A-band Region Research Articles

A recommended line list for water vapor in the 12,969–13,418cm-1 interval

We have recently devoted two reports to the very weak absorption spectrum of water vapor in the 12,969 – 13,417 cm-1 region by high sensitivity cavity ring down spectroscopy (CRDS) [Vasilchenko et al. J. Quant. Spectrosc. Radiat. Transfer 275 (2021) 107,847; doi.org/10.1016/j.jqsrt.2021.107847; Vasilchenko et al. Molecular Physics e2051762; doi.org/10.1080/00,268,976.2022.2051762]. This spectral interval is important because it includes the region of the oxygen A-band near 760 nm. The achieved sensitivity allowed us to measure lines with intensity on the order of 10–29 cm molecule-1, about two orders of magnitude lower than the detectivity threshold of previous studies. A total of about 240 energy levels of H216O were newly determined. Our comparison to the W2020 empirical levels [Furtenbacher et al. J. Phys. Chem. Ref. Data 49 (2020) 043,103; doi.org/10.1063/5.0030680] led us to identify and correct about 300 W2020 energy levels of the main isotopologue differing from our energy value by more than 5 × 10–3 cm-1. In the present contribution, we consider in details the sources of the W2020 energy levels to identify the origin of these deviations. We confirm that more than 260 energy levels should be corrected but for the remaining thirty-two levels, the W2020 energy values are in fact, more reliable. As a result, we propose an improved list of energy levels retrieved from the CRDS data and a recommended line list for water vapor in natural isotopic abundance in the 12,969 – 13,417 cm-1 region, mostly based on the CRDS line parameters.

Molecular insights into titin’s A-band

The thick filament-associated A-band region of titin is a highly repetitive component of the titin chain with important scaffolding properties that support thick filament assembly. It also has a demonstrated link to human disease. Despite its functional significance, it remains a largely uncharacterized part of the titin protein. Here, we have performed an analysis of sequence and structure conservation of A-band titin, with emphasis on poly-FnIII tandem components. Specifically, we have applied multi-dimensional sequence pairwise similarity analysis to FnIII domains and complemented this with the crystallographic elucidation of the 3D-structure of the FnIII-triplet A84-A86 from the fourth long super-repeat in the C-zone (C4). Structural models serve here as templates to map sequence conservation onto super-repeat C4, which we show is a prototypical representative of titin’s C-zone. This templating identifies positionally conserved residue clusters in C super-repeats with the potential of mediating interactions to thick-filament components. Conservation localizes to two super-repeat positions: Ig domains in position 1 and FnIII domains in position 7. The analysis also allows conclusions to be drawn on the conserved architecture of titin’s A-band, as well as revisiting and expanding the evolutionary model of titin’s A-band.

Different phosphorylation states of native titin filaments visualized by atomic force microscopy.

During muscle stretch, elastic or “passive” force develops which is mainly determined by the giant protein titin that forms the third filament system of muscle sarcomeres. The magnitude of this force is mainly dependent on the elasticity of the titin filaments, which is primarily determined by the structure of the polypeptide chain. However, it is suggested that post-translational modifications of titin, such as phosphokinase activity, regulate the sarcomeric passive force development. Mechanical studies on single myofibrils revealed that different protein kinases alter the passive tension of muscle, surprisingly, in opposite ways. This suggests that phosphorylation of sarcomeric proteins by various kinases is an essential regulatory mechanism of passive force. However, the extrapolation of these findings to titin's phosphorylation has not been studied so far at the single-molecule level. To reveal titin's structural alterations due to phosphorylation, single-molecule experiments must be carried out on individual titin molecules, where the effect of phosphorylation can be tested individually. In our work, we isolated individual titin molecules from rabbit and mouse skeletal muscle. The isolated native titin molecules showed high levels of phosphorylation, when stained with phosphoprotein gel stain. The isolated molecules were treated with lambda protein phosphatase to decrease the in situ phosphorylation level of the polymer. To investigate the effect of the treatment on titin's structure, we visualized surface-bound titin molecules by atomic force microscope and found that the C-terminal region of dephosphorylated titins collapses into a compact, coiled structure. Our findings suggest that the structure of the A-band region of titin is highly sensitive for phospohrylation which might play a role in controlling protein-titin interactions in the A-band.

Effect of Novel High-Intensity Ultrasound Technique on Physio-Chemical, Sensory Attributes, and Microstructure of Bovine Semitendinosus Muscle.

The present study aimed to evaluate the effects of high-intensity ultrasound (HIU) application on meat quality traits, sensory parameters, and the microstructure of semitendinosus muscle from Hanwoo cattle. The samples were treated in an ultrasonic bath (35 kHz) at an intensity of 800 W/cm2 for 60 min, followed by aging at 1°C for 0, 3, and 7 days. The application of ultrasound resulted in lower Warner-Bratzler shear force and higher myofibrillar fragmentation index values during the storage period. HIU also enhanced the tenderness, flavor, umami, and overall acceptability of cooked beef muscle. However, the electronic tongue evaluation results showed higher umami values in the control treatment on the seventh day of storage. The microstructure of sonicated meat showed disorganized myofibrillar architecture and swelling in the A-band region of sarcomeres during the storage period, which led to greater meat tenderness. The heatmap illustrated the high abundance of α-linolenic acid (C20:5n3) and eicosapentaenoic acid (C18:3n3) in sonicated meat samples on the third day of the storage. These results showed that HIU is a potential method for tenderizing and improving the sensory attributes of beef without compromising other quality aspects.

Study of the expression of Cronos titin in TTN-truncation cardiomyopathy and heart development

Cronos is a recently discovered isoform of the giant protein titin driven by an alternative internal promoter. Cronos spans the half-A-band of a sarcomere and binds to myosin filaments, but its role in healthy or diseased hearts is largely unknown. Upregulation of Cronos has been suggested as a possible compensatory mechanism in titin-truncation cardiomyopathy, to help explain why truncations in titin's I-band region are less frequent and often cause a milder disease phenotype than truncations in the A-band region.

Water vapor absorption in the region of the oxygen A-band near 760 nm

The oxygen A-band near 760 nm is used to determine the air-mass along the line of sight from ground or space borne atmospheric spectra. This band is located in a spectral region of very weak absorption of water vapor. The increased requirements on the determination of the air columns make suitable to accurately characterize water absorption spectrum in the region. In the present work, we use a cavity ring down spectrometer newly developed in Tomsk, to measure with unprecedented sensitivity and accuracy the water spectrum in the 12969 - 13172 cm−1 region. While about fifty transitions were previously detected in the region, a total of about 580 water lines are measured by CRDS and rovibrationally assigned, leading to the determination of 103 new levels and correction of 134 levels of H216O. Spectroscopic line lists available in the region (HITRAN, W2020 and theoretical line lists) show some important deviations compared to observations. In particular, line intensities are poorly predicted by available ab initio calculations for transitions involving a highly bending excitation.

A Novel Titin Truncation Variant Linked to Familial Dilated Cardiomyopathy Found in a Japanese Family and Its Functional Analysis in Genome-Edited Model Cells

Dilated cardiomyopathy (DCM) is a common cause of heart failure. TTN, which encodes titin protein, is a representative causative gene of DCM, and is presented mainly as a truncation variant. However, TTN truncation variants are also found in healthy individuals, and it is therefore important to evaluate the pathogenicity of each variant. In this study, we analyzed 67 cardiomyopathy-associated genes in a male Japanese patient who was hospitalized for recurrent severe heart failure and identified a novel truncation variant, TTN Ser17456Arg fs*14. This TTN truncation variant was located in the A-band region. Moreover, the patient's mother with heart failure harbored the same variant, whereas the father and brother without heart failure did not harbor the variant. To examine the functional changes associated with the truncation variant, H9c2 cells were subjected to genome editing to generate cells with a homologous truncation variant. The cells were differentiated using all-trans-retinoic acid, and the mRNA expression of skeletal actin and cardiac actin were found to be increased and decreased, respectively, consistent with known changes in patients with DCM or heart failure. In contrast, another cell with the titin truncation variant used as a control showed no changes in heart failure-related genes. In summary, we found a novel TTN truncation variant in familial DCM patients and confirmed its functional changes using a relatively simple cell model. The novel truncation variant was identified as a pathogenic and disease-causing mutation.

Fine mapping titin's C-zone: Matching cardiac myosin-binding protein C stripes with titin's super-repeats

Titin is largely comprised of serially-linked immunoglobulin (Ig) and fibronectin type-III (Fn3) domains. Many of these domains are arranged in an 11 domain super-repeat pattern that is repeated 11 times, forming the so-named titin C-zone in the A-band region of the sarcomere. Each super-repeat is thought to provide binding sites for thick filament proteins, such as cMyBP-C (cardiac myosin-binding protein C). However, it remains to be established which of titin's 11 C-zone super-repeats anchor cMyBP-C as titin contains 11 super-repeats and cMyBP-C is found in 9 stripes only. To study the layout of titin's C-zone in relation to MyBP-C, immunolabeling studies were performed on mouse skinned myocardium with antibodies to titin and cMyBP-C, using both immuno-electron microscopy and super-resolution optical microscopy. Results indicate that cMyBP-C locates near the interface between titin's C-zone super-repeats. Studies on a mouse model in which two of titin's C-zone repeats have been genetically deleted support that the first Ig domain of a super-repeat is important for anchoring cMyBP-C but also Fn3 domains located at the end of the preceding repeat. Furthermore, not all super-repeat interfaces are equal as the interface between super-repeat 1 and 2 (close to titin's D-zone) does not contain cMyBP-C. Finally, titin's C-zone does not extend all the way to the bare zone but instead terminates at the level of the second myosin crown. This study enhances insights in the molecular layout of the C-zone of titin, its relation to cMyBP-C, and its possible roles in cardiomyopathies.

Relevance of Titin Missense and Non-Frameshifting Insertions/Deletions Variants in Dilated Cardiomyopathy

Recent advancements in next generation sequencing (NGS) technology have led to the identification of the giant sarcomere gene, titin (TTN), as a major human disease gene. Truncating variants of TTN (TTNtv) especially in the A-band region account for 20% of dilated cardiomyopathy (DCM) cases. Much attention has been focused on assessment and interpretation of TTNtv in human disease; however, missense and non-frameshifting insertions/deletions (NFS-INDELs) are difficult to assess and interpret in clinical diagnostic workflow. Targeted sequencing covering all exons of TTN was performed on a cohort of 530 primary DCM patients from three cardiogenetic centres across Europe. Using stringent bioinformatic filtering, twenty-nine and two rare TTN missense and NFS-INDELs variants predicted deleterious were identified in 6.98% and 0.38% of DCM patients, respectively. However, when compared with those identified in the largest available reference population database, no significant enrichment of such variants was identified in DCM patients. Moreover, DCM patients and reference individuals had comparable frequencies of splice-region missense variants with predicted splicing alteration. DCM patients and reference populations had comparable frequencies of rare predicted deleterious TTN missense variants including splice-region missense variants suggesting that these variants are not independently causative for DCM. Hence, these variants should be classified as likely benign in the clinical diagnostic workflow, although a modifier effect cannot be excluded at this stage.

The Giant Protein Titin Regulates the Length of the Striated Muscle Thick Filament-Titin Rules

The contractile machinery of heart and skeletal muscles that powers life has as its most essential component the thick filament, comprised of the molecular motor myosin. The thick filament is of a precisely controlled length, defining thereby the force level that muscles generate and how this force varies with muscle length. It has been speculated that the mechanism by which the length of the thick filament is so exquisitely controlled involves the giant protein titin, but no conclusive support for or against exists. Here we show studies on a mouse model in which we deleted two of titin's C-zone super-repeats, located in the A-band region of the sarcomere. Structural studies using immunoelectron microscopy and super-resolution optical microscopy in both cardiac and skeletal muscles reveal a thick filament length that is reduced by ∼170 nm; functional studies reveal reduced force generation and a dilated cardiomyopathy (DCM) phenotype. Our studies show for the first time the important role of titin in regulating thick filament length, with each of titin's C-zone repeats being responsible for a quantal 43 nm thick filament length. We conclude that thick filament length regulation is titin-based and that this mechanism is crucial for maintaining muscle health.

Titin-truncating mutations in dilated cardiomyopathy: the long and short of it.

Truncating variants in the TTN gene (TTNtv) are frequently identified in patients with dilated cardiomyopathy (DCM) but are also present in apparently healthy people in the general population. Consequently, there is considerable uncertainty about what it means for any single individual if a TTNtv is found. The aim of this review is to summarize current evidence implicating TTNtv in DCM pathogenesis and to provide some interpretative guidelines for clinical management. Next-generation sequencing studies have recently demonstrated that TTNtv are present in approximately one in five patients with DCM but also in up to 3% of individuals in the general population. These observations question whether TTNtv are sufficient alone to cause DCM and whether some TTNtv may be more deleterious than others. It has been suggested that functional effects of TTNtv can be predicted by their location in the titin protein, with DCM-associated variants typically occurring in the A-band region and/or in exons that are highly utilized across the range of titin isoforms. Recent data from animal and cell models suggest that developmental defects in the structural assembly of titin-deficient sarcomeres provide a template for mechanical stress-induced myocardial dysfunction during later life. Not all TTNtv are equal, and variants in constitutively expressed exons have the greatest likelihood of pathogenicity. The clinical significance of high-impact TTNtv is likely to differ according to patient context and each individual's unique suite of background genetic factors, comorbidities, and lifestyle factors. TTNtv identified in patients with DCM can be expected to have a major role in disease pathogenesis, but whether unaffected individuals with TTNtv will develop DCM is less certain.

Structural and Biophysical Analysis of Hypertrophic Cardiomyopathy-Linked Titin Missense Variants

Cardiomyopathies are an important cause of premature death from heart failure, ventricular arrhythmia and stroke. The most common subtype, hypertrophic cardiomyopathy (HCM), is an inherited disease that results in the thickening of the myocardium, whilst at the cellular level the sarcomere - the contractile unit of cardiac and skeletal muscle - is disrupted. The advent of high throughput sequencing has enabled the identification of variants in physiologically relevant genes, aiding the search for causative mutations of HCM. One gene that is now amenable to sequencing in HCM patients is TTN, which encodes Titin, a multidomain, multifunctional filament that spans half a sarcomere in striated muscle. Titin is essential for sarcomere assembly and plays a variety of roles in the heart, including providing passive muscle elasticity during the contraction-relaxation cycle and signaling via its kinase domain. The protein contains over 300 immunoglobulin and fibronectin III domains, the majority of which are located along the myosin-associated A-band region of the sarcomere. Whole exome sequencing of a cohort of HCM patients has identified a large number of missense variants in Titin, particularly in the A-band region of the protein. Whilst many of the variants are likely neutral, a number are enriched when compared to their frequency in control cohorts. We present here the structural and biophysical analysis of five Titin A-band fibronectin III domains and their associated variants enriched in either the HCM cohort or in the general population. The crystal structures of two wild type and HCM-linked variant domains have been determined, and thermal denaturation experiments demonstrate both variants have a reduced stability when compared to their wild type counterparts, suggesting a causal role in HCM and hinting at a method to distinguish pathogenic from non-pathogenic Titin missense variants.

Targeted Next-Generation Sequencing Reveals Novel TTN Mutations Causing Recessive Distal Titinopathy.

Tibial muscular dystrophy (TMD) is the first described human titinopathy. It is a mild adult-onset slowly progressive myopathy causing weakness and atrophy in the anterior lower leg muscles. TMD is caused by mutations in the last two exons, Mex5 and Mex6, of the titin gene (TTN). The first reported TMD mutations were dominant, but the Finnish founder mutation FINmaj, an 11-bp insertion/deletion in Mex6, in homozygosity caused a completely different severe early-onset limb-girdle muscular dystrophy 2J (LGMD2J). Later, we reported that not all TMD mutations cause LGMD when homozygous or compound heterozygous with truncating mutation, but some of them rather cause a more severe TMD-like distal disease. We have now performed targeted next-generation sequencing of myopathy-related genes on seven families from Albania, Bosnia, Iran, Tunisia, Belgium, and Spain with juvenile or early adult onset recessive distal myopathy. Novel mutations in TTN Mex5, Mex6 and A-band exon 340 were identified in homozygosity or compound heterozygosity with a frameshift or nonsense mutation in TTN I- or A-band region. Family members having only one of these TTN mutations were healthy. Our results add yet another entity to the list of distal myopathies: juvenile or early adult onset recessive distal titinopathy.

Relevance of truncating titin mutations in dilated cardiomyopathy.

Dilated cardiomyopathy (DCM), a genetically heterogeneous cardiac disease characterized by left ventricular dilatation and systolic dysfunction, is caused majorly by truncations of titin (TTN), especially in A-band region. Clinical interpretation of TTN-truncating variants (TTNtv) has been challenged by the existing inaccurate variant assessment strategies and uncertainty in the true frequency of TTNtv across the general population. We aggregated TTNtv identified in 1788 DCM patients and compared the variants with those reported in over 60,000 Exome Aggregation Consortium reference population. We implemented our current variant assessment strategy that prioritizes TTNtv affecting all transcripts of the gene, and observed a decline in the prevalence of TTNtv in DCM. Despite this decline, TTNtv are more prevalent in DCM patients compared with reference population (p = 4.1 × 10(-295) ). Moreover, our extended analyses confirmed the enrichment of TTNtv not only in the A-band but also in the I/A-band junction of TTN. We estimated the probability of pathogenicity of TTNtv affecting all transcripts of TTN, identified in unselected DCM patients to be 97.8% (likelihood ratio (LR) = 42.2). We emphasize that identifying a TTNtv, especially in the A-band region, has a higher risk of being disease-causing than previously anticipated, and recommend prioritizing TTNtv affecting at least five transcripts of the gene.

Exercise Mitigates Increased Diastolic Stiffness in the Titin IA KO Mouse

The giant sarcomeric protein titin spans the length of the half sarcomere and consists of an I-band region that functions as a molecular spring and an A-band region. Here we use a mouse model in which a portion of titin near the IA junction has been removed (IA KO) resulting in diastolic dysfunction and increased total and titin-based passive tension. We test the hypothesis that exercise ameliorates increased titin-based stiffness. In this study, 3 month old male IA KO and WT mice were allowed free access to a running wheel for 28 days and distance, speed, and duration were recorded each night. After 28 days mice were sacrificed and the LV was used for skinned muscle mechanics, analysis gels, and western blots. WT and IA KO mice ran for the same duration but IA KO mice ran slower and therefore less distance than their WT counterparts (WT mice ran ∼6 km per night while IA KOs ran ∼4.5 km). Stiffness in both WT and IA KO mice was reduced after exercise; titin-based tension at a sarcomere length of 2.3 μm was reduced by ∼16% and ∼20% respectively. No changes in titin isoform ratios were observed but exercised mice showed hypo-phosphorylation of S26, a PKC site in titin's PEVK region. S26 phosphorylation is known to increase titin's stiffness so hypo-phosphorylation is consistent with the reduction in stiffness observed in exercised mice. We conclude that exercise improves diastolic dysfunction though modulation of titin phosphorylation.

Prevalence of Titin Truncating Variants in General Population.

BackgroundTruncating titin (TTN) mutations, especially in A-band region, represent the most common cause of dilated cardiomyopathy (DCM). Clinical interpretation of these variants can be challenging, as these variants are also present in reference populations. We carried out systematic analyses of TTN truncating variants (TTNtv) in publicly available reference populations, including, for the first time, data from Exome Aggregation Consortium (ExAC). The goal was to establish more accurate estimate of prevalence of different TTNtv to allow better clinical interpretation of these findings.Methods and ResultsUsing data from 1000 Genomes Project, Exome Sequencing Project (ESP) and ExAC, we estimated the prevalence of TTNtv in the population. In the three population datasets, 52–54% of TTNtv were not affecting all TTN transcripts. The frequency of truncations affecting all transcripts in ExAC was 0.36% (0.32% - 0.41%, 95% CI) and 0.19% (0.16% - 0.23%, 95% CI) for those affecting the A-band. In the A-band region, the prevalences of frameshift, nonsense and essential splice site variants were 0.057%, 0.090%, and 0.047% respectively. Cga/Tga (arginine/nonsense–R/*) transitional change at CpG mutation hotspots was the most frequent type of TTN nonsense mutation accounting for 91.3% (21/23) of arginine residue nonsense mutation (R/*) at TTN A-band region. Non-essential splice-site variants had significantly lower proportion of private variants and higher proportion of low-frequency variants compared to essential splice-site variants (P = 0.01; P = 5.1 X 10−4, respectively).ConclusionA-band TTNtv are more rare in the general population than previously reported. Based on this analysis, one in 500 carries a truncation in TTN A-band suggesting the penetrance of these potentially harmful variants is still poorly understood, and some of these variants do not manifest as autosomal dominant DCM. This calls for caution when interpreting TTNtv in individuals and families with no history of DCM. Considering the size of TTN, expertise in DNA library preparation, high coverage NGS strategies, validated bioinformatics approach, accurate variant assessment strategy, and confirmatory sequencing are prerequisites for reliable evaluation of TTN in clinical settings, and ideally with the inclusion of mRNA and/or protein level assessment for a definite diagnosis.

G.P.323 - Targeted next-generation sequencing reveals novel TTN mutations causing recessive distal titinopathy

Tibial muscular dystrophy (TMD) is the first described human titinopathy. It is a mild adult-onset slowly progressive myopathy causing weakness and atrophy in the anterior lower leg muscles. TMD is caused by mutations in the last two exons, Mex5 and Mex6, of the titin gene (TTN). The first reported TMD mutations were dominant, but the Finnish founder mutation FINmaj, an 11-bp insertion/deletion in Mex6, in homozygosity caused a completely different severe early-onset limb-girdle muscular dystrophy 2J (LGMD2J). Later, we reported that not all TMD mutations cause LGMD when homozygous or compound heterozygous with truncating mutation, but some of them rather cause a more severe TMD-like distal disease. We have now performed targeted next-generation sequencing of myopathy-related genes on seven families from Albania, Bosnia, Iran, Tunisia, Belgium, and Spain with juvenile or early adult onset recessive distal myopathy. Novel mutations in TTN Mex5, Mex6 and A-band exon 340 were identified in homozygosity or compound heterozygosity with a frameshift or nonsense mutation in TTN I- or A-band region. Family members having only one of these TTN mutations were healthy. Our results add yet another entity to the list of distal myopathies: juvenile or early adult onset recessive distal titinopathy.

High pressure Cavity Ring Down Spectroscopy: Application to the absorption continuum of CO2 near 1.7 µm

A Cavity Ring Down Spectrometer has been developed for high sensitivity absorption spectroscopy in the near infrared at pressure up to 10bar. In order to strictly avoid perturbations of the optical alignment by pressure forces, the pre-aligned CRDS cavity is inserted inside the high pressure cell. The stability of the spectra baseline has been validated by filling the CRDS cell with Ar and N2 up to 10bar.We present here the first application of this CW-CRDS spectrometer to the study of the high pressure spectrum of CO2 at room temperature. The spectra were recorded between 5850 and 5960cm−1 for a series of pressure values up to 6400Torr. The studied spectral interval corresponds to the high energy range of the 1.75µm transparency window of CO2 of particular interest for Venus.The CO2 absorption coefficient at a given pressure value was obtained as the increase of the CRDS loss rate from its value at zero pressure taking into account the small contribution due to Rayleigh scattering. The CO2 absorption spectrum includes the contribution of the self broadened local rovibrational lines together with a broad and weak continuum. The CO2 continuum was obtained as the difference of the CO2 absorption coefficient and of a local lines simulation using the CO2 HITRAN line list and a truncated Voigt profile. A quadratic pressure dependence of the absorption continuum was observed, with an average binary absorption coefficient increasing smoothly from 3.7 to 11.5×10−9cm−1amagat−2 between 5850 and 5960cm−1. The derived continuum shows a spectral feature located in the region of a band of 16O12C18O (present in natural abundance) which dominates the spectrum in the region. This spectral feature was found to arise from collisional interferences between local lines and quantitatively accounted for using a theoretical approach based on the impact and Energy Corrected Sudden (ECS) approximations. The impact of the chosen far wings CO2 line shape model on the retrieved continuum absorption is discussed.

Role of Mitofusin-2 in mitochondrial localization and calcium uptake in skeletal muscle

As muscle contraction requires ATP and Ca2+, skeletal muscle function is highly dependent on communication between two major intracellular organelles: mitochondria and sarcoplasmic reticulum (SR). In adult skeletal muscle, mitochondria located within the I-band of the sarcomere are connected to the SR by small ∼10nm electron dense tethers that bridge the outer mitochondrial membrane to the region of SR that is ∼130nm from the site of Ca2+ release. However, the molecular composition of tethers and their precise impact on mitochondrial Ca2+ uptake in skeletal muscle is unclear. Mitofusin-2 (Mfn2) is a transmembrane GTPase present in both mitochondria and ER/SR membranes that forms trans-dimers and participates in mitochondrial fusion. Here we evaluated the role Mfn2 plays in mitochondrial morphology, localization, and functional SR-mitochondrial Ca2+ crosstalk in adult skeletal muscle. Compared to a non-targeting (CTRL) siRNA, in vivo electroporation of 400nM Mfn2 siRNA (Mfn2 KD) into mouse footpads resulted in a marked acute reduction (67±3%) in Mfn2 protein levels in flexor digitorum brevis (FDB) muscles that occurred without a change in other key Ca2+ regulatory proteins. Electron microscopy analyses revealed that Mfn2 knockdown resulted in a change in mitochondria morphology and mis-localization of some mitochondria from the I-band to the A-band region of the sarcomere. To assess the role of Mfn2 in SR-mitochondrial crosstalk, we measured mitochondrial Ca2+ uptake and myoplasmic Ca2+ transients with rhod-2 and mag-fluo-4, respectively, during repetitive high frequency tetanic stimulation (5×100Hz tetani, 500ms/tetani, 0.2 duty cycle) in CTRL and Mfn2 KD fibers. Mitochondrial Ca2+ uptake during repetitive tetanic stimulation was significantly reduced (40%) in Mfn2 KD FDB fibers, which was accompanied by a parallel elevation in the global electrically evoked myoplasmic Ca2+ transient. Mfn2 KD also resulted in a reduction of the mitochondrial membrane potential, which contributed to the observed decrease in activity-dependent mitochondrial Ca2+ uptake. Consistent with this idea, a similar decrease in mitochondrial Ca2+ uptake was also observed in wild type fibers following a comparable reduction in mitochondrial membrane potential induced by acute exposure to a low concentration (50nM) of carbonylcyanide-p-trifluoromethoxyphenylhydrazone (FCCP). In addition, both global and mitochondrial Ca2+ transients during repetitive tetanic stimulation were similarly reduced by both slow (EGTA) and fast (BAPTA) Ca2+ chelating agents. Together, these results indicate that Mfn2 promotes proper mitochondrial morphology, localization, and membrane potential required for optimal activity-dependent mitochondrial Ca2+ uptake and buffering of the global myoplasmic Ca2+ transient in adult skeletal muscle.

A.P.8: Necklace cytoplasmic bodies in hereditary myopathy with early respiratory failure (HMERF)

Hereditary myopathy with early respiratory failure (HMERF) is an adult-onset progressive myopathy characterized by early presentation of respiratory disturbance usually during ambulant stage. The disease is caused by mutations in the exon 343 of TTN that encodes the fibronectin-3 119 domain in A-band region (ENS0589042). Pathologically, HMERF is categorized into myofibrillar myopathy (MFM). Interestingly, cytoplasmic bodies (CBs) often localize in subsarcolemmal regions, with a necklace-like alignment in muscle fibers. Then we supposed that the necklace CBs might be useful in the diagnosis of HMERF. Here we aim to elucidate the diagnostic value of the necklace CBs for HMERF. We screened exon343 of TTN in genomic DNA samples from 187 subjects from 175 different families who were pathologically categorized as MFM. Furthermore we re-evaluated muscle pathology of the subjects to assess the sensitivity and specificity of the necklace CBs in the diagnosis of HMERF. TTN mutations were identified in 17 subjects from 14 families, whose phenotypes were consistent with HMERF. Among them, 14 had the necklaces CBs. In contrast, no other MFM patient had necklace CBs except for only one patient with reducing body myopathy. Thus the sensitivity and specificity of the necklace CBs for HMERF were 82% (14/17) and 99% (169/170), respectively. Positive predictive value was calculated as 93% in the MFM cohort. The necklace CBs should be a useful pathological marker for HMERF. When muscle pathology reveals the presence of the necklace CBs, exon 343 of TTN can be considered as a primary target for genetic diagnosis.