Titin Mutations Research Articles

HEART DISEASE. Titin mutations in iPS cells define sarcomere insufficiency as a cause of dilated cardiomyopathy.

Human mutations that truncate the massive sarcomere protein titin [TTN-truncating variants (TTNtvs)] are the most common genetic cause for dilated cardiomyopathy (DCM), a major cause of heart failure and premature death. Here we show that cardiac microtissues engineered from human induced pluripotent stem (iPS) cells are a powerful system for evaluating the pathogenicity of titin gene variants. We found that certain missense mutations, like TTNtvs, diminish contractile performance and are pathogenic. By combining functional analyses with RNA sequencing, we explain why truncations in the A-band domain of TTN cause DCM, whereas truncations in the I band are better tolerated. Finally, we demonstrate that mutant titin protein in iPS cell-derived cardiomyocytes results in sarcomere insufficiency, impaired responses to mechanical and β-adrenergic stress, and attenuated growth factor and cell signaling activation. Our findings indicate that titin mutations cause DCM by disrupting critical linkages between sarcomerogenesis and adaptive remodeling.

76 Comprehensive Assessment of Rare Genetic Variation in Dilated Cardiomyopathy Genes in Patients and Controls: Abstract 76 Table 1

<h3>Introduction</h3> Dilated cardiomyopathy (DCM) is a genetically heterogenous condition with mutations reported in at least 50 associated genes.¹ Our consortium has demonstrated the presence of truncating mutations in the giant sarcomeric gene titin (TTN) in up to 27% of DCM² making this the commonest genetic cause of DCM. Here we conducted a comprehensive analysis of the frequency of rare coding variants in cardiomyopathy genes in DCM patients and ethnically-matched healthy controls. <h3>Methods</h3> We sequenced 64 cardiomyopathy genes in 651 individuals, comprising 234 patients referred for prospective evaluation of DCM with cardiac MRI (54 ± 14 yrs at scan, 70.5% males), 98 end-stage DCM (39 ± 16 yrs on date of transplant/left-ventricular assist device implantation, 83.7% males), and 319 prospectively recruited adult healthy volunteers (HVOL, 42 ± 14 yrs at scan, 46.1% males), who underwent detailed phenotyping with cardiac MRI. Next-generation targeted exon sequencing was performed on the SOLiD 5500xl platform. Variant calling was performed using Lifescope v2.5.1. Data were mapped to the Hg19 (GRCh37) human genome reference. Caucasian ethnicity was confirmed using Principal Component Analysis. Burden testing for potentially disease-causing variation was performed on each gene. <h3>Results</h3> DCM patients were enriched for novel protein-altering variants in 28 genes (Table 1: top 7 genes), but only 5 achieved nominal significance. Truncating variants in TTN (TTNtv) remained significant after multiple testing correction (12% DCM, 1.9% controls, p = 8.1 × 10–6). Rare variation in SCN5A and MYH6, previously reported to play a key role in DCM, were not enriched in DCM (SCN5A – 2.1% DCM, 2.8% controls, p = 0.80; MYH6 –0.9% DCM, 2.2% controls, p = 0.95) and nor were non-synonymous SNPs in TTN (TTNns, 25.6% DCM, 25.7% controls, p = 0.55). Importantly, an additive effect of variation in multiple DCM genes on DCM risk was identified using logistic regression models (p = 5.7 × 10 x -4), demonstrating a multi-genic basis for DCM in some cases. <h3>Conclusions</h3> To the best of our knowledge, this is the first comprehensive study of the genetic architecture of DCM compared to an ethnically-matched healthy control cohort. The majority of genes reported to contain rare variants in DCM have similar burdens of variation in controls. Some variants may contribute to disease, but excluding TTNtv the majority of such variants are uninterpretable in the absence of functional or segregation data. <h3>References</h3> Mestroni L, Taylor MR. Genetics and genetic testing of dilated cardiomyopathy: a new perspective. Discov Med. 2013;15(80):43-9 Herman DS, Lam L, Taylor MR, Wang L, Teekakirikul P, Christodoulou D, <i>et al</i>. Truncations of titin causing dilated cardiomyopathy. N Engl J Med. 2012;366(7):619-28

Titin mutations: the fall of Goliath.

Titin (TTN), the largest protein in the human body, forms powerful elastic filaments along the sarcomere of cardiomyocytes. This multifunctional protein is involved in numerous cellular processes, including sarcomeric assembly, stabilization and mechanosensing. Along physiological sarcomere lengths, TTN is also the most important determinant of the passive tension of cardiac muscle. However, as the giant Goliath was brought down by David's slingshot, so single-base-pair mutations in the gene encoding TTN (TTN) can ultimately impair to some degree a normal cardiac function. Since the first report on the involvement of TTN mutations in the development of hypertrophic cardiomyopathy, in 1999, dozens of other mutations have been described and associated with the onset of cardiac disease. In this review, we aim to explore some of the mechanisms underlying the functions of TTN, as well as the pathophysiological consequences arising from the expression of abnormal TTN isoforms resulting from mutations located along TTN.

Pressure Overload by Transverse Aortic Constriction Induces Maladaptive Hypertrophy in a Titin-Truncated Mouse Model.

Mutations in the giant sarcomeric protein titin (TTN) are a major cause for inherited forms of dilated cardiomyopathy (DCM). We have previously developed a mouse model that imitates a TTN truncation mutation we found in a large pedigree with DCM. While heterozygous Ttn knock-in mice do not display signs of heart failure under sedentary conditions, they recapitulate the human phenotype when exposed to the pharmacological stressor angiotensin II or isoproterenol. In this study we investigated the effects of pressure overload by transverse aortic constriction (TAC) in heterozygous (Het) Ttn knock-in mice. Two weeks after TAC, Het mice developed marked impairment of left ventricular ejection fraction (p < 0.05), while wild-type (WT) TAC mice did not. Het mice also trended toward increased ventricular end diastolic pressure and volume compared to WT littermates. We found an increase in histologically diffuse cardiac fibrosis in Het compared to WT in TAC mice. This study shows that a pattern of DCM can be induced by TAC-mediated pressure overload in a TTN-truncated mouse model. This model enlarges our arsenal of cardiac disease models, adding a valuable tool to understand cardiac pathophysiological remodeling processes and to develop therapeutic approaches to combat heart failure.

G.P.34: The expanding spectrum of congenital titinopathies

Congenital core myopathies (CM) remain genetically unexplained in many cases. Heart disease is not considered part of the typical CM spectrum. Childhood-onset dilated cardiomyopathy (DCM) was reported in two families with minicores and homozygous TTN mutations. TTN encodes the giant sarcomere protein titin. Relatively few titin mutations (mostly heterozygous and associated to adult-onset cardiac or skeletal muscle disease) have been reported, TTN size and complexity precluding full screening before NGS. Thus, the prevalence and spectrum of titinopathies is probably underestimated. Here we report the analysis of 23 families with CM and primary heart disease using TTN M-line targeted sequencing followed in selected patients by whole-exome sequencing and functional studies. We found 7 novel homozygous or compound heterozygous TTN mutations (5 in the M-line, 5 truncating) in 5 patients. All heterozygous parents were healthy. We identified 4 novel phenotypes previously unreported or non-associated with TTN , including several congenital cardiopathies. The first antenatal-onset titinopathy presented with multi-minicore disease (MmD), arthrogryposis, left ventricular non-compaction and ventricular septal defect, and was associated with the first-reported absence of a functional titin kinase domain in humans. Other novel phenotypes associate MmD with auricular and/or ventricular septal defects, Rigid Spine, Emery-Dreifuss phenotype and/or adult-onset DCM. The histological pattern consistently showed minicores, central nuclei and structural lesions. Conversely, the spectrum of severity and heart disease was large. Thus, we demonstrate that TTN M-line truncating mutations are typically recessive and represent a significant cause of MmD with heart disease. Our results represent the first series of recessive titinopathies, expand TTN mutational and phenotypic spectrum, and identify TTN as a candidate gene in arthrogryposis, EDMD and pediatric heart defects.

A.P.2: Proteomic profile of cytoplasmic bodies (CB) compared to non-CB aggregates in HMERF associated with mutations in A-band titin

Hereditary myopathy with early respiratory failure (HMERF) is caused by A-band titin mutations and characterized by myofibrillar alterations and protein aggregation in skeletal muscle fibers. Our previous studies revealed that the composition of myotilin-positive aggregates is comparable to that seen in myofibrillar myopathies (MFM). However, there are two different types of protein aggregates in HMERF: subsarcolemmal cytoplasmic bodies (CB) with a dense filamentous core and non-CB aggregates. The aim of this study was to compare the proteomic profile of these protein deposits. We used Gomori trichrome stained skeletal muscle sections from two HMERF patients to separately collect CB, non-CB aggregates and intraindividual control samples by laser microdissection and performed a highly sensitive label-free mass spectrometry approach for protein detection and relative quantification based on spectral counting. The proteomic profiles of CB and non-CB aggregates showed a high degree of similarity: XIRP2, desmin, filamin C, N-RAP, nestin, COL6A3, myotilin, sarcosin, αB-crystallin, dystrophin, XIN and aciculin ranked among the most abundant over-represented proteins (ratio >1.5 compared to control samples) in both aggregate types. Peptides assigned to these twelve proteins accounted for 69% (CB) and 75% (non-CB aggregates) of all peptides assigned to over-represented proteins and the individual proportions of these proteins were almost the same. We also detected similarities in less abundant over-represented proteins. Titin was under-represented in CB (ratio 0.81) and in non-CB aggregates (ratio 0.80). In conclusion, our analysis revealed that the proteomic profiles of CB and non-CB aggregates are highly similar in HMERF, especially regarding abundant over-represented proteins, and typical of MFM aggregates.

A.P.7: Misfolding of fibronectin III 119 subdomain in titin results in hereditary myopathy with early respiratory failure

Hereditary myopathy with early respiratory failure (HMERF) is a rare disease with muscle weakness and respiratory failure as early symptoms. Muscle pathology is characterized by the presence of multiple cytoplasmic bodies and other protein aggregates in muscle fibers. These protein aggregates are composed of multiple proteins including titin, aB-crystallin, actin, myotilin, desmin and others. Previous reports have described mutations in A-band titin ( TTN ) gene associated with HMERF. All patients harbor mutations located in exon 343 in the TTN gene that codes for the fibronectin III domain 119 (FN3 119) in the 10th motif of the 11-element motif A-band super-repeat. Based on the pathological findings in HMERF of protein aggregates in muscle fibers, we investigated if disease-causing mutations affect the biochemical behavior of this titin domain. All five disease-causing amino acid changes analyzed by us (p.P30068R, p.C30071R, p.W30088R, p.W30088C and p.P30091L) resulted in impaired FN3 119 domain solubility. In contrast, amino acid changes associated with common SNPs (p.V30076I, p.R30107C and p.S30125F) did not have this effect. In silico analyses further support the notion that disease-causing mutations impair proper folding of the FN3 119 domain. Our results suggest that HMERF is caused by defective protein folding. We speculate that the FN3 119 domain could be important for protein–protein interaction that could explain why all identified mutations causing HMERF are located in this particular domain.

Abstract 279: A Deletion In The N2A Region Of Titin Carried By Muscular Dystrophy With Myositis (mdm) Mice Severely Affects Skeletal Muscle, But Not The Heart

20% of dilated cardiomyopathy patients carry mutations in the giant protein titin. Mutations are predominant in A band but also occur in I band, a domain that regulates passive tension and myocyte signaling. A recessive mouse mutation in titin I band N2A region (mdm) causes early onset muscular dystrophy with myositis and death. We assessed cardiac morphology, function, and transcriptional profiles (RNAseq) in mdm mice. Young homozygous mdm mice (n&gt;6) have reduced body weight (7gms) vs. heterozygous (20gm) or WT (17gm) littermates, with severe skeletal muscle dystrophy. Four-week old homozygous mdm mice have higher left ventricular (LV): body weight ratios. Echocardiography revealed thinner LV posterior wall and septum (LVPWd and IVSd) and normal LV diameter (LVDd); when normalized for body weight, cardiac dimensions were increased compared to WT or heterozygous mdm mice. Fractional shortening was reduced in homozygous Mdm mice (35%) vs. WT (40-41%, p&lt;0.01); histology showed neither overt pathology nor fibrosis. Titin gels showed lack of difference in cardiac titin isoform pattern, consistent with RNAseq, which showed the mdm titin transcript excluded exons 107 and 108, deleting in frame 48 amino acids. 240 transcripts (0.8%) were differentially expressed (fold change &gt;1.5 and &lt;0.75, p&lt;0.001) in homozygous vs. heterozygous mdm hearts; ANP and BNP were mildly upregulated (2- and 1.2-fold). Altered transcripts participated in extracellular and immune signaling pathways. Among titin binding partners, only calpain-3 that interacts with N2A was changed (0.6-fold), consistent with previous reports in skeletal muscle. As humans have heterozygous mutations, we stressed adult heterozygous mdm and WT mice (2 weeks of angiotensin II infusion): both had comparable hypertrophic responses (increased LVPWd and IVSd). Aged (89 week old) unstressed heterozygous mdm mice had normal cardiac dimensions and function. The N2A region, I-band titin mdm mutation causes minimal cardiac dysfunction in mice, unlike the severe skeletal muscle phenotype. Human I-band mutations are unlikely to cause dilated cardiomyopathy.

The zebrafish as a tool to identify novel therapies for human cardiovascular disease.

Over the past decade, the zebrafish has become an increasingly popular animal model for the study of human cardiovascular disease. Because zebrafish embryos are transparent and their genetic manipulation is straightforward, the zebrafish has been used to recapitulate a number of cardiovascular disease processes ranging from congenital heart defects to arrhythmia to cardiomyopathy. The use of fluorescent reporters has been essential to identify two discrete phases of cardiomyocyte differentiation necessary for normal cardiac development in the zebrafish. These phases are analogous to the differentiation of the two progenitor heart cell populations in mammals, termed the first and second heart fields. The small size of zebrafish embryos has enabled high-throughput chemical screening to identify small-molecule suppressors of fundamental pathways in vasculogenesis, such as the BMP axis, as well as of common vascular defects, such as aortic coarctation. The optical clarity of zebrafish has facilitated studies of valvulogenesis as well as detailed electrophysiological mapping to characterize the early cardiac conduction system. One unique aspect of zebrafish larvae is their ability to oxygenate through diffusion alone, permitting the study of mutations that cause severe cardiomyopathy phenotypes such as silent heart and pickwickm171, which mimic titin mutations observed in human dilated cardiomyopathy. Above all, the regenerative capacity of zebrafish presents a particularly exciting opportunity to discover new therapies for cardiac injury, including scar formation following myocardial infarction. This Review will summarize the current state of the field and describe future directions to advance our understanding of human cardiovascular disease.

Gigantic Business

The giant protein titin forms a unique filament network in cardiomyocytes, which engages in both mechanical and signaling functions of the heart. TTN, which encodes titin, is also a major human disease gene. In this review, we cover the roles of cardiac titin in normal and failing hearts, with a special emphasis on the contribution of titin to diastolic stiffness. We provide an update on disease-associated titin mutations in cardiac and skeletal muscles and summarize what is known about the impact of protein-protein interactions on titin properties and functions. We discuss the importance of titin-isoform shifts and titin phosphorylation, as well as titin modifications related to oxidative stress, in adjusting the diastolic stiffness of the healthy and the failing heart. Along the way we distinguish among titin alterations in systolic and in diastolic heart failure and ponder the evidence for titin stiffness as a potential target for pharmacological intervention in heart disease.

Gene Expression Profiling in Tibial Muscular Dystrophy Reveals Unfolded Protein Response and Altered Autophagy

Tibial muscular dystrophy (TMD) is a late onset, autosomal dominant distal myopathy that results from mutations in the two last domains of titin. The cascade of molecular events leading from the causative Titin mutations to the preterm death of muscle cells in TMD is largely unknown. In this study we examined the mRNA and protein changes associated with the myopathology of TMD. To identify these components we performed gene expression profiling using muscle biopsies from TMD patients and healthy controls. The profiling results were confirmed through quantitative real-time PCR and protein level analysis. One of the pathways identified was activation of endoplasmic reticulum (ER) stress response. ER stress activates the unfolded protein response (UPR) pathway. UPR activation was supported by elevation of the marker genes HSPA5, ERN1 and the UPR specific XBP1 splice form. However, UPR activation appears to be insufficient to correct the protein abnormalities causing its activation because degenerative TMD muscle fibres show an increase in ubiquitinated protein inclusions. Abnormalities of VCP-associated degradation pathways are also suggested by the presence of proteolytic VCP fragments in western blotting, and VCP's accumulation within rimmed vacuoles in TMD muscle fibres together with p62 and LC3B positive autophagosomes. Thus, pathways controlling turnover and degradation, including autophagy, are distorted and lead to degeneration and loss of muscle fibres.

Cardiac Titin and Heart Disease

The giant sarcomeric protein titin is a key determinant of myocardial passive stiffness and stress-sensitive signaling. Titin stiffness is modulated by isoform variation, phosphorylation by protein kinases, and, possibly, oxidative stress through disulfide bond formation. Titin has also emerged as an important human disease gene. Early studies in patients with dilated cardiomyopathy (DCM) revealed shifts toward more compliant isoforms, an adaptation that offsets increases in passive stiffness based on the extracellular matrix. Similar shifts are observed in heart failure with preserved ejection fraction. In contrast, hypophosphorylation of PKA/G sites contributes to a net increase in cardiomyocyte resting tension in heart failure with preserved ejection fraction. More recently, titin mutations have been recognized as the most common etiology of inherited DCM. In addition, some DCM-causing mutations affect RBM20, a titin splice factor. Titin mutations are a rare cause of hypertrophic cardiomyopathy and also underlie some cases of arrhythmogenic right ventricular dysplasia. Finally, mutations of genes encoding proteins that interact with and/or bind to titin are responsible for both DCM and hypertrophic cardiomyopathy. Targeting titin as a therapeutic strategy is in its infancy, but it could potentially involve manipulation of isoforms, posttranslational modifications, and upregulation of normal protein in patients with disease-causing mutations.

Mechano-signaling in heart failure

Mechanosensation and mechanotransduction are fundamental aspects of biology, but the link between physical stimuli and biological responses remains not well understood. The perception of mechanical stimuli, their conversion into biochemical signals, and the transmission of these signals are particularly important for dynamic organs such as the heart. Various concepts have been introduced to explain mechanosensation at the molecular level, including effects on signalosomes, tensegrity, or direct activation (or inactivation) of enzymes. Striated muscles, including cardiac myocytes, differ from other cells in that they contain sarcomeres which are essential for the generation of forces and which play additional roles in mechanosensation. The majority of cardiomyopathy causing candidate genes encode structural proteins among which titin probably is the most important one. Due to its elastic elements, titin is a length sensor and also plays a role as a tension sensor (i.e., stress sensation). The recent discovery of titin mutations being a major cause of dilated cardiomyopathy (DCM) also underpins the importance of mechanosensation and mechanotransduction in the pathogenesis of heart failure. Here, we focus on sarcomere-related mechanisms, discuss recent findings, and provide a link to cardiomyopathy and associated heart failure.

Atypical phenotypes in titinopathies explained by second titin mutations

Several patients with previously reported titin gene (TTN) mutations causing tibial muscular dystrophy (TMD) have more complex, severe, or unusual phenotypes. This study aimed to clarify the molecular cause of the variant phenotypes in 8 patients of 7 European families. Clinical, histopathological, and muscle imaging data of patients and family members were reanalyzed. The titin protein was analyzed by Western blotting and TTN gene by reverse transcription polymerase chain reaction (RT-PCR) and Sanger sequencing. Western blotting showed more pronounced C-terminal titin abnormality than expected for heterozygous probands, suggesting the existence of additional TTN mutations. RT-PCR indicated unequal mRNA expression of the TTN alleles in biopsies of 6 patients, 3 with an limb-girdle muscular dystrophy type 2J (LGMD2J) phenotype. Novel frameshift mutations were identified in 5 patients. A novel A-band titin mutation, c.92167C>T (p.P30723S), was found in 1 patient, and 1 Portuguese patient with a severe TMD phenotype proved to be homozygous for the previously reported Iberian TMD mutation. The unequal expression levels of TTN transcripts in 5 probands suggested severely reduced expression of the frameshift mutated allele, probably through nonsense-mediated decay, explaining the more severe phenotypes. The Iberian TMD mutation may cause a more severe TMD rather than LGMD2J when homozygous. The Finnish patient compound heterozygous for the FINmaj TMD mutation and the novel A-band titin missense mutation showed a phenotype completely different from previously described titinopathies. Our results further expand the complexity of muscular dystrophies caused by TTN mutations and suggest that the coexistence of second mutations may constitute a more common general mechanism explaining phenotype variability.

Posttranslational Modifications as Modulators of Mechanical Protein Folding

Understanding the molecular basis of muscle elasticity is an important challenge in medicine. For example, mutations in the giant elastic protein titin are implicated in the etiology of dilated cardiomyopathies. We use single molecule force-clamp spectroscopy to study the folding and unfolding dynamics of single polyproteins composed of tandem repeats of titin Ig domains. Protein elasticity is due to the diffusional extension and collapse of a polyprotein along its free energy landscape. Using single molecule force-clamp traces we calculate the free energy of a polyprotein along its stretching coordinate under different mechanical force regimes. At high stretching forces the free energy is dominated by changes in entropy as the molecule unfolds and extends. When the force is quenched, enthalpic interactions become important as the polyprotein slowly progresses from collapsed states to molten globules and then to the mechanically stable native state. We also show that while the polyprotein is in the extended state, cysteine residues that are normally buried become exposed to the solution where they can be post-translationally modified. We have found that S-glutathionylation of such cryptic cysteines greatly decreases the mechanical stability of an Ig domain as well as its ability to fold, favoring more extensible states in titin. Thus, the free energy landscape of a protein extending and folding under force is strongly modulated by the redox state of a cell. We propose that posttranslational modification of cryptic cysteine residues is a major regulatory pathway of tissue elasticity. Accurate predictions of elastic phenotypes in titin will be possible after we fully understand how cellular chemistry modulates the physics of a polypeptide extending under force.

Use of a murine Advanced Intercross Line combined with whole genome sequencing and transcriptome analysis confirms previously discovered QTL and identifies candidate genetic loci for cardiac atrial septal morphology

truncating Titin (TTN) mutations are present in 25% of familial DCM cases; however, it is unclear whether they are sufficient to cause disease or modify disease susceptibility. The aim of this study was to investigate the prevalence of TTN mutations in an Australian DCM cohort. Targeted re-sequencing of 69 cardiomyopathy genes was conducted on DNA from 60 probands with familial DCM. Truncating TTN mutations were identified in 18 probands (30%) and detailed analysis of genotype-phenotype correlation was performed in six of these families. There was good correlation between truncating TTNmutations and disease in three families with TTN mutations present in all the affected and none of the unaffected family members tested. In the remaining three families, six individualswith TTNmutations but normal echocardiograms were identified. However, these individuals were all younger than 50 years of age at testing suggesting the possibility of age-dependent penetrance. Varying age of diagnosis and disease severity suggest the involvement of additional genetic or acquired factors in at least one family. Truncating TTN mutations are present in a significant proportion of our familial DCM cases. However, further work is required to classify the segregation of these mutations with disease in larger cohorts. Inclusion of TTN in genetic screening should improve the yield from genetic testing to greater than 50% and may potentially improve the management and treatment for these families.

Truncating Titin mutations: Are they a primary cause of dilated cardiomyopathy or a susceptibility factor?

Truncating Titin mutations: Are they a primary cause of dilated cardiomyopathy or a susceptibility factor?

Determining the Molecular Mechanisms that Link a Titin Mutation to Cardiomyopathy

A mutation in the giant elastic protein titin has recently been linked to arrhythmogenic cardiomyopathy (AC), a disease primarily characterized by fibrofatty infiltration of the myocardium. Titin is the first sarcomeric protein linked to AC. In addition to largely unstructured segments, the extensible I-band region of titin contains ∼40 immunoglobulin(Ig)-like domains that are natively folded as highly stable beta barrels. One of these domains, Ig10, contains a single Thr→Ile mutation that leads to AC. The first step in determining how this mutation leads to severe cardiac disease was to study the mutation at the single molecule level using atomic force microscopy (AFM) and in vitro degradation assays. With AFM individual proteins are mechanically stretched, and it was found that the mutation significantly reduces the force needed to unfold Ig10. Although no difference in refolding rate was found, by comparing the experimental AFM unfolding forces with computer simulated Ig unfolding, it was found that the mutation increases Ig10's unfolding rate fourfold. This effect predicts increased Ig10 unfolding (i.e. transition into a non-native state) under physiological conditions. It was also found that mutant Ig10 is more prone to protease degradation due to compromised local structure at the point mutation. This suggests that accelerated titin turnover in cardiomyocytes instigates the cardiac remodeling process in patients with titin-linked AC. This is the first time an Ig domain in titin's elastic I-band region has been linked to cardiac disease, and this study proposes a novel disease mechanism contingent on Ig domain unfolding.

What's in the Literature?

In amyotrophic lateral sclerosis (ALS), a recent double-blind placebo-controlled trial of acetyl-L-carnitine along with riluzole showed probable benefit in 42 patients compared with 40 patients who received placebo. Using an electrophysiologic measure devised to differentiate ALS from other neuromuscular conditions, a "splint-hand index" was devised and is reviewed. Analysis of skin in ALS may also be of interest, and there was a report of accumulation of fused in sarcoma protein in the epidermis of ALS patients. With regard to myasthenia gravis, there is another report that early treatment of ocular symptoms with corticosteroids may prevent the development of generalized symptoms. A new study of thymus histopathology in muscle-specific tyrosine kinase (MuSK) myasthenia gravis is covered and another article on the use of thymectomy. In Duchenne muscular dystrophy, the best method of administrating corticosteroids is still being debated, and a long-term study of daily versus intermittent prednisolone is reviewed. The study showed less sustained benefit from the intermittent prednisolone but with a better side effect profile. According to 2 recent reports, it seems that titin mutations may be an underrecognized cause of myopathy with early respiratory failure in adults. Keeping with the respiratory failure theme, there was also an interesting article on the long-term benefits and side effects of cyclosporine in patients with interstitial lung disease from antisynthetase syndrome. Finally, the spectrum of small fiber neuropathy may be expanding to include a causative role in some patients with fibromyalgia syndrome and in juveniles with diffuse pain and a possible autoimmune predisposition.

O.4 Proteomic analysis confirms that HMERF associated with mutations in A-band titin is a new subtype of myofibrillar myopathies

Hereditary myopathy with early respiratory failure (HMERF) caused by A-band TTN mutations shows a clinical and histopathological overlap with myofibrillar myopathies (MFM) characterized by disintegration of myofibrils and protein aggregation in muscle fibers. We applied a proteomic approach to decipher the aggregate composition in HMERF and compared the proteomic profile with findings in MFM subtypes. Nine skeletal muscle samples from HMERF patients with three different mutations in A-band titin and histopathological findings consistent with MFM were analyzed. Protein aggregates and intraindividual control samples (from aggregate-free muscle fibers) were collected by laser microdissection and analysed by a label-free mass spectrometric approach for identification and relative quantification of proteins. Fifty-eight proteins showed a statistically significant accumulation in aggregates with a ratio > 1.8 compared to control samples. The over-represented proteins desmin, filamin C, Xirp2, N-RAP, alphaB-crystallin, nestin, myotilin, Xin and Hsp27 were highly abundant in aggregates. The same was found in different MFM subtypes. The detection of further Z-disc proteins, chaperones, proteins involved in protein degradation and sarcolemmal proteins was also typical of MFM. In addition, we identified an over-representation of proteins that are involved in signaling pathways (e.g. inhibitors of the Rho/ROCK signaling pathway and ankyrin repeat domain-containing proteins). Titin was slightly but significant under-represented in aggregates (ratio 0.83). In conclusion, the proteomic profile of aggregates in HMERF associated with mutations in A-band titin is typical of MFM and confirms that this type of titinopathy is a new MFM subtype. The finding that titin is under-represented in aggregates indicates that aggregate formation is mediated by secondary effects. The detected over-representation of proteins involved in signaling pathways may play a role in pathogenesis.