Добірка наукової літератури з теми "Z-disc proteins"

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.

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.

Intense exercise has been shown to produce pathological changes in normal skeletal muscle ultrastructure. Eccentric exercise (muscle lengthening during active tension development) in particular has been shown to cause the most severe muscle damage, and studies of both human and animal tissue following eccentric exercise have documented disruption to the contractile apparatus. The disruption originates at the Z-disc, which appears broadened, smeared, or totally disrupted, with Z-discs of adjacent myofibrils out of register and running a “zig-zag” course transversely across the fiber. This condition is known as Z-line streaming. Several researchers have implicated the disruption of the intermediate filament system in the etiology of exercise-induced Z-line streaming, as these filaments are believed to link adjacent myofibrils at the level of the Z-disc. The intermediate filaments are composed predominantly of the proteins desmin and vimentin. This study utilized immunoelectron microscopic localization of desmin in order to elucidate the role of the intermediate filament system in Zline streaming of eccentrically-exercised skeletal muscle.

We define here a previously unrecognized structural element close to the heart muscle plasma membrane at the intercalated disc where the myofibrils lead into the adherens junction. At this location, the plasma membrane is extensively folded. Immunofluorescence and immunogold electron microscopy reveal a spectrin-rich domain at the apex of the folds. These domains occur at the axial level of what would be the final Z-disc of the terminal sarcomere in the myofibril, although there is no Z-disc-like structure there. However, a sharp transitional boundary lies between the myofibrillar I-band and intercalated disc thin filaments, identifiable by the presence of Z-disc proteins, α-actinin, and N-terminal titin. This allows for the usual elastic positioning of the A-band in the final sarcomere, whereas the transduction of the contractile force normally associated with the Z-disc is transferred to the adherens junctions at the plasma membrane. The axial conjunction of the transitional junction with the spectrin-rich domains suggests a mechanism for direct communication between intercalated disc and contractile apparatus. In particular, it provides a means for sarcomeres to be added to the ends of the cells during growth. This is of particular relevance to understanding myocyte elongation in dilated cardiomyopathy.

The main purpose of the present study was to investigate the role(s) of 25-kDa heat shock protein (HSP25) in the regulation and integration of myofibrillar Z-disc structure during down- or upregulation of the size in rat soleus muscle fibers. Hindlimb unloading by tail suspension was performed in adult rats for 7 days, and reloading was allowed for 5 days after the termination of suspension. Interaction of HSP25 and Z-disc proteins, phosphorylation status, distribution, and complex formation of HSP25 were investigated. Non- and single-phosphorylated HSP25s were generally expressed in the cytoplasmic fraction of normal muscle. The level of total HSP25, as well as the phosphorylation ratio, did not change significantly in response to atrophy. Increased expressions of HSP25, phosphorylated at serine 15 (p-Ser15) and dual-phosphorylated form, were noted, when atrophied muscles were reloaded. Myofibrillar HSP25 was also noted in reloaded muscle. Histochemical analysis further indicated the localization of p-Ser15 in the regions with disorganization of Z-disc structure in reloaded muscle fibers. HSP25 formed a large molecular complex in the cytoplasmic fraction of normal muscle, whereas dissociation of free HSP25 with Ser15 phosphorylation was noted in reloaded muscle. The interaction of p-Ser15 with desmin and actinin was detected in Z-discs by proximity ligation assay. Strong interaction between p-Ser15 and desmin, but not actinin, was noted in the disorganized areas. These results indicated that HSP25 contributed to the desmin cytoskeletal organization following the phosphorylation at Ser15 during reloading and regrowing of soleus muscle.

The Z-disc of striated muscle cells is a highly specialized three-dimensional structure which delineates the boundary of the individual sarcomeres. It accomplishes a unique role by anchoring actin filaments and acts as a molecular trigger for contraction. Beyond a well-defined structural role, in recent years it is emerging the hypothesis that Z-disc may be directly involved in the perception and transmission of muscular stress signals. To achieve these complex functions, many Z-disc proteins are involved in multiple protein interactions. The importance of these interactions is indicated by the fact that mutations in several Z-disc proteins can result in muscular dystrophies and/or cardiomyopathies in human and mice. The knowledge of Z-disc interactome and its regulation would improve by far the comprehension of the Z-disc biology and the onset of muscular disorders. The main goal of my project was to understand the complex network of protein-protein interactions occurring at the Z-disc of skeletal and cardiac muscle. In particular, my work was focused on two groups of Z-disc proteins: the FATZ and myotilin protein families on one hand, and some proteins belonging to the enigma family on the other hand. This work led to the identification of a specific interaction between the PDZ domains of enigma family members and the C-terminal five amino acids of the FATZ and myotilin families. The work of this thesis is part of a wider project involving the groups of Dr. G. Faulkner at ICGEB, Trieste, and Prof. O. Carpen at University of Turku, Finland. Together with our collaborators we noted that the C-terminal five amino acids of FATZ-1 (ETEEL), FATZ-2 (ESEDL), FATZ-3 (ESEEL), myotilin (ESEEL), palladin (ESEDL) and myopalladin (ESDEL) are highly similar. Searches in protein sequence database revealed that this E-[S/T]-[D/E]-[D/E]-L motif is restricted in Vertebrates to the FATZ and myotilin families of proteins, and it is evolutionary conserved from zebrafish to humans, indicating its importance for their biological function. The ELM program (a source for predicting functional sites in eukaryotic proteins) predicted that the terminal four amino acids of the FATZ family, myotilin, palladin and myopalladin constitute a binding motif for class III PDZ domain proteins (X-[D/E]-X-[V/I/L]). The first object of my work was to determine if the proteins with this new type of class III PDZ binding motif at their C-terminal could effectively bind PDZ domains. We knew from the literature that ZASP binds to all the three members of the FATZ family by means of its N-terminal PDZ domain, and that the C-terminal region of myotilin interacts with ZASP. In addition to ZASP, other two members of the enigma family of PDZ proteins, ALP and CLP-36, were included in this study. Both the full-length and the truncated (lacking the last five amino acids) version of the FATZ and myotilin families were produced as native proteins and tested for PDZ binding using the AlphaScreen (Amplified Luminescence Proximity Homogeneous Assay) technique. Biotinylated phosphorylated and non-phosphorylated peptides corresponding to the C-terminal five amino acids of the FATZ family, myotilin, palladin and myopalladin were also used in AlphaScreen interaction experiments, as well as a control peptide having E instead of L as its last amino acid (ESEEE). The results presented in this thesis show that the final five amino acids of the FATZ and myotilin families of proteins are responsible for the binding to the PDZ domains of ZASP, ALP and CLP-36, and that the nature of the last amino acid of the motif is crucial for the interaction. We also show that phosphorylation of the ligand sequence modulates the ability of the peptides to bind to the PDZ domains of the enigma family. ?-actinin-2 was included in this study as its C-terminus (GESDL) is classified as a class I PDZ binding motif that is able to bind to ZASP and ALP PDZ domains. AlphaScreen experiments confirm the binding of both the full-length and the C-terminal phosphorylated and non-phosphorylated peptides of ?-actinin-2 to the PDZ domains of ZASP and ALP, and they also reveal an interaction with the PDZ domain of CLP-36. These interactions were verified using another in vitro binding technique, the TranSignal PDZ Domain Array. Based on the results of the PDZ arrays, RIL was found to be another member of the enigma family capable to bind to the E-[S/T]-[D/E]-[D/E]-L motif. Therefore, these final five amino acids can be considered a novel type of class III PDZ binding motif specific for the PDZ domains of enigma proteins. To better quantify the strength of the noted interactions, SPR (Surface Plasmon Resonance) experiments were performed in the laboratory of Dr. A. Baines at University of Kent, UK. The affinities of the interactions between the PDZ domain of ZASP and some of the phosphorylated and non-phosphorylated peptides of the FATZ and myotilin families result to be in the nM range. The SPR results also demonstrate a new interaction between the PDZ domain of ZASP and ANKRD2. This protein is a member of the MARP family and it is thought to be involved in muscle stress response pathways. ANKRD2 localizes both in the sarcomeric I-band and the nucleus, and it is able to bind to several transcription factors, including YB-1, PML and p53. This interaction strengthens the hypothesis that, besides a structural function, Z-disc could have a role in cell signalling. The fact that at the Z-disc many proteins can interact with the same partners, it would be helpful to define the pattern and level of expression of the individual proteins in different muscle tissues. Another aim of my work was to measure the abundance of mRNAs of some Z-disc proteins using the Real-Time PCR technique. Four different muscles from adult mice were considered: tibialis (a fast-twitch skeletal muscle), soleus (a slow-twitch skeletal muscle), gastrocnemius (a skeletal muscle with mixed fibers) and heart (cardiac muscle). The different distribution of the FATZ proteins, myotilin and the alternatively spliced variants of ZASP suggest that, at least in mouse, the interactions between these proteins could be compartmentalized in distinct fiber types.
Il disco-Z del muscolo striato è una struttura molecolare altamente specializzata a livello della quale si instaurano numerose interazioni proteina-proteina. Il disco-Z delinea il confine dei singoli sarcomeri, fornendo un punto di ancoraggio per i filamenti sottili di actina; il loro scorrimento sui filamenti spessi di miosina produce la forza meccanica responsabile della contrazione. Uno dei ruoli chiave del disco-Z, dunque, è quello di trasmettere la tensione generata dalla struttura seriale dei sarcomeri lungo le miofibrille e, di conseguenza, lungo tutto il muscolo. Al di là di un evidente significato strutturale, negli ultimi anni sta diventando sempre più consistente l’ipotesi di un suo coinvolgimento anche nella percezione e nella trasmissione di segnali. L’importanza delle interazioni tra le proteine del disco-Z è indicata dal fatto che mutazioni in molte di queste proteine possono risultare in distrofie muscolari e/o cardiomiopatie sia in uomo sia in topo. Una più ampia conoscenza delle interazioni che si articolano a livello del disco-Z e, in generale, degli eventi che le regolano, aiuterebbe a chiarire la biologia del disco-Z e l’insorgenza di eventuali patologie associate. Il mio progetto di Dottorato è stato incentrato su due gruppi di proteine sarcomeriche e sulle loro interazioni: le proteine delle famiglie FATZ e miotilina da un lato, e alcune proteine appartenenti alla famiglia enigma dall’altro. Questo lavoro ha portato all’identificazione di un’interazione specifica tra i domini PDZ delle proteine della famiglia enigma e gli ultimi cinque residui aminoacidici presenti nelle proteine delle famiglie FATZ e miotilina. Il lavoro di questa tesi fa parte di un progetto più ampio che coinvolge i gruppi coordinati dalla Dr.ssa G. Faulkner dell’ICGEB, Trieste, e il Prof. O. Carpen dell’Università di Turku, Finlandia. Grazie alla loro collaborazione, è stato possibile notare che i cinque residui C-terminali delle proteine FATZ-1 (ETEEL), FATZ-2 (ESEDL), FATZ-3 (ESEEL), miotilina (ESEEL), palladina (ESEDL) e miopalladina (ESDEL) sono molto simili. Una ricerca effettuata in database di sequenze proteiche ha rivelato che questo motivo, E-[S/T]-[D/E]-[D/E]-L, è quasi esclusivamente ristretto nei Vertebrati alle proteine delle famiglie FATZ e miotilina; inoltre, esso sembra essere conservato da zebrafish ad uomo, suggerendo la sua importanza per le proteine che lo contengono. Il programma ELM (che predice siti funzionali in proteine eucariotiche) ha predetto che gli ultimi quattro amino acidi delle proteine FATZ, miotilina, palladina e miopalladina costituiscono un motivo di legame per le proteine con domini PDZ di classe III (X-[D/E]-X-[V/I/L]). Il mio primo obiettivo è stato quello di verificare se le proteine caratterizzate da questo nuovo motivo C-terminale potessero effettivamente legare domini PDZ. E’ noto dalla letteratura che tutti e tre i componenti della famiglia FATZ legano il PDZ di ZASP, e che l’interazione tra ZASP e miotilina è mediata dalla regione C-terminale di quest’ultima. Oltre a ZASP, altri due membri della famiglia enigma, ALP e CLP-36, sono stati inclusi nello studio. Le proteine della famiglia FATZ e miotilina sono state prodotte sia in versione full-length sia priva degli ultimi cinque amino acidi per essere utilizzate in saggi di interazione AlphaScreen (Amplified Luminescence Proximity Homogeneous Assay). Peptidi biotinilati, fosforilati e non, corrispondenti ai cinque amino acidi finali delle FATZ, miotilina, palladina e miopalladina sono stati inoltre impiegati nei saggi AlphaScreen, così come un peptide di controllo avente in ultima posizione un acido glutammico (E) invece che una leucina (L). I risultati riportati in questa tesi dimostrano che gli ultimi cinque amino acidi delle proteine delle famiglie FATZ e miotilina sono responsabili del legame ai domini PDZ di ZASP, ALP e CLP-36, e che la natura dell’ultimo residuo aminoacidico è cruciale per questa interazione. Inoltre, la fosforilazione del residuo di serina o treonina del ligando C-terminale può influenzare il legame dei peptidi nei confronti dei domini PDZ della famiglia enigma. La proteina ?-actinina-2 è stata introdotta nello studio, poiché la sua sequenza C-terminale (GESDL) è classificata come motivo di legame per i domini PDZ di classe I (X-[S/T]-X-[V/I/L]). Gli esperimenti AlphaScreen hanno confermato l’interazione di ?-actinina-2 (sia della forma full-length sia dei peptidi C-terminali, fosforilati e non) con i PDZ di ZASP e ALP, e hanno fatto emergere una nuova interazione con il PDZ di CLP-36. Molte di queste interazioni sono state verificate con un altro metodo di interazione proteina-proteina in vitro, il TranSignal PDZ Domain Array. Sulla base dei risultati di PDZ array è stato possibile identificare un altro membro della famiglia di proteine enigma, RIL, in grado di legare il motivo E-[S/T]-[D/E]-[D/E]-L. Possiamo considerare questi cinque amino acidi C-terminali come un nuovo motivo di legame per le proteine con domini PDZ di classe III, specifico per i domini PDZ delle proteine enigma. Per poter meglio quantificare la forza delle interazioni studiate, alcuni esperimenti di SPR (Surface Plasmon Resonance) sono stati eseguiti nel laboratorio del Dr. A. Baines all’Università di Kent, UK. Le affinità delle interazioni tra il dominio PDZ di ZASP e alcuni dei peptidi fosforilati e non-fosforilati delle famiglie di proteine FATZ e miotilina risultano essere nell’ordine del nM. Gli esperimenti di SPR hanno portato anche all’identificazione di un’interazione tra il PDZ di ZASP e ANKRD2. Si pensa che questa proteina, membro della famiglia MARP, sia coinvolta nelle vie di risposta a stress muscolari. ANKRD2 può trovarsi sia nella banda-I del sarcomero sia nel nucleo ed è in grado di legare diversi fattori di trascrizione, come YB-1, PML e p53. La scoperta di questa interazione rafforza l’ipotesi che il disco-Z, oltre ad un ruolo specificamente strutturale, potrebbe essere coinvolto in vie di segnalazione. Dal momento che a livello del disco-Z molte proteine hanno più di un partner proteico, sarebbe utile cercare di definire il livello e il profilo di espressione delle singole proteine in tessuti muscolari con diverse caratteristiche. Un altro obiettivo del mio lavoro è stato quindi quello di valutare l’abbondanza degli mRNA di alcune delle proteine del disco-Z da me studiate con la Real-Time PCR. Allo scopo sono stati presi in considerazione quattro tessuti muscolari di topo adulto: il tibiale (un muscolo scheletrico a contrazione rapida), il soleo (un muscolo scheletrico a contrazione lenta), il gastrocnemio (un muscolo scheletrico con fibre miste) e il muscolo cardiaco. La differente distribuzione delle FATZ, miotilina e ZASP (con le sue varianti di splicing) suggerisce che, almeno in topo, le interazioni tra queste proteine potrebbero essere compartimentalizzate in distinte fibre muscolari.

Ziel dieser Arbeit war, in einem experimentellen Ansatz der Frage nachzugehen, welche pathophysiologischen Veränderungen in Bezug auf Hypertrophie und Funktionalität der Herzmuskulatur nach einem Myokardinfarkt durch Calsarcin-1 hervorgerufen werden und welchen Einfluss das Z-Scheiben-Protein in-vivo auf den Kalzium-Calmodulin Signalweg besitzt. Für die dafür durchgeführten Untersuchungen konnte auf drei verschiedene Mauslinien zurückgegriffen werden (Calsarcin-1 knockout-Mäuse, Calsarcin-1 transgene Mäuse, Wildtypmäuse). Die vorliegende Arbeit baut auf den in-vitro Ergebnissen von Frey et al. (2004) auf. Insgesamt wurden 278 Mäuse einer Infarkt- oder Scheinoperation unterzogen. Fünf Wochen nach ihrer Operation wurde das Herz jeder Maus mittels Ultraschall vermessen und auf seine Funktionstüchtigkeit untersucht. Anschließend wurden die Tiere getötet. Die entnommenen Herzen wurden gewogen, die entnommenen Unterschenkel vermessen. Insgesamt 60 Herzen wurden nach konventionellen histologischen Verfahren HE-gefärbt. 39 Mäuse wurden 24 Stunden nach ihrer Infarktoperation getötet. Ihre Herzen wurden mit Evans-blue und Tetrazoliumchlorid gefärbt. Insgesamt gingen Gewebeproben von 67 Herzen in die Untersuchungen auf RNA-Ebene (Real-Time PCR, Dot Blot) ein. Die Herzen von 40 Tieren konnten auf Proteinebene (Western Blot) untersucht werden. Die echokardiologische Untersuchung der Mäuse nach fünf Wochen zeigte eine deutliche Dilatation des linken Ventrikels derjenigen Tiere, die einer Infarktoperation unterzogen worden waren. Die größte Dilatation der drei Infarktgruppen wiesen die Mäuse auf, die nicht in der Lage sind, das Z-Scheiben-Protein Calsarcin-1 auszubilden (0,558 cm (ko Mi) vs. 0,494 cm (Wt Mi); p < 0,001). Diese Mäuse zeigten auch gegenüber den anderen beiden Infarktgruppen die ausgeprägteste systolische Dysfunktion (FS von 0,238% (ko Mi) vs. 0,376% (Wt Mi) und 0,353% (tg Mi); jeweils p < 0,001). Keine Unterschiede bestanden zwischen den Gruppen der scheinoperierten Mäuse. Morphometrische Analysen belegten eine deutliche Hypertrophie der Calsarcin-1 defizienten Mäuse, die durch die Infarktoperation einer biomechanischen Stresssituation ausgesetzt wurden. Als Hypertrophiemaß wurde der Quotient aus Herz- und Körpergewicht gewählt, zusätzlich wurde der Quotient aus Herzgewicht und Tibialänge bestimmt. Bei beiden Messungen unterschied sich das Herzgewicht der knockout-Mäuse mit Infarkt signifikant von den anderen beiden Infarktgruppen. Für das Verhältnis von Herz- zu Körpergewicht wurde für die drei Mäusegruppen ermittelt: 7,55 ± 0,6mg/g (ko Mi ), 5,56 ± 0,23mg/g (WtMi) und 5,73 ± 0,4mg/g (tgMi), wobei p < 0,01 bei ko Mi/Wt Mi und p < 0,86 bei tg Mi / Wt Mi. Für das Verhältnis von Herzgewicht zu Tibialänge ergab sich: 12,4mg/mm (ko Mi), 10,11mg/mm (Wt Mi) und 10,02mg/mm (tg Mi) (p < 0,001 koMi / WtMi, p < 0,27 tg Mi / WtMi). Zwischen den Gruppen der scheinoperierten Mäuse wurden keine signifikanten Unterschiede festgestellt. Auch auf zellulärer Ebene wiesen die Calsarcin-1 knockout-Mäuse mit Myokardinfarkt eine deutliche Hypertrophie auf verglichen mit den Wildtyp-Mäusen mit Infarkt und den Calsarcin-1 transgenen Tieren (Zellgrößenzunahme um 43,12% (koMi), 34,85% (WtMi) und 29,12% (tgMi); jeweils p < 0,001). Von allen drei Infarktgruppen zeigten die knockout-Mäuse nach fünf Wochen die ausgeprägteste Narbenbildung (Fläche der Infarktnarbe in % der Fläche des linken Ventrikels: 73,41±7,85% (ko-Mi), 53,71±3,81% (WtMi) und 48,60±6,04% (tgMi)). Übereinstimmend dazu wiesen die knockout-Mäuse mit Myokardinfarkt eine übermäßige Steigerung der ANP Produktion auf mRNA-Ebene auf. Auf Proteinebene konnte eine Steigerung der Produktion von MCIP nachgewiesen werden (ko Mi 4,3 ± 0,5 vs. Wt Mi 2,3 ± 0,3 ; p < 0,01). Zusammenfassend lassen die Ergebnisse auf eine gesteigerte Aktivität von Calcineurin und auf ein pathologisches Remodeling in der Abwesenheit von Calsarcin-1 schließen. Die Überexpression von Calsarcin-1 scheint dagegen eine pathologische Hypertrophie des Herzmuskels abmildern zu können.

Abstract The Z-disk is a sophisticated structure that connects adjacent sarcomeres in striated muscle myofibrils. α-Actinin provides strength to the Z-disks by crosslinking the actin filaments of adjacent sarcomeres. α-Actinin is an antiparallel homodimer, composed of an N-terminal actin binding domain (ABD), the central rod domain, and two pairs of C-terminal EF-hands. The PDZ-LIM domain proteins interact with α-actinin at the Z-disk. Of these proteins, only the actinin-associated LIM protein (ALP), Z-band alternatively spliced PDZ-containing protein (ZASP/Cypher) and C-terminal LIM protein (CLP36) have a ZASP/Cypher-like (ZM) motif consisting of 26-27 conserved residues in the internal region between the PDZ and LIM domains. The aim of this work was to understand the molecular interplay between the ZM-motif containing members of the PDZ-LIM proteins and α-actinin. To unveil the biological relevance of the interaction between the PDZ-LIM proteins and α-actinin, naturally occurring human ZASP/Cypher mutations were analyzed. Two interaction sites were found between ALP, CLP36 and α-actinin using recombinant purified proteins in surface plasmon resonance (SPR) analysis. The PDZ domain of ALP and CLP36 recognized the C-terminus of α-actinin, whereas the internal regions bound to the rod domain. Further characterization showed that the ALP internal region adopts and extended conformation when interacting with α-actinin and that the ZM-motif partly mediated the interaction, but did not define the entire interaction area. ZASP/Cypher also interacted and competed with ALP in binding to the rod domain. The internal fragments containing the ZM-motif were important for co-localization of ALP and ZASP/Cypher with α-actinin at the Z-disks and on stress fibers. The absence of ALP and ZASP/Cypher in focal contacts indicates that other interacting molecules, for instance vinculin and integrin, may compete in binding to the rod in these areas or additional proteins are required in targeting to these locations. The co-localization of the ZASP/Cypher with α-actinin could be released by disrupting the stress fibers leading to an accumulation of α-actinin in the cell periphery, whereas ZASP/Cypher was not in these areas. This suggests that an intact cytoskeleton is important for ZASP/Cypher interaction with α-actinin. Earlier studies have shown that mutations in the ZASP/Cypher internal region are associated with muscular diseases. These mutations, however, did not affect ZASP/Cypher co-localization with α-actinin or the stability of ZASP/Cypher proteins. The Z-disk possesses a stretch sensor, which is involved in triggering hypertrophic growth as a compensatory mechanism to increased workloads. α-Actinin is a docking site of molecules that are involved in hypertrophic signaling cascades mediated by calsarcin-calcineurin and protein kinase C (PKC) isoforms. The internal interaction site may be involved in targeting PKCs, which bind to the LIM domains of ZASP/Cypher, to the Z-disks. The similar location of the internal interaction site with calsarcin on the rod suggests that ZASP/Cypher, ALP and CLP36 may regulate calsarcin-mediated hypertrophic signaling.

Ziel dieser Arbeit war, in einem experimentellen Ansatz der Frage nachzugehen, welche pathophysiologischen Veränderungen in Bezug auf Hypertrophie und Funktionalität der Herzmuskulatur nach einem Myokardinfarkt durch Calsarcin-1 hervorgerufen werden und welchen Einfluss das Z-Scheiben-Protein in-vivo auf den Kalzium-Calmodulin Signalweg besitzt. Für die dafür durchgeführten Untersuchungen konnte auf drei verschiedene Mauslinien zurückgegriffen werden (Calsarcin-1 knockout-Mäuse, Calsarcin-1 transgene Mäuse, Wildtypmäuse). Die vorliegende Arbeit baut auf den in-vitro Ergebnissen von Frey et al. (2004) auf. Insgesamt wurden 278 Mäuse einer Infarkt- oder Scheinoperation unterzogen. Fünf Wochen nach ihrer Operation wurde das Herz jeder Maus mittels Ultraschall vermessen und auf seine Funktionstüchtigkeit untersucht. Anschließend wurden die Tiere getötet. Die entnommenen Herzen wurden gewogen, die entnommenen Unterschenkel vermessen. Insgesamt 60 Herzen wurden nach konventionellen histologischen Verfahren HE-gefärbt. 39 Mäuse wurden 24 Stunden nach ihrer Infarktoperation getötet. Ihre Herzen wurden mit Evans-blue und Tetrazoliumchlorid gefärbt. Insgesamt gingen Gewebeproben von 67 Herzen in die Untersuchungen auf RNA-Ebene (Real-Time PCR, Dot Blot) ein. Die Herzen von 40 Tieren konnten auf Proteinebene (Western Blot) untersucht werden. Die echokardiologische Untersuchung der Mäuse nach fünf Wochen zeigte eine deutliche Dilatation des linken Ventrikels derjenigen Tiere, die einer Infarktoperation unterzogen worden waren. Die größte Dilatation der drei Infarktgruppen wiesen die Mäuse auf, die nicht in der Lage sind, das Z-Scheiben-Protein Calsarcin-1 auszubilden (0,558 cm (ko Mi) vs. 0,494 cm (Wt Mi); p < 0,001). Diese Mäuse zeigten auch gegenüber den anderen beiden Infarktgruppen die ausgeprägteste systolische Dysfunktion (FS von 0,238% (ko Mi) vs. 0,376% (Wt Mi) und 0,353% (tg Mi); jeweils p < 0,001). Keine Unterschiede bestanden zwischen den Gruppen der scheinoperierten Mäuse. Morphometrische Analysen belegten eine deutliche Hypertrophie der Calsarcin-1 defizienten Mäuse, die durch die Infarktoperation einer biomechanischen Stresssituation ausgesetzt wurden. Als Hypertrophiemaß wurde der Quotient aus Herz- und Körpergewicht gewählt, zusätzlich wurde der Quotient aus Herzgewicht und Tibialänge bestimmt. Bei beiden Messungen unterschied sich das Herzgewicht der knockout-Mäuse mit Infarkt signifikant von den anderen beiden Infarktgruppen. Für das Verhältnis von Herz- zu Körpergewicht wurde für die drei Mäusegruppen ermittelt: 7,55 ± 0,6mg/g (ko Mi ), 5,56 ± 0,23mg/g (WtMi) und 5,73 ± 0,4mg/g (tgMi), wobei p < 0,01 bei ko Mi/Wt Mi und p < 0,86 bei tg Mi / Wt Mi. Für das Verhältnis von Herzgewicht zu Tibialänge ergab sich: 12,4mg/mm (ko Mi), 10,11mg/mm (Wt Mi) und 10,02mg/mm (tg Mi) (p < 0,001 koMi / WtMi, p < 0,27 tg Mi / WtMi). Zwischen den Gruppen der scheinoperierten Mäuse wurden keine signifikanten Unterschiede festgestellt. Auch auf zellulärer Ebene wiesen die Calsarcin-1 knockout-Mäuse mit Myokardinfarkt eine deutliche Hypertrophie auf verglichen mit den Wildtyp-Mäusen mit Infarkt und den Calsarcin-1 transgenen Tieren (Zellgrößenzunahme um 43,12% (koMi), 34,85% (WtMi) und 29,12% (tgMi); jeweils p < 0,001). Von allen drei Infarktgruppen zeigten die knockout-Mäuse nach fünf Wochen die ausgeprägteste Narbenbildung (Fläche der Infarktnarbe in % der Fläche des linken Ventrikels: 73,41±7,85% (ko-Mi), 53,71±3,81% (WtMi) und 48,60±6,04% (tgMi)). Übereinstimmend dazu wiesen die knockout-Mäuse mit Myokardinfarkt eine übermäßige Steigerung der ANP Produktion auf mRNA-Ebene auf. Auf Proteinebene konnte eine Steigerung der Produktion von MCIP nachgewiesen werden (ko Mi 4,3 ± 0,5 vs. Wt Mi 2,3 ± 0,3 ; p < 0,01). Zusammenfassend lassen die Ergebnisse auf eine gesteigerte Aktivität von Calcineurin und auf ein pathologisches Remodeling in der Abwesenheit von Calsarcin-1 schließen. Die Überexpression von Calsarcin-1 scheint dagegen eine pathologische Hypertrophie des Herzmuskels abmildern zu können.

The genetic background of dilated cardiomyopathy is characterized by heterogeneity. Truncating mutations in titin (TTN), responsible for around 20% of cases, have recently been recognized as the most prevalent genetic cause of dilated cardiomyopathy. Other important causal genes are LMNA (coding for the nuclear envelope protein, lamin A/C) and sarcomere protein genes, such as beta-myosin heavy chain (MYH7) and troponin T (TNNT2). Other loci, including genes that code for cytoskeleton, Z-disc, and membrane-associated proteins, are each responsible for a lower percentage of cases. Current consensus recommendations propose genetic testing in the presence of familial forms of the disease or when certain phenotype characteristics, such as conduction disease or a family history of sudden cardiac death, are present. Some causal genes are associated with a worse prognosis. This is most strongly established for LMNA, where the presence of a disease-causing mutation, together with certain clinical risk factors, is an indication for an implantable cardioverter defibrillator.

The sarcomeres represent the essential contractile units of the cardiac myocyte and are bordered by two Z-lines (disks) that are made by various proteins. The cardiac Z-disk is recognized as one of the nodal points in cardiomyocyte structural organization, mechano-sensation and signal transduction. Rapid progress in molecular and cellular biology has significantly improved the knowledge about pathogenic mechanisms and signaling pathways involved in the development of inherited cardiomyopathies. Genetic insult resulting in expression of mutated proteins that maintain the structure of the heart can perturb cardiac function. The primary mutation in the cardiac contractile apparatus or other subcellular complexes can lead to cardiac pathology on a tissue level, resulting in organ and organism level pathophysiology. The “final common pathway” hypothesis interpreting the genetic basis and molecular mechanisms involved in the development of cardiomyopathies suggests that mutations in cardiac genes encoding proteins with similar structure, function, or location and operating in the same pathway, are responsible for a particular phenotype of cardiomyopathy with unique morpho-histological remodeling of the heart. This chapter will describe genetic abnormalities of cardiac Z-disk and related “final common pathways” that are triggered by a Z-disk genetic insult leading to heart muscle diseases. In addition, animal models carrying mutations in Z-disk proteins will be described.

Cardiomyopathies (CMs) encompass a heterogeneous group of structural and functional (systolic and diastolic) abnormalities of the myocardium and are either confined to the cardiovascular system or are part of a systemic disorder. CMs represent a leading cause of morbidity and mortality and account for a significant percentage of death and cardiac transplantation. The 2006 American Heart Association (AHA) classification grouped CMs into primary (genetic, mixed, or acquired) or secondary (i.e., infiltrative or autoimmune). In 2008, the European Society of Cardiology classification proposed subgrouping CM into familial or genetic and nonfamilial or nongenetic forms. In 2013, the World Heart Federation recommended the MOGES nosology system, which incorporates a morpho-functional phenotype (M), organ(s) involved (O), the genetic inheritance pattern (G), an etiological annotation (E) including genetic defects or underlying disease/substrates, and the functional status (S) of a particular patient based on heart failure symptoms. Rapid advancements in the biology of cardio-genetics have revealed substantial genetic and phenotypic heterogeneity in myocardial disease. Given the variety of disciplines in the scientific and clinical fields, any desired classification may face challenges to obtaining consensus. Nonetheless, the heritable phenotype-based CM classification offers the possibility of a simple, clinically useful diagnostic scheme. In this chapter, we will describe the genetic basis of dilated cardiomyopathy (DCM), hypertrophic cardiomyopathy (HCM), arrhythmogenic cardiomyopathy (ACM), LV noncompaction cardiomyopathy (LVNC), and restrictive cardiomyopathy (RCM). Although the descriptive morphologies of these types of CM differ, an overlapping phenotype is frequently encountered within the CM types and arrhythmogenic pathology in clinical practice. CMs appear to originate secondary to disruption of “final common pathways.” These disruptions may have purely genetic causes. For example, single gene mutations result in dysfunctional protein synthesis causing downstream dysfunctional protein interactions at the level of the sarcomere and a CM phenotype. The sarcomere is a complex with multiple protein interactions, including thick myofilament proteins, thin myofilament proteins, and myosin-binding proteins. In addition, other proteins are involved in the surrounding architecture of the sarcomere such as the Z-disk and muscle LIM proteins. One or multiple genes can exhibit tissue-specific function, development, and physiologically regulated patterns of expression for each protein. Alternatively, multiple mutations in the same gene (compound heterozygosity) or in different genes (digenic heterozygosity) may lead to a phenotype that may be classic, more severe, or even overlapping with other disease forms.