Adjacent Sarcomeres Research Articles

The non-muscle functions of actinins: an update

α-Actinins are a major class of actin filament cross-linking proteins expressed in virtually all cells. In muscle, actinins cross-link thin filaments from adjacent sarcomeres. In non-muscle cells, different actinin isoforms play analogous roles in cross-linking actin filaments and anchoring them to structures such as cell-cell and cell-matrix junctions. Although actinins have long been known to play roles in cytokinesis, cell adhesion and cell migration, recent studies have provided further mechanistic insights into these functions. Roles for actinins in synaptic plasticity and membrane trafficking events have emerged more recently, as has a 'non-canonical' function for actinins in transcriptional regulation in the nucleus. In the present paper we review recent advances in our understanding of these diverse cell biological functions of actinins in non-muscle cells, as well as their roles in cancer and in genetic disorders affecting platelet and kidney physiology. We also make two proposals with regard to the actinin nomenclature. First, we argue that naming actinin isoforms according to their expression patterns is problematic and we suggest a more precise nomenclature system. Secondly, we suggest that the α in α-actinin is superfluous and can be omitted.

Cryo-Em of Z-Discs Isolated from Honeybee Flight Muscle

Muscle Z-discs were originally thought to have the purely mechanical function of transmitting contractile force along myofibrils, connecting thin filaments in adjacent sarcomeres through Z-bridges composed principally of α-actinin. However, the Z-disc is now known to be considerably more complex, with ∼40 different proteins, some of which are transient. Other Z-disc functions identified include stress sensing into signalling pathways involving muscle growth, remodelling and degradation. Z-discs vary widely in different muscles, including differences in thickness and symmetry. Electron microscope transverse sections of vertebrate muscle show Z-discs with tetragonal symmetry, but with two appearances, called small-square and basketweave, with the basketweave lattice 10% smaller. The relative proportions of these states can be modulated by several factors affecting the state of the muscle, but the significance of this transition is not known. Invertebrate Z-discs have hexagonal symmetry and have been most studied in insect flight muscle. The structure of the Z-disc is known only in outline, to ∼7 nm resolution, whereas ∼2 nm resolution is required to recognise protein shapes and accurately dock crystal structures. Isolated Z-discs are potentially useful for EM studies because they are naturally thin and do not have to be cut into thin sections, which causes damage and loss of resolution. Reports of methods to prepare and purify Z-discs date back 50 years, but such preparations have not been examined by cryo-EM or tomography, which may improve resolution. Isolated Z-discs may also be valuable for composition studies, perhaps including genetic modification. We have obtained preliminary cryo-EM data from honeybee flight muscle Z-discs prepared by high salt treatment of myofibrils.

Mathematical modeling of the dynamic mechanical behavior of neighboring sarcomeres in actin stress fibers.

Actin stress fibers (SFs) in live cells consist of series of dynamic individual sarcomeric units. Within a group of consecutive SF sarcomeres, individual sarcomeres can spontaneously shorten or lengthen without changing the overall length of this group, but the underlying mechanism is unclear. We used a computational model to test our hypothesis that this dynamic behavior is inherent to the heterogeneous mechanical properties of the sarcomeres and the cytoplasmic viscosity. Each sarcomere was modeled as a discrete element consisting of an elastic spring, a viscous dashpot and an active contractile unit all connected in parallel, and experiences forces as a result of actin filament elastic stiffness, myosin II contractility, internal viscoelasticity, or cytoplasmic drag. When all four types of forces are considered, the simulated dynamic behavior closely resembles the experimental observations, which include a low-frequency fluctuation in individual sarcomere length and compensatory lengthening and shortening of adjacent sarcomeres. Our results suggest that heterogeneous stiffness and viscoelasticity of actin fibers, heterogeneous myosin II contractility, and the cytoplasmic drag are sufficient to cause spontaneous fluctuations in SF sarcomere length. Our results shed new light to the dynamic behavior of SF and help design experiments to further our understanding of SF dynamics.

P.15.4 ZASP–sZM mutations in myofibrillar myopathy cause skeletal muscle Z-disc disruption by disassembling α-actinin cross-linked skeletal actin filaments

Myofibrillar myopathies (MFM) are characterized by early and prominent disruption of the Z-disc with focal dissolution of myofibrils and ectopic accumulation of myofibrillar proteins. The molecular mechanisms underlying the Z-disc disruption in MFM are not yet delineated. The Z-disc is a key element for structural integrity of the sarcomere, the basic contractile unit in skeletal muscle. The core of the Z-disc consists of actin filaments (F-actin) from adjacent sarcomeres cross-linked by α-actinin. We explored the mechanism of skeletal muscle Z-disc disruption in zaspopathy, a prototype MFM caused by ZASP mutations (A147T and A165V) at or within a highly conserved motif (sZM) that is expressed in striated muscle. Our studies show that ZASP is a novel skeletal actin binding protein and that the sZM domain is important for ZASP-actin interaction in skeletal muscle. Both wild type and mutant ZASP interact with skeletal actin, but only mutant proteins cause disassembly of actinin-crosslinked F-actin in vitro and the Z-disc disruption with F-actin accumulation in electroporated mouse skeletal muscle, as in the human disease. These results show that ZASP–sZM mutations have deleterious effects on the core structure of the Z-discs in striated muscle and support a toxic gain-of-function disease mechanism. Other MFM gene products (myotilin, desmin, BAG3, αβ-Crystallin, and filamin C) are known to regulate the organization and stability of F-actin in skeletal muscle. It is possible that the molecular pathways leading to skeletal muscle Z-disc disruption are shared by these myopathies. Alteration of F-actin dynamics, by either direct or indirect association of mutant proteins with actin filaments, may emerge as a unifying disease mechanism in this group of degenerative myopathies.

Short Read (Next-Generation) Sequencing

Rapid advances in DNA sequencing technologies have made it increasingly cost-effective to obtain accurate and timely large-scale genomic sequence data on individuals (short read massively parallel or next generation [next-gen]). A next-gen molecular diagnostic approach that has seen rapid deployment in the clinic over the last year is exome sequencing. Whole exome sequencing covers all protein-coding genes in the genome (≈1.1% of genome), and an exome test for a single patient generates ≈6 gigabases (109 bp) of DNA sequence data. A key challenge facing routine use of next-gen data in patient diagnosis and management is data interpretation. What sequence variant findings are relevant to diagnosis (pathogenic mutations)? What sequence variant findings are relevant to clinical care but not necessarily to patient diagnosis (clinically actionable incidental data)? What sequence information should be stored, and where can it be stored? This review provides a tutorial on current approaches to answering these questions. A recent landmark study showed that application of next-gen sequencing to a large cohort of idiopathic dilated cardiomyopathy patients found ≈27% of patients to show mutations of the titin gene, the most complex gene in the genome (363 exons). We use titin in cardiomyopathy as an exemplar for explaining next-gen sequencing approaches and data interpretation. Decreasing sequencing costs and broad dissemination of next-generation (next-gen) equipment and expertise are increasing availability of massively parallel sequencing of patient DNA samples (short read massively parallel or next-gen sequencing).1,2 Most rapidly expanding is exome sequencing, where all protein-coding sequences (exons) are selected from total genomic DNA and selectively sequenced.3 Alternative approaches to next-gen sequencing include targeted sequencing (TS) and whole genome (complete genome) sequencing. Currently, marketed targeted Sanger sequencing panels using traditional individual exon-by-exon sequencing remain expensive and time consuming, and massively parallel next-gen approaches are beginning to supplant …

Stress Fibers Get a Makeover

Stress Fibers Get a Makeover

Lateral Communication between Stress Fiber Sarcomeres Facilitates a Local Remodeling Response

Actin stress fibers (SFs) are load-bearing and mechanosensitive structures. To our knowledge, the mechanisms that enable SFs to sense and respond to strain have not been fully defined. Acute local strain events can involve a twofold extension of a single SF sarcomere, but how these dramatic local events affect the overall SF architecture is not believed to be understood. Here we have investigated how SF architecture adjusts to episodes of local strain that occur in the cell center. Using fluorescently tagged zyxin to track the borders of sarcomeres, we characterize the dynamics of resting sarcomeres and strain-site sarcomeres. We find that sarcomeres flanking a strain site undergo rapid shortening that directly compensates for the strain-site extension, illustrating lateral communication of mechanical information along the length of a stress fiber. When a strain-site sarcomere extends asymmetrically, its adjacent sarcomeres exhibit a parallel asymmetric shortening response, illustrating that flanking sarcomeres respond to strain magnitude. After extension, strain-site sarcomeres become locations of new sarcomere addition, highlighting mechanical strain as a trigger of sarcomere addition and revealing a, to our knowledge, novel type of SF remodeling. Our findings provide evidence to suggest SF sarcomeres act as strain sensors and are interconnected to support communication of mechanical information.

Analyses of Sarcomeric Self-Oscillatory Properties of Rat Neonatal Cardiomyocytes

It is generally believed that the rhythm of heartbeat is controlled by electric stimulations produced by pacemaker cells. However, sarcomeres in cardiac myofibrils exhibit spontaneous rhythmic oscillations of sarcomere length (SL) and force under partial activation conditions (i.e., SPOC). SPOC occurs either at pCa∼6.0 (Ca-SPOC ) or at the coexistence of MgADP and Pi in the absence of Ca2+ (ADP-SPOC) (Ishiwata et al., 2011). We have reported that the SPOC period correlates strongly with the resting heart rate in various animal species (Sasaki et al., 2006). In the present study, to test whether SPOC plays a key role in the regulation of heartbeat, we performed analyses on the waveform properties of SPOC by using rat neonatal cardiomyocytes expressing AcGFP in the Z-lines. First, we found that SPOC occurs in the neonatal myocytes treated with ionomycin at pCa ∼6.0 (pCa adjusted by Ca/EGTA), viz., in the presence of the inner-membrane system. We termed this phenomenon “Cell-SPOC ”. Our careful analysis revealed that the shortening time depends strongly on the amplitude during Cell-SPOC, whereas the dependence was relatively weak for the lengthening time. Next, we electrically activated the intact, untreated myocytes at various frequencies (1 - 5 Hz) in Tyrode's solution containing 2 mM Ca2+, and compared the waveform properties of SL oscillations to those obtained during Cell-SPOC. We found that the waveform properties at physiologically high electric frequencies (4 - 5Hz) were statistically indistinguishable from those obtained during Cell-SPOC occurring at the similar frequencies. These findings support the notion that SPOC plays a key role in propagating the rhythm of shortening/relengthening to adjacent sarcomeres along myofibrils, thereby producing efficient, periodic contractions of cardiac muscle in vivo.

The Sarcomeric Protein Nebulin: Another Multifunctional Giant in Charge of Muscle Strength Optimization

The sliding filament model of the sarcomere was developed more than half a century ago. This model, consisting only of thin and thick filaments, has been successful in explaining many, but not all, features of skeletal muscle. Work during the 1980s revealed the existence of two additional filaments: the giant filamentous proteins titin and nebulin. Whereas the role of titin rapidly progressed, nebulin’s role in muscle structure and function remained long nebulous. An important feature of muscle structure and function that has remained relatively obscure concerns the mechanisms that are involved in regulating thin filament length. Filament length is an important aspect of muscle function as force production is proportional to the amount of overlap between thick and thin filaments. Recent advances, due in part to the generation of nebulin KO models, reveal that nebulin plays an important role in the regulation of thin filament length, most likely by stabilizing F-actin assemblies. Another structural feature of skeletal muscle that has been incompletely understood concerns the mechanisms involved in maintaining Z-disk structure and the regular lateral alignment of adjacent sarcomeres during contraction. Recent studies indicate that nebulin is part of a protein complex that mechanically links adjacent myofibrils. In addition to these structural roles in support of myofibrillar force generation, nebulin has been also shown to regulate directly muscle contraction at the level of individual crossbridges: cycling kinetics and the calcium sensitivity of force producing crossbridges is enhanced in the presence of nebulin. Thus, these recent data all point to nebulin being important for muscle force optimization. Consequently, muscle weakness as the lead symptom develops in the case of patients with nemaline myopathy that have mutations in the nebulin gene. Here, we discuss these important novel insights into the role of nebulin in skeletal muscle function.

Electron Microscopy and X-Ray Diffraction Evidence for Two Z-Band Structural States

In vertebrate muscles, Z-bands connect adjacent sarcomeres, incorporate several cell signaling proteins, and may act as strain sensors. Previous electron microscopy (EM) showed Z-bands reversibly switch between a relaxed, “small-square” structure, and an active, “basketweave” structure, but the mechanism of this transition is unknown. Here, we found the ratio of small-square to basketweave in relaxed rabbit psoas muscle varied with temperature, osmotic pressure, or ionic strength, independent of activation. By EM, the A-band and both Z-band lattice spacings varied with temperature and pressure, not ionic strength; however, the basketweave spacing was consistently 10% larger than small-square. We next sought evidence for the two Z-band structures in unfixed muscles using x-ray diffraction, which indicated two Z-reflections whose intensity ratios and spacings correspond closely to the EM measurements for small-square and basketweave if the EM spacings are adjusted for 20% shrinkage due to EM processing. We conclude that the two Z-reflections arise from the small-square and basketweave forms of the Z-band as seen by EM. Regarding the mechanism of transition during activation, the effects of Ca 2+ in the presence of force inhibitors suggested that the interconversion of Z-band forms was correlated with tropomyosin movement on actin.

Reduced myofibrillar connectivity and increased Z-disk width in nebulin-deficient skeletal muscle

A prominent feature of striated muscle is the regular lateral alignment of adjacent sarcomeres. An important intermyofibrillar linking protein is the intermediate filament protein desmin, and based on biochemical and structural studies in primary cultures of myocytes it has been proposed that desmin interacts with the sarcomeric protein nebulin. Here we tested whether nebulin is part of a novel biomechanical linker complex, by using a recently developed nebulin knockout (KO) mouse model and measuring Z-disk displacement in adjacent myofibrils of both extensor digitorum longus (EDL) and soleus muscle. Z-disk displacement increased as sarcomere length (SL) was increased and the increase was significantly larger in KO fibers than in wild-type (WT) fibers; results in 3-day-old and 10-day-old mice were similar. Immunoelectron microscopy revealed reduced levels of desmin in intermyofibrillar spaces adjacent to Z-disks in KO fibers compared with WT fibers. We also performed siRNA knockdown of nebulin and expressed modules within the Z-disk portion of nebulin (M160-M170) in quail myotubes and found that this prevented the mature Z-disk localization of desmin filaments. Combined, these data suggest a model in which desmin attaches to the Z-disk through an interaction with nebulin. Finally, because nebulin has been proposed to play a role in specifying Z-disk width, we also measured Z-disk width in nebulin KO mice. Results show that most Z-disks of KO mice were modestly increased in width (approximately 80 nm in soleus and approximately 40 nm in EDL fibers) whereas a small subset had severely increased widths (up to approximately 1 microm) and resembled nemaline rod bodies. In summary, structural studies on a nebulin KO mouse show that in the absence of nebulin, Z-disks are significantly wider and that myofibrils are misaligned. Thus the functional roles of nebulin extend beyond thin filament length regulation and include roles in maintaining physiological Z-disk widths and myofibrillar connectivity.

New Insights into the Structural Roles of Nebulin in Skeletal Muscle

One important feature of muscle structure and function that has remained relatively obscure is the mechanism that regulates thin filament length. Filament length is an important aspect of muscle function as force production is proportional to the amount of overlap between thick and thin filaments. Recent advances, due in part to the generation of nebulin KO models, reveal that nebulin plays an important role in the regulation of thin filament length. Another structural feature of skeletal muscle that is not well understood is the mechanism involved in maintaining the regular lateral alignment of adjacent sarcomeres, that is, myofibrillar connectivity. Recent studies indicate that nebulin is part of a protein complex that mechanically links adjacent myofibrils. Thus, novel structural roles of nebulin in skeletal muscle involve the regulation of thin filament length and maintaining myofibrillar connectivity. When these functions of nebulin are absent, muscle weakness ensues, as is the case in patients with nemaline myopathy with mutations in nebulin. Here we review these new insights in the role of nebulin in skeletal muscle structure.

Sarcomere Mechanics in Capillary Endothelial Cells

Tension generation in endothelial cells of the aorta, spleen, and eye occurs in actin stress fibers, and is necessary for normal cell function. Sarcomeres are the tension-generating units of actin stress fibers in endothelial cells. How sarcomeres generate and maintain tension in stress fibers is not well understood. Using femtosecond laser ablation, we severed living stress fibers and measured sarcomere contraction under zero tension. The length of the sarcomere decreased in two phases: an instantaneous initial response, followed by a slower change in length attributed to myosin activity. The latter phase ceased abruptly after a minimum sarcomere length was reached, suggesting a rigid resistance that prevents further contraction. Furthermore, severed, contracted stress fibers did not relax when treated with myosin inhibitors, indicating that contracted stress fibers do not store elastic potential energy. These novel measurements combined with modeling suggest that myosin-generated forces in adjacent sarcomeres are directly in balance, and argue against sarcomere models with springlike elements in parallel with myosin contractile elements. We propose a new model for tension generation in the sarcomere, which provides a mechanistic interpretation for our observations and previous observations of inhomogeneous sarcomere contraction and apparent stress fiber viscoelastic behavior.

Inter-sarcomere coordination in muscle revealed through individual sarcomere response to quick stretch

The force generation and motion of muscle are produced by the collective work of thousands of sarcomeres, the basic structural units of striated muscle. Based on their series connection to form a myofibril, it is expected that sarcomeres are mechanically and/or structurally coupled to each other. However, the behavior of individual sarcomeres and the coupling dynamics between sarcomeres remain elusive, because muscle mechanics has so far been investigated mainly by analyzing the averaged behavior of thousands of sarcomeres in muscle fibers. In this study, we directly measured the length-responses of individual sarcomeres to quick stretch at partial activation, using micromanipulation of skeletal myofibrils under a phase-contrast microscope. The experiments were performed at ADP-activation (1 mM MgATP and 2 mM MgADP in the absence of Ca(2+)) and also at Ca(2+)-activation (1 mM MgATP at pCa 6.3) conditions. We show that under these activation conditions, sarcomeres exhibit 2 distinct types of responses, either "resisting" or "yielding," which are clearly distinguished by the lengthening distance of single sarcomeres in response to stretch. These 2 types of sarcomeres tended to coexist within the myofibril, and the sarcomere "yielding" occurred in clusters composed of several adjacent sarcomeres. The labeling of Z-line with anti-alpha-actinin antibody significantly suppressed the clustered sarcomere "yielding." These results strongly suggest that the contractile system of muscle possesses the mechanism of structure-based inter-sarcomere coordination.

Nebulin Interacts with CapZ and Regulates Thin Filament Architecture within the Z-Disc

The barbed ends of actin filaments in striated muscle are anchored within the Z-disc and capped by CapZ; this protein blocks actin polymerization and depolymerization in vitro. The mature lengths of the thin filaments are likely specified by the giant "molecular ruler" nebulin, which spans the length of the thin filament. Here, we report that CapZ specifically interacts with the C terminus of nebulin (modules 160-164) in blot overlay, solid-phase binding, tryptophan fluorescence, and SPOTs membrane assays. Binding of nebulin modules 160-164 to CapZ does not affect the ability of CapZ to cap actin filaments in vitro, consistent with our observation that neither of the two C-terminal actin binding regions of CapZ is necessary for its interaction with nebulin. Knockdown of nebulin in chick skeletal myotubes using small interfering RNA results in a reduction of assembled CapZ, and, strikingly, a loss of the uniform alignment of the barbed ends of the actin filaments. These data suggest that nebulin restricts the position of thin filament barbed ends to the Z-disc via a direct interaction with CapZ. We propose a novel molecular model of Z-disc architecture in which nebulin interacts with CapZ from a thin filament of an adjacent sarcomere, thus providing a structural link between sarcomeres.

Differentially expressed genes and morphological changes during lengthened immobilization in rat soleus muscle

To examine the effect of lengthened immobilization on the expression of genes and concomitant morphological changes in soleus muscle, rat hindlimbs were immobilized at the ankle in full dorsiflexion by plaster cast. After removing the muscle (after 1 hr, 1, 4, and 7 days of immobilization), morphology and differential gene expression were analyzed through electron microscopy and differential display reverse transcription-polymerase chain reaction (DDRT-PCR), respectively. At the myotendinous junction (MTJ), a large cytoplasmic space appeared after 1 hr of immobilization and became enlarged over time, together with damaged Z lines. Interfibrillar space was detected after 1 day of immobilization, but diminished after 7 days. At the muscle belly, Z-line streaming and widening were observed following 1 hr of immobilization. Disorganization of myofilaments (misalignment of adjacent sarcomeres, distortion, or absence of Z lines) was detected after 4 days. Furthermore, mitochondrial swelling and cristae disruption were observed after 1 day of stretching. A set of 15 differentially expressed candidate genes was identified through DDRT-PCR. Of 11 known genes, seven ( Atp5g3, TOM22, INrf2, Slc25a4, Hdac6, Tpm1, and Sv2b) were up and three ( Podxl, Myh1, and Surf1) were down-regulated following immobilization. In the case of Acyp2, 1-day stretching-specific expression was observed. Atp5g3, Slc25a4, TOM22, and Surf1 are mitochondrial proteins related to energy metabolism, except TOM22, which has a chaperone-like activity located in the mitochondrial outer membrane. Together with these, INrf2, Hdac6, Podxl, and Acyp2 are related more or less to stress-induced apoptosis, indicating the responses to apoptotic changes in mitochondria caused by stretching. The expression of both Tpm1 and Myh1, fast twitch isoforms, suggests adaption to the immobilization. These results altogether indicate that lengthened immobilization regulates the expression of several stress/apoptosis-related and muscle-specific genes responsible for the slow-to-fast transition in soleus muscle despite profound muscle atrophy.

Role of Protein-tyrosine Phosphatase SHP2 in Focal Adhesion Kinase Down-regulation during Neutrophil Cathepsin G-induced Cardiomyocytes Anoikis

Inflammatory cells and their proteases contribute to tissue reparation at site of inflammation. Although beneficial at early stages, excessive inflammatory reaction leads to cell death and tissue damage. Cathepsin G (Cat.G), a neutrophil-derived serine protease, has been shown to induce neonatal rat cardiomyocyte detachment and apoptosis by anoikis through caspase-3 dependent pathway. However the early mechanisms that trigger Cat.G-induced caspase-3 activation are not known. This study identifies focal adhesion kinase (FAK) tyrosine dephosphorylation as an early mechanism that regulates Cat.G-induced anoikis in cardiomyocytes. Both FAK tyrosine phosphorylation at Tyr-397 and kinase activity decrease rapidly upon Cat.G treatment and was associated with a decrease of FAK association with adapter and cytoskeletal proteins, p130(Cas) and paxillin, respectively. FAK-decreased tyrosine phosphorylation is required for Cat.G-induced myocyte anoikis as concurrent expression of phosphorylation-deficient FAK mutated at Tyr-397 or pretreatment with a protein-tyrosine phosphatase (PTP) inhibitor, pervanadate, blocks Cat.G-induced FAK tyrosine dephosphorylation, caspase-3 activation and DNA fragmentation. Analysis of PTPs activation shows that Cat.G treatment induces an increase of SHP2 and PTEN phosphorylation; however, only SHP2 forms a complex with FAK in response to Cat.G. Expression of dominant negative SHP2 mutant markedly attenuates FAK tyrosine dephosphorylation induced by Cat.G and protects myocytes to undergo apoptosis. In contrast, increased SHP2 expression exacerbates Cat.G-induced FAK tyrosine dephosphorylation and myocyte apoptosis. Taken together, these results show that Cat.G induces SHP2 activation that leads to FAK tyrosine dephosphorylation and promotes cardiomyocyte anoikis.

A Drosophila model of oculopharyngeal muscular dystrophy reveals intrinsic toxicity of PABPN1

Oculopharyngeal muscular dystrophy (OPMD) is an adult-onset syndrome characterized by progressive degeneration of particular muscles. OPMD is caused by short GCG repeat expansions within the gene encoding the nuclear poly(A)-binding protein 1 (PABPN1) that extend an N-terminal polyalanine tract in the protein. Mutant PABPN1 aggregates as nuclear inclusions in OMPD patient muscles. We have created a Drosophila model of OPMD that recapitulates the features of the human disorder: progressive muscle degeneration, with muscle defects proportional to the number of alanines in the tract, and formation of PABPN1 nuclear inclusions. Strikingly, the polyalanine tract is not absolutely required for muscle degeneration, whereas another domain of PABPN1, the RNA-binding domain and its function in RNA binding are required. This demonstrates that OPMD does not result from polyalanine toxicity, but from an intrinsic property of PABPN1. We also identify several suppressors of the OPMD phenotype. This establishes our OPMD Drosophila model as a powerful in vivo test to understand the disease process and develop novel therapeutic strategies.

Characterization of Functional Abnormalities in Hearts of Transgenic Mice Expressing Forms of Actin Capping Protein Defective in Attaching Actin Filaments to Z‐lines

In striated muscle, the barbed ends of actin filaments are attached to Z lines. Biochemical and cell biological studies suggest that actin capping protein (CP) mediates this attachment by binding the barbed ends of actin filaments and alpha-actinin crosslinks overlapping filaments from adjacent sarcomeres. Previous transgenic studies revealed that defective interaction between CP and actin filaments causes major structural defects in sarcomere organization, leading to cardiac hypertrophy and lethality. To determine the basis of the myofibril defect, we have examined the hearts of transgenic mice using histological and immunofluorescence microscopy. The hearts of transgenic mice were hypertrophied relative to body size, with an enlargement of the ventricle wall. Immunofluorescence studies revealed an altered actin organization with disruption of myofibril architecture. To understand how this defect affects cardiac function, blood pressure was measured using an indirect tail pressure assay and ECG measurements determined. The transgenic mice had a lower systolic pressure and an increased heart rate compared to wildtype mice. In ECG studies, a significant increase for both the PR and R wave height was detected, indicative of atrial and ventricular hypertrophy, validating histological studies. This research was funded by a Faculty Development Grant, Minnesota State University-Mankato, MN.

Peroxynitrite-induced α-actinin nitration and contractile alterations in isolated human myocardial cells

Peroxynitrite-mediated myocardial protein nitration has been associated with a depressed cardiac pump function. In the present study, an attempt was made to elucidate the molecular background of peroxynitrite-evoked alterations in the human myocardium. Isometric force generation was measured in permeabilized human ventricular myocytes and biochemical methods were employed to identify the proteins affected by peroxynitrite-induced nitrotyrosine formation. The maximal Ca(2+)-activated isometric force (pCa=4.75) decreased to zero with increasing concentrations of peroxynitrite in a concentration-dependent manner (IC50: 55+/-4 microM; based on a total of 75 myocytes). However, there were no differences before and after the application of 50 microM peroxynitrite in the Ca(2+)-sensitivity of force production (pCa50: 5.89+/-0.02 and 5.86+/-0.04), in the steepness of the Ca(2+)-force relationship (nHill: 2.22+/-0.11 and 2.42+/-0.25), and in the actin-myosin turnover kinetics (k(tr) at saturating [Ca2+]: 1.14+/-0.03 1/s and 1.05+/-0.07 1/s) (P>0.05). Nevertheless, 50 muM peroxynitrite greatly deteriorated the cross-striation pattern and induced a slight, but significant, increase in the passive force component (from 2.1+/-0.1 to 2.5+/-0.2 kN/m2; n=57 cells), reflecting ultrastructural alterations. Western immunoblots revealed that 50 microM peroxynitrite selectively induced the nitration of a protein with an apparent molecular mass of about 100 kDa. Subsequent immunoprecipitation assays identified this nitrated protein as alpha-actinin, a major Z-line protein. These results suggest alpha-actinin as a novel target for peroxynitrite in the human myocardium; its nitration induces a contractile dysfunction, presumably by decreasing the longitudinal transmission of force between adjacent sarcomeres.