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Telethonin (also known as titin-cap or t-cap) is a 19 kDa Z-disk protein with a unique β-sheet structure, hypothesized to assemble in a palindromic way with the N-terminal portion of titin and to constitute a signalosome participating in the process of cardio-mechanosensing. In addition, a variety of telethonin mutations are associated with the development of several different diseases; however, little is known about the underlying molecular mechanisms and telethonin’s in vivo function.
Here we aim to investigate the role of telethonin in vivo and to identify molecular mechanisms underlying disease as a result of its mutation.
By using a variety of different genetically altered animal models and biophysical experiments we show that, contrary to previous views, telethonin is not an indispensable component of the titin-anchoring system, nor is deletion of the gene or cardiac specific overexpression associated with a spontaneous cardiac phenotype. Rather, additional titin-anchorage sites, such as actin-titin crosslinks via α-actinin, are sufficient to maintain Z-disk stability despite the loss of telethonin. We demonstrate that a main novel function of telethonin is to modulate the turnover of the pro-apoptotic tumor suppressor p53 after biomechanical stress in the nuclear compartment, thus linking telethonin, a protein well known to be present at the Z-disk, directly to apoptosis (“mechanoptosis”). In addition, loss of telethonin mRNA and nuclear accumulation of this protein is associated with human heart failure, an effect which may contribute to enhanced rates of apoptosis found in these hearts.
Telethonin knockout mice do not reveal defective heart development or heart function under basal conditions, but develop heart failure following biomechanical stress, owing at least in part to apoptosis of cardiomyocytes, an effect which may also play a role in human heart failure.
The heart is a dynamic organ able to self-adapt to mechanical demands, but the underlying molecular mechanisms remain poorly understood. We have previously shown that the sarcomeric Z-disk, which serves as an important anchorage site for titin and actin molecules, is not only important for mechanical force transduction but harbours as well a pivotal mechanosensitive signalosome where muscle LIM protein (MLP) and telethonin play major roles in the perception of mechanical stimuli.1–3 Here we focus on telethonin, a striated-muscle specific protein with a unique β–sheet structure (and no direct homologue genes), enabling it to bind in an antiparallel (2:1) sandwich complex to the titin Z1-Z2 domains, essentially “gluing” together the N-termini of two adjacent titin molecules.4 Interestingly the telethonin – titin interaction represents the strongest protein – protein interaction observed to date.5 Beside being phosphorylated by protein kinase D,6 telethonin is also an in vitro substrate of the titin kinase, an interaction thought to be critical during myofibril growth.7 The giant elastic protein titin extends across half the length of a sarcomere and is thought to stabilize sarcomere assembly by serving as a scaffold to which other contractile, regulatory, and structural proteins attach.8
Telethonin was shown to interact with MLP, hypothesized to be part of a macromolecular mechanosensor complex and to play a role in a subset of human cardiomyopathies.2 In this context, telethonin interacts with calsarcin-1 (also known as FATZ-2 or myozenin-2, a gene recently shown to cause cardiomyopathy9), ankyrin repeat protein 2, small ankyrin-1 (a transmembrane protein of the sarcoplasmic reticulum)10 and minK, a potassium channel β subunit.11–14 In addition, telethonin was shown to interact with MDM215 and MuRF116 - E3 ubiquitin ligases with strong impact on cardiac protein turnover as well as with the proapoptotic protein Siva17. Recessive nonsense mutations in the telethonin gene are associated with limb-girdle muscular dystrophy type 2 G18–20 and heterozygous missense mutations with dilated and hypertrophic forms of cardiomyopathy1, 21–22 as well as with intestinal pseudo-obstruction.23 Interestingly, a naturally occurring telethonin variant that has a Glu13 deletion (E13del telethonin) was initially found in patients affected by hypertrophic cardiomyopathy21 and then later in healthy, unaffected individuals.24–25 However, the molecular consequences of the E13del variant, especially on the telethonin-titin interaction, as well as telethonin mediated pathways in general remain unclear.
Please see also the detailed methods description in the online supplement material.
Myofibrils were prepared from telethonin-deficient or wildtype tissue as described previously.26
Z1Z2 titin, MLP, telethonin as well as its mutants were expressed and purified as previously described.27 Z1Z2-telethonin complexes were formed and analyzed on native gels and gel filtration columns as previously described.4, 27
U-2H,15N-labelled p53DBD for NMR studies was prepared using M9-medium supplemented with 1g/l 15NH4Cl, 2g/l 2H,13C glucose in 99.9% D2O (Eurisotop, Saarbrücken, Germany). Nuclear magnetic resonance (NMR) experiments were done at 293K on a Bruker Avance900 spectrometer (Bruker Biospin, Rheinstetten, Germany).
In the current project we employed two different anti telethonin antibodies: a mouse anti-telethonin polyclonal antibody raised against a recombinant His-tagged human full-length telethonin (western blots, immune precipitations, mouse heart and human heart sections) and a rat polyclonal anti-telethonin antibody (immunofluorescence in neonatal rat cardiac myocytes). Both the mouse and rat antibodies to human telethonin were produced by immunizing respectively Balb/C mice or LOU/Nmir rats with purified recombinant full-length telethonin protein (1-167 aa) and their specificity checked by their ability to detect telethonin on Western blots of human heart and skeletal muscle protein. Anti-Z1Z2 titin antibody was a kind gift of Prof. S. Labeit. We used as well p21WAF1 EA10 (Calbiochem), Mdm2 2A9 and 2A10, myc 4A6 (Upstate) and actin AC15 (Abcam), anti-p53 (DO-1, FL 393, Santa Cruz), and/or mouse monoclonal p53 (1C12, Cell signalling), mouse monoclonal anti α-actinin (Sigma) and phalloidin conjugated Alexa 350 antibody. The secondary antibodies used were Alexa-labeled 633 anti-rat, Alexa- labelled 488 anti-rabbit and Alexa- labelled 488 anti-mouse (Invitrogen) antibody (please see also the Online Supplement Material for additional information).
All animals used in the experiments were age and sex matched. All assays were analyzed in “double blind” fashion. T-tests were used to analyze differences in echocardiography (n = 8 – 9 animals per group) and for the analysis of Z-disks following sarcomere stretch. Whenever more than 2 groups were compared, ANOVA tests followed by Bonferroni’s Multiple Comparison test were applied. Statistical significance was reached at p<0.05.
To be able to perform a detailed functional analysis of cardiac performance, we generated telethonin-deficient mice by homologous recombination, replacing exons 1 and 2 with a Lac Z-neomycin cassette (Figure 1). Using this approach, telethonin was found to be transcribed as early as embryonic day 10.5 (not shown). Telethonin is a late-in protein, as such it is not a surprise that telethonin-deficient mice are born in the expected Mendelian ratios and that this protein is apparently not required during heart development.28–29
In contrast to recently published zebrafish and xenopus knock down models30–31 as well as what was expected based on the available knowledge, the analysis of myocardial function by echocardiography (online table I) as well as by in vivo heart catheterization using 3 – 4 months old telethonin−/− mice under basic conditions did not reveal any abnormal parameters. Histological analysis of the spontaneous cardiac phenotype of telethonin−/− mice revealed no alterations, including the amount of extracellular matrix deposition (fig. 3A and B), and changes in titin-isoform composition that could be excluded based on gel electrophoresis (Online Figure I). Epifluorescence experiments showed unaltered global intracellular Ca2+ handling (Online Figure II) and immunohistochemistry as well as immunogold electron microscopy did not reveal any defects in telethonin-deficient Z-disks (Online Figure III).
Telethonin was shown to interact directly with the potassium channel subunit minK13, as well as with different sodium channels such as SCN5A23 – as a consequence we performed extensive analyses of electrocardiograms (ECG) in vivo as well as patch-clamp experiments in vitro, but did not find any significant differences in ECG parameters such as PQ interval, QRS width, QT interval, or action-potential repolarisation between control littermates and telethonin-deficient animals, without any occurrence of early or delayed after depolarisations in either group. The telethonin-minK or telethonin-SCN5A interaction may thus have little physiological relevance in the heart, at least in the mouse model (Online Figure IV). This remarkable mild cardiac phenotype despite loss of telethonin is supported by another recently published telethonin knock out mouse, where the same approach has been used to inactivate telethonin (i.e. exons 1 and 2 have been replaced by a Lac Z neomycin cassette) and where the skeletal muscle phenotype has been analyzed but almost no pathology has been detected under spontaneous conditions.32
Telethonin was shown to interact with the N-terminal Z1Z2 titin and as such, might have an important function in mechanically linking two titin molecules together.4 Again, surprisingly to what we expected, stretch of single isolated myofibrils obtained from telethonin−/− heart or skeletal muscle did not cause any changes in Z-disk architecture or displacement of the titin N-terminus from the Z-disk, even when the sarcomeres were extended stepwise to (unphysiological) lengths of >3.2 μm to reach very high passive forces of tens of mN/mm2 (Figure 2A, B). In contrast, compromised anchorage of the titin N-terminus was observed after removal of actin from cardiac sarcomeres (using a Ca2+-independent gelsolin fragment26), suggesting that telethonin is mechanically relevant only when there is additional disturbance of the Z-disk (Figure 2A, B), such as impairment of the α-actinin-mediated titin-actin crosslinks.
Moreover, we reconstituted in vitro a complex consisting of telethonin and the N-terminal (Z1-Z2) titin domains and analyzed the effects of different human telethonin mutations on this complex formation. In contrast to several point mutations tested previously,4 the E13del variant, which due to its presence in healthy unaffected individuals has been regarded as a polymorphism24–25 rather than a disease causing mutation,21 lost the ability to bind the titin N-terminus (Figure 2E–I). Consistent with previous data,4 the deletion of this residue in telethonin leads to a loss of proper formation of the telethonin β-hairpin structure, which forms the basis for the titin binding. Given the available information on heterozygous and homozygous telethonin deficiency reported here and the fact that heterozygous loss of telethonin is not associated with any phenotype (Figure 2H), one possible conclusion is that E13del telethonin is probably a harmless naturally occurring variant unable to bind titin, hence supporting our view that telethonin, at least in mammals, performs no important structural functions. However additional effects of the E13del telethonin variant cannot be excluded and homozygous patients have not been reported.
The fulminant defects observed after actin removal in the myofibril stretch experiments led us to increase the biomechanical load under in vivo conditions by transverse aortic constriction (TAC). Two to three weeks after this intervention, telethonin−/− animals developed maladaptive cardiac hypertrophy and severe heart failure as judged by clinical signs and echocardiography (Figure 2H).
Moreover, we found an increase in focal fibrosis as well as a significant increase in TUNEL positive cells in the telethonin−/− animals following the TAC intervention pointing to apoptosis as a possible cause of cell death (Figure 3A–D). A detailed analysis revealed that primarily cardiac myocytes were TUNEL positive and gene expression analysis revealed differential expression of several genes involved in the apoptotic pathway(Online Figure V and VI).
Cardiac apoptosis can be efficiently induced by the tumor suppressor gene product p53,33 a protein known to be polyubiquitinylated and marked for degradation by the E3 ubiquitin ligase MDM2.34 Western blot analysis revealed increased p53 levels in the telethonin−/− animals following TAC (Figure 3E, F), whereas the apoptosis repressor with caspase recruitment domain (ARC) – another important heart specific survival factor – remained unchanged (not shown). We also found significant increases in p21 and Caspase 8 mRNA expression, both of which are p53 target genes (Figure 3 G, Online Figure VI), and a significant increase in nuclear p53 (Figure 5), supporting the finding of enhanced p53 protein levels.
Interestingly, myostatin has been implicated in the regulation of p53 and p21 expression; it is a negative regulator of cardiac growth35–38 and is upregulated under stress.39–41 Moreover, myostatin has also been associated with fibrosis.42 Most importantly, telethonin has been shown previously to interact with myostatin and to inhibit its expression.43 Thus myostatin might be able to cause the observed effects, but we did not detect any significant changes in myostatin mRNA or protein expression (Online Figure VII and VIII).
We also performed in vitro experiments in neonatal rat cardiomyocytes, where we knocked down telethonin, and found a strong induction of p53 after additional doxorubicin treatment (a drug known to cause oxidative cellular stress and to induce stress responsive genes,44 Figure 4A). In addition, we found evidence of telethonin being present in the nuclei of neonatal rat cardiomyocytes at early stages of culture (up to 2 days after plating, not shown).
Transient over-expression of telethonin in U2OS cells led to a strong down-regulation of endogenous p53 (fig. 4B, lane 2 vs. lane 4). These effects were not observed in the presence of nutlin-3, a compound preventing the interaction between p53 and MDM2, suggesting that MDM2 is required for the effects of telethonin on p53 degradation. Accordingly, expression of classical p53-responsive genes, p21 and MDM2, were suppressed due to the diminished p53 levels. This prompted us to investigate the underlying molecular mechanism in more detail and we found direct interaction of telethonin and MDM2 by co-immunoprecipitation assays (Online Figure IX), supporting earlier observations by Tian and co-workers.15 In addition, a direct interaction of p53 and telethonin was detectable by co-immunoprecipitation experiments (Figure 4C) as well as pull down assays (Figure 4D). Native and SDS-PAGE gel electrophoresis indicated the formation of a high-molecular weight complex (~ 150 kDa) between full-length telethonin (wild type and E13del) and full length p53 in vitro (Figure 4D, right panels).
Static light scattering and NMR analysis additionally showed that the interaction involves the p53 DNA binding domain (p53DBD). Static light scattering indicated a molecular weight of 163 kDa for this complex suggesting that it might consist of multiple telethonin and p53DBD molecules (Figure 4E). NMR spectroscopy further confirms that the telethonin interaction involves the p53DBD (Figure 4F). NMR chemical shift differences between the free and telethonin-bound p53DBD reveal that telethonin contacts the β-sheet of p53DBD, at a site that is remote from the DNA binding interface (Figure 4F, Online Figure X). Fluorescence polarization (FP) experiments (Figure 4G) and Surface Plasmon Resonance (SPR, Biacore) experiments (Online Figure X) show that the interaction between telethonin and p53DBD has a low micromolar dissociation constant (KD = 2.2 ± 0.2 μM and 0.765 ± 0.03 μM for FP and SPR, respectively, Online Figure X). These values are comparable to other protein-protein interactions that have been mapped to p53DBD.45–47 It is interesting to note that the interaction of telethonin with titin (Figure 2G) also preferentially involves the β-sheets of the titin Z1-Z2 domains forming intermolecular β-strand contacts. Similar interactions might contribute to the stabilization of the telethonin-p53DBD complex.
We performed as well a series of F-actin, α-actinin, telethonin and p53 co-localization studies and found that p53, in contrast to the Z-disk localization of telethonin, is not clearly detectable under spontaneous conditions, neither in the in vivo setting nor in isolated neonatal rat cardiomyocytes in vitro (Online Figure XI, XII). However, after biomechanical or oxidative stress in vivo, such as TAC (Figure 5), or doxorubicin treatment in vitro and in vivo (Online Figure XIII, XIV), we observed a strong induction of p53 in cardiomyocyte nuclei, which is well in accordance with previously published data on p53.48–49 Moreover, under both stress conditions telethonin co-localized with p53 in cardiomyocyte nuclei (Online Figure XIII, XIV). However, an even stronger increase in p53 nuclear expression was observed after TAC in telethonin deficient animals, supporting the results of our previous Western blot analysis (Figure 3 and Figure 5). Telethonin/p53 colocalization was also observed when we transfected neonatal rat cardiomyocytes in vitro using a GFP-telethonin construct (Online Figure XV).
Based on these data we assumed that telethonin at least supports MDM2 mediated p53 degradation. As a consequence we aimed to analyze the effects of telethonin overexpression on myocardial function under in vivo conditions and generated telethonin transgenic animals. We used the myocardium specific alpha myosin heavy chain promoter and a FLAG-tagged mouse telethonin cDNA (Figure 6). Again, to our surprise these animals did not exhibit any spontaneous phenotype (online table II).50 Of note, they develop less apoptosis as well as less p53 expression in comparison to wildtype littermate controls after TAC (Figure 6). Particularly the decrease in apoptotic (TUNEL positive) cells in telethonin transgenic animals is interesting and might indicate potential protective effects of telethonin overexpression.
We then assumed that p53 determines the negative outcome in telethonin deficient animals following biomechanical stress and we used a transgenic line overexpressing a well characterized dominant negative p53 mutant (i. e. the Arg193Pro mutation)51–52 to inactivate this protein in the telethonin−/−background (Figure 7 A). dnp53/telethonin−/− double transgenic animals did not develop any spontaneous phenotype and they did not exhibit any significant change in myocardial function following TAC. However, genetic inhibition of p53 in the telethonin deficient background significantly inhibited the increase in apoptosis found after biomechanical stress in telethonin−/−animals alone (Figure 7 B).
In order to study telethonin mRNA expression in the human heart we analyzed myocardial samples from end-stage heart failure patients and found significant down-regulation compared to normal donor hearts (Figure 7 C), as well as an increase in nuclear telethonin (Figure 7D). This may have implications for p53 expression and p53-related apoptosis both of which have previously been shown to be elevated in these patients. We also found down-regulation of telethonin in acute donor organ failure suggesting this effect is not restricted to the setting of chronic end-stage failure.
Here we demonstrate a model where a primary defect in an integral Z-disk component is not associated with any cardiac phenotype or functional abnormality under basal conditions.53 However, pressure overload causes a maladaptive response in homozygous telethonin−/− hearts, ultimately leading to global heart failure in vivo. Loss of the p53-ligand telethonin is associated with an increase in p53 as well as elevated apoptosis following an increase in afterload – which is the first description of a Z-disk component to do so. Moreover, by binding to p53’s DNA binding domain, telethonin is potentially able to repress the function of this important transcription factor.
Telethonin, which was shown to be phosphorylated in vitro by the titin kinase,7 does not seem to have a function during embryonic development in vivo. A recent study54 reported a defect in C2C12 myoblast differentiation when telethonin was downregulated by the use of siRNAs. It remains to be elucidated whether there are differences in vivo and in vitro or whether the telethonin siRNAs per se exhibit off target effects that account for the observed differences. In addition loss of telethonin in zebrafish or xenopus is associated with a spontaneous defect30–31 as such it will be important to elucidate in future whether telethonin in mammalian hearts acquired additional functions during evolution or if so far unknown telethonin homologue genes are upregulated or if differences in Z-disk structure account for the observed differences. Our data are generally consistent with a recent report, whereby telethonin binds in an antiparallel (2:1) sandwich complex to the titin Z1-Z2 domains.4 However, telethonin clearly is not required to stabilize the sarcomere structure. Instead, telethonin may serve as a pivotal element in cardiac signalling by controlling apoptosis and cell death via p53. Our data are compatible with a direct molecular link between the sarcomeric Z-disk, cardiac performance, as well as gene transcription and cell survival (“mechano-transcriptional coupling - MTC”), although additional data need to be provided to entirely support such a functional link. It might well be that Z-disk proteins carry at least two different functions: 1) a structural function which might be dismissible particularly in “peripheral” Z-disk proteins and 2) a regulatory function which, as in this case, might be much more important. One implication of this could be that cardiomyopathy and associated heart failure, which can be caused by mutations in Z-disk components (now regarded as a “hot spot” for these mutations55) might be seen as a disease caused by “defects in cardiac regulation” or of “defects in mechano-transcriptional coupling”.
In conclusion, this study might change the previous concept on Z-disk structure, which we now suggest to also be a pivotal node for apoptosis – essentially by linking telethonin to p53 (Figure 8). In contrast to previous views, telethonin is not an indispensable component of the cardiac titin anchoring system and cardiac specific telethonin overexpression is not immediately associated with Z-disk pathology and as such is compatible with life. Instead, under normal conditions, actin crosslinking may be sufficient to keep the sarcomere structure viable, despite loss of telethonin. With an increase in hemodynamic load or an increase in biomechanical or oxidative stress, however, telethonin deficiency leads directly to enhanced p53 levels and as such promotes an increase in apoptosis and cell death, thus initiating the development of heart failure, an effect which might be called “mechanoptosis”.
Telethonin mutations are associated with several diseases but the underlying molecular mechanisms remain not well understood. To analyze the in vivo function of telethonin we generated genetically altered mouse models and found that telethonin is a dispensable component of the sarcomeric Z-disk. Deletion or cardiac-specific overexpression of telethonin was not associated with a spontaneous cardiac phenotype. However, our results showed that telethonin modulates the turnover of the pro-apoptotic protein p53 after biomechanical stress. This novel finding links telethonin directly to apoptosis (“mechanoptosis”), which is considered a new cell death associated pathway. We also observed a reduction in the eexpression of telethonin and an increase in its nuclear abundance in myocardial samples from end-stage heart failure patients, indicating that changes in telethoninmay contribute to cardiac maladaptation. These findings suggest that telethonin, together with other Z-disk associated proteins, might have novel functions in anti-apoptotic cell survival pathways.
Prof. J. Robbins is acknowledged for providing the αMHC promoter. Dr. B. North, Dept of Biostatistics, Imperial College, London, is gratefully acknowledged for his support with regard to the statistics.
Sources of Funding
Dr. R. Knöll is supported by DFG Kn 448/9-1, DFG Kn 448 10-1, Fritz Thyssen Stiftung, British Heart Foundation (PG11/34/28793) and FP7-PEOPLE-2011-IRSES, Proposal No 291834 – Acronym: SarcoSi. Dr. W. Linke (Li 690/7-1) and Dr. L. Maier (MA 1982/2-2, MA 1982/4-1) are funded by the DFG. Dr. G. Faulkner and Dr. S. Miocic are supported by grant GGP04088 from the Telethon Foundation-Italy and Dr. Faulkner acknowledges support from the Fondazione Cariparo, Italy (Progetto Eccellenza 2010 CROMUS). Dr. H. Granzier acknowledges grant HL062881. Pof. Dr. Dr. h.c. H. Kessler, Dr. P. Zou, Dr. F. Hagn and Prof. M. Sattler acknowledge support by the Elitenetzwerk Bayern and the DFG (SFB594). Prof. M. Wilmanns acknowledges funding from FWF/DFG (P1906). Dr. P. Barton is supported by the NIHR Cardiovascular Biomedical Research Unit of Royal Brompton and Harefield NHS Foundation Trust and Imperial College London.