Connective Tissue Architecture Research Articles

Morphological variation of the soleus muscle: Determining general patterns and characteristics of the connective tissue architecture

BackgroundThe tendons within the soleus muscle are commonly utilized to delineate location of injury for soleus muscle strains. Strains within the soleus frequently involve the myotendinous (MTJ) or myofascial (MFJ) junctions, although spatial relationship between these tendinous structures are not well understood. This study aimed to structurally identify the MTJs and MFJs within the soleus as a starting point to understanding the distribution of connective tissue for further anatomical classification. MethodsForty (n = 40) soleus muscles, representing left and right sides from twenty (n = 20) formalin-embalmed cadavers (average age 78 years old; 10 males, 10 females) were dissected in-situ to analyze the distribution and orientation of the MTJs and MFJs within classified morphological variants. Muscles were cut in cross-section at three measured locations, proximal, middle, and distal, which allowed for analyzation of tendons through the course of the muscle. Additionally, anterior surfaces of morphological variants were visualized and reconstructed in three dimensions using a handheld blue light 3D scanner. ResultsThe study revealed five morphological variants. Bipennate-Midline (n = 25), Bipennate-Medial Deviation (n = 6), Bipennate-Lateral Deviation (n = 3), Unipennate (n = 3), and Hypopennate (n = 3). Muscles included an anterior aponeurosis that was split into medial and lateral components, with each side made up of interconnections between the MTJ and MJF. The average width of the medial aponeurosis was greatest in the middle location, while the average lateral aponeurotic width decreased from proximal to distal. Regression analysis at the middle location revealed that 65 % of the change in width of the medial aponeuroses is due to the width of the medial MFJ. ConclusionsProximal-to-distal interconnections between the lateral and medial anterior aponeuroses and their corresponding MTJs and MFJs likely play a role in soleus injury patterns, especially in morphological variants. Awareness of anatomical variations in the location and orientation of these tendinous relationships is crucial for understanding lesions on diagnostic imaging.

STRUCTURAL ORGANIZATION OF COLLAGEN-CONTAINING CONNECTIVE TISSUE AS A PREDICTOR OF METASTATIC PROGRESSION OF PROSTATE CANCER

Summary. Aim: to determine at the optical level the role of the fibrillar organization of the collagen-containing connective tissue component of the primary tumor focus in prostate cancer (PCa) progression. Objects and methods: the morphological study is based on the analysis of the histological material of the primary tumor foci of 55 PCa patients without tumor progression in the postoperative period and bone metastases. The tumors were graded Gleason 6–9 at surgery. The features of the architecture of collagen-containing connective tissue in tumors were determined in histological slides stained by Van-Gieson. Results: the remodeling of the collagen-containing stromal component located both around the glandular structures (increase in the percentage of straight and aligned collagen fibers compared to the curved ones) and in the stroma itself has been found. Such remodeling is manifested by an increase in the total mass of fibrous structures, an increase in the width of collagen fibrils, their compaction in relation to the density of their location, peculiarities of desmoplasia, alignment and elongation. The specified features are consistent with the Gleason score and the postoperative disease course, namely, the occurrence of metastases. Conclusions: the desmoplastically changed collagen-containing connective tissue component of the PCa creates favorable conditions for the unimpeded migration and realization of the invasive potential of cancer cells, in particular those expressing bone tissue remodeling proteins.

α-Lipoic Acid Protects against Cyclosporine A-Induced Hepatic Toxicity in Rats: Effect on Oxidative Stress, Inflammation, and Apoptosis.

The clinical application of cyclosporine A (CsA) as an immunosuppressive agent is limited by its organ toxicity. We aimed to evaluate the effectiveness of α-lipoic acid against CsA-induced hepatotoxicity and to delineate the underlying molecular mechanisms. Male Wistar rats (n = 24, 8 per each group) received the vehicle, CsA (25 mg/kg) and/or ALA (100 mg/kg, p.o.) for 3 weeks. Biochemical markers of liver function (serum ALT, AST, ALP < GGT), oxidative stress (MDA, TAC, SOD, GSH, Nrf2/HO-1), inflammation (NF-κB, CD68, iNOS, NO, COX-2), and apoptosis (caspase-3) were assessed in serum and tissue. Liver histological analysis using H&E and Sirius red was performed. The development of liver injury in CsA-treated animals was indicated by elevated levels of liver enzymes, oxidants/antioxidants imbalance, inflammatory cells infiltration, up-regulated expression of inflammatory mediators, and apoptosis. These changes were associated with altered architecture of hepatic cells and fibrous connective tissue. ALA co-administration protected against CsA-induced liver damage and ameliorated biochemical changes and cellular injury. In conclusion, ALA demonstrated hepatoprotective potential against CsA-induced liver injury through combating oxidative stress, inflammation, and apoptosis, highlighting ALA as a valuable adjunct to CsA therapy.

Striae Distensae: Scars without Wounds.

Striae distensae, or stretch marks, are common linear lesions of atrophic skin characterized histologically by epidermal atrophy, absent rete ridges, and alterations in connective tissue architecture. Hormonal excess, mechanical stress, and genetic predisposition are all associated with striae distensae, but their exact pathogenesis remains unknown. Despite a multitude of options, no single treatment has yet proven effective. In this article, the authors describe an up-to-date overview of striae distensae in terms of their etiology, pathophysiology, and therapeutic options. Further research is required to better elucidate their pathophysiology and to develop targeted effective treatments.

Biological insights in the pathogenesis of hypermobile Ehlers-Danlos syndrome from proteome profiling of patients' dermal myofibroblasts

Hypermobile Ehlers-Danlos syndrome (hEDS), mainly characterized by generalized joint hypermobility and its complications, minor skin changes, and apparently segregating with an autosomal dominant pattern, is still without a known molecular basis. Hence, its diagnosis is only clinical based on a strict set of criteria defined in the revised EDS nosology. Moreover, the hEDS phenotypic spectrum is wide-ranging and comprises multiple associated signs and symptoms shared with other heritable or acquired connective tissue disorders and chronic inflammatory diseases. In this complex scenario, we previously demonstrated that hEDS patients' skin fibroblasts show phenotypic features of myofibroblasts, widespread extracellular matrix (ECM) disarray, perturbation of ECM-cell contacts, and dysregulated expression of genes involved in connective tissue architecture and related to inflammatory and pain responses. Herein, the cellular proteome of 6 hEDS dermal myofibroblasts was compared to that of 12 control fibroblasts to deepen the knowledge on mechanisms involved in the disease pathogenesis. Qualitative and quantitative differences were assessed based on top-down and bottom-up approaches and some differentially expressed proteins were proofed by biochemical analyses. Proteomics disclosed the differential expression of proteins principally implicated in cytoskeleton organization, energy metabolism and redox balance, proteostasis, and intracellular trafficking. Our findings offer a comprehensive view of dysregulated protein networks and related pathways likely associated with the hEDS pathophysiology. The present results can be regarded as a starting point for future in-depth investigations aimed to decipher the functional impact of potential bioactive molecules for the development of targeted management and therapies.

Fiber Metabolism, Procollagen and Collagen Type III Immunoreactivity in Broiler Pectoralis Major Affected by Muscle Abnormalities.

Simple SummaryThe impressive production performances achieved by the modern chicken hybrids selected for meat production have indirectly predisposed the pectoral muscle to the onset and progression of abnormalities (i.e., white striping, wooden breast, and spaghetti meat). These myopathies affect the pectoralis major, with a high incidence rate and result in relevant economic losses for the poultry industry due to downgrading of the affected meat. These muscular abnormalities have been often associated with changes in ether the metabolism of the fibers (i.e., a shift from type IIB towards type IIA fibers) or to an increased deposition of collagen up to fibrosis. As collagen type III is typically observed in regenerating muscles, this and its precursor (procollagen type III) might play a role in the cellular processes, resulting in the development of white striping, wooden breast, and spaghetti meat abnormalities. According to our findings, both morphology and metabolism of the fibers were remarkably affected by the occurrence muscular abnormalities that are also associated with a profound modification in the connective tissue architecture. Intriguingly, an altered metabolism and an evident difference in the presence and distribution of procollagen and collagen type III was even observed in pectoralis major muscle from cases classified as unaffected.The present study aimed to evaluate the muscle fiber metabolism and assess the presence and distribution of both procollagen and collagen type III in pectoralis major muscles affected by white striping (WS), wooden breast (WB), and spaghetti meat (SM), as well as in those with macroscopically normal appearance (NORM). For this purpose, 20 pectoralis major muscles (five per group) were selected from the same flock of fast-growing broilers (Ross 308, males, 45-days-old, 3.0 kg live weight) and were used for histochemical (nicotinamide adenine dinucleotide tetrazolium reductase (NADH-TR) and alpha-glycerophosphate dehydrogenase (α-GPD)) and immunohistochemical (procollagen and collagen type III) analyses. When compared to NORM, we found an increased proportion (p < 0.001) of fibers positively stained to NADH-TR in myopathic muscles along with a relevant decrease (p < 0.001) in the percentage of those exhibiting a positive reaction to α-GPD. In addition, an increased proportion of fibers exhibiting a positive reaction to both stainings was observed in SM, in comparison with NORM (14.3 vs. 7.2%; p < 0.001). After reacting to NADH-TR, SM exhibited the lowest (p < 0.001) cross-sectional area (CSA) of the fibers (−12% with respect to NORM). On the other hand, after reacting to α-GPD, the CSA of WS was found to be significantly larger (+10%) in comparison with NORM (7480 vs. 6776 µm2; p < 0.05). A profound modification of the connective tissue architecture involving a different presence and distribution of procollagen and collagen type III was observed. Intriguingly, an altered metabolism and differences in the presence and distribution of procollagen and collagen type III were even observed in pectoralis major muscle classified as NORM.

The Architecture of Connective Tissue in the Musculoskeletal System—Often Overlooked Functional Parameter as to the Proprioception of the Locomotor Apparatus

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Architecture of connective tissue regenerated by enamel matrix derivative around hydroxyapatite implanted into tooth extraction sockets in the rat maxilla.

We investigated the architecture of periodontal ligament regenerated by an enamel matrix derivative (EMD, Emdogain®) coating on the surface of hydroxyapatite (EMD-HA). Immediately after extraction of the maxillary first molar in rats, HA alone or EMD-HA was implanted into the socket. At 5days, and 2 and 4weeks after implantation, the specimens were examined by light and transmission electron microscopy, and immunohistochemistry for periostin and matrix metalloproteinase (MMP)-13. Histological observations revealed a large number of fibroblasts and well-developed blood capillaries in the fibrous connective tissue surrounding EMD-HA at 5days. Ultrastructural analysis showed a distinct difference in the architecture of the fibrous connective tissue. As compared with the poorly constructed architecture of HA, EMD-HA had an orderly alignment of fibroblasts and bundled collagen fibers, with some fibroblasts in the cytoplasm showing collagen fiber phagocytosis. Periostin immunoreactivity was observed in the fibrous connective tissue around EMD-HA at each time point, but was not seen in HA at 5days and 2weeks. MMP-13 immunoreactivity was intensely localized in fibroblasts at 5days and 2weeks in EMD-HA. The present results indicate that EMD may greatly contribute to a well-developed architecture accompanied by orderly alignment of fibroblasts and bundled collagen fibers, through accelerated induction of periostin, maintenance of fibrillogenesis, and degradation of collagen fibers by extracellular proteinase and phagocytosis.

Bidirectional skin tension creates subcutaneous fascia movement

Introduction: Can manual bidirectional tension to the skin create subcutaneous movement? The skin and fascia exhibit tensegrity (intrinsic tension). Gracovetsky has considered the issues relating to the transfer of forces from the skin surface to deeper tissues, and suggests “While it is not known how much energy can be transferred from the skin surface to the deeper layers, it can be speculated that at least some of the therapist’s energy, applied to the skin, will end up being transferred”. Gracovetsky points to video illustrations of Jean Claude Guimberteau that demonstrate how a force applied to the surface of the skin ends up being dissipated deep into the tissues via a densely interconnected network of collagenous tissues (Gracovetsky 2016). Viscoelastic responses to mechanical forces are determined by their connective tissue (CT) extracellular matrix (ECM) composition and architecture. Mechanical tension can induce changes in the ECM subsequently modulating biological functions (Guimberteau 2012). CT fibroblasts play a pivotal role in both immediate and long-term CT responses to mechanical forces. Ultrasound elasticity imaging is emerging as a powerful non-invasive technique to quantify biomechanical tissue behavior (Wong et al 2011).

The cytokine secretion profile of mesenchymal stromal cells is determined by surface structure of the microenvironment

Mesenchymal stromal cells (MSC) secrete factors that contribute to organ homeostasis and repair in a tissue specific manner. For instance, kidney perivascular mesenchymal stromal cells (kPSCs) can facilitate renal epithelial repair through secretion of hepatocyte growth factor (HGF) while the secretome of bone marrow MSCs gives rise to immunosuppression. Stromal cells function in a complex 3-dimensional (3D) connective tissue architecture that induces conformational adaptation. Here we tested the hypothesis that surface topography and associated cell adaptations dictate stromal cell function through tuning of the cytokines released. To this end, we cultured human bone marrow and kidney perivascular stromal cells in the TopoWell plate, a custom-fabricated multi-well plate containing 76 unique bioactive surface topographies. Using fluorescent imaging, we observed profound changes in cell shape, accompanied by major quantitative changes in the secretory capacity of the MSCs. The cytokine secretion profile was closely related to cell morphology and was stromal cell type specific. Our data demonstrate that stromal cell function is determined by microenvironment structure and can be manipulated in an engineered setting. Our data also have implications for the clinical manufacturing of mesenchymal stromal cell therapy, where surface topography during bioreactor expansion should be taken into account to preserve therapeutic properties.

Clinical Aspects and Cellular Mechanisms of Fascia, their Interactions, and Importance in Medicine

The fascial system is a 3D continuum of soft, collagen‐containing, loose and dense fibrous connective tissues, widely including adipose tissue, adventitiae and neurovascular sheaths, aponeuroses, deep and superficial fasciae, epineurium, membranes, joint capsules, ligaments, meninges, myofascial expansions, periostea, retinaculae, septa, tendons, visceral fasciae, and all the intramuscular and intermuscular connective tissues including endo‐/peri‐/epimysium. These muscle layers may seem distinct in 2D sections, but the borders are somewhat arbitrary when viewed as an extensive 3 D network, with connective tissue fibers extending both along and across muscle fibers. The fascial system interpenetrates and surrounds all organs, muscles, bones and nerve fibers, endowing the body with a functional structure, and providing an environment that enables all body systems to operate in an integrated manner. Fascia is traditionally named according to the discrete anatomic structure that it surrounds which obscures its continuity and distinct layers.Fascial tissue provides a structural framework, continuously adapting and transmitting mechanical and chemical signals to differentiate tissue. The continuum of fascia throughout the body allows it to serve as a body‐wide mechanosensitive signaling system. The extracellular matrix plays an essential role in the development of skeletal musculature in the embryo. Fascia is a composite material of collagen fibers and matrix. In outer layers of the extremities and neck, fibers are found in parallel directions oriented at 55 degrees to the layer underneath, similar to a garden hose, which resists pressure and remains flexible. In other layers, collagen fibers are less regular and may have a wavy or helical structure. Fascia may also vary in proportions of collagen type I, III, IV and V and in rate of turnover. Fascial structure has functional implications. For example, when one muscle contracts against a thick, resistant fascia layer, the compartment pressure increases, and this pumps blood and lymphatic fluid against gravity and raises the contractile efficiency of other muscles in the same compartment. A fiber connects a muscle to the nitric oxide receptor of its arteriole, which is pulled open during contraction to increase blood flow. Thoracolumbar fascia balances abdominal and spinal muscle tension.Fascia is gaining interest from researchers, physicians and many types of therapists. Manual therapy techniques treat the fascial layers by altering density, tonus, viscosity, and the arrangement of fascia. Manual stimulation of sensory nerve endings may lead to tonus changes in muscle. The fascial system is now being recognized in the etiology of pain and proprioception. Fascia research can help understand aspects of musculoskeletal problems such as myofascial trigger points, low back pain, and fibromyalgia. Connective tissue influences the normal or pathological processes in a wide variety of organ systems e.g. intramuscular connective tissue architecture changes after immobilization of an extremity.The mechanical interactions among muscle, tendon and fascia in humans developed over many thousands of years and allow us to adapt to a wide range of activities. We are just beginning to understand these enough to develop specific fascial exercises and activities.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Etiology of Inguinal Hernias: A Comprehensive Review.

The etiology of inguinal hernias remains uncertain even though the lifetime risk of developing an inguinal hernia is 27% for men and 3% for women. The aim was to summarize the evidence on hernia etiology, with focus on differences between lateral and medial hernias. Lateral and medial hernias seem to have common as well as different etiologies. A patent processus vaginalis and increased cumulative mechanical exposure are risk factors for lateral hernias. Patients with medial hernias seem to have a more profoundly altered connective tissue architecture and homeostasis compared with patients with lateral hernias. However, connective tissue alteration may play a role in development of both subtypes. Inguinal hernias have a hereditary component with a complex inheritance pattern, and inguinal hernia susceptible genes have been identified that also are involved in connective tissue homeostasis. The etiology of lateral and medial hernias are at least partly different, but the final explanations are still lacking on certain areas. Further investigations of inguinal hernia genes may explain the altered connective tissue observed in patients with inguinal hernias. The precise mechanisms why processus vaginalis fails to obliterate in certain patients should also be clarified. Not all patients with a patent processus vaginalis develop a lateral hernia, but increased intraabdominal pressure appears to be a contributing factor.

Biomechanics of the Levator Aponeurosis.

To investigate biomechanical properties of the levator aponeurosis. Patients undergoing external levator resection for primary or revision correction of acquired ptosis were analyzed as primary and revision groups. Immediately postoperatively, the resected segments of the levator aponeurosis were stressed by adding sequential masses to the tissue. Specimen length was recorded at each stress level. Stress-strain diagrams were used to summarize how the tissue elongated in response to the external forces, because these diagrams correct for differences in specimen size. Representative specimens were analyzed histologically. Twenty-two specimens from 14 patients in the primary group and 10 specimens from 7 patients in the revision group met inclusion criteria. In the primary group, the mean age was 66 years; 8 patients were women. In the revision group, the mean age was 69 years; 6 patients were women. Levator aponeurosis specimens in both the primary and revision group demonstrated proportional increases in length with increasing stress, demonstrating that the levator aponeurosis is extensile. Given the same amount of stress, the levator aponeurosis from primary specimens elongated more than revision specimens. Histologically, revision specimens exhibited increased collagen and haphazard, tangled elastin fibers. The levator aponeurosis obtained during surgical correction of acquired ptosis elongates in response to nominal external forces. This biomechanical property is important because the length of the aponeurosis may vary intraoperatively if variable forces are applied to the eyelid. This property might be related to connective tissue architecture and, specifically, fibrosis. Surgeons performing levator aponeurosis resection should be mindful to maintain a consistent amount of force on the levator aponeurosis when performing the resection to maximize intercase consistency.

Dynamics and thermal sensitivity of ballistic and non-ballistic feeding in salamanders.

Low temperature reduces the performance of muscle-powered movements, but in movements powered by elastic recoil mechanisms, this effect can be mitigated and performance can be increased. To better understand the morphological basis of high performance and thermal robustness of elastically powered movements, we compared feeding dynamics at a range of temperatures (5-25°C) in two species of terrestrial plethodontid salamanders, Plethodon metcalfi and Ensatina eschscholtzii, which differ in tongue muscle architecture and the mechanism of tongue projection. We found that Ensatina is capable of ballistic projection with a mean muscle mass-specific power of 2100 W kg(-1), revealing an elastic mechanism. Plethodon, in contrast, projected its tongue non-ballistically with a mean power of only 18 W kg(-1), indicating it is muscle powered. Ensatina projected its tongue significantly farther than Plethodon and with dynamics that had significantly lower thermal sensitivity at temperatures below 15°C. These performance differences were correlated with morphological differences, namely elongated collagenous aponeuroses in the projector muscle of Ensatina as compared with Plethodon, which are likely the site of energy storage, and the absence in Ensatina of projector muscle fibers attaching to the tongue skeleton that allows projection to be truly ballistic. These findings demonstrate that, in these otherwise similar species, the presence in one species of elaborated connective tissue in series with myofibers confers not only 10-fold greater absolute performance but also greater thermal robustness of performance. We conclude that changes in muscle and connective tissue architecture are sufficient to alter significantly the mechanics, performance and thermal robustness of musculoskeletal systems.

Oral ingestion of collagen peptide causes change in width of the perimysium of the chicken iliotibialis lateralis muscle.

Skeletal muscle is mainly composed of myofibers and intramuscular connective tissue. Bundles composed of many myofibers, with each myofiber sheathed in connective tissue called the endomysium, are packed in the perimysium, which occupies the vast bulk of the intramuscular connective tissue. The perimysium is a major determination factor for muscle texture. Some studies have reported that collagen peptide (Col-Pep) ingestion improves the connective tissue architecture, such as the tendon and dermis. The present study evaluated the effects of Col-Pep ingestion on the chicken iliotibialis lateralis (ITL) muscle. Chicks were allocated to three groups: the 0.15% or 0.3% Col-Pep groups and a control group. Col-Pep was administered by mixing in with commercial food. On day 49, the ITL muscles were analyzed by morphological observation and the textural property test. The width of the perimysium in the 0.3% Col-Pep group was significantly larger than other two groups. Although scanning electron microscopic observations did not reveal any differences in the architecture of the endomysium, elastic improvement of the ITL muscle was observed as suggested by an increase of the width of perimysium and improved rheological properties. Our results indicate that ingestion of Col-Pep improves the textural property of ITL muscle of chickens by changing structure of the perimysium.

Insights in the etiopathology of galactosyltransferase II (GalT-II) deficiency from transcriptome-wide expression profiling of skin fibroblasts of two sisters with compound heterozygosity for two novel B3GALT6 mutations

Mutations in B3GALT6, encoding the galactosyltransferase II (GalT-II) involved in the synthesis of the glycosaminoglycan (GAG) linkage region of proteoglycans (PGs), have recently been associated with a spectrum of connective tissue disorders, including spondyloepimetaphyseal dysplasia with joint laxity type 1 (SEMDJL1) and Ehlers–Danlos-like syndrome. Here, we report on two sisters compound heterozygous for two novel B3GALT6 mutations that presented with severe short stature and progressive kyphoscoliosis, joint hypermobility and laxity, hyperextensible skin, platyspondyly, short ilia, and elbow malalignment. Microarray-based transcriptome analysis revealed the differential expression of several genes encoding extracellular matrix (ECM) structural components, including COMP, SPP1, COL5A1, and COL15A1, enzymes involved in GAG synthesis and in ECM remodeling, such as CSGALNACT1, CHPF, LOXL3, and STEAP4, signaling transduction molecules of the TGFβ/BMP pathway, i.e., GDF6, GDF15, and BMPER, and transcription factors of the HOX and LIM families implicated in skeletal and limb development. Immunofluorescence analyses confirmed the down-regulated expression of some of these genes, in particular of the cartilage oligomeric matrix protein and osteopontin, encoded by COMP and SPP1, respectively, and showed the predominant reduction and disassembly of the heparan sulfate specific GAGs, as well as of the PG perlecan and type III and V collagens. The key role of GalT-II in GAG synthesis and the crucial biological functions of PGs are consistent with the perturbation of many physiological functions that are critical for the correct architecture and homeostasis of various connective tissues, including skin, bone, cartilage, tendons, and ligaments, and generates the wide phenotypic spectrum of GalT-II-deficient patients.

Periostin is temporally expressed as an extracellular matrix component in skeletal muscle regeneration and differentiation

The transcriptional events and pathways responsible for the acquisition of the myogenic phenotype during regeneration and myogenesis have been studied extensively. The modulators that shape the extracellular matrix in health and disease, however, are less understood. Understanding the components and pathways of this remodeling will aid the restoration of the architecture and prevent deterioration under pathological conditions such as fibrosis. Periostin, a matricellular protein associated with remodeling of the extracellular matrix and connective tissue architecture, is emerging in pathological conditions associated with fibrosis in adult life. Periostin also complicates fibrosis in degenerative skeletal muscle conditions such as dystrophies. This study primarily addresses the spatial and temporal involvement of periostin along skeletal muscle regeneration. In the acute skeletal muscle injury model that shows recovery without fibrosis, we show that periostin is rapidly disrupted along with the extensive necrosis and periostin mRNA is transiently upregulated during the myotube maturation. This expression is stringently initiated from the newly regenerating fibers. However, this observation is contrasting to a model that displays extensive fibrosis where upregulation of periostin expression is stable and confined to the fibrotic compartments of endomysial and perimysial space. In vitro myoblast differentiation further supports the claim that upregulation of periostin expression is a function of extracellular matrix remodeling during myofiber differentiation and maturation. We further seek to identify the expression kinetics of various periostin isoforms during the differentiation of rat and mouse myoblasts. Results depict that a singular periostin isoform dominated the rat muscle, contrasting to multiple isoforms in C2C12 myoblast cells. This study shows that periostin, a mediator with deleterious impact on conditions exhibiting fibrosis, is also produced and secreted by myoblasts and regenerating myofibers during architectural remodeling in the course of development and regeneration.

Changes in Cx43 and NaV1.5 Expression Precede the Occurrence of Substantial Fibrosis in Calcineurin-Induced Murine Cardiac Hypertrophy

In mice, the calcium-dependent phosphatase calcineurin A (CnA) induces a transcriptional pathway leading to pathological cardiac hypertrophy. Interestingly, induction of CnA has been frequently noticed in human hypertrophic and failing hearts. Independently, the arrhythmia vulnerability of such hearts has been regularly associated with remodeling of parameters determining electrical conduction (expression level of connexin43 (Cx43) and NaV1.5, connective tissue architecture), for which the precise molecular basis and sequence of events is still unknown. Recently, we observed reduced Cx43 and NaV1.5 expression in 4-week old mouse hearts, overexpressing a constitutively active form of CnA (MHC-CnA model), but the order of events is still unknown. Therefore, three key parameters of conduction (Cx43, NaV1.5 and connective tissue expression) were characterized in MHC-CnA ventricles versus wild-type (WT) during postnatal development on a weekly basis. At postnatal week 1, CnA overexpression induced cardiac hypertrophy in MHC-CnA. Moreover, protein and RNA levels of both Cx43 and NaV1.5 were reduced by at least 50% as compared to WT. Cx43 immunoreactive signal was reduced at week 2 in MHC-CnA. At postnatal week 3, Cx43 was less phosphorylated and RNA level of Cx43 normalized to WT values, although the protein level was still reduced. Additionally, MHC-CnA hearts displayed substantial fibrosis relative to WT, which was accompanied by increased RNA levels for genes previously associated with fibrosis such as Col1a1, Col1a2, Col3a1, Tgfb1, Ctgf, Timp1 and microRNA miR-21. In MHC-CnA, reduction in Cx43 and NaV1.5 expression thus coincided with overexpression of CnA and hypertrophy development and preceded significant presence of fibrosis. At postnatal week 4 the alterations in conductional parameters observed in the MHC-CnA model lead to abnormal conduction and arrhythmias, similar to those observed in cardiac remodeling in heart failure patients. The MHC-CnA model, therefore, provides for a unique model to resolve the molecular origin of conductional remodeling in detail.

A custom image-based analysis tool for quantifying elastin and collagen micro-architecture in the wall of the human aorta from multi-photon microscopy

The aorta possesses a micro-architecture that imparts and supports a high degree of compliance and mechanical strength. Alteration of the quantity and/or arrangement of the main load-bearing components of this micro-architecture – the elastin and collagen fibers – leads to mechanical, and hence functional, changes associated with aortic disease and aging. Therefore, in the future, the ability to rigorously characterize the wall fiber micro-architecture could provide insight into the complicated mechanisms of aortic wall remodeling in aging and disease. Elastin and collagen fibers can be observed using state-of-the-art multi-photon microscopy. Image-analysis algorithms have been effective at characterizing fibrous constructs using various microscopy modalities. The objective of this study was to develop a custom MATLAB-language automated image-based analysis tool to describe multiple parameters of elastin and collagen micro-architecture in human soft fibrous tissue samples using multi-photon microscopy images. Human aortic tissue samples were used to develop the code. The tool smooths, cleans and equalizes fiber intensities in the image before segmenting the fibers into a binary image. The binary image is cleaned and thinned to a fiber skeleton representation of the image. The developed software analyzes the fiber skeleton to obtain intersections, fiber orientation, concentration, porosity, diameter distribution, segment length and tortuosity. In the future, the developed custom image-based analysis tool can be used to describe the micro-architecture of aortic wall samples in a variety of conditions. While this work targeted the aorta, the software has the potential to describe the architecture of other fibrous materials, tube-like networks and connective tissues.

Effect of Age on Structural Properties of Intramuscular Connective Tissue, Muscle Fibre, Collagen Content and Meat Tenderness in Pig longissimus lumborum muscle

Changes in the structure and properties of the intramuscular connective tissue, muscle fibre size, collagen content and meat tenderness of m. longissimus lumborum during growth was studied in 45 Polish Large White (PLW) pigs slaughtered at 90, 150 and 210 days of age. The results show that the endomysial sheath in m. longissimus lumborum consists of collagen fibrils of wavy appearance which run in all directions and form a loose network. The arrangement of collagen fibrils in the endomysium and perimysium becomes denser and more regular with increasing age of pigs. In addition, the increase in endomysium and perimysium thickness was paralleled by a significant increase in muscle fibre diameter, as well as an increase in shear force value with chronological aging. In contrast, the percentage of collagen area in muscle structure and the amount of total collagen and amount of soluble collagen decreased gradually with age of pigs. In conclusion, the structural changes in the arrangement of collagen fibres in the architecture of intramusclular connective tissue, as well as the decrease in soluble collagen content in m. longissimus lumborum during growth of pigs are important factors influencing shear force value, and thus raw meat tenderness.