Biomedical subjects

Aikaterini Kontrogianni-Konstantopoulos

Publications and source records attributed to Aikaterini Kontrogianni-Konstantopoulos.

11 recordsLinked to original sources

PubMed2006

Obscurin is required for the lateral alignment of striated myofibrils in zebrafish.

Obscurin/obscurin-MLCK is a giant sarcomere-associated protein with multiple isoforms whose interactions with titin and small ankyrin-1 suggest that it has an important role in myofibril assembly, structural support, and the sarcomeric alignment of the sarcoplasmic reticulum. In this study, we characterized the zebrafish orthologue of obscurin and examined its role in striated myofibril assembly. Zebrafish obscurin was expressed in the somites and central nervous system by 24 hours post-fertilization (hpf) and in the heart by 48 hpf. Depletion of obscurin using two independent morpholino antisense oligonucleotides resulted in diminished numbers and marked disarray of skeletal myofibrils, impaired lateral alignment of adjacent myofibrils, disorganization of the sarcoplasmic reticulum, somite segmentation defects, and abnormalities of cardiac structure and function. This is the first demonstration that obscurin is required for vertebrate cardiac and skeletal muscle development. The diminished capacity to generate and organize new myofibrils in response to obscurin depletion suggests that it may have a vital role in the causation of or adaptation to cardiac and skeletal myopathies.

Amino Acid Sequence↗

PubMed2006

Obscurin modulates the assembly and organization of sarcomeres and the sarcoplasmic reticulum.

Obscurin (approximately 800 kDa) in striated muscle closely surrounds sarcomeres at the level of the M-band and Z-disk where, we hypothesize, it participates in the assembly of the contractile apparatus and membrane systems required for Ca2+ homeostasis. In this study, we used small inhibitory RNA (siRNA) technology to reduce the levels of obscurin in primary cultures of skeletal myotubes to study its role in myofibrillogenesis and the organization of the sarcoplasmic reticulum (SR). siRNA-treated myotubes showed a specific and dramatic reduction in the approximately 800 kDa form of obscurin by reverse transcription-polymerase chain reaction, immunoblotting, and immunofluorescence. M-bands and A-bands, but not Z-disks or I-bands, were disrupted when the synthesis of obscurin was inhibited. Small ankyrin 1, an integral protein of the network SR that binds to obscurin, also failed to align around developing sarcomeres in treated myotubes. Myosin and myomesin levels were significantly reduced in treated myotubes but alpha-actinin was not, suggesting that down-regulation of obscurin destabilizes proteins of the M-band and A-band but not of the Z-disk. Our findings suggest that obscurin is required for the assembly of the M-band and A-band and for the regular alignment of the network SR around the contractile apparatus.

Animals↗

PubMed2005

Essential role of obscurin in cardiac myofibrillogenesis and hypertrophic response: evidence from small interfering RNA-mediated gene silencing.

Obscurin is a recently identified giant multidomain muscle protein (approximately 800 kDa) whose structural and regulatory functions remain to be defined. The goal of this study was to examine the effect of obscurin gene silencing induced by RNA interference on the dynamics of myofibrillogenesis and hypertrophic response to phenylephrine in cultured rat cardiomyocytes. We found that that the adenoviral transfection of short interfering RNA (siRNA) constructs targeting the first coding exon of obscurin sequence resulted in progressive depletion of cellular obscurin. Confocal microscopy demonstrated that downregulation of obscurin expression led to the impaired assembly of new myofibrillar clusters and considerable aberrations of the normal structure of the contractile apparatus. While the establishment of the initial periodic pattern of alpha-actinin localization remained mainly unaffected in siRNA-transfected cells, obscurin depletion did cause the defective lateral alignment of myofibrillar bundles, leading to their abnormal bifurcation, dispersal and multiple branching. Bending of immature myofibrils, apparently associated with the loss of their rigidity, a modified titin pattern, the absence of well-formed A-bands in newly formed contractile structures as documented by a diffuse localization of sarcomeric myosin labeling, and an occasional irregular periodicity of sarcomere spacing were typical of obscurin siRNA-treated cells. These results suggest that obscurin is indispensable for spatial positioning of contractile proteins and for the structural integration and stabilization of myofibrils, especially at the stage of myosin filament incorporation and A-band assembly. This demonstrates a vital role for obscurin in myofibrillogenesis and hypertrophic growth.

Actinin↗

PubMed2005

De novo myofibrillogenesis in C2C12 cells: evidence for the independent assembly of M bands and Z disks.

We studied the distribution of the giant sarcomeric protein obscurin during de novo myofibrillogenesis in C(2)C(12) myotubes to learn when it is integrated into developing sarcomeres. Obscurin becomes organized first at the developing M band and later at the mature Z disk. Primordial M bands consisting of obscurin, myomesin, and M band epitopes of titin assemble before adult fast-twitch sarcomeric myosin is organized periodically and nearly concurrently with primitive Z disks, which are composed of alpha-actinin and Z disk epitopes of titin. Z disks and M bands can assemble independently at spatially distant sites. As sarcomerogenesis proceeds, these structures interdigitate to produce a more mature organization. Fast-twitch muscle myosin accumulates in the myoplasm and assembles into A bands only after Z disks and M bands assume their typical interdigitated striations. The periodicities of M bands remain constant at approximately 1.8 microm throughout sarcomerogenesis, whereas distances between Z disks increase from approximately 1.1 microm in early sarcomeres to approximately 1.8 microm in more mature structures. Our findings indicate for the first time that primitive M bands self-assemble independently of Z disks, that obscurin is a component of these primitive M bands in skeletal muscle cells, and that A bands assemble only after M bands and Z disks integrate into maturing sarcomeres.

Actinin↗

PubMed2005

Array lessons from the heart: focus on the genome and transcriptome of cardiomyopathies.

Our understanding of the cardiovascular system has evolved through the years by extensive studies emphasizing the identification of the molecular and physiological mechanisms involved in its normal function and disease pathogenesis. Major discoveries have been made along the way. However, the majority of this work has focused on specific genes or pathways rather than integrative approaches. In cardiomyopathies alone, over 30 different loci have shown mutations with varying inheritance patterns, yet mostly coding for structural proteins. The emergence of microarrays in the early 1990s paved the way to a new era of cardiovascular research. Microarrays dramatically accelerated the rhythm of discoveries by giving us the ability to simultaneously study thousands of genes in a single experiment. In the field of cardiovascular research, microarrays are having a significant contribution, with the majority of work focusing on end-stage cardiomyopathies that lead to heart failure. Novel molecular mechanisms have been identified, known pathways are seen under new light, disease subgroups begin to emerge, and the effects of various drugs are molecularly dissected. This cross-study data comparison concludes that consistent energy metabolism gene expression changes occur across dilated, hypertrophic, and ischemic cardiomyopathies, while Ca2+ homeostasis changes are prominent in the first two cardiomyopathies, and structural gene expression changes accompany mostly the dilated form. Gene expression changes are further correlated to disease genetics. The future of microarrays in the cardiomyopathy field is discussed with an emphasis on optimum experimental design and on applications in diagnosis, prognosis, and drug discovery.

Calcium↗

PubMed2005

Obscurin: a multitasking muscle giant.

Obscurin (approximately 800 kDa) is the third member of a family of giant proteins expressed in vertebrate striated muscle, along with titin (3-3.7 MDa) and nebulin (approximately 800 kDa). Like its predecessors, it is a multidomain protein composed of tandem adhesion modules and signaling domains. Unlike titin and nebulin, which are integral components of sarcomeres, obscurin is concentrated at the peripheries of Z-disks and M-lines, where it is appropriately positioned to communicate with the surrounding myoplasm. This unique distribution allows obscurin to bind small ankyrin 1, an integral component of the sarcoplasmic reticulum (SR) membrane. Obscurin also associates with the contractile apparatus through its binding to titin, sarcomeric myosin and perhaps other proteins of the contractile apparatus. Overexpression of the COOH-terminus of obscurin in primary myotubes has a dramatic and specific effect on the organization of sarcomeric myosin, indicating a role in the organization and regular assembly of A-bands. Given its ability to associate tightly, selectively and periodically with the periphery of the myofibril, its high affinity for an integral membrane protein of the SR and its close association with thick filaments, we speculate that obscurin is ideally suited to play key roles in modulating the organization and assembly of both the myofibril and the SR.

Animals↗

PubMed2004

Obscurin regulates the organization of myosin into A bands.

Obscurin is a giant sarcomeric protein composed of adhesion modules and signaling domains. It surrounds myofibrils at the level of the Z disk and the M line. To study the role of obscurin during myofibrillogenesis, we used adenovirus-mediated gene delivery to overexpress part of its COOH terminus in primary cultures of postnatal day 1 (P1) skeletal myotubes. Examination of the subcellular distribution of a number of sarcomeric proteins revealed that the organization of myosin into A bands was dramatically reduced. Myosin assembled into A bands normally in mock- or control-infected P1 myotubes. Overexpression of the COOH terminus of obscurin did not affect the organization of other sarcomeric markers, including actin, alpha-actinin, titin, and myomesin. Assembly of myomesin into nascent M lines in treated myotubes suggests that these structures can form independently of A bands. Immunoblot analysis indicated that there was a small ( approximately 20%) but consistent decrease in the amount of myosin expressed in cells infected with the COOH terminus of obscurin. Coimmunoprecipitation experiments in which we used adult skeletal muscle homogenates demonstrated that obscurin exists in a complex with myosin. Thus our findings suggest that the COOH-terminal region of obscurin interacts with sarcomeric myosin and may play a critical role in its ability to assemble into A bands in striated muscle.

Animals↗

PubMed2004

Dynamics of obscurin localization during differentiation and remodeling of cardiac myocytes: obscurin as an integrator of myofibrillar structure.

Obscurin is a newly identified giant muscle protein whose functions remain to be elucidated. In this study we used high-resolution confocal microscopy to examine the dynamics of obscurin localization in cultures of rat cardiac myocytes during the assembly and disassembly of myofibrils. Double immunolabeling of neonatal and adult rat cells for obscurin and sarcomeric alpha-actinin, the major protein of Z-lines, demonstrated that, during myofibrillogenesis, obscurin is intensely incorporated into M-band areas of A-bands and, to a lesser extent, in Z-lines of newly formed sarcomeres. Presarcomeric structural precursors of myofibrils were intensely immunopositive for alpha-actinin and, unlike mature myofibrils, weakly immunopositive or immunonegative for obscurin. This indicates that most of the obscurin assembles in developing myofibrils after abundant incorporation of alpha-actinin and that massive integration of obscurin occurs at more advanced stages of sarcomere assembly. Immunoreactivity for obscurin in the middle of A-bands and in Z-lines of sarcomeres bridged the gaps between individual bundles of newly formed myofibrils, suggesting that this protein appears to be directly involved in their primary lateral connection and registered alignment into larger clusters. Close sarcomeric localization of obscurin and titin suggests that they may interact during myofibril assembly. Interestingly, the laterally aligned striated pattern of obscurin formed at a stage when desmin, traditionally considered as a molecular linker responsible for the lateral binding and stabilization of myofibrils at the Z-bands, was still diffusely localized. During the disassembly of the contractile system in adult myocytes, disappearance of the cross-striated pattern of obscurin preceded the disorganization of registered alignment and intense breakdown of myofibrils. The cross-striated pattern of desmin typical of terminally differentiated myocytes disappeared before or simultaneously with obscurin. During redifferentiation, as in neonatal myocytes, sarcomeric incorporation of obscurin closely followed that of alpha-actinin and occurred earlier than the striated arrangement of desmin intermediate filaments. The presence of obscurin in the Z-lines and its later assembly into the A/M-bands indicate that it may serve to stabilize and align sarcomeric structure when myosin filaments are incorporated. Our data suggest that obscurin, interacting with other muscle proteins and possibly with the sarcoplasmic reticulum, may have a role as a flexible structural integrator of myofibrils during assembly and adaptive remodeling of the contractile apparatus.

Actins↗

PubMed2003

Rapid response of cardiac obscurin gene cluster to aortic stenosis: differential activation of Rho-GEF and MLCK and involvement in hypertrophic growth.

Obscurin and obscurin myosin light chain kinase (MLCK) are two recently identified muscle proteins encoded by the same gene cluster. The production of obscurin, which contains a Rho-guanine exchange factor (GEF)-like sequence, and obscurin-MLCK by this cluster suggests that these novel genes may be involved in signal transduction cascades that control adaptive and compensatory responses of the heart. The goal of the present study was to investigate the transcriptional response of the obscurin gene cluster to the initiation of myocardial hypertrophy induced in mice by aortic constriction. The transcriptional activity of the obscurin genes was examined using reverse-transcriptase primed quantitative PCR. We found that the transcripts encoding the obscurin Rho-GEF and the obscurin-MLCK internal serine-threonine kinase II (SK II) domains were significantly upregulated following aortic constriction. The expression of Rho-GEF-containing transcripts at different stages of the hypertrophic growth exceeded the control levels by 2- to 6-fold. Following the induction of hypertrophy, the quantity of the SK II-encoding transcripts increased 10-fold by 24h and 16-fold by 48h, then decreased by day 7, and returned to the control level by day 56. The quantity of the carboxy terminal obscurin-MLCK transcripts encoding for SK I increased 2-fold by day 2 and returned to the control values at later stages. Immunolocalization of obscurin, which contains Rho-GEF domain, in cardiomyocytes during pharmacologically induced hypertrophic growth in vitro demonstrated that the expression was topographically associated with the growing myofibrils and with the sites of initiation and progression of myofibrillogenesis at the periphery of the sarcoplasm. This suggests that upregulation of obscurin synthesis is associated with the formation of additional amounts of contractile structures during cardiac hypertrophy. Thus, the obscurin gene cluster represents a new example of an operon that encodes differentially regulated structural and signaling proteins implicated in the control of assembly and adaptive remodeling of myofibrils during normal and hypertrophic growth.

Animals↗

PubMed2003

Obscurin is a ligand for small ankyrin 1 in skeletal muscle.

The factors that organize the internal membranes of cells are still poorly understood. We have been addressing this question using striated muscle cells, which have regular arrays of membranes that associate with the contractile apparatus in stereotypic patterns. Here we examine links between contractile structures and the sarcoplasmic reticulum (SR) established by small ankyrin 1 (sAnk1), a approximately 17.5-kDa integral protein of network SR. We used yeast two-hybrid to identify obscurin, a giant Rho-GEF protein, as the major cytoplasmic ligand for sAnk1. The binding of obscurin to the cytoplasmic sequence of sAnk1 is mediated by a sequence of obscurin that is C-terminal to its last Ig-like domain. Binding was confirmed in two in vitro assays. In one, GST-obscurin, bound to glutathione-matrix, specifically adsorbed native sAnk1 from muscle homogenates. In the second, MBP-obscurin bound recombinant GST-sAnk1 in nitrocellulose blots. Kinetic studies using surface plasmon resonance yielded a K(D) = 130 nM. On subcellular fractionation, obscurin was concentrated in the myofibrillar fraction, consistent with its identification as sarcomeric protein. Nevertheless, obscurin, like sAnk1, concentrated around Z-disks and M-lines of striated muscle. Our findings suggest that obscurin binds sAnk1, and are the first to document a specific and direct interaction between proteins of the sarcomere and the SR.

Amino Acid Sequence↗

PubMed2002

The hydrophilic domain of small ankyrin-1 interacts with the two N-terminal immunoglobulin domains of titin.

Little is known about the mechanisms that organize the internal membrane systems in eukaryotic cells. We are addressing this question in striated muscle, which contains two novel systems of internal membranes, the transverse tubules and the sarcoplasmic reticulum (SR). Small ankyrin-1 (sAnk1) is an approximately 17-kDa transmembrane protein of the SR that concentrates around the Z-disks and M-lines of each sarcomere. We used the yeast two-hybrid assay to determine whether sAnk1 interacts with titin, a giant myofibrillar protein that organizes the sarcomere. We found that the hydrophilic cytoplasmic domain of sAnk1 interacted with the two most N-terminal Ig domains of titin, ZIg1 and ZIg2, which are present at the Z-line in situ. Both ZIg1 and ZIg2 were required for binding activity. sAnk1 did not interact with other sequences of titin that span the Z-disk or with Ig domains of titin near the M-line. Titin ZIg1/2 also bound T-cap/telethonin, a 19-kDa protein of the Z-line. We show that titin ZIg1/2 could form a three-way complex with sAnk1 and T-cap. Our results indicate that titin ZIg1/2 can bind sAnk1 in muscle homogenates and suggest a role for these proteins in organizing the SR around the contractile apparatus at the Z-line.

Ankyrins↗