Sarcomeric Cytoskeleton Research Articles

Abstract Or105: Constitutive Deletion of the Obscurin Ig58/59 Domains Induces Structural and Electrophysiological Remodeling in Atria

Introduction: Obscurin is a giant cytoskeletal protein that supports muscle development, tethers intracellular compartments and regulates contraction. In the ObscnΔIg58/59 mouse model, expressing obscurin lacking Immunoglobulin (Ig) domains 58 and 59, aging males exhibit irregular heart rhythm with prominent atrial fibrillation, atrial enlargement, and progressive remodeling of the ventricles. A mechanistic basis for the emergence of arrhythmia early on could not be identified in ObscnΔIg58/59 ventricles, suggesting that the atria are preferentially impacted by deletion of the obscurin Ig58/59 module prior to the ventricles. Hypothesis: We hypothesize that Ig58/59 deletion elicits unique structural, electrical, and functional consequences in the atria preceding ventricular maladaptation. Aims: I. Examine the structural organization of ObscnΔIg58/59 atria II. Assess Ca 2+ handling in isolated atrial cardiomyocytes and determine molecular alterations. Methods: Electron microscopy and super resolution microscopy were utilized to visualize sarcomeric ultrastructure and the transverse-axial tubule (TAT) network, respectively. Ca 2+ handling activity and synchrony were also resolved, while Phos-Tag gels were used to quantify T-cap phospho-species. Results: ObscnΔIg58/59 atria exhibited misalignment of Z-disks. Spontaneous and stimulated Ca 2+ cycling behavior were [AB1] differentially disrupted in atrial cardiomyocytes in 6- and 12-month ObscnΔIg58/59 males. Relatedly, atrial cells showed an age-dependent deterioration in TAT architecture. Finally, as a function of aging, ObscnΔIg58/59 atria displayed alterations in the expression and phosphorylation of T-cap, a Z-disk protein implicated in the integration of t-tubules with the sarcomeric cytoskeleton. Conclusions: The structural and electrical alterations that arise in ObscnΔIg58/59 atria precede ventricular dysfunction and coincide with the emergence of atrial fibrillation. Collectively, our work indicates that the atria are principally affected by Ig58/59 elimination, instigating arrhythmias and morphological alterations with age. The ObscnΔIg58/59 mouse model has thus emerged as a proxy for atrial cardiomyopathy.

How Localized Z-Disc Damage Affects Force Generation and Gene Expression in Cardiomyocytes.

The proper function of cardiomyocytes (CMs) is highly related to the Z-disc, which has a pivotal role in orchestrating the sarcomeric cytoskeletal function. To better understand Z-disc related cardiomyopathies, novel models of Z-disc damage have to be developed. Human pluripotent stem cell (hPSC)-derived CMs can serve as an in vitro model to better understand the sarcomeric cytoskeleton. A femtosecond laser system can be applied for localized and defined damage application within cells as single Z-discs can be removed. We have investigated the changes in force generation via traction force microscopy, and in gene expression after Z-disc manipulation in hPSC-derived CMs. We observed a significant weakening of force generation after removal of a Z-disc. However, no significant changes of the number of contractions after manipulation were detected. The stress related gene NF-kB was significantly upregulated. Additionally, α-actinin (ACTN2) and filamin-C (FLNc) were upregulated, pointing to remodeling of the Z-disc and the sarcomeric cytoskeleton. Ultimately, cardiac troponin I (TNNI3) and cardiac muscle troponin T (TNNT2) were significantly downregulated. Our results allow a better understanding of transcriptional coupling of Z-disc damage and the relation of damage to force generation and can therefore finally pave the way to novel therapies of sarcomeric disorders.

Стабильный уровень гигантских белков саркомерного цитоскелета в поперечнополосатых мышцах сони-полчка Glis glis при гибернации

The changes in the content of giant proteins of the sarcomeric cytoskeleton titin (3000-3700 kDa) and nebulin (770 kDa) in skeletal muscles (m. soleus, m. gastrocnemius), titin in the myocardium (left ventricle), as well as membrane cytoskeletal protein dystrophin (427 kDa) in m. soleus and m. extensor digitorum longus of edible dormouse Glis glis during hibernation have been studied. The animals were of two experimental groups: “Summer activity” and “Hypothermia”. It was found that the development of atrophic changes in the skeletal muscles of the animal during hibernation is accompanied by a decrease in the content of dystrophin. In particular, the intensity of fluorescence on cross-sections of skeletal muscles stained with primary antibodies to dystrophin and secondary antibodies conjugated to the Alexa488 fluorochrome decreased 2.7 times (p < 0.05) and 2.0 times (p < 0.05) in m. soleus and m. EDL, respectively, in animals of the “Hypothermia” group. SDS electrophoresis of proteins in agarose-strengthened macroporous 2.2%-polyacrylamide gel revealed a slight decrease (by 15%, p ≤ 0.01) in the titin content in relation to the content of myosin heavy chains in m. gastrocnemius of animals of the “Hypothermia” group. Titin content in m. soleus and cardiac muscle, as well as the nebulin content in the studied skeletal muscles did not decrease during hibernation. These results are consistent with our earlier data for other hibernates: long-tailed ground squirrel, brown and Himalayan black bears. It can be assumed that in the process of evolution in hibernating animals, molecular mechanisms were developed that are responsible for maintaining a stable level of giant proteins of the sarcomeric cytoskeleton during hibernation.

MUSCLE FUNCTION & HOMEOSTASIS / MOLECULAR THERAPEUTIC APPROACHES: P.68 O-GlcNAcylation and its interplay with phosphorylation on desmin behaviour: focus on its partition and its interaction with alphaB-crystallin

O-GlcNAcylation, a dynamic, reversible and atypical glycosylation akin to phosphorylation modifies a plethora of myofibrillar proteins, especially structural and contractile proteins. We have previously demonstrated that O-GlcNAcylation regulates sarcomeric cytoskeleton since the sarcomere morphometry is modified consecutively to O-GlcNAcylation changes. Interestingly, desmin, a key protein of striated muscle cells cytoarchitecture and its molecular chaperone alphaB-crystallin seems to be involved in sarcomere morphometry changes through the modification of their interaction.

Predominant synthesis of giant myofibrillar proteins in striated muscles of the long-tailed ground squirrel Urocitellus undulatus during interbout arousal

Molecular mechanisms underlying muscle-mass retention during hibernation have been extensively discussed in recent years. This work tested the assumption that protein synthesis hyperactivation during interbout arousal of the long-tailed ground squirrel Urocitellus undulatus should be accompanied by increased calpain-1 activity in striated muscles. Calpain-1 is known to be autolysed and activated in parallel. Western blotting detected increased amounts of autolysed calpain-1 fragments in the heart (1.54-fold, p < 0.05) and m. longissimus dorsi (1.8-fold, p < 0.01) of ground squirrels during interbout arousal. The total protein synthesis rate determined by SUnSET declined 3.67-fold in the heart (p < 0.01) and 2.96-fold in m. longissimus dorsi (p < 0.01) during interbout arousal. The synthesis rates of calpain-1 substrates nebulin and titin in muscles did not differ during interbout arousal from those in active summer animals. A recovery of the volume of m. longissimus dorsi muscle fibres, a trend towards a heart-muscle mass increase and a restoration of the normal titin content (reduced in the muscles during hibernation) were observed. The results indicate that hyperactivation of calpain-1 in striated muscles of long-tailed ground squirrels during interbout arousal is accompanied by predominant synthesis of giant sarcomeric cytoskeleton proteins. These changes may contribute to muscle mass retention during hibernation.

Abstract 477: Role of Telomere Dysfunction in Duchenne Muscular Dystrophy Cardiomyopathy

Duchenne muscular dystrophy (DMD) is a devastating X-linked genetic disorder that affects 1 in 3500 males. Characterized by progressive muscle degeneration that culminates in respiratory failure and dilated cardiomyopathy, DMD is caused by a lack of dystrophin, a protein that provides structural support between the sarcomeric cytoskeleton and the extracellular matrix. Loss of dystrophin leads to a leaky plasma membrane, contractile stress, and disruption of cellular homeostasis. However, the molecular mechanism that eventually leads to cell death remains to be explored. Recently, the Blau lab discovered a pathogenic link between DMD cardiomyopathy and telomere dysfunction. While mdx mice that lack functional dystrophin do not exhibit dilated cardiomyopathy as in human patients, when crossed with mTR mice that lack the RNA component of telomerase (TERC), mdx mice with “humanized” telomere lengths fully manifested the severe muscle wasting and cardiac failure seen in patients. Notably, the longer telomeres, characteristic of mice, appear to be cardioprotective. Importantly, we observed telomere shortening in cardiomyocytes, but not other cell types, of DMD patients compared to age-matched controls. Preliminary data suggests that contractile stress due to the lack of dystrophin leads to a pathogenic feed-forward loop which ultimately culminates in cardiomyocyte cell death. Using human iPS cells derived from DMD patients, we have modeled telomere shortening and aspects of cardiomyocyte dysfunction characteristic of DMD, including aberrant calcium transients, mechanical stress, and arrhythmia. By comparing cardiomyocytes derived from DMD iPS cells with those from CRISPR-corrected isogenic controls on patterned bioengineered hydrogel platforms of varying stiffness, we can study the role of fibrotic stiffening in the myocardium in the premature demise of DMD cardiomyocytes. Pinpointing the early molecular events that trigger the pathogenic feed-forward loop will provide strategies for intervention to ameliorate DMD cardiomyopathy.

O-GlcNAcylation as a regulator of the functional and structural properties of the sarcomere in skeletal muscle: An update review.

Although the O-GlcNAcylation process was discovered in 1984, its potential role in the physiology and physiopathology of skeletal muscle only emerged 20years later. An increasing number of publications strongly support a key role of O-GlcNAcylation in the modulation of important cellular processes which are essential for skeletal muscle functions. Indeed, over a thousand of O-GlcNAcylated proteins have been identified within skeletal muscle since 2004, which belong to various classes of proteins, including sarcomeric proteins. In this review, we focused on these myofibrillar proteins, including contractile and structural proteins. Because of the modification of motor and regulatory proteins, the regulatory myosin light chain (MLC2) is related to several reports that support a key role of O-GlcNAcylation in the fine modulation of calcium activation parameters of skeletal muscle fibres, depending on muscle phenotype and muscle work. In addition, another key function of O-GlcNAcylation has recently emerged in the regulation of organization and reorganization of the sarcomere. Altogether, this data support a key role of O-GlcNAcylation in the homeostasis of sarcomeric cytoskeleton, known to be disturbed in many related muscle disorders.

Change in the content of titin and nebulin and their phosphorylation level in the quadriceps femoris muscle in chronic alcoholic myopathy

Objective : to assess the structural and functional state of skeletal muscles in the hip, as well as changes in the content of the sarcomere cytoskeleton proteins titin and nebulin and their phosphorylation level in patients with chronic alcohol intoxication. Patients and methods . Thirteen patients (4 men and 9 women; mean age, 38.9±9.9 years) with chronic alcohol intoxication were examined. The average duration of regular consumption of alcoholic beverages was 7.6±3.7 years. The mean amount of alcohol consumed per week was 48.2±13.1 units. A control group included 10 healthy volunteers matched for age and sex. A neurological examination was performed according to the generally accepted scheme. Laboratory tests involved blood biochemical analysis estimating the levels of liver enzymes, creatine phosphokinase (CPK), and insulin-like growth factor-1 (IGF-1). All the patients underwent hip magnetic resonance imaging (MRI), followed by assessment of the degree of muscle tissue damage and by determination of the volume of anterior and posterior thigh muscles. The content of titin and nebulin and their phosphorylation level were determined in the muscle tissue samples obtained by an open biopsy from the lateral head of the quadriceps femoris muscle. Results and discussion . Four (30.8%) patients were found to have proximal leg muscle weakness; the degree of paresis was the same in the anterior and posterior thigh muscles. There was a significant increase in the plasma level of liver enzymes; the CPK level remained within the reference values; there was a tendency towards lower IGF-1 levels. Analysis of MRI data showed that 7 (53.8%) patients had fatty degeneration in the thigh muscles. Quantitative evaluation ascertained a significant symmetrical decrease in the volume of anterior thigh muscles and a tendency towards a symmetrical reduction in that in the posterior thigh muscle compared to the control. Analysis of the content of titin and nebulin in the lateral head of the quadriceps femoris muscle revealed a significant decrease in the percentage of nebulin (81.1%; p < 0.01) and intact titin-1 (T1) isoforms (83.6%; p < 0.01). The percentage of proteolytic titin-2 (T2) fragments in the muscle of patients did not differ significantly from that in the control group. Estimating the phosphorylation level of the structural muscle proteins showed no significant differences when compared to the control. Conclusion . Anterior and posterior thigh muscle weakness should be considered as the main clinical manifestation of chronic alcoholic myopathy (CAM) in the absence of biochemical and neurophysiological markers of the disease. Lower extremity muscle MRI that can reveal a lower muscle volume concurrent with fatty degeneration is a non-invasive informative diagnostic technique for CAM. The pathogenesis of skeletal muscle atrophy in chronic alcohol intoxication involves the sarcomere structural proteins titin and nebulin, which regulate the interaction of the major contractile proteins actin and myosin, and whole muscle contraction.

Slik phosphorylation of Talin T152 is crucial for proper Talin recruitment and maintenance of muscle attachment in Drosophila.

Talin is the major scaffold protein linking integrin receptors with the actin cytoskeleton. In Drosophila, extended Talin generates a stable link between the sarcomeric cytoskeleton and the tendon matrix at muscle attachment sites. Here, we identify phosphorylation sites on Drosophila Talin by mass spectrometry. Talin is phosphorylated in late embryogenesis when muscles differentiate, especially on T152 in the exposed loop of the F1 domain of the Talin head. Localization of a mutated version of Talin (Talin-T150/T152A) is reduced at muscle attachment sites and can only partially rescue muscle attachment compared with wild-type Talin. We also identify Slik as the kinase phosphorylating Talin at T152. Slik localizes to muscle attachment sites, and the absence of Slik reduces the localization of Talin at muscle attachment sites causing phenotypes similar to Talin-T150/T152A. Thus, our results demonstrate that Talin phosphorylation by Slik plays an important role in fine-tuning Talin recruitment to integrin adhesion sites and maintaining muscle attachment.

Differences in Titin and Nebulin Gene Expression in Skeletal Muscles of Rats Chronically Alcoholized by Different Methods

This work studied the changes in the levels of the main proteins of the calpain system (μ-calpain, Ca^(2+)-dependent protease, and fragments of its autolysis, inhibitor calpastatin) and μ-calpain substrates (giant proteins of the sarcomere cytoskeleton, titin and nebulin) in skeletal muscle (m. gastrocnemius, m. soleus, m. longissimus dorsi) of rats alcoholized for three months by different methods using agar containing 30% ethanol and nutrient-balanced liquid feed containing 5% ethanol using gel electrophoresis methods under denaturing conditions and immunoblotting. No decrease in the muscle mass/body weight ratio, indicating the development of atrophy, no increase in autolysis of μ-calpain, indicating an increase in the activity of this enzyme, no changes in the content of intact titin (T1), nebulin, μ-calpain and calpastatin, as well as the total calpain activity measured using Calpain Activity Assay Kit were detected in alcoholized rats of both groups. No changes in the total level of titin phosphorylation in the rat muscles of alcoholized groups were detected using Pro-Q Diamond fluorescent dye for phosphate groups of proteins. No statistically significant differences in the content of titin and nebulin mRNA in skeletal muscles of control rats and rats alcoholized using agar were detected. In rats, alcoholized by the method of liquid feed, the levels of titin and nebulin mRNA were increased 1.5-2.5 times possibly due to a higher fat content in such a diet. The presented data may be useful for choosing a chronic alcoholization model for animals.

Knockout of human muscle genes revealed by large scale whole-exome studies

Large scale whole-exome sequence studies have revealed that a number of individuals from different populations have predicted loss-of-function of different genes due to nonsense, frameshift, or canonical splice-site mutations. Surprisingly, many of these mutations do not apparently show the deleterious phenotypic consequences expected from gene knockout. These homozygous null mutations, when confirmed, can provide insight into human gene function and suggest novel approaches to correct gene dysfunction, as the lack of the expected disease phenotype may reflect the existence of modifier genes that reveal potential therapeutic targets. Human knockouts complement the information derived from mouse knockouts, which are not always good models of human disease. We have examined human knockout datasets searching for genes expressed exclusively or predominantly in striated muscle. A number of well-known muscle genes was found in one or more datasets, including genes coding for sarcomeric myosins, components of the sarcomeric cytoskeleton, sarcoplasmic reticulum and plasma membrane, and enzymes involved in muscle metabolism. The surprising absence of phenotype in some of these human knockouts is critically discussed, focusing on the comparison with the corresponding mouse knockouts.

Binding of Myomesin to Obscurin-Like-1 at the Muscle M-Band Provides a Strategy for Isoform-Specific Mechanical Protection

SummaryThe sarcomeric cytoskeleton is a network of modular proteins that integrate mechanical and signaling roles. Obscurin, or its homolog obscurin-like-1, bridges the giant ruler titin and the myosin crosslinker myomesin at the M-band. Yet, the molecular mechanisms underlying the physical obscurin(-like-1):myomesin connection, important for mechanical integrity of the M-band, remained elusive. Here, using a combination of structural, cellular, and single-molecule force spectroscopy techniques, we decode the architectural and functional determinants defining the obscurin(-like-1):myomesin complex. The crystal structure reveals a trans-complementation mechanism whereby an incomplete immunoglobulin-like domain assimilates an isoform-specific myomesin interdomain sequence. Crucially, this unconventional architecture provides mechanical stability up to forces of ∼135 pN. A cellular competition assay in neonatal rat cardiomyocytes validates the complex and provides the rationale for the isoform specificity of the interaction. Altogether, our results reveal a novel binding strategy in sarcomere assembly, which might have implications on muscle nanomechanics and overall M-band organization.

A novel dominant D109A CRYAB mutation in a family with myofibrillar myopathy affects αB-crystallin structure

Myofibrillar myopathy (MFM) is a group of inherited muscular disorders characterized by myofibrils dissolution and abnormal accumulation of degradation products. So far causative mutations have been identified in nine genes encoding Z-disk proteins, including αB-crystallin (CRYAB), a small heat shock protein (also called HSPB5).Here, we report a case study of a 63-year-old Polish female with a progressive lower limb weakness and muscle biopsy suggesting a myofibrillar myopathy, and extra-muscular multisystemic involvement, including cataract and cardiomiopathy. Five members of the proband's family presented similar symptoms. Whole exome sequencing followed by bioinformatic analysis revealed a novel D109A mutation in CRYAB associated with the disease.Molecular modeling in accordance with muscle biopsy microscopic analyses predicted that D109A mutation influence both structure and function of CRYAB due to decreased stability of oligomers leading to aggregate formation. In consequence disrupted sarcomere cytoskeleton organization might lead to muscle pathology. We also suggest that mutated RQDE sequence of CRYAB could impair CRYAB chaperone-like activity and promote aggregation of lens crystallins.

Role of dystroglycan in limiting contraction-induced injury to the sarcomeric cytoskeleton of mature skeletal muscle

Dystroglycan (DG) is a highly expressed extracellular matrix receptor that is linked to the cytoskeleton in skeletal muscle. DG is critical for the function of skeletal muscle, and muscle with primary defects in the expression and/or function of DG throughout development has many pathological features and a severe muscular dystrophy phenotype. In addition, reduction in DG at the sarcolemma is a common feature in muscle biopsies from patients with various types of muscular dystrophy. However, the consequence of disrupting DG in mature muscle is not known. Here, we investigated muscles of transgenic mice several months after genetic knockdown of DG at maturity. In our study, an increase in susceptibility to contraction-induced injury was the first pathological feature observed after the levels of DG at the sarcolemma were reduced. The contraction-induced injury was not accompanied by increased necrosis, excitation-contraction uncoupling, or fragility of the sarcolemma. Rather, disruption of the sarcomeric cytoskeleton was evident as reduced passive tension and decreased titin immunostaining. These results reveal a role for DG in maintaining the stability of the sarcomeric cytoskeleton during contraction and provide mechanistic insight into the cause of the reduction in strength that occurs in muscular dystrophy after lengthening contractions.

O-GlcNAcylation is a key modulator of skeletal muscle sarcomeric morphometry associated to modulation of protein–protein interactions

BackgroundThe sarcomere structure of skeletal muscle is determined through multiple protein–protein interactions within an intricate sarcomeric cytoskeleton network. The molecular mechanisms involved in the regulation of this sarcomeric organization, essential to muscle function, remain unclear. O-GlcNAcylation, a post-translational modification modifying several key structural proteins and previously described as a modulator of the contractile activity, was never considered to date in the sarcomeric organization. MethodsC2C12 skeletal myotubes were treated with Thiamet-G (OGA inhibitor) in order to increase the global O-GlcNAcylation level. ResultsOur data clearly showed a modulation of the O-GlcNAc level more sensitive and dynamic in the myofilament-enriched fraction than total proteome. This fine O-GlcNAc level modulation was closely related to changes of the sarcomeric morphometry. Indeed, the dark-band and M-line widths increased, while the I-band width and the sarcomere length decreased according to the myofilament O-GlcNAc level. Some structural proteins of the sarcomere such as desmin, αB-crystallin, α-actinin, moesin and filamin-C have been identified within modulated protein complexes through O-GlcNAc level variations. Their interactions seemed to be changed, especially for desmin and αB-crystallin. ConclusionsFor the first time, our findings clearly demonstrate that O-GlcNAcylation, through dynamic regulations of the structural interactome, could be an important modulator of the sarcomeric structure and may provide new insights in the understanding of molecular mechanisms of neuromuscular diseases characterized by a disorganization of the sarcomeric structure. General significanceIn the present study, we demonstrated a role of O-GlcNAcylation in the sarcomeric structure modulation.

The sarcomeric cytoskeleton: from molecules to motion.

Highly ordered organisation of striated muscle is the prerequisite for the fast and unidirectional development of force and motion during heart and skeletal muscle contraction. A group of proteins, summarised as the sarcomeric cytoskeleton, is essential for the ordered assembly of actin and myosin filaments into sarcomeres, by combining architectural, mechanical and signalling functions. This review discusses recent cell biological, biophysical and structural insight into the regulated assembly of sarcomeric cytoskeleton proteins and their roles in dissipating mechanical forces in order to maintain sarcomere integrity during passive extension and active contraction. α-Actinin crosslinks in the Z-disk show a pivot-and-rod structure that anchors both titin and actin filaments. In contrast, the myosin crosslinks formed by myomesin in the M-band are of a ball-and-spring type and may be crucial in providing stable yet elastic connections during active contractions, especially eccentric exercise.

Structure and Molecular Dynamics of the Ig58/58 Domains of Obscurin

Obscurin (800-900 kDa) is a giant muscle protein vital to muscle cell maintenance and organization. It is the only known connection between the contractile apparatus and the sarcoplasmic reticulum and also binds to specific cytoskeletal, signaling, or membrane-associated proteins. Obscurin domains Ig58/59 bind to titin ZIg9/10, which is hypothesized to stabilize the sarcomeric cytoskeleton. Mutations in this obscurin region lead to malformed muscle architecture and, eventually, to hypertrophic cardiomyopathy (HCM). For obscurin/titin binding to occur, all four of these domains must be present. In order to fully characterize this physiologically important region of obscurin, and by extension determine the molecular factors that drive HCM, here we present the Ig58 and Ig59 structure using both X-ray crystallography and heteronuclear multidimensional NMR spectra. Low-resolution structure and dynamics of these tandem domains, along with how these domains react to physical stress, are also discussed.

An ongoing role for structural sarcomeric components in maintaining Drosophila melanogaster muscle function and structure.

Animal muscles must maintain their function while bearing substantial mechanical loads. How muscles withstand persistent mechanical strain is presently not well understood. The basic unit of muscle is the sarcomere, which is primarily composed of cytoskeletal proteins. We hypothesized that cytoskeletal protein turnover is required to maintain muscle function. Using the flight muscles of Drosophila melanogaster, we confirmed that the sarcomeric cytoskeleton undergoes turnover throughout adult life. To uncover which cytoskeletal components are required to maintain adult muscle function, we performed an RNAi-mediated knockdown screen targeting the entire fly cytoskeleton and associated proteins. Gene knockdown was restricted to adult flies and muscle function was analyzed with behavioural assays. Here we analyze the results of that screen and characterize the specific muscle maintenance role for several hits. The screen identified 46 genes required for muscle maintenance: 40 of which had no previously known role in this process. Bioinformatic analysis highlighted the structural sarcomeric proteins as a candidate group for further analysis. Detailed confocal and electron microscopic analysis showed that while muscle architecture was maintained after candidate gene knockdown, sarcomere length was disrupted. Specifically, we found that ongoing synthesis and turnover of the key sarcomere structural components Projectin, Myosin and Actin are required to maintain correct sarcomere length and thin filament length. Our results provide in vivo evidence of adult muscle protein turnover and uncover specific functional defects associated with reduced expression of a subset of cytoskeletal proteins in the adult animal.

Remodeling of the sarcomeric cytoskeleton in cardiac ventricular myocytes during heart failure and after cardiac resynchronization therapy

Sarcomeres are the basic contractile units of cardiac myocytes. Recent studies demonstrated remodeling of sarcomeric proteins in several diseases, including genetic defects and heart failure. Here we investigated remodeling of sarcomeric α-actinin in two models of heart failure, synchronous (SHF) and dyssynchronous heart failure (DHF), as well as a model of cardiac resynchronization therapy (CRT). We applied three-dimensional confocal microscopy and quantitative methods of image analysis to study isolated cells from our animal models. 3D Fourier analysis revealed a decrease of the spatial regularity of the α-actinin distribution in both SHF and DHF versus control cells. The spatial regularity of α-actinin in DHF cells was reduced when compared with SHF cells. The spatial regularity of α-actinin was partially restored after CRT. We found longitudinal depositions of α-actinin in SHF, DHF and CRT cells. These depositions spanned adjacent Z-disks and exhibited a lower density of α-actinin than in the Z-disk. Differences in the occurrence of depositions between the SHF, CRT and DHF models versus control were significant. Also, CRT cells exhibited a higher occurrence of depositions versus SHF, but not DHF cells. Other sarcomeric proteins did not accumulate in the depositions to the same extent as α-actinin. We did not find differences in the expression of α-actinin protein and its encoding gene in our animal models. In summary, our studies indicate that HF is associated with two different types of remodeling of α-actinin and only one of those was reversed after CRT. We suggest that these results can guide us to an understanding of remodeling of structures and function associated with sarcomeres.

Novel Locations; Familiar Functions: Obscurin at the Cardiac Intercalated Disc

The intercalated disc (ID) of cardiac muscle embodies a highly ordered, multifunctional network, which is essential for the transmission of electrical stimuli and mechanical force resulting in the synchronous contraction of the heart. Recently, a plethora of proteins have been identified as novel components of the ID. The challenge now lies in their characterization as it relates to the mechanical and electrical coupling of neighbouring cardiomyocytes. Here we focus on the molecular and functional description of one of these novel members, obscurin-90.Obscurins are a family of proteins expressed in striated muscles where they localize to distinct subdomains, i.e. the sarcoplasmic reticulum, the sarcomeric cytoskeleton, and the sarcolemma, and function in their assembly and integration. Previous studies have shown that transcripts arising from the single OBSCN gene are extensively spliced, resulting in several obscurin isoforms. Complex splicing at its 3' end gives rise to obscurin-90 (obsc90), the isoform that preferentially localizes to the ID. Obsc90 contains tandem RhoGEF and PH motifs, two Ig domains, and a non-modular COOH-terminus with ankyrin-binding sites and ERK phosphorylation cassettes. Using immunofluorescence and immunoelectron microscopy, we show that obsc90 localizes to the ID, and specifically at the outer edge of the transition zone. Consistent with this, biochemical assays demonstrated that obsc90 is in a complex with major ID proteins, including N-cadherin, connexin-43, vinculin, and ankyrinG. Obsc90 is targeted to the ID through the direct binding of its PH domain to membrane lipids, and its functions are likely regulated by phosphorylation of its COOH-terminus. Preliminary evidence indicates that the phosphorylation profile of obsc90 is altered in hypertrophic cardiomyopathy.. Further experiments are underway to examine the function of obsc90 at the ID and its regulation via phosphorylation in health and disease.