Affimers targeting proteins in the cardiomyocyte Z-disc: Novel tools that improve imaging of heart tissue

Abstract

Dilated Cardiomyopathy is a common form of heart failure. Determining how this disease affects the structure and organization of cardiomyocytes in the human heart is important in understanding how the heart becomes less effective at contraction. Here we isolated and characterised Affimers (small non-antibody binding proteins) to Z-disc proteins ACTN2 (α-actinin-2), ZASP (also known as LIM domain binding protein 3 or LDB3) and the N-terminal region of the giant protein titin (TTN Z1-Z2). These proteins are known to localise in both the sarcomere Z-discs and the transitional junctions, found close to the intercalated discs that connect adjacent cardiomyocytes. We use cryosections of left ventricles from two patients diagnosed with end-stage Dilated Cardiomyopathy who underwent Orthotopic Heart Transplantation and were whole genome sequenced. We describe how Affimers substantially improve the resolution achieved by confocal and STED microscopy compared to conventional antibodies. We quantified the expression of ACTN2, ZASP and TTN proteins in two patients with dilated cardiomyopathy and compared them with a sex- and age-matched healthy donor. The small size of the Affimer reagents, combined with a small linkage error (the distance from the epitope to the dye label covalently bound to the Affimer) revealed new structural details in Z-discs and intercalated discs in the failing samples. Affimers are thus useful for analysis of changes to cardiomyocyte structure and organisation in diseased hearts.

Segmenting cardiac muscle Z-disks with deep neural networks

“…Dataset. The images used in this work were generated from staining cryo-sections of human cardiac tissue from normal donors as described in Parker, et al 17 The tissue samples were obtained from the Sydney heart bank with consent obtained from St Vincent's Hospital, Darlinghurst (HREC #H91/048/1a) and the University of Sydney (HREC #2016/923), prepared according to a standardised protocol 17 and stained using a dye-labelled Affimer to highlight the Z-disk proteins α-actinin-2, 19 Z1Z2 or to ZASP, 17 followed by mouse monoclonal antibody to human desmoglein-2 (CCSTEM28; eBiosciences from Thermo Scientific, 1:200). Sections were imaged using a Zeiss LSM 880 Airyscan confocal, using a x40 objective (NA 1.4) in Airyscan mode.…”

“…To test generalisability, an additional test set was created containing 10 images of heart tissue with a titin mutation,. 17 Methods. The study evaluates the performance of established deep segmentation networks for segmenting Z-disks in Airyscan confocal microscopy images.…”

“…To verify generalisability, we tested all networks on an additional dataset containing images of patient with titin mutation. 17 All networks were trained on a workstation with a 12th Gen Intel(R) Core(TM) i7-12700H CPU and NVIDIA T600 GPU. Metrics used to evaluate the performance of all segmentation models included the Dice score and pixel-wise recall (sensitivity).…”

“…Recently, novel binders termed Affimers were isolated for specific Z-disk proteins including alpha-actinin, ZASP and titin. 17 In combination with super-resolution imaging techniques such as Airyscan confocal and STED microscopy, Affimers showed improved resolution and revealed new structural details in the Z-disks, 17 enabling investigation of cardiomyocyte structure and identification of disease phenotypes. Segmentation of Z-disks.…”

Biomarkers of heart failure: advances in omics studies

“…29 Conversely, ACTN2 encodes cardiac a-actinin-2, which functions as the structural backbone of the Z-disc, and so variants in this loci may make individuals susceptible to HF. 30 Further characterization and precise positioning of the ACTN2 locus revealed potential causal variants in cardiac-specific regulatory regions during cardiomyocyte differentiation. 21,31 There is definitive evidence to support the relationship between ACTN2 and intrinsic cardiomyopathy.…”