Ephrin-B1 is a novel specific component of the lateral membrane of the cardiomyocyte and is essential for the stability of cardiac tissue architecture cohesion.

RATIONALE: Cardiac tissue cohesion relying on highly ordered cardiomyocytes (CM) interactions is critical because most cardiomyopathies are associated with tissue remodeling and architecture alterations. OBJECTIVE: Eph/ephrin system constitutes a ubiquitous system coordinating cellular communications which recently emerged as a major regulator in adult organs. We examined if eph/ephrin could participate in cardiac tissue cyto-organization. METHODS AND RESULTS: We reported the expression of cardiac ephrin-B1 in both endothelial cells and for the first time in CMs where ephrin-B1 localized specifically at the lateral membrane. Ephrin-B1 knock-out (KO) mice progressively developed cardiac tissue disorganization with loss of adult CM rod-shape and sarcomeric and intercalated disk structural disorganization confirmed in CM-specific ephrin-B1 KO mice. CMs lateral membrane exhibited abnormal structure by electron microscopy and notably increased stiffness by atomic force microscopy. In wild-type CMs, ephrin-B1 interacted with claudin-5/ZO-1 complex at the lateral membrane, whereas the complex disappeared in KO/CM-specific ephrin-B1 KO mice. Ephrin-B1 deficiency resulted in decreased mRNA expression of CM basement membrane components and disorganized fibrillar collagen matrix, independently of classical integrin/dystroglycan system. KO/CM-specific ephrin-B1 KO mice exhibited increased left ventricle diameter and delayed atrioventricular conduction. Under pressure overload stress, KO mice were prone to death and exhibited striking tissue disorganization. Finally, failing CMs displayed downregulated ephrin-B1/claudin-5 gene expression linearly related to the ejection fraction. CONCLUSIONS: Ephrin-B1 is necessary for cardiac tissue architecture cohesion by stabilizing the adult CM morphology through regulation of its lateral membrane. Because decreased ephrin-B1 is associated with molecular/functional cardiac defects, it could represent a new actor in the transition toward heart failure.

Ultrastructural maturation of human-induced pluripotent stem cell-derived cardiomyocytes in a long-term culture.

BACKGROUND: In the short- to mid-term, cardiomyocytes generated from human-induced pluripotent stem cells (hiPSC-CMs) have been reported to be less mature than those of adult hearts. However, the maturation process in a long-term culture remains unknown. METHODS AND RESULTS: A hiPSC clone generated from a healthy control was differentiated into CMs through embryoid body (EB) formation. The ultrastructural characteristics and gene expressions of spontaneously contracting EBs were analyzed through 1-year of culture after cardiac differentiation was initiated. The 14-day-old EBs contained a low number of myofibrils, which lacked alignment, and immature high-density Z-bands lacking A-, H-, I-, and M-bands. Through the long-term culture up to 180 days, the myofibrils became more tightly packed and formed parallel arrays accompanied by the appearance of mature Z-, A-, H-, and I-bands, but not M-bands. Notably, M-bands were finally detected in 360-day-old EBs. The expression levels of the M-band-specific genes in hiPSC-CMs remained lower in comparison with those in the adult heart. Immunocytochemistry indicated increasing number of MLC2v-positive/MLC2a-negative cells with decreasing number of MLC2v/MLC2a double-positive cells, indicating maturing of ventricular-type CMs. CONCLUSIONS: The structural maturation process of hiPSC-CMs through 1-year of culture revealed ultrastructural sarcomeric changes accompanied by delayed formation of M-bands. Our study provides new insight into the maturation process of hiPSC-CMs.

JunB-CBFbeta signaling is essential to maintain sarcomeric Z-disc structure and when defective leads to heart failure.

In muscle cells, a complex network of Z-disc proteins allows proper reception, transduction and transmission of mechanical and biochemical signals. Mutations in genes encoding different Z-disc proteins such as integrin-linked kinase (ILK) and nexilin have recently been shown to cause heart failure by distinct mechanisms such as disturbed mechanosensing, altered mechanotransduction or mechanical Z-disc destabilization. We identified core-binding factor ß (CBFß) as an essential component for maintaining sarcomeric Z-disc and myofilament organization in heart and skeletal muscle. In CBFß-deficient cardiomyocytes and skeletal-muscle cells, myofilaments are thinned and Z-discs are misaligned, leading to progressive impairment of heart and skeletal-muscle function. Transcription of the gene encoding CBFß mainly depends on JunB activity. In JunB-morphant zebrafish, which show a heart-failure phenotype similar to that of CBFß-deficient zebrafish, transcript and protein levels of CBFß are severely reduced. Accordingly, ectopic expression of CBFß can reconstitute cardiomyocyte function and rescue heart failure in JunB morphants, demonstrating for the first time an essential role of JunB-CBFß signaling for maintaining sarcomere architecture and function.

Myosin head orientation: a structural determinant for the Frank-Starling relationship.

The cellular mechanism underlying the Frank-Starling law of the heart is myofilament length-dependent activation. The mechanism(s) whereby sarcomeres detect changes in length and translate this into increased sensitivity to activating calcium has been elusive. Small-angle X-ray diffraction studies have revealed that the intact myofilament lattice undergoes numerous structural changes upon an increase in sarcomere length (SL): lattice spacing and the I(1,1)/I(1,0) intensity ratio decreases, whereas the M3 meridional reflection intensity (I(M3)) increases, concomitant with increases in diastolic and systolic force. Using a short (∼10 ms) X-ray exposure just before electrical stimulation, we were able to obtain detailed structural information regarding the effects of external osmotic compression (with mannitol) and obtain SL on thin intact electrically stimulated isolated rat right ventricular trabeculae. We show that over the same incremental increases in SL, the relative changes in systolic force track more closely to the relative changes in myosin head orientation (as reported by I(M3)) than to the relative changes in lattice spacing. We conclude that myosin head orientation before activation determines myocardial sarcomere activation levels and that this may be the dominant mechanism for length-dependent activation.

Weaving hypothesis of cardiomyocyte sarcomeres: discovery of periodic broadening and narrowing of intercalated disk during volume-load change.

To investigate how cardiomyocytes change their length, echocardiographic and morphological studies were performed on rabbit hearts that were subjected to volume overload, overload removal, and repeated cycles of overload and overload removal. These conditions were created by arterio-venous fistula between the carotid artery and jugular vein, closure of the fistula, and cycles of repeatedly forming and closing fistula, respectively. After overload, hearts dilated and myocytes elongated. Intercalated disks repeatedly broadened and narrowed with a 2-day cycle, which continued for 8 weeks in many animals. The cycle consisted of shifts between five modes characterized by two interdigitation elongation-and-shortenings as follows: (I) flat with short ( approximately 1/4 to approximately 1/3 sarcomere long) interdigitations; (II) flat with long (one sarcomere long) interdigitations; (III) grooved with short interdigitations; (IV) grooved with long interdigitations; (V) flat with short interdigitations intermingled by sporadic long interdigitations; and return to (I). After overload removal, hearts contracted and myocytes shortened with similar 2-day broadening and narrowing cycle of intercalated disks, in which the five modes were reversed. Repeated overload and overload removal resulted in the repetition of myocyte elongation and shortening. We hypothesize that a single elongation-and-shortening event creates or disposes one sarcomere layer, and the two consecutive elongation-and-shortenings occur complementarily to each other so that the disks return to their original state after each cycle. Our hypothesis predicts that intercalated disks weave and unravel one sarcomere per myocyte per day.

Effects of eccentric strength training on biceps femoris muscle architecture and knee joint range of movement.

The aim was to determine whether eccentric strengthening changed the muscle architecture of human biceps femoris and consequently, knee range of motion. Twenty-two subjects were randomly assigned to control and experimental groups. The experimental group completed an eccentric strengthening programme for 8 weeks. Outcome measures included hamstring muscle strength (one repetition maximum), the passive knee extension test (PKE) (knee joint angle at which the onset of passive tension occurs), fascicle length (FL) and pennation angle (PA). One repetition maximum increased by 34% (P < 0.01), the PKE test revealed a 5% increase in joint range of motion (P = 0.01), FL increased by 34% (P = 0.01) and PA did not change (P = 0.38). This is the first report of an increase in FL in the biceps femoris following eccentric resistance training. In addition, the results might imply that this fascicle lengthening could lead to an increase in the range of motion of the knee. Clinical implications for rehabilitation and injury prevention are discussed.

High resolution muscle measurements provide insights into equinus contractures in patients with cerebral palsy.

Muscle contractures that occur after upper motor neuron lesion are often surgically released or lengthened. However, surgical manipulation of muscle length changes a muscle's sarcomere length (Ls ), which can affect force production. To predict effects of surgery, both macro- (fascicle length (Lf )) and micro- (Ls ) level structural measurements are needed. Therefore, the purpose of this study was to quantify both Ls and Lf in patients with cerebral palsy (CP) as well as typically developing (TD) children. Soleus ultrasound images were obtained from children with CP and TD children. Lf was determined and, with the joint in the same position, CP biopsies were obtained and formalin fixed, and Ls was measured by laser diffraction. Since soleus Ls values were not measurable in TD children, TD Ls values were obtained using three independent methods. While average Lf did not differ between groups (CP=3.6±1.2 cm, TD=3.5±0.9 cm; p>0.6), Ls was dramatically longer in children with CP (4.07±0.45 µm vs. TD=2.17±0.24 µm; p<0.0001). While Lf values were similar between children with CP and TD children, this was due to highly stretched sarcomeres within the soleus muscle. Surgical manipulation of muscle-tendon unit length will thus alter muscle sarcomere length and change force generating capacity of the muscle.

AM-FM texture segmentation in electron microscopic muscle imaging.

This paper describes the application of an amplitude modulation-frequency modulation (AM-FM) image representation in segmenting electron micrographs of skeletal muscle for the recognition of: 1) normal sarcomere ultrastructural pattern and 2) abnormal regions that occur in sarcomeres in various myopathies. A total of 26 electron micrographs from different myopathies were used for this study. It is shown that the AM-FM image representation can identify normal repetitive structures and sarcomeres, with a good degree of accuracy. This system can also detect abnormalities in sarcomeres which alter the normal regular pattern, as seen in muscle pathology, with a recognition accuracy of 75%-84% as compared to a human expert.

Permanent cardiac sarcomere changes in a rabbit model of intrauterine growth restriction.

BACKGROUND: Intrauterine growth restriction (IUGR) induces fetal cardiac remodelling and dysfunction, which persists postnatally and may explain the link between low birth weight and increased cardiovascular mortality in adulthood. However, the cellular and molecular bases for these changes are still not well understood. We tested the hypothesis that IUGR is associated with structural and functional gene expression changes in the fetal sarcomere cytoarchitecture, which remain present in adulthood. METHODS AND RESULTS: IUGR was induced in New Zealand pregnant rabbits by selective ligation of the utero-placental vessels. Fetal echocardiography demonstrated more globular hearts and signs of cardiac dysfunction in IUGR. Second harmonic generation microscopy (SHGM) showed shorter sarcomere length and shorter A-band and thick-thin filament interaction lengths, that were already present in utero and persisted at 70 postnatal days (adulthood). Sarcomeric M-band (GO: 0031430) functional term was over-represented in IUGR fetal hearts. CONCLUSION: The results suggest that IUGR induces cardiac dysfunction and permanent changes on the sarcomere.

Structural adaptations of rat lateral gastrocnemius muscle-tendon complex to a chronic stretching program and their quantification based on ultrasound biomicroscopy and optical microscopic images.

BACKGROUND: A chronic regimen of flexibility training can increase range of motion, with the increase mechanisms believed to be a change in the muscle material properties or in the neural components associated with this type of training. METHODS: This study followed chronic structural adaptations of lateral gastrocnemius muscle of rats submitted to stretching training (3 times a week during 8weeks), based on muscle architecture measurements including pennation angle, muscle thickness and tendon length obtained from ultrasound biomicroscopic images, in vivo. Fiber length and sarcomere number per 100µm were determined in 3 fibers of each muscle (ex vivo and in vitro, respectively), using conventional optical microscopy. FINDINGS: Stretching training resulted in a significant pennation angle reduction of the stretched leg after 12 sessions (25%, P=0.002 to 0.024). Muscle thickness and tendon length presented no significant changes. Fiber length presented a significant increase for the stretched leg (8.5%, P=0.00006), with the simultaneous increase in sarcomere length (5%, P=0.041) since the stretched muscles presented less sarcomeres per 100µm. INTERPRETATION: A stretching protocol with characteristics similar to those applied in humans was sufficient to modify muscle architecture of rats with absence of a sarcomerogenesis process. The results indicate that structural adaptations take place in skeletal muscle tissue submitted to moderate-intensity stretching training.

The external anal sphincter operates at short sarcomere length in humans.

BACKGROUND: The length at which a muscle/sarcomere operates in vivo (operational length) and the length at which it generates maximal stress (optimal length) can be quite different. In a previous study, we found that the rabbit external anal sphincter (EAS) operates on the ascending limb of the length-tension curve, in other words at lengths shorter than its optimal length (short sarcomere length). In this study, we tested whether the human EAS muscle also operates at a short sarcomere length. METHODS: The length-tension relationship of the EAS muscle was studied in vivo in 10 healthy nullipara women. EAS muscle length was altered by anal distension using custom-designed probes of 5, 10, 15, and 20mm diameter. Probes were equipped with a sleeve sensor to measure anal canal pressure. The EAS muscle electromyograph (EMG) was recorded using wire electrodes. Ultrasound images of anal canal were obtained to measure EAS muscle thickness and anal canal diameter. EAS muscle stress was calculated from the anal canal pressure, inner radius, and thickness of the EAS muscle. KEY RESULTS: Rest and squeeze stress of the anal canal increased with the increase in probe size. Similarly, the change in anal canal stress, i.e. the difference between the rest and the squeeze, which represents the active contribution of EAS to the anal canal stress, increased with the increase in probe size. However, increase in probe size was not associated with an increase in the external anal sphincter EMG activity. CONCLUSIONS & INFERENCES: Increase in EAS muscle stress with the increase in probe size, in the presence of constant EMG (neural input), demonstrates that the human EAS muscle operates on the ascending limb of the length-tension curve or at low sarcomere lengths. We propose that surgically adjusting EAS sarcomere length may represent a novel strategy to treat fecal incontinence in humans.

Patterns of muscular strain in the embryonic heart wall.

The hypothesis that inner layers of contracting muscular tubes undergo greater strain than concentric outer layers was tested by numerical modeling and by confocal microscopy of strain within the wall of the early chick heart. We modeled the looped heart as a thin muscular shell surrounding an inner layer of sponge-like trabeculae by two methods: calculation within a two-dimensional three-variable lumped model and simulated expansion of a three-dimensional, four-layer mesh of finite elements. Analysis of both models, and correlative microscopy of chamber dimensions, sarcomere spacing, and membrane leaks, indicate a gradient of strain decreasing across the wall from highest strain along inner layers. Prediction of wall thickening during expansion was confirmed by ultrasonography of beating hearts. Degree of stretch determined by radial position may thus contribute to observed patterns of regional myocardial conditioning and slowed proliferation, as well as to the morphogenesis of ventricular trabeculae and conduction fascicles. Developmental Dynamics 238:1535-1546, 2009. (c) 2009 Wiley-Liss, Inc.

Force-length characteristics of in vivo human skeletal muscle.

In the present study, the in vivo force-length relations of the human soleus (SOL) and tibialis anterior (TA) muscles were estimated. Measurements were taken in six men at ankle angles from 30 degrees of dorsiflexion to 45 degrees of plantarflexion in steps of 15 degrees, and involved dynamometry, electrical stimulation, ultrasonography and magnetic resonance imaging (MRI). For each muscle and ankle angle studied the following three measurements were carried out: (1) dynamometry-based measurement of maximal voltage tetanic moment, (2) ultrasound-based measurement of pennation angle and fibre length and (3) MRI-based measurement of tendon moment arm length. Tendon forces were calculated dividing moments by moment arm lengths, and muscle forces were calculated dividing tendon forces by the cosine of pennation angles. In the transition from 30 degrees of dorsiflexion to 45 degrees of plantarflexion the SOL muscle fibre length decreased from 3.8 to 2.4 cm and its force decreased from 3330 to 290 N. Over the same range of ankle angles the TA muscle fibre length increased from 3.7 to 6 cm and its force increased from 157 to 644 N. Over the longest muscle fibre lengths reached the force of both muscles remained approximately constant. These results indicate that the intact human SOL and TA muscles operate in the ascending limb and plateau region of the force-length relationship. Similar conclusions were reached when calculating the theoretical operating range of the two muscle sarcomeres in the study.