establishes the Obscn-ΔIg58/59 model as a reputable genetic model for atrial cardiomyopathy and provides mechanistic insights into atrial fibrillation and remodeling.

Authors

Alyssa Grogan, Annie Brong, Humberto C. Joca, Liron Boyman, Aaron D. Kaplan, Christopher W. Ward, Maura Greiser, Aikaterini Kontrogianni-Konstantopoulos

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Figure 5

The expression and phosphorylation of T-cap are altered in Obscn-ΔIg58/59 atria.

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The expression and phosphorylation of T-cap are altered in Obscn-ΔIg58/5...
(A and B) Representative immunoblots (A) and relative quantifications (B) revealed significantly increased T-cap expression in Obscn-ΔIg5859 atria compared with wild-type at 6 months but not at 3.5 or 12 months. (C and D) Representative phosphorylated Phos-tag acrylamide immunoblots (C) and relative quantifications (D) did not indicate significant differences in normalized pT-cap at 6 months, but revealed increased levels of biphosphorylated T-cap (2P) and a corresponding decrease in the lower molecular weight monophosphorylated (1P1) T-cap species with no statistically significant differences in the higher molecular weight (1P2) or nonphosphorylated (0P) T-cap species in Obscn-ΔIg5859 atria compared with wild-type at both 3.5 and 12 months. Nonphosphorylated T-cap species were not reliably detected at 3.5 months and therefore were not quantified; t test, *P < 0.05; (A): n = 3 animals per genotype for the 3.5- and 6-month time points and n = 6 animals per genotype for the 12-month time point; (B): n = 6 animals per genotype for the 3.5-month time point, n = 3 animals per genotype for the 6-month time point, and n = 5 animals per genotype for the 12-month time point; data points represent the average of at least 3 technical replicates per animal; quantifications of pT-cap were normalized to the summed intensity of all species for a given sample. (E) Schematic depicting the decrease in 1P1 T-cap species and corresponding increase in 2P T-cap observed in Obscn-ΔIg5859 atria at 12 months. Figure generated with BioRender.com (License OT27PC68YH). (F–H) Representative immunoblots (F) and relative quantifications (G and H) revealed significantly increased total uncleaved junctophilin-2 (JPH2), but not cleaved JPH2 NT1, in Obscn-ΔIg5859 atria compared with wild-type at 12 months; t test, *P < 0.05; n = 3 animals per group (3.5 and 6 months), n = 6 animals per group (12 months). Numbers on right of blots represent kilodaltons; data points represent the average of at least 3 technical replicates per animal.