Characterization of the roles of obscurin in intercellular adhesion and development of hypertrophic cardiomyopathy

Abstract

Obscurins comprise a family of giant (~870-600kDa) and small (~250-55kDa) proteins that play important roles in myofibrillogenesis and cytoskeletal organization, and are implicated in hypertrophic cardiomyopathy (HCM) and tumorigenesis. Giant obscurins are composed of tandem structural and signaling motifs, including two serine/threonine kinase domains, SK1 and SK2, present at the COOH-terminus of giant obscurin-B. Through biochemical and cellular approaches, my studies show for the first time that both SK1 and SK2 possess kinase activities, with SK2 phosphorylating the cytoplasmic domain of N-cadherin and SK1 autophosphorylating. In addition to SK2 interacting with an adhesion protein, SK1 interacts with the extracellular domain of the &beta₁-subunit of the Na<super>+</super>/K<super>+</super>-ATPase, another protein residing in adherens junctions. Immunostaining of non-permeabilized myofibers and cardiocytes revealed that some obscurin-kinase isoforms localize extracellularly, and treatment of heart lysates with Peptide:N-Glycosidase F revealed that at least one obscurin kinase isoform is glycosylated. Collectively, these studies demonstrate that the obscurin-kinase domains are enzymatically active with some portions localized extracellularly. Sequence analysis of a patient with HCM uncovered a missense mutation, R4344Q, present in the immunoglobulin (Ig) domain 58 of giant obscurins. This mutation results in diminished binding between domains Ig58/59 of obscurins and Ig9/10 of titin located at the periphery of Z-disks. To examine the physiological significance of the R4344Q missense mutation and of the Ig58/59 module, our laboratory generated two animal models: a knock-in model that expresses full-length obscurins carrying the R4344Q mutation, and a partial knock-out model that lacks Ig58/59. While both mutant and truncated obscurins are properly expressed and incorporated into cardiac sarcomeres, homozygous partial knock-out mice develop overt cardiac hypertrophy by 12 months of age, as measured by echocardiography; notably, hypertrophy was exacerbated in the female homozygous animals. Importantly, transverse aortic constriction of ~2 month-old knock-in and partial knock-out mice led to severe cardiac hypertrophy and reduced ejection fractions within 4-8 weeks post-surgery, while the wild-type animals exhibited hypertrophy without functional defects. Our studies therefore reveal that animals with mutant or truncated giant obscurins are more susceptible to developing HCM as a result of aging or stress.