Dataset Information

Combined loss of obscurin and obscurin-like 1 in murine hearts results in diastolic dysfunction, altered metabolism and deregulated mitophagy.

ABSTRACT: Background: Muscle proteins of the obscurin protein family play important roles in sarcomere organization, sarcoplasmic reticulum (SR) and T-tubule architecture and function. However, their precise molecular functions and redundancies between protein family members as well as their involvement in cardiac diseases remain to be fully understood. Methods: To investigate the functional roles of obscurin and its close homologue obscurin-like 1 (Obsl1) in the heart, we generated and analyzed knockout mice for obscurin, Obsl1, as well as obscurin/Obsl1 double-knockouts (dKO). Results: We show that dKO mice are viable but show postnatal deficits in cardiac muscle SR and mitochondrial architecture and function at the microscopic, biochemical, and cellular level. Altered SR structure resulted in perturbed calcium cycling, while mitochondrial ultrastructure deficits were linked to decreased levels of Chchd3, a Micos complex protein. Hearts of dKO mice also show altered levels of Atg4 proteins, novel Obsl1 interactors, resulting in abnormal mitophagy, and increased unfolded protein response. At the physiological level, loss of obscurin and Obsl1 resulted in a profound delay of cardiac relaxation, associated with metabolic signs of heart failure.

INSTRUMENT(S):

ORGANISM(S): Mus Musculus (mouse)

TISSUE(S): Heart

SUBMITTER: Majid Ghassemian

LAB HEAD: Stephan Lange

PROVIDER: PXD058697 | Pride | 2025-05-07

REPOSITORIES: Pride

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Combined Loss of Obsc and Obsl1 in Murine Hearts Results in Diastolic Dysfunction, Altered Metabolism, and Deregulated Mitophagy.

Fujita Kyohei K Desmond Patrick P Blondelle Jordan J Soták Matúš M Rajan Meenu Rohini MR Clark Madison M Estève Éric É Chan Yunghang Y Gu Yusu Y Actis Dato Virginia V Marrocco Valeria V Dalton Nancy D ND Ghassemian Majid M Do Aryanne A Klos Matthew M Peterson Kirk L KL Sheikh Farah F Cho Yoshitake Y Börgeson Emma E Lange Stephan S

Circulation. Heart failure 20250311 4

<h4>Background</h4>Muscle proteins of the obscurin protein family play important roles in sarcomere organization and sarcoplasmic reticulum and T-tubule architecture and function. However, their precise molecular functions and redundancies between protein family members as well as their involvement in cardiac diseases remain to be fully understood.<h4>Methods</h4>To investigate the functional roles of Obsc (obscurin) and its close homolog Obsl1 (obscurin-like 1) in the heart, we generated and an ...[more]

PMID: 40066567

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Project description:Obscurins are giant, modular, cytoskeletal proteins that regulate striated muscle structure and function. Immunoglobulin domains 58/59 (Ig58/59) of obscurin interact with diverse proteins including titin variants and phospholamban. Mutations within Ig58/59 that alter these binding interactions have been linked to the development of myopathy in humans, underscoring the pathophysiological significance of this module. We previously generated a constitutive deletion mouse model, Obscn-ΔIg58/59, that expresses obscurin lacking Ig58/59 and comprehensively characterized the effects of this deletion on cardiac morphology and function through aging. Our findings demonstrated that Obscn-ΔIg58/59 male animals develop severe arrhythmia characterized by spontaneous atrial fibrillation/junctional escape by 6-months of age, with atrial enlargement and ventricular remodeling manifesting by 12-months. Herein, we aim to mechanistically evaluate the impact of the Ig58/59 deletion in atria at the cellular level and determine the molecular basis for atrial enlargement and arrhythmia due to the physiological process of aging. Ultrastructural evaluation of sedentary aging male Obscn-ΔIg58/59 atria revealed prominent Z-disk streaming and misalignment. More importantly, Obscn-ΔIg58/59 atrial cardiomyocytes exhibited increased Ca2+ spark frequency and age-specific alterations in Ca2+ cycling kinetics and sarcoplasmic reticulum Ca2+ content that preceded those observed in Obscn-ΔIg58/59 ventricular cardiomyocytes. Proteomic and phospho-proteomic analyses revealed extensive and novel alterations in the expression and phosphorylation profile of major cytoskeletal proteins, Ca2+ regulators, and Z-disk associated protein complexes in Obscn-ΔIg58/59 atria through aging that likely underlie the observed atrial pathologies. These studies are the first to evaluate the role of obscurin in atria and to reveal chamber-specific molecular alterations due to Ig58/59 deletion. Moreover, our findings provide new molecular insights into a genetic model of spontaneous atrial fibrillation and remodeling where atrial dysfunction precedes ventricular maladaptation.

Project description:Cardiac sarcoplasmic reticulum (SR) plays a central role in cellular Ca homeostasis, as does endoplasmic reticulum (ER) in the nonmuscle cell. The importance of SR Ca-handling function has led to detailed understanding of Ca-release and re-uptake protein complexes, but relatively little information regarding other known ER functions. We isolated cardiac membranes based upon their ability to efficiently accumulate Ca by the SR,ER-ATPase (SERCA-positive membranes), a process known to produce two characteristic SR membrane fractions: those enriched in known junctional SR markers, and those enriched in proteins originating from classic ER subdomains. Using standard proteomic techniques, we determined the SR proteins in three membrane preparations: 1) the highest-density SERCA-positive microsomes (HighSR); 2) the slightly less dense SERCA-positive microsomes that are constrained by a ryanodine-dependent leak (MedSR); and the original crude cardiac microsomes. Only a third of all crude microsomal proteins in heart were enriched in SERCA-positive membranes at least two fold. This protocol completely excluded known contractile, mitochondrial, sarcolemmal, and other major microsomal proteins, producing a far cleaner preparation of SR. Each SERCA-positive protein was distributed between HighSR and MedSR membrane vesicles in accordance with relative densities of SERCA2a versus ryanodine receptor. The distributions reinforced past findings on the distributions for several of the major known proteins. When combined with values for relative abundance, and enrichment over crude membranes, our protocol conveniently and effectively exposed multiple domains of the cardiac ER/SR. Most of the highly enriched and abundant cardiac SR/ER proteins represent proteins that have received little research attention. The data generate a useful quantitative analysis of intracellular membrane structure and function within cardiomyocytes, and may provide a reliable way of gauging broader changes in cardiac SR.

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