The Z-disc acts as a protein-rich structure to tether thin filament in the contractile units, the sarcomeres, of striated muscle cells. Proteins found in the Z-disc are integral for maintaining the architecture of the sarcomere. They also enable it to function as a (bio-mechanical) signalling hub. Numerous proteins interact in the Z-disc to facilitate force transduction and intracellular signalling in both cardiac and skeletal muscle. This review will focus on six key Z-disc proteins: α-actinin 2, filamin C, myopalladin, myotilin, telethonin and Z-disc alternatively spliced PDZ-motif (ZASP), which have all been linked to myopathies and cardiomyopathies. We will summarise pathogenic variants identified in the six genes coding for these proteins and look at their involvement in myopathy and cardiomyopathy. Listing the Minor Allele Frequency (MAF) of these variants in the Genome Aggregation Database (GnomAD) version 3.1 will help to critically re-evaluate pathogenicity based on variant frequency in normal population cohorts.

Titin truncating variants are a well-established cause of cardiomyopathy; however, the role of titin missense variants is less well understood. Here we describe the generation of a mouse model to investigate the underlying disease mechanism of a previously reported titin A178D missense variant identified in a family with non-compaction and dilated cardiomyopathy. Heterozygous and homozygous mice carrying the titin A178D missense variant were characterised in vivo by echocardiography. Heterozygous mice had no detectable phenotype at any time point investigated (up to 1 year). By contrast, homozygous mice developed dilated cardiomyopathy from 3 months. Chronic adrenergic stimulation aggravated the phenotype. Targeted transcript profiling revealed induction of the foetal gene programme and hypertrophic signalling pathways in homozygous mice, and these were confirmed at the protein level. Unsupervised proteomics identified downregulation of telethonin and four-and-a-half LIM domain 2, as well as the upregulation of heat shock proteins and myeloid leukaemia factor 1. Loss of telethonin from the cardiac Z-disc was accompanied by proteasomal degradation; however, unfolded telethonin accumulated in the cytoplasm, leading to a proteo-toxic response in the mice.We show that the titin A178D missense variant is pathogenic in homozygous mice, resulting in cardiomyopathy. We also provide evidence of the disease mechanism: because the titin A178D variant abolishes binding of telethonin, this leads to its abnormal cytoplasmic accumulation. Subsequent degradation of telethonin by the proteasome results in proteasomal overload, and activation of a proteo-toxic response. The latter appears to be a driving factor for the cardiomyopathy observed in the mouse model.

Titin, the largest protein known, has attracted a lot of interest in the cardiovascular field in recent years, since the discovery that truncating variants in titin are commonly found in patients with dilated cardiomyopathy. This review will discuss the contribution of variants in titin to inherited cardiac conditions (cardiomyopathies) and how model systems, such as animals and cellular systems, can help to provide insights into underlying disease mechanisms. It will also give an outlook onto exciting technological developments, such as in the field of CRISPR, which may facilitate future research on titin variants and their contributions to cardiomyopathies.

Pathogenic variants in ACTN2, coding for alpha-actinin 2, are known to be rare causes of Hypertrophic Cardiomyopathy. However, little is known about the underlying disease mechanisms. Adult heterozygous mice carrying the Actn2 p.Met228Thr variant were phenotyped by echocardiography. For homozygous mice, viable E15.5 embryonic hearts were analysed by High Resolution Episcopic Microscopy and wholemount staining, complemented by unbiased proteomics, qPCR and Western blotting. Heterozygous Actn2 p.Met228Thr mice have no overt phenotype. Only mature males show molecular parameters indicative of cardiomyopathy. By contrast, the variant is embryonically lethal in the homozygous setting and E15.5 hearts show multiple morphological abnormalities. Molecular analyses, including unbiased proteomics, identified quantitative abnormalities in sarcomeric parameters, cell-cycle defects and mitochondrial dysfunction. The mutant alpha-actinin protein is found to be destabilised, associated with increased activity of the ubiquitin-proteasomal system. This missense variant in alpha-actinin renders the protein less stable. In response, the ubiquitin-proteasomal system is activated; a mechanism that has been implicated in cardiomyopathies previously. In parallel, a lack of functional alpha-actinin is thought to cause energetic defects through mitochondrial dysfunction. This seems, together with cell-cycle defects, the likely cause of the death of the embryos. The defects also have wide-ranging morphological consequences.

Mechanical properties are cues for many biological processes in health or disease. Likewise, in the heart it is becoming clearer that mechanical signals are critically involved in the disease progression. Cardiomyocytes sense the mechanical properties of their environment at costameres through integrins and associated proteins, including the mechanosensitive protein talin as an integral component. Our previous work indicated different modes of talin tension, depending on the extracellular matrix stiffness. Here, we wanted to study how this leads to downstream mechanotransduction changes, further influencing the cardiomyocyte phenotype. Combining immunoprecipitations and Fluorescence Recovery after Photobleaching (FRAP) experiments, we identify that the talin interacting proteins DLC1, RIAM and paxillin each preferentially bind to talin at specific extracellular matrix stiffness and this interaction is preserved even in absence of tension. This demonstrates a mechanical memory, which we confirm further in vivo in mouse hearts. The mechanical memory is regulated through adhesion related kinase pathways. Optogenetic experiments using the LOVTRAP systems confirm direct competition between the individual proteins, which again is altered through phosphorylation. DLC1 regulates RhoA activity in a stiffness dependent way and both loss and overexpression of DLC1 results in myofibrillar disarray. Together the study demonstrates a mechanism of imprinting mechanical information into the talin-interactome to finetune RhoA activity, with impacts on cardiac health and disease.

No abstract

Funding Acknowledgements Type of funding sources: Foundation. Main funding source(s): Medical Research Council (MR/V009540/1), Wellcome Trust (201543/B/16/Z) British Heart Foundation (FS/12/40/29712). Hypertrophic cardiomyopathy (HCM) is an inherited cardiac condition associated with diastolic dysfunction and sudden cardiac death. Disease genes for HCM are traditionally coding for proteins involved in force generation. More recently, it has emerged that variants in genes coding for proteins involved in biomechanical stress-signalling can also cause HCM. One such protein is filamin C, with proposed mechano-sensing functions in the heart. Within the protein, the immunoglobulin-like domain 20 (Ig20) may play a crucial role in mediating binding to muscle specific ligands. While the mechano-sensing functions of filamin C have been investigated well in skeletal muscle, the underlying cardiac disease mechanisms are not completely understood. Aim This work attempts to provide insights into the role of filamin C in cardiac mechano-sensing and dissect disease pathways leading to HCM in the presence of the FLNC variants in Ig20. Methods Using mass spectrometry, we aimed to provide a detailed analysis of the proteome of mice carrying the filamin C variant, using ventricular tissue samples from 14wk old homozygous mice. Samples were subject to molecular biology technical and underwent subcellular fractionation (n = 6 per genotype) and were investigated by label-free mass spectrometry. Results Utilising whole genome sequencing, a heterozygous FLNC missense variant in Ig20 was identified in a three-generation family affected by HCM. Mice carrying this variant recapitulate molecular features of HCM in the homozygous setting. Three proteins (FLNC, MYH7, MYOT) were found to be upregulated in the myofilament-enriched fraction. Up-regulations of key proteins were found to relocalise towards load-baring sites. Conclusion Our data indicate that changes in filamin C and its binding partners expression and localisation are involved in the pathogenesis of HCM in this mouse model.