The vertebrate muscle Z-disc: sarcomere anchor for structure and signalling

The Z-disc, appearing as a fine dense line forming sarcomere boundaries in striated muscles, when studied in detail reveals crosslinked filament arrays that transmit tension and house myriads of proteins with diverse functions. At the Z-disc the barbed ends of the antiparallel actin filaments from adjoining sarcomeres interdigitate and are crosslinked primarily by layers of α-actinin. The Z-disc is therefore the site of polarity reversal of the actin filaments, as needed to interact with the bipolar myosin filaments in successive sarcomeres. The layers of α-actinin determine the Z-disc width: fast fibres have narrow (~30–50 nm) Z-discs and slow and cardiac fibres have wide (~100 nm) Z-discs. Comprehensive reviews on the roles of the numerous proteins located at the Z-disc in signalling and disease have been published; the aim here is different, namely to review the advances in structural aspects of the Z-disc

Analysing the lattice transition of thin filaments in striated muscle

Thin filaments, through interaction with thick filaments, form the contractile apparatus of striated muscle. Therefore, the length and arrangement of the thin filaments are of key importance to the function of the muscle. The thin filaments from adjacent sarcomeres are anchored at the Z-disc. In 1968 Pringle predicted that thin filament are organised in the Z-disc in a rhomboid lattice rather than a square lattice. Previous experimental evidence has been insufficient to verify Pringle’s suggestion. In the A-band the thin filaments interdigitate with the thick filaments on a hexagonal lattice, hence from the Z-disc to the A-band, there is a transition of the lattice from square to hexagonal. In this project, I have firstly used Fourier analysis and electron tomography to investigate the thin filament lattice in the Z-disc. I have used electron tomography to determine how the lattice transition occurs between the Z-disc and the A-band. Electron tomography of these samples also allowed me to determine the lengths of thin filaments, showing unequivocally that they are of variable lengths in cardiac muscle

The oxoglutarate dehydrogenase complex is involved in myofibril growth and Z-disc assembly in Drosophila

Myofibrils are long intracellular cables specific to muscles, composed mainly of actin and myosin filaments. The actin and myosin filaments are organized into repeated units called sarcomeres, which form the myofibrils. Muscle contraction is achieved by the simultaneous shortening of sarcomeres, which requires all sarcomeres to be the same size. Muscles have a variety of ways to ensure sarcomere homogeneity. We have previously shown that the controlled oligomerization of Zasp proteins sets the diameter of the myofibril. Here, we looked for Zasp-binding proteins at the Z-disc to identify additional proteins coordinating myofibril growth and assembly. We found that the E1 subunit of the oxoglutarate dehydrogenase complex localizes to both the Z-disc and the mitochondria, and is recruited to the Z-disc by Zasp52. The three subunits of the oxoglutarate dehydrogenase complex are required for myofibril formation. Using super-resolution microscopy, we revealed the overall organization of the complex at the Z-disc. Metabolomics identified an amino acid imbalance affecting protein synthesis as a possible cause of myofibril defects, which is supported by OGDH-dependent localization of ribosomes at the Z-disc

The Sarcomeric Z-Disc and Z-Discopathies

The sarcomeric Z-disc defines the lateral borders of the sarcomere and has primarily been seen as a structure important for mechanical stability. This view has changed dramatically within the last one or two decades. A multitude of novel Z-disc proteins and their interacting partners have been identified, which has led to the identification of additional functions and which have now been assigned to this structure. This includes its importance for intracellular signalling, for mechanosensation and mechanotransduction in particular, an emerging importance for protein turnover and autophagy, as well as its molecular links to the t-tubular system and the sarcoplasmic reticulum. Moreover, the discovery of mutations in a wide variety of Z-disc proteins, which lead to perturbations of several of the above-mentioned systems, gives rise to a diverse group of diseases which can be termed Z-discopathies. This paper provides a brief overview of these novel aspects as well as points to future research directions

Arrangement and structure of α-actinins in striated muscle

The smallest contractile unit within striated muscle cells are called sarcomeres. The boundary regions between sarcomeres are called Z-discs, which contain over 30 different proteins, organised within a narrow ~100 nm wide structure. Standard fluorescence microscopy approaches do not reveal the arrangement of Z-disc proteins, as the width of the Z-disc is below the resolution limit (~250 nm). The arrangement of the actin filaments and the cross-linking proteins α-actinin in the Z-discs are well characterised by electron microscopy (EM) studies, however other Z-disc proteins are not. With the development of super-resolution fluorescence microscopy techniques, it is now possible to obtain Z-disc protein localisation information. Here, dSTORM (direct Stochastic Optical Reconstruction Microscopy) was used, to investigate the arrangement of α–actinins in Z-discs of cardiomyocytes, and then the arrangement of the N-terminal ends of the giant protein titin in the Z-discs. Affimers were generated to bind α–actinin 2 and the N-terminal titin domains (Z1/Z2), to use as binders in dSTORM. Affimers are small (~12 kDa) non-antibody binding proteins, about 1/10th the size of antibodies, that can be selected to bind to a specific protein. The localisation data of dSTORM using the Affimer binders showed the same regular arrangement of α-actinins observed in EM studies. The use of dSTORM with Affimers also suggests the titin Z1/Z2 domains do not only localise at the edges of the Z-discs but arranged throughout the Z-disc with regular spacing (~25 nm) in the transverse plane of the Z-discs. Also, three mutations located in the actin binding domain of α-actinin 2 associated to hypertrophic cardiomyocytes (G111V, A119T and M228T) were characterised by in vitro co-sedimentation assays with actin. The mutants G111V and A119T did not show a significant difference in binding affinity to actin compared to the wild-type. The co-sedimentation assays did however suggest the mutation M228T significantly increases the binding affinity of α–actinin 2

Evaluation of laser induced sarcomere microdamage: Role of damage extent and location in cardiomyocytes

Whereas it is evident that a well aligned and regular sarcomeric structure in cardiomyocytes is vital for heart function, considerably less is known about the contribution of individual elements to the mechanics of the entire cell. For instance, it is unclear whether altered Z-disc elements are the reason or the outcome of related cardiomyopathies. Therefore, it is crucial to gain more insight into this cellular organization. This study utilizes femtosecond laserbased nanosurgery to better understand sarcomeres and their repair upon damage. We investigated the influence of the extent and the location of the Z-disc damage. A single, three, five or ten Z-disc ablations were performed in neonatal rat cardiomyocytes. We employed image-based analysis using a self-written software together with different already published algorithms. We observed that cardiomyocyte survival associated with the damage extent, but not with the cell area or the total number of Z-discs per cell. The cell survival is independent of the damage position and can be compensated. However, the sarcomere alignment/orientation is changing over time after ablation. The contraction time is also independent of the extent of damage for the tested parameters. Additionally, we observed shortening rates between 6-7% of the initial sarcomere length in laser treated cardiomyocytes. This rate is an important indicator for force generation in myocytes. In conclusion, femtosecond laser-based nanosurgery together with image-based sarcomere tracking is a powerful tool to better understand the Z-disc complex and its force propagation function and role in cellular mechanisms. Copyright

Inhibition of DNAJ-HSP70 interaction improves strength in muscular dystrophy

Dominant mutations in the HSP70 cochaperone DNAJB6 cause a late-onset muscle disease termed limb-girdle muscular dystrophy type D1 (LGMDD1), which is characterized by protein aggregation and vacuolar myopathology. Disease mutations reside within the G/F domain of DNAJB6, but the molecular mechanisms underlying dysfunction are not well understood. Using yeast, cell culture, and mouse models of LGMDD1, we found that the toxicity associated with disease-associated DNAJB6 required its interaction with HSP70 and that abrogating this interaction genetically or with small molecules was protective. In skeletal muscle, DNAJB6 localizes to the Z-disc with HSP70. Whereas HSP70 normally diffused rapidly between the Z-disc and sarcoplasm, the rate of diffusion of HSP70 in LGMDD1 mouse muscle was diminished, probably because it had an unusual affinity for the Z-disc and mutant DNAJB6. Treating LGMDD1 mice with a small-molecule inhibitor of the DNAJ-HSP70 complex remobilized HSP70, improved strength, and corrected myopathology. These data support a model in which LGMDD1 mutations in DNAJB6 are a gain-of-function disease that is, counterintuitively, mediated via HSP70 binding. Thus, therapeutic approaches targeting HSP70-DNAJB6 may be effective in treating this inherited muscular dystrophy

The cardiac-restricted protein ADP-ribosylhydrolase-like 1 is essential for heart chamber outgrowth and acts on muscle actin filament assembly

AbstractAdprhl1, a member of the ADP-ribosylhydrolase protein family, is expressed exclusively in the developing heart of all vertebrates. In the amphibian Xenopus laevis, distribution of its mRNA is biased towards actively growing chamber myocardium. Morpholino oligonucleotide-mediated knockdown of all Adprhl1 variants inhibits striated myofibril assembly and prevents outgrowth of the ventricle. The resulting ventricles retain normal electrical conduction and express markers of chamber muscle differentiation but are functionally inert. Using a cardiac-specific Gal4 binary expression system, we show that the abundance of Adprhl1 protein in tadpole hearts is tightly controlled through a negative regulatory mechanism targeting the 5′-coding sequence of Xenopus adprhl1. Over-expression of full length (40kDa) Adprhl1 variants modified to escape such repression, also disrupts cardiac myofibrillogenesis. Disarrayed myofibrils persist that show extensive branching, with sarcomere division occurring at the actin-Z-disc boundary. Ultimately, Adprhl1-positive cells contain thin actin threads, connected to numerous circular branch points. Recombinant Adprhl1 can localize to stripes adjacent to the Z-disc, suggesting a direct role for Adprhl1 in modifying Z-disc and actin dynamics as heart chambers grow. Modelling the structure of Adprhl1 suggests this cardiac-specific protein is a pseudoenzyme, lacking key residues necessary for ADP-ribosylhydrolase catalytic activity