Dataset Information

Cronos Titin Is Expressed in Human Cardiomyocytes and Necessary for Normal Sarcomere Function.

ABSTRACT: BACKGROUND:The giant sarcomere protein titin is important in both heart health and disease. Mutations in the gene encoding for titin (TTN) are the leading known cause of familial dilated cardiomyopathy. The uneven distribution of these mutations within TTN motivated us to seek a more complete understanding of this gene and the isoforms it encodes in cardiomyocyte (CM) sarcomere formation and function. METHODS:To investigate the function of titin in human CMs, we used CRISPR/Cas9 to generate homozygous truncations in the Z disk (TTN-Z-/-) and A-band (TTN-A-/-) regions of the TTN gene in human induced pluripotent stem cells. The resulting CMs were characterized with immunostaining, engineered heart tissue mechanical measurements, and single-cell force and calcium measurements. RESULTS:After differentiation, we were surprised to find that despite the more upstream mutation, TTN-Z-/--CMs had sarcomeres and visibly contracted, whereas TTN-A-/--CMs did not. We hypothesized that sarcomere formation was caused by the expression of a recently discovered isoform of titin, Cronos, which initiates downstream of the truncation in TTN-Z-/--CMs. Using a custom Cronos antibody, we demonstrate that this isoform is expressed and integrated into myofibrils in human CMs. TTN-Z-/--CMs exclusively express Cronos titin, but these cells produce lower contractile force and have perturbed myofibril bundling compared with controls expressing both full-length and Cronos titin. Cronos titin is highly expressed in human fetal cardiac tissue, and when knocked out in human induced pluripotent stem cell derived CMs, these cells exhibit reduced contractile force and myofibrillar disarray despite the presence of full-length titin. CONCLUSIONS:We demonstrate that Cronos titin is expressed in developing human CMs and is able to support partial sarcomere formation in the absence of full-length titin. Furthermore, Cronos titin is necessary for proper sarcomere function in human induced pluripotent stem cell derived CMs. Additional investigation is necessary to understand the molecular mechanisms of this novel isoform and how it contributes to human cardiac disease.

SUBMITTER: Zaunbrecher RJ

PROVIDER: S-EPMC6911360 | biostudies-literature | 2019 Nov

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Cronos Titin Is Expressed in Human Cardiomyocytes and Necessary for Normal Sarcomere Function.

Zaunbrecher Rebecca J RJ Abel Ashley N AN Beussman Kevin K Leonard Andrea A von Frieling-Salewsky Marion M Fields Paul A PA Pabon Lil L Reinecke Hans H Yang Xiulan X Macadangdang Jesse J Kim Deok-Ho DH Linke Wolfgang A WA Sniadecki Nathan J NJ Regnier Michael M Murry Charles E CE

Circulation 20191007 20

<h4>Background</h4>The giant sarcomere protein titin is important in both heart health and disease. Mutations in the gene encoding for titin (<i>TTN</i>) are the leading known cause of familial dilated cardiomyopathy. The uneven distribution of these mutations within <i>TTN</i> motivated us to seek a more complete understanding of this gene and the isoforms it encodes in cardiomyocyte (CM) sarcomere formation and function.<h4>Methods</h4>To investigate the function of titin in human CMs, we used ...[more]

PMID: 31587567

Similar Datasets

Project description:Myopathies encompass a wide variety of acquired and hereditary disorders. The pathomechanisms include structural and functional changes affecting, e.g., myofiber metabolism and contractile properties. In this study, we observed increased passive tension (PT) of skinned myofibers from patients with myofibrillar myopathy (MFM) caused by FLNC mutations (MFM-filaminopathy) and limb-girdle muscular dystrophy type-2A due to CAPN3 mutations (LGMD2A), compared to healthy control myofibers. Because the giant protein titin determines myofiber PT, we measured its molecular size and the titin-to-myosin ratio, but found no differences between myopathies and controls. All-titin phosphorylation and site-specific phosphorylation in the PEVK region were reduced in myopathy, which would be predicted to lower PT. Electron microscopy revealed extensive ultrastructural changes in myofibers of various hereditary myopathies and also suggested massive binding of proteins to the sarcomeric I-band region, presumably heat shock proteins (HSPs), which can translocate to elastic titin under stress conditions. Correlative immunofluorescence and immunoelectron microscopy showed that two small HSPs (HSP27 and αB-crystallin) and the ATP-dependent chaperone HSP90 translocated to the titin springs in myopathy. The small HSPs, but not HSP90, were upregulated in myopathic versus control muscles. The titin-binding pattern of chaperones was regularly observed in Duchenne muscular dystrophy (DMD), LGMD2A, MFM-filaminopathy, MFM-myotilinopathy, titinopathy, and inclusion body myopathy due to mutations in valosin-containing protein, but not in acquired sporadic inclusion body myositis. The three HSPs also associated with elastic titin in mouse models of DMD and MFM-filaminopathy. Mechanical measurements on skinned human myofibers incubated with exogenous small HSPs suggested that the elevated PT seen in myopathy is caused, in part, by chaperone-binding to the titin springs. Whereas this interaction may be protective in that it prevents sarcomeric protein aggregation, it also has detrimental effects on sarcomere function. Thus, we identified a novel pathological phenomenon common to many hereditary muscle disorders, which involves sarcomeric alterations.

Dataset Information

Cronos Titin Is Expressed in Human Cardiomyocytes and Necessary for Normal Sarcomere Function.

Publications

Cronos Titin Is Expressed in Human Cardiomyocytes and Necessary for Normal Sarcomere Function.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets