Generation of two iPSC clones (MHHi021-A and MHHi021-B) from a patient with hypertrophic cardiomyopathy with p.Arg723Gly mutation in the MYH7 gene.

Hypertrophic cardiomyopathy (HCM) is the most common form of genetic heart disease and is characterized by abnormal thickening of the left ventricular wall and interventricular septum. Here we describe the generation of two induced pluripotent stem cell (iPSC) clones from a HCM patient, heterozygous for the p.Arg723Gly (c.2169C > G) mutation in the MYH7 gene. The generated iPSC clones may provide a useful resource for disease modelling to study the mechanisms underlying HCM pathogenesis in iPSC derived progenies, in particular cardiomyocytes.

Hypertrophic cardiomyopathy MYH7 mutation R723G alters mRNA secondary structure.

Point mutation R723G in the MYH7 gene causes hypertrophic cardiomyopathy (HCM). Heterozygous patients with this mutation exhibit a comparable allelic imbalance of the MYH7 gene. On average 67% of the total MYH7 mRNA are derived from the MYH7R723G-allele and 33% from the MYH7WT allele. Mechanisms underlying mRNA allelic imbalance are largely unknown. We suggest that a different mRNA lifetime of the alleles may cause the allelic drift in R723G patients. A potent regulator of mRNA lifetime is its secondary structure. To test for alterations in the MYH7R723G mRNA structure we used selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE) analysis. We show significantly different SHAPE reactivity of wild-type and MYH7R723G RNA, which is in accordance with bioinformatically predicted structures. Thus, we provide the first experimental evidence for mRNA secondary structure alterations by the HCM point mutation. We assume that this may result in a prolonged lifetime of MYH7R723G mRNA in vivo and subsequently in the determined allelic imbalance.

Successful knock-in of Hypertrophic Cardiomyopathy-mutation R723G into the MYH7 gene mimics HCM pathology in pigs.

Familial Hypertrophic Cardiomyopathy (HCM) is the most common inherited cardiac disease. About 30% of the patients are heterozygous for mutations in the MYH7 gene encoding the ß-myosin heavy chain (MyHC). Hallmarks of HCM are cardiomyocyte disarray and hypertrophy of the left ventricle, the symptoms range from slight arrhythmias to sudden cardiac death or heart failure. To gain insight into the underlying mechanisms of the diseases' etiology we aimed to generate genome edited pigs with an HCM-mutation. We used TALEN-mediated genome editing and successfully introduced the HCM-point mutation R723G into the MYH7 gene of porcine fibroblasts and subsequently cloned pigs that were heterozygous for the HCM-mutation R723G. No off-target effects were determined in the R723G-pigs. Surprisingly, the animals died within 24 h post partem, probably due to heart failure as indicated by a shift in the a/ß-MyHC ratio in the left ventricle. Most interestingly, the neonatal pigs displayed features of HCM, including mild myocyte disarray, malformed nuclei, and MYH7-overexpression. The finding of HCM-specific pathology in neonatal R723G-piglets suggests a very early onset of the disease and highlights the importance of novel large animal models for studying causative mechanisms and long-term progression of human cardiac diseases.

Crop wild relative populations of Beta vulgaris allow direct mapping of agronomically important genes.

Rapid identification of agronomically important genes is of pivotal interest for crop breeding. One source of such genes are crop wild relative (CWR) populations. Here we used a CWR population of <200 wild beets (B. vulgaris ssp. maritima), sampled in their natural habitat, to identify the sugar beet (Beta vulgaris ssp. vulgaris) resistance gene Rz2 with a modified version of mapping-by-sequencing (MBS). For that, we generated a draft genome sequence of the wild beet. Our results show the importance of preserving CWR in situ and demonstrate the great potential of CWR for rapid discovery of causal genes relevant for crop improvement. The candidate gene for Rz2 was identified by MBS and subsequently corroborated via RNA interference (RNAi). Rz2 encodes a CC-NB-LRR protein. Access to the DNA sequence of Rz2 opens the path to improvement of resistance towards rhizomania not only by marker-assisted breeding but also by genome editing.

Evaluation of multi-locus models for genome-wide association studies: a case study in sugar beet.

Association mapping has become a widely applied genomic approach to dissect the genetic architecture of complex traits. A major issue for association mapping is the need to control for the confounding effects of population structure, which is commonly done by mixed models incorporating kinship information. In this case study, we employed experimental data from a large sugar beet population to evaluate multi-locus models for association mapping. As in linkage mapping, markers are selected as cofactors to control for population structure and genetic background variation. We compared different biometric models with regard to important quantitative trait locus (QTL) mapping parameters like the false-positive rate, the QTL detection power and the predictive power for the proportion of explained genotypic variance. Employing different approaches we show that the multi-locus model, that is, incorporating cofactors, outperforms the other models, including the mixed model used as a reference model. Thus, multi-locus models are an attractive alternative for association mapping to efficiently detect QTL for knowledge-based breeding.

Inflammatory response to burn trauma: nicotine attenuates proinflammatory cytokine levels.

OBJECTIVE: The immune response to an inflammatory stimulus is balanced and orchestrated by stimulatory and inhibitory factors. After a thermal trauma, this balance is disturbed and an excessive immune reaction with increased production and release of proinflammatory cytokines results. The nicotine-stimulated anti-inflammatory reflex offsets this. The goal of this study was to verify that transdermal administration of nicotine downregulates proinflammatory cytokine release after burn trauma. METHODS: A 30% total body surface area full-thickness rat burn model was used in Sprague Dawley rats (n = 35, male). The experimental animals were divided into a control group, a burn trauma group, a burn trauma group with additional nicotine treatment, and a sham + nicotine group with 5 experimental animals per group. The last 2 groups received a transdermal nicotine administration of 1.75 mg. The concentrations of tumor necrosis factor alpha, interleukin 1 beta, and interleukin 6 were determined in homogenates of hearts, livers, and spleens 12 or 24 hours after burn trauma. RESULTS: Experimental burn trauma resulted in a significant increase in cytokine levels in hearts, livers, and spleens. Nicotine treatment led to a decrease of the effect of the burn trauma with significantly lower concentrations of tumor necrosis factor alpha, interleukin 1 beta, and interleukin 6 compared to the trauma group. CONCLUSIONS: This study confirms in a standardized burn model that stimulation of the nicotinic acetylcholine receptor is involved in the regulation of effectory molecules of the immune response. Looking at the results of our study, further experiments designed to explore and evaluate the potency and mechanisms of the immunomodulating effects of this receptor system are warranted.

Cross-validation in association mapping and its relevance for the estimation of QTL parameters of complex traits.

Association mapping has become a widely applied genomic approach to identify quantitative trait loci (QTL) and dissect the genetic architecture of complex traits. However, approaches to assess the quality of the obtained QTL results are lacking. We therefore evaluated the potential of cross-validation in association mapping based on a large sugar beet data set. Our results show that the proportion of the population that should be used as estimation and validation sets, respectively, depends on the size of the mapping population. Generally, a fivefold cross-validation, that is, 20% of the lines as independent validation set, appears appropriate for commonly used population sizes. The predictive power for the proportion of genotypic variance explained by QTL was overestimated by on average 38% indicating a strong bias in the estimated QTL effects. The cross-validated predictive power ranged between 4 and 50%, which are more realistic estimates of this parameter for complex traits. In addition, QTL frequency distributions can be used to assess the precision of QTL position estimates and the robustness of the detected QTL. In summary, cross-validation can be a valuable tool to assess the quality of QTL parameters in association mapping.

Cardiomyopathy mutations reveal variable region of myosin converter as major element of cross-bridge compliance.

The ability of myosin to generate motile forces is based on elastic distortion of a structural element of the actomyosin complex (cross-bridge) that allows strain to develop before filament sliding. Addressing the question, which part of the actomyosin complex experiences main elastic distortion, we suggested previously that the converter domain might be the most compliant region of the myosin head domain. Here we test this proposal by studying functional effects of naturally occurring missense mutations in the beta-myosin heavy chain, 723Arg --> Gly (R723G) and 736Ile --> Thr (I736T), in comparison to 719Arg --> Trp (R719W). All three mutations are associated with hypertrophic cardiomyopathy and are located in the converter region of the myosin head domain. We determined several mechanical parameters of single skinned slow fibers isolated from Musculus soleus biopsies of hypertrophic cardiomyopathy patients and healthy controls. Major findings of this study for mutation R723G were i), a >40% increase in fiber stiffness in rigor with a 2.9-fold increase in stiffness per myosin head (S( *)(rigor R723G) = 0.84 pN/nm S( *)(rigor WT) = 0.29 pN/nm); and ii), a significant increase in force per head (F( *)(10 degrees C), 1.99 pN vs. 1.49 pN = 1.3-fold increase; F( *)(20 degrees C), 2.56 pN vs. 1.92 pN = 1.3-fold increase) as well as stiffness per head during isometric steady-state contraction (S( *)(active10 degrees C), 0.52 pN/nm vs. 0.28 pN/nm = 1.9-fold increase). Similar changes were found for mutation R719W (2.6-fold increase in S( *)(rigor); 1.8-fold increase in F( *)(10 degrees C), 1.6-fold in F( *)(20 degrees C); twofold increase in S( *)(active10 degrees C)). Changes in active cross-bridge cycling kinetics could not account for the increase in force and active stiffness. For the above estimates the previously determined fraction of mutated myosin in the biopsies was taken into account. Data for wild-type myosin of slow soleus muscle fibers support previous findings that for the slow myosin isoform S( *) and F( *) are significantly lower than for fast myosin e.g., of rabbit psoas muscle. The data indicate that two mutations, R723G and R719W, are associated with an increase in resistance to elastic distortion of the individual mutated myosin heads whereas mutation I736T has essentially no effect. The data strongly support the notion that major elastic distortion occurs within the converter itself. Apparently, the compliance depends on specific residues, e.g., R719 and R723, presumably located at strategic positions near the long alpha-helix of the light chain binding domain. Because amino acids 719 and 723 are nonconserved residues, cross-bridge stiffness may well be specifically tuned for different myosins.

Force-generating cross-bridges during ramp-shaped releases: evidence for a new structural state.

Mechanical and two-dimensional (2D) x-ray diffraction studies suggest that during isometric steady-state contraction, strongly bound cross-bridges mostly occupy early states in the power stroke, whereas rigor or rigor-like cross-bridges could not be detected. However, it remained unclear whether cross-bridges accumulate, at least transiently, in rigor or rigor-like states in response to rapid-length releases. We addressed this question using time-resolved recording of 2D x-ray diffraction patterns of permeabilized fibers from rabbit psoas muscles during isometric contraction and when small, ramp-shaped length-releases were applied to these fibers. This maneuver allows a transient accumulation of cross-bridges in states near the end of their power stroke. By lowering the temperature to 5 degrees C, force transients were slowed sufficiently to record diffraction patterns in several 2-4-ms time frames before and during such releases, using the RAPID detector (Refined ADC Per Input Detector) at beam line ID02 of the European Synchrotron Radiation Facility (Grenoble, France). The same sequence of frames was recorded in relaxation and rigor. Comparisons of 2D patterns recorded during isometric contraction, with patterns recorded at different [MgATPgammaS] and at 1 degrees C, showed that changes in intensity profiles along the first and sixth actin layer lines (ALL1 and ALL6, respectively) allowed for discernment of the formation of rigor or rigor-like cross-bridges. During ramp-shaped releases of activated fibers, intensity profiles along ALL1 and ALL6 did not reveal evidence for the accumulation of rigor-like cross-bridges. Instead, changes in the ALL6-profile suggest that during ramp-shaped releases, cross-bridges transiently accumulate in a structural state that, to our knowledge, was not previously seen, but that could well be a strongly bound state with the light-chain binding domain in a conformation between a near prepower-stroke (isometric) orientation and the orientation in rigor.

Recommended guidelines for reporting on emergency medical dispatch when conducting research in emergency medicine: the Utstein style.

OBJECTIVES: To establish a uniform framework describing the system and organisation of emergency medical response centres and the process of emergency medical dispatching (EMD) when reporting results from studies in emergency medicine and prehospital care. DESIGN AND RESULTS: In September 2005 a task force of 22 experts from 12 countries met in Stavanger; Norway at the Utstein Abbey to review data and establish a common terminology for medical dispatch centres including core and optional data to be used for health monitoring, benchmarking and future research.

[Severe Class III malocclusion: long-term stability. Retrospective analysis of 12 cases]. / Classes III sévères: stabilité à long terme. Etude rétrospective: à propos de 12 cas.

PURPOSE: Long-term stability of severe class III is rarely evaluated in the literature. We present our findings with 12 patients who underwent surgery from June 1995 to December 1997 and analyze cephalometric superpositions. MATERIALS AND METHODS: Twelve operated patients were reviewed to analyze long-term results (follow-up 3 years 8 months). The sex ratio was well balanced. Mean age was 23 years. All patients were given pre- and postoperative orthodontic care and underwent bimaxillary surgery with Lefort 1 osteotomy and sagittal osteotomy of the rami. Delaire cephalometry on preoperative and early and late postoperative films was used to analyze outcome. Skeletal instability was defined as displacement greater than 4 mm. Recurrence was defined as secondary loss of the functional and esthetic result. RESULTS: Four patients (33%) developed skeletal instability. All patients achieved long-term stability. DISCUSSION: To detail the cause of skeletal instability, study of the principal factors of instability is presented together with a discussion of data in the literature.

[Brain abscess and diffuse cervico-facial cellulitis: complication after mandibular third molar extraction]. / Abcès intracérébral et cellulite cervico-faciale diffuse: complications d'une extraction de dent de sagesse: à propos d'un cas.

The authors report a case of cervico-facial cellulitis with brain abscess after mandibular third molar removal. This is the observation of a 26 years old boy surgically treated for a cervico-facial cellulitis ten days after a third molar's removal. He was given anti-inflammatory drugs after removal for analgesia. After a phase of clinical improving, the patient developed pulmonary and brain abscess with neurological signs. He needed neurosurgery in emergency. After eight weeks of antibiotic treatment, the patient was cured with aftereffects (jaw constriction and sensory disorders of the right thigh). Cerebro-meningeal complications of diffuse cervico-facial cellulitis are exceptional but are responsible for heavy aftereffects. This observation confirms that using anti-inflammatory drugs for analgesia is associated with a higher rate of complications after dental removal.

In vitro engineering of heart muscle: artificial myocardial tissue.

INTRODUCTION: Myocardial infarction followed by heart failure represents one of the major causes of morbidity and mortality, particularly in industrialized countries. Engineering and subsequent transplantation of contractile artificial myocardial tissue and, consequently, the replacement of ischemic and infarcted areas of the heart provides a potential therapeutic alternative to whole organ transplantation. METHODS: Artificial myocardial tissue samples were engineered by seeding neonatal rat cardiomyocytes with a commercially available 3-dimensional collagen matrix. The cellular engraftment within the artificial myocardial tissues was examined microscopically. Force development was analyzed in spontaneously beating artificial myocardial tissues, after stretching, and after pharmacologic stimulation. Moreover, electrocardiograms were recorded. RESULTS: Artificial myocardial tissues showed continuous, rhythmic, and synchronized contractions for up to 13 weeks. Embedded cardiomyocytes were distributed equally within the 3-dimensional matrix. Application of Ca(2+) and epinephrine, as well as electrical stimulation or stretching, resulted in enhanced force development. Electrocardiographic recording was possible on spontaneously beating artificial myocardial tissue samples and revealed physiologic patterns. CONCLUSIONS: Using a clinically well-established collagen matrix, contractile myocardial tissue can be engineered in vitro successfully. Mechanical and biologic properties of artificial myocardial tissue resemble native cardiac tissue. Use of artificial myocardial tissues might be a promising approach to reconstitute degenerated or failing cardiac tissue in many disease states and therefore provide a reasonable alternative to whole organ transplantation.

[Lactic acid polymer implants in the repair of traumatic defects of the orbital floor]. / Intérêt des implants en polymères d'acide lactique dans la réparation des pertes de substance traumatiques du plancher de l'orbite.

BACKGROUND: The criteria determining the choice of the technique used to repair traumatic orbital floor defects include the quality of the expected result, morbidity, ease of use and plasticity of the method, and its cost and availability. Among the different methods proposed, lactic acid polymer implants are particularly interesting. MATERIAL AND METHODS: Eighteen patients with an isolated blow-out fracture of the orbital floor were treated with a lactic acid polymer implant between 1995 and 1996. Ten of these patients were reviewed at 24 to 43 months follow-up. RESULTS: The mean age of the patients was 35 years (20-52 years). No residual diplopia was observed. None of the patients had an anomalous orbital volume or ocular dystopia. None of the implants migrated. One patient experienced episodes of palpebral inflammation that resolved spontaneously. The ten patients reviewed were satisfied with the outcome. DISCUSSION: The properties of lactic acid polymer implants facilitate their use, avoid morbidity and provide a quality result. This is the procedure of choice for repairing tissue loss of the orbital floor. It has several advantages over alternative methods (the implants are rigid, thin, resorbable and well tolerated) without having their defects (thickness, fragility, roughness, predetermined form, rapid alteration, specific instrumentation, iatrogenic disorders). In addition, lactic acid polymer plates can be remodeled when heated, allowing a precise adaptation of the implant to the orbital structures. Finally, the cost, compared with the advantages, is not a barrier for routine use.

Growth factors regulate phototransduction in retinal rods by modulating cyclic nucleotide-gated channels through dephosphorylation of a specific tyrosine residue.

Illumination of vertebrate rod photoreceptors leads to a decrease in the cytoplasmic cGMP concentration and closure of cyclic nucleotide-gated (CNG) channels. Except for Ca(2+), which plays a negative feedback role in adaptation, and 11-cis-retinal, supplied by the retinal pigment epithelium, all of the biochemical machinery of phototransduction is thought to be contained within rod outer segments without involvement of extrinsic regulatory molecules. Here we show that insulin-like growth factor-I (IGF-I), a paracrine factor released from the retinal pigment epithelium, alters phototransduction by rapidly increasing the cGMP sensitivity of CNG channels. The IGF-I-signaling pathway ultimately involves a protein tyrosine phosphatase that catalyzes dephosphorylation of a specific residue in the alpha-subunit of the rod CNG channel protein. IGF-I conjointly accelerates the kinetics and increases the amplitude of the light response, distinct from events that accompany adaptation. These effects of IGF-I could result from the enhancement of the cGMP sensitivity of CNG channels. Hence, in addition to long-term control of development and survival of rods, growth factors regulate phototransduction in the short term by modulating CNG channels.

Linkage disequilibrium mapping of the bolting gene in sea beet using AFLP markers.

The possibility of using linkage disequilibrium mapping in natural plant populations was assessed. In studying linkage disequilibrium among 137 mapped AFLP markers in four populations of sea beet (Beta vulgaris ssp. maritima (L.) Arcang.) it was shown that tightly linked loci could be detected by screening for associations. It was hypothesized that the short distances spanned by linkage disequilibrium enable markers that are very tightly linked to a target gene to be identified. The hypothesis was tested by whole-genome screening of AFLP markers for association with the gene for the annual growth habit, the B gene, in a sample of 106 sea beets. Despite the dominant nature of AFLP, two markers showing significant linkage disequilibrium with the B gene were detected. The results indicate the potential use of linkage disequilibrium for gene mapping in natural plant populations.

Plastid position in Arabidopsis columella cells is similar in microgravity and on a random-positioning machine.

In order to study gravity effects on plant structure and function, it may become necessary to remove the g-stimulus. On Earth, various instruments such as clinostats have been used by biologists in an attempt to neutralize the effects of gravity. In this study, the position of amyloplasts was assayed in columella cells in the roots of Arabidopsis thaliana (L.) Heynh. seedlings grown in the following conditions: on Earth, on a two-dimensional clinostat at 1 rpm, on a three-dimensional clinostat (also called a random-positioning machine, or an RPM), and in space (true microgravity). In addition, the effects of these gravity treatments on columella cell area and plastid area also were measured. In terms of the parameters measured, only amyloplast position was affected by the gravity treatments. Plastid position was not significantly different between spaceflight and RPM conditions but was significantly different between spaceflight and the classical two-dimensional clinostat treatments. Flanking columella cells showed a greater susceptibility to changes in gravity compared to the central columella cells. In addition, columella cells of seedlings that were grown on the RPM did not exhibit deleterious effects in terms of their ultrastructure as has been reported previously for seedlings grown on a two-dimensional clinostat. This study supports the hypothesis that the RPM provides a useful simulation of weightlessness.

Coupling of creatine kinase to glycolytic enzymes at the sarcomeric I-band of skeletal muscle: a biochemical study in situ.

The specific interaction of muscle type creatine-kinase (MM-CK) with the myofibrillar M-line was demonstrated by exchanging endogenous MM-CK with an excess of fluorescently labeled MM-CK in situ, using chemically skinned skeletal muscle fibers and confocal microscopy. No binding of labeled MM-CK was noticed at the I-band of skinned fibers, where the enzyme is additionally located in vivo, as shown earlier by immunofluorescence staining of cryosections of intact muscle. However, when rhodamine-labeled MM-CK was diffused into skinned fibers that had been preincubated with phosphofructokinase (PFK), a glycolytic enzyme known to bind to actin, a striking in vivo-like interaction of Rh-MM-CK with the I-band was found, presumably mediated by binding of Rh-MM-CK to the glycolytic enzyme. Aldolase, another actin-binding glycolytic enzyme was also able to bind Rh-MM-CK to the I-band, but formation of the complex occurred preferably at long sarcomere length (> 3.0 microm). Neither pyruvate kinase, although known for its binding to actin, nor phosphoglycerate kinase (PGK), not directly interacting with the I-band itself, did mediate I-band targeting of MM-CK. Anchoring of MM-CK to the I-band via PFK, but not so via aldolase, was strongly pH-dependent and occurred below pH 7.0. Labeling performed at different sarcomere length indicated that the PFK/MM-CK complex bound to thin filaments of the I-band, but not within the actomyosin overlap zones. The physiological consequences of the structural interaction of MM-CK with PFK at the I-band is discussed with respect to functional coupling of MM-CK to glycolysis, metabolic regulation and channeling in multi-enzyme complexes. The in situ binding assay with skinned skeletal muscle fibers described here represents a useful method for further studies of specific protein-protein interactions in a structurally intact contractile system under various precisely controlled conditions.

Thin filament activation probed by fluorescence of N-((2-(iodoacetoxy)ethyl)-N-methyl)amino-7-nitrobenz-2-oxa-1,3-diazole-labeled troponin I incorporated into skinned fibers of rabbit psoas muscle.

A method is described for the exchange of native troponin of single rabbit psoas muscle fibers for externally applied troponin complexes without detectable impairment of functional properties of the skinned fibers. This approach is used to exchange native troponin for rabbit skeletal troponin with a fluorescent label (N-((2-(iodoacetoxy)ethyl)-N-methyl)amino-7-nitrobenz-2-oxa-1, 3-diazole, IANBD) on Cys(133) of the troponin I subunit. IANBD-labeled troponin I has previously been used in solution studies as an indicator for the state of activation of reconstituted actin filaments (. Proc. Natl. Acad. Sci. USA. 77:7209-7213). In the skinned fibers, the fluorescence of this probe is unaffected when cross-bridges in their weak binding states attach to actin filaments but decreases either upon the addition of Ca(2+) or when cross-bridges in their strong binding states attach to actin. Maximum reduction is observed when Ca(2+) is raised to saturating concentrations. Additional attachment of cross-bridges in strong binding states gives no further reduction of fluorescence. Attachment of cross-bridges in strong binding states alone (low Ca(2+) concentration) gives only about half of the maximum reduction seen with the addition of calcium. This illustrates that fluorescence of IANBD-labeled troponin I can be used to evaluate thin filament activation, as previously introduced for solution studies. In addition, at nonsaturating Ca(2+) concentrations IANBD fluorescence can be used for straightforward classification of states of the myosin head as weak binding (nonactivating) and strong binding (activating), irrespective of ionic strength or other experimental conditions. Furthermore, the approach presented here not only can be used as a means of exchanging native skeletal troponin and its subunits for a variety of fluorescently labeled or mutant troponin subunits, but also allows the exchange of native skeletal troponin for cardiac troponin.

Sarcomeric binding pattern of exogenously added intact caldesmon and its C-terminal 20-kDa fragment in skinned fibers of skeletal muscle.

Intact caldesmon and particularly the actin-binding C-terminal fragment (20-kDa) of caldesmon have been shown in skeletal muscle fibers to selectively displace low affinity, weakly bound cross-bridges from actin without significantly altering the actin attachment of force producing, strong binding cross-bridges (Brenner et al., 1991; Kraft et al., 1995a). However, the sarcomeric distribution and the specific binding of externally added caldesmon to the myofilaments of skeletal muscle fibers was not known. It was e.g., unclear whether caldesmon binds along actin in a manner similar to tropomyosin or whether it also binds to myosin. In this study, we determined the binding pattern of exogenously added intact caldesmon and its C-terminal 20-kDa fragment, respectively, in MgATP-relaxed rabbit skeletal muscle fibers using electron (EM) and confocal fluorescence microscopy (CFM). EM showed that similar to what has been demonstrated earlier for smooth muscle thin filaments (Lehman et al., 1989), intact caldesmon binds periodically every 38 nm along the thin filaments. CFM revealed that rhodamine-labeled intact caldesmon and the 20-kDa caldesmon fragment bind along nearly the entire length of the thin filaments. A portion of the I-band near the Z-line appears unlabeled, both when equilibrated at normal and long sarcomere lengths. The width of the unlabeled region seems to depend on ionic strength. The 20-kDa C-terminal caldesmon fragment binds in essentially the same pattern as intact caldesmon. This indicates that the high fluorescence intensity in the overlap region seen with intact caldesmon does not depend on caldesmon binding to myosin. X-ray diffraction was used to monitor the effects of filament lattice. Intact caldesmon at > 0.3 mg/ml induced disorder in the myofilament lattice. No such disordering was observed, however, when fibers were equilibrated with up to 0.8 mg/ml of the 20-kDa caldesmon fragment.