Effect of nonsense-mediated mRNA decay factor SMG9 deficiency on premature aging in zebrafish.

SMG9 is an essential component of the nonsense-mediated mRNA decay (NMD) machinery, a quality control mechanism that selectively degrades aberrant transcripts. Mutations in SMG9 are associated with heart and brain malformation syndrome (HBMS). However, the molecular mechanism underlying HBMS remains unclear. We generated smg9 mutant zebrafish (smg9oi7/oi7) that have a lifespan of approximately 6 months or longer, allowing for analysis of the in vivo function of Smg9 in adults in more detail. smg9oi7/oi7 zebrafish display congenital brain abnormalities and reduced cardiac contraction. Additionally, smg9oi7/oi7 zebrafish exhibit a premature aging phenotype. Analysis of NMD target mRNAs shows a trend toward increased mRNA levels in smg9oi7/oi7 zebrafish. Spermidine oxidase (Smox) is increased in smg9oi7/oi7 zebrafish, resulting in the accumulation of byproducts, reactive oxygen species, and acrolein. The accumulation of smox mRNA due to NMD dysregulation caused by Smg9 deficiency leads to increased oxidative stress, resulting in premature aging.

DHCR7 links cholesterol synthesis with neuronal development and axonal integrity.

The DHCR7 enzyme converts 7-DHC into cholesterol. Mutations in DHCR7 can block cholesterol production, leading to abnormal accumulation of 7-DHC and causing Smith-Lemli-Opitz syndrome (SLOS). SLOS is an autosomal recessive disorder characterized by multiple malformations, including microcephaly, intellectual disability, behavior reminiscent of autism, sleep disturbances, and attention-deficit/hyperactivity disorder (ADHD)-like hyperactivity. Although 7-DHC affects neuronal differentiation in ex vivo experiments, the precise mechanism of SLOS remains unclear. We generated Dhcr7 deficient (dhcr7-/-) zebrafish that exhibited key features of SLOS, including microcephaly, decreased neural stem cell pools, and behavioral phenotypes similar to those of ADHD-like hyperactivity. These zebrafish demonstrated compromised myelination, synaptic anomalies, and neurotransmitter imbalances. The axons of the dhcr7-/- zebrafish showed increased lysosomes and attenuated autophagy, suggesting that autophagy-related neuronal homeostasis is disrupted.

Association between interatrial septum adiposity and atrial fibrillation: transesophageal echocardiography imaging and autopsy study.

Recent clinical evidence has suggested that interatrial septal (IAS) adiposity contributes to atrial fibrillation (AF). The present study aimed to confirm the usefulness of transesophageal echocardiography (TEE) to estimate IAS adiposity in patients with AF. The histological IAS analysis based on autopsy samples sought to clarify characteristics that underlie the contribution of IAS adiposity to AF. The imaging study analyzed the TEE results in patients with AF (n = 184) in comparison with transthoracic echocardiography (TTE) and computed tomography (CT) results. The autopsy study histologically analyzed IAS in subjects with (n = 5) and without (n = 5) history of AF. In the imaging study, the ratio of interatrial septum adipose tissue (IAS-AT) volume per epicardial adipose tissue (EpAT) volume was greater in patients with persistent AF compared (PerAF) to those with paroxysmal AF (PAF). Multivariable analysis revealed that both TEE-assessed IAS thickness and TTE-assessed left atrial dimension were predicted by CT-assessed IAS-AT volume. In the autopsy study, the histologically-assessed IAS section thickness was greater in the AF group than that in the non-AF group and was positively correlated with the IAS-AT area percentage. In addition, the size of adipocytes in IAS-AT was smaller, compared to EpAT and subcutaneous adipose tissue (SAT). IAS-AT infiltrated into the IAS myocardium, as if adipose tissue split the myocardium (designated as myocardial splitting by IAS-AT). The number of island-like myocardium pieces as a result of myocardial splitting by IAS-AT was greater in the AF group than in the non-AF group and was positively correlated with the IAS-AT area percentage. The present imaging study confirmed the usefulness of TEE to estimate IAS adiposity in patients with AF without radiation exposure. The autopsy study suggested that the myocardial splitting by IAS-AT may contribute to atrial cardiomyopathy leading to AF.

Microvascular system of hip joint constituents with special reference to ultrastructural findings and early arteriosclerosis.

This study aimed to examine the ultrastructure of the arteries of the synovium, acetabular labrum, and ligamentum teres of the hip joint using light, scanning electron, and transmission electron microscopes to identify features of early arteriosclerosis. Tissues collected from three patients (under 40 years of age) with osteonecrosis of the femoral head were immersed in 8 N HCl at 60 °C for 20 min to digest collagen fibers for scanning electron microscopy. Tortuous arterioles and arteries were noted in the joint components, including the synovium, acetabular labrum, and ligamentum teres. The ultrastructure of the arterioles appeared normal; however, intimal thickening was found in most arteries. The thickened intima had abundant elastic fibers and many smooth muscle cells (which were of a synthetic phenotype because they had a few actin filaments and well-developed rough endoplasmic reticulum). This study illustrates that arteriosclerotic changes are present in tortuous arteries in the synovium, acetabular labrum, and ligamentum teres of the hip joint even from a relatively young age and suggests that meandering blood vessels may be the preferred foci of arteriosclerosis.

Detection of fibrotic remodeling of epicardial adipose tissue in patients with atrial fibrillation: Imaging approach based on histological observation.

BACKGROUND: Fibrotic remodeling of epicardial adipose tissue (EAT) is crucial for proinflammatory atrial myocardial fibrosis, which leads to atrial fibrillation (AF). OBJECTIVES: We tested the hypothesis that the ratio of central to marginal adipocyte diameter in EAT represents its fibrotic remodeling. Based on a similar concept, we also tested whether the percent (%) change in EAT fat attenuation determined using computed tomographic (CT) images can detect this remodeling. METHODS: Left atrial appendages were obtained from 76 consecutive AF patients during cardiovascular surgery. EAT in the central area (central EAT: C-EAT) and that adjacent to the atrial myocardium (Marginal EAT: M-EAT) were evaluated histologically. CT images for all of the 76 patients were also analyzed. RESULTS: The adipocyte diameter was smaller, fibrotic remodeling of EAT (EAT fibrosis) was more severe, and infiltration of macrophages and myofibroblasts was more extensive in M-EAT than in C-EAT. EAT fibrosis was positively correlated with adipocyte diameter in C-EAT and negatively correlated in M-EAT, resulting in a positive correlation between EAT fibrosis and the ratio of central to marginal adipocyte diameter (C/M diameter ratio; r = 0.73, P < .01). The C/M diameter ratio was greater in patients with persistent AF than in those with paroxysmal AF. CT images demonstrated that the %change in EAT fat attenuation was positively correlated with EAT fibrosis. CONCLUSION: Our results suggest that the central-to-marginal adipocyte diameter ratio is tightly associated with fibrotic remodeling of EAT. In addition, the %change in EAT fat attenuation determined using CT imaging can detect remodeling noninvasively.

Effect of age and sex on femoral curvature in the Japanese population: three-dimensional computed tomography findings.

Age- or sex-related differences in femoral curvature affect the onset of trauma or degenerative diseases of the hip joints. This study aimed to investigate femoral curvature in detail in Japanese individuals using three-dimensional computed tomography, evaluate its effects on the position of proximal femur, and assess differences in femoral curvature according to age and sex. We measured sagittal and coronal femoral bowing in 40 elderly (mean age 85.2 years) and 40 adult (mean age 30.1 years) Japanese individuals using computed tomography. In adult individuals, the radii of the femoral curvatures of the distal end and shaft in the coronal planes were significantly smaller in women than in men. In contrast, no significant difference was observed in femoral curvature between the sexes in the elderly group. Furthermore, the radius of femoral curvature was significantly smaller in elderly individuals than in adult individuals, regardless of the sex and location of the measurement. The highest point of the greater trochanter of the femoral head center was 7.3 ± 5.6 mm in the elderly group and 2.2 ± 4.6 mm in the adult group (p < 0.05). Thus, the femoral curvature varies with age and sex in Japanese individuals. In addition, the femoral curvature could cause positional changes in the proximal femur, such as the highest point of the greater trochanter. Therefore, further studies investigating the biomechanical effects of these morphological changes are warranted.

Exosc2 deficiency leads to developmental disorders by causing a nucleotide pool imbalance in zebrafish.

Exosc2 is one of the components of the exosome complex involved in RNA 3' end processing and degradation of various RNAs. Recently, EXOSC2 mutation has been reported in German families presenting short stature, hearing loss, retinitis pigmentosa, and premature aging. However, the in vivo function of EXOSC2 has been elusive. Herein, we generated Exosc2 knockout (exosc2-/-) zebrafish that showed larval lethality 13 days post fertilization, with microcephaly, loss of spinal motor neurons, myelin deficiency, and retinitis pigmentosa. Mechanistically, Exosc2 deficiency caused impaired mRNA turnover, resulting in a nucleotide pool imbalance. Rapamycin, which modulated mRNA turnover by inhibiting the mTOR pathway, improved nucleotide pool imbalance in exosc2-/- zebrafish, resulting in prolonged survival and partial rescue of neuronal defects. Taken together, our findings offer new insights into the disease pathogenesis caused by Exosc2 deficiency, and might help explain fundamental molecular mechanisms in neuronal diseases, such as Alzheimer's disease, amyotrophic lateral sclerosis, and spinal muscular atrophy.

Role of angiopoietin-like protein 2 in atrial fibrosis induced by human epicardial adipose tissue: Analysis using an organo-culture system.

BACKGROUND: We have recently reported that peri-left atrial epicardial adipose tissue (EAT) is associated with atrial myocardial fibrosis, in which angiopoietin-like protein 2 (Angptl2) protein content in EAT is associated with atrial myocardial fibrosis. OBJECTIVE: This study aimed to examine whether Angptl2 contained in peri-left atrial EAT can induce atrial myocardial fibrosis. METHODS: Human peri-left atrial EAT and abdominal subcutaneous adipose tissue (SAT) were collected from 9 autopsy cases. EAT- or SAT-conditioned medium was dropped onto the rat left atrial epicardial surface using an organo-culture system. Conditioned medium, recombinant Angptl2, and its antibody effects on organo-cultured rat atrial myocardial fibrosis were evaluated. Angptl2 effects on cultured neonatal rat fibroblasts were also investigated. RESULTS: EAT-conditioned medium induced atrial fibrosis in organo-cultured rat atrium with a progressive increase in the number of myofibroblasts. The profibrotic effect of EAT was greater than that of SAT. EAT in patients with atrial fibrillation induced a more significant atrial fibrosis than in those without. Treatment with human recombinant Angptl2 induced fibrosis in organo-cultured rat atrium, which was suppressed by the concomitant treatment with Angptl2 antibody. In cultured fibroblasts, Angptl2 upregulated the expression of α-smooth muscle actin, transforming growth factor-ß1, phospho-extracellular signal-regulated kinase,phospho-inhibitor of κBα, and phospho-p38 mitogen-activated protein kinase. CONCLUSION: This study demonstrated that Angptl2 contained in EAT played a crucial role in EAT-induced inflammatory atrial fibrosis. The results also suggested that antagonizing the expression of Angptl2 in EAT can be a novel therapeutic approach to prevent atrial fibrillation.

Histological evaluation of the acetabular labrum after bipolar hip hemiarthroplasty: a case report.

We report herein the microstructure of the acetabular labrum obtained from a patient with stem loosening but without bipolar cup migration who had undergone hemiarthroplasty for femoral neck fracture 18 years ago. We used light and scanning electron microscopy to investigate the influence of bipolar cup on acetabular labrum in vivo. Deparaffinized blocks were treated with 2 N NaOH to digest the cell matrix, allowing the collagen fibers, constituting the acetabular labrum, to be observed under scanning electron microscopy. Although chondrocyte atrophy was seen, the basic structure was not different from the normal tissue images of the elderly. However, in the deep part of the acetabular labrum, there was an area that was not stained with Alcian blue observed with light microscopy, and there was an amorphous tissue without type II collagen fibrils observed with scanning electron microscopy. These findings proved that the acetabular labrum has partially degenerated over the long term after bipolar hemiarthroplasty, and that the acetabular labrum can survive in vivo in such a condition. Given that hemiarthroplasty has a possibility to preserve the face-to-face tissue in the long term in vivo, it may be one of the valuable options for modern or future joint reconstruction surgery.

Ultrastructure and three-dimensional architecture of the anterior cruciate ligament in the knee joints of young and old monkeys.

We examined the ultrastructure of the anterior cruciate ligament and assessed age-related changes by comparing the ligaments of young and old monkeys. Ultrathin sections of the anterior cruciate ligament were observed by transmission electron microscopy. The three-dimensional architecture of collagen fibers in the ligament was examined by scanning electron microscopy after tissue specimens were treated with 2 N NaOH to digest the extracellular matrix. At the surface layer of the cruciate ligament in young monkeys, fusiform-shaped fibroblasts actively produced collagen fibrils. The ligament consisted of parallel bundles of dense collagen fibrils of approximately 200 nm in diameter. Collagen fibrils appeared to run linearly. Ligament fibrocytes in the deep layer had a stellate form. Ligament fibrocytes decreased in number and showed marked atrophy in old age. Collagen fibrils had a looser configuration in older monkeys. Despite atrophy of fibroblasts in the deep layer of the anterior cruciate ligament, the area with atrophic fibroblasts in the ligament expands with age, which can likely cause deterioration of and a reduction in collagen fibers. This information can be applied in studies on the cause of the low repair ability of and aging-related changes in the anterior cruciate ligament in humans.

Three-dimensional architecture of the acetabular labrum in the human hip joint.

The acetabular labrum is frequently damaged with advancing age. As collagen fibers are the main sources of strength, knowledge of their ultrastructure is important to determine the cause of age-induced changes. We aimed to investigate the ultrastructure of collagen fibers constituting the acetabular labrum using scanning electron microscopy (SEM). Acetabular labrum samples obtained during total hip arthroplasty were studied. The samples were specially prepared to observe the steric construction of collagen fibrils constituting the acetabular labrum under light microscopy followed by SEM. The acetabular labrum was mostly composed of cartilage tissue, consisting of chondrocytes and collagen type II, with a layer of collagen type I. In adults, chondrocytes with a rich cytoplasm were surrounded by a dense network of fine type II collagen fibrils, and small bundles of type I collagen fibrils were interposed in the cartilage layer. In elderly individuals, the chondrocytes atrophied and both type I and II collagen fibrils were sparse. We suggest that cartilage has three to five layers, consisting of type I and type II collagen fibrils with a solid cartilage substrate. In elderly individuals, the density of chondrocytes decreases and the cellular shape and architecture of collagen fibrils also changes.

Association of fibrotic remodeling and cytokines/chemokines content in epicardial adipose tissue with atrial myocardial fibrosis in patients with atrial fibrillation.

BACKGROUND: Epicardial adipose tissue (EAT) is associated with atrial fibrillation (AF), but the underlying mechanisms remain to be fully elucidated. OBJECTIVE: The purpose of this study was to examine, using human left atrial appendage (LAA) samples, the interactive relationship between the EAT profile and atrial myocardial fibrosis through histologic and biochemical analyses. METHODS: LAA samples were obtained from 59 consecutive AF patients during cardiovascular surgery. In histologic analysis, adipose tissue, atrial myocardial fibrosis, EAT fibrosis, macrophage infiltration, and matrix metalloproteinase-2 and hypoxia-inducible factor-1α (Hif-1α) expression were evaluated in LAA sections. In biochemical analysis, proinflammatory/fibrotic proteins in EAT, total collagen in left atrial (LA) myocardium, angiopoietin-like protein-2 (Angptl2)-related proteins in EAT, and proinflammatory/fibrotic proteins in serum were evaluated. RESULTS: Histology revealed that the severity of fibrotic remodeling of EAT was associated with LA myocardial fibrosis. Immunohistochemical and electron microscopic findings revealed that fibrotic remodeling of EAT was associated with infiltration of macrophages and myofibroblasts. Protein concentration analysis demonstrated that the total collagen in the LA myocardium was positively correlated with proinflammatory and profibrotic cytokines/chemokines, including interleukin-6, monocyte chemoattractant protein-1, tumor necrosis factor-α, vascular endothelial growth factor, and matrix metalloproteinase-2 and matrix metalloproteinase-9 in EAT. The proinflammatory and profibrotic cytokines/chemokines in EAT and the total collagen in the LA were also positively correlated with Angptl2 in EAT. CONCLUSION: Our study demonstrated that fibrotic remodeling and cytokines/chemokines in peri-LA EAT were associated with atrial myocardial fibrosis as a substrate of AF. Our results also suggested that overexpression of Hif-1α and Angptl2 may be involved in these processes.

Three-dimensional architecture of the ligamentum teres in the human hip joint.

BACKGROUND: We aimed to investigate the three-dimensional structure of the collagenous fibers of the ligamentum teres (LT) of the human hip and clarify the LT micro-anatomy at the attachment of the femoral head. METHODS: Femoral heads and LT were collected during hip arthroplasty. Specimens were cut into 5-10-mm squares, prepared, developed, and observed under a light microscope. Next, specimens were prepared and examined under a scanning electron microscope (SEM). RESULTS: Under optical microscope, LT adhered to the artificial cartilage at the attachment of the femoral head. Under SEM, LT comprised parallelly arranged collagenous fibers and the fine collagenous fibrils were twisted. While the central collagenous fibers of the LT at the attachment of the femoral head penetrated the articular cartilage tissue and reached the ring-shaped bone, fibers at the margin traversed and adhered to the cartilage surface. CONCLUSION: Articular cartilage and subchondral bone are present at the LT attachment to the femoral head. Although collagenous fibers of the LT show parallel arrangement at the main trunk, they are dispersed at the cartilage surface and not all reach the thin subchondral bone of the femoral head. This could possibly weaken ligament strength at the attachment of the femoral head. LEVEL OF EVIDENCE: IV.

Role of atrial endothelial cells in the development of atrial fibrosis and fibrillation in response to pressure overload.

BACKGROUND: Monocyte chemoattractant protein-1 (MCP-1)-mediated inflammatory mechanisms have been shown to play a crucial role in atrial fibrosis induced by pressure overload. In the present study, we investigated whether left atrial endothelial cells would quickly respond structurally and functionally to pressure overload to trigger atrial fibrosis and fibrillation. METHODS AND RESULTS: Six-week-old male Sprague-Dawley rats underwent suprarenal abdominal aortic constriction (AAC) or a sham operation. By day 3 after surgery, macrophages were observed to infiltrate into the endocardium. The expression of MCP-1 and E-selectin in atrial endothelium and the expression of intercellular adhesion molecule-1, vascular cell adhesion molecule-1, and ED1 in left atrial tissue were enhanced. Atrial endothelial cells were irregularly hypertrophied with the disarrangement of lines of cells by scanning electron microscopy. Various-sized gap formations appeared along the border in atrial endothelial cells, and several macrophages were located just in the endothelial gap. Along with the development of heterogeneous interstitial fibrosis, interatrial conduction time was prolonged and the inducibility of atrial fibrillation by programmed extrastimuli was increased in the AAC rats compared to the sham-operated rats. CONCLUSIONS: Atrial endothelium responds rapidly to pressure overload by expressing adhesion molecules and MCP-1, which induce macrophage infiltration into the atrial tissues. These processes could be an initial step in the development of atrial remodeling for atrial fibrillation.

Role of Indoxyl Sulfate as a Predisposing Factor for Atrial Fibrillation in Renal Dysfunction.

BACKGROUND: Renal dysfunction is a major risk factor for atrial fibrillation (AF). The uremic toxin indoxyl sulfate may contribute to the progression of cardiac fibrosis and AF substrate in renal dysfunction. METHODS AND RESULTS: Male Sprague-Dawley rats were assigned randomly to the following groups: 5/6 nephrectomy (5/6Nx) with vehicle, 5/6Nx with AST-120, sham procedure with vehicle, and sham procedure with AST-120. Vehicle and AST-120 were administered for 4 weeks. Serum levels of IS were significantly increased in 5/6Nx groups. Expression of malondialdehyde, an indicator of oxidative stress, was upregulated in the left atrium of 5/6Nx groups and was accompanied by an increase in expression of NADPH oxidase 2 and 4. Monocyte-mediated inflammatory signals such as CD68, monocyte chemoattractant protein 1, and vascular cell adhesion molecule 1 were also upregulated in 5/6Nx groups. Interstitial fibrosis was promoted heterogeneously, and expression of profibrotic indicators such as transforming growth factor ß1, α-smooth muscle actin, and collagen type 1 was upregulated in left atrium tissue of 5/6Nx groups. In cultured atrial fibroblasts, incubation with IS upregulated expression of the markers of oxidative stress, inflammation, and profibrotic factors. These results suggest the direct effects of IS on the progression of AF substrate. AF was consistently and invariably induced by atrial extrastimuli in 5/6Nx groups in electrophysiological experiments. AST-120 treatment significantly alleviated renal dysfunction-induced oxidative stress, inflammation, and atrial fibrosis and, consequently, attenuated AF inducibility. CONCLUSIONS: Indoxyl sulfate facilitates atrial fibrosis and AF and thus is a novel therapeutic target for prevention of renal dysfunction-induced AF.

A clinical approach to brown adipose tissue in the para-aortic area of the human thorax.

BACKGROUND: Human thoracic brown adipose tissue (BAT), composed of several subdivisions, is a well-known target organ of many clinical studies; however, the functional contribution of each part of human thoracic BAT remains unknown. The present study analyzed the significance of each part of human thoracic BAT in the association between regional distribution, cellularity, and factors involved in the functional regulation of thoracic BAT. METHODS: We analyzed 1550 healthy adults who underwent medical check-ups by positron-emission tomography and computed tomography (PET-CT) imaging, 8 cadavers, and 78 autopsy cases in an observational study. We first characterized the difference between the mediastinum and the supraclavicular areas using counts of BAT detection and conditions based on PET-CT outcomes. The measurable important area was then subjected to systematic anatomical and immunohistochemical analyses using anti-uncoupling protein 1 (UCP1) antibody to characterize the cellularity in association with age and sex. RESULTS: In PET-CT scanning, the main site of thoracic BAT was the mediastinum rather than the supraclavicular area (P < 0.05). Systemic macroanatomy revealed that the thumb-sized BAT in the posterior mediastinal descending para-aortic area (paBAT) had feeding vessels from the posterior intercostal arteries and veins and sympathetic/parasympathetic innervation from trunks of the sympathetic and vagus nerves, respectively. Immunohistochemical analysis indicated that the paBAT exhibited immunoreactivity for tyrosine hydroxylase and vesicular acetylcholine transporter located in the pericellular nervous fibers and intracellular UCP1. The brown adipose cells of paBAT showed age-dependent decreases in UCP1 expression (P < 0.05), accompanied by a significant increase in vacuole formation, indicating fat accumulation (P < 0.05), from 10 to 37 years of age (P < 0.01). CONCLUSIONS: paBAT may be one of the essential sites for clinical application in BAT study because of its visible anatomy with feeding vessels and sympathetic/parasympathetic innervation functionally affected by outer condition and senescence.

Dual targeting of heat shock proteins 90 and 70 promotes cell death and enhances the anticancer effect of chemotherapeutic agents in bladder cancer.

Heat shock proteins (HSPs), which are molecular chaperones that stabilize numerous vital proteins, may be attractive targets for cancer therapy. The aim of the present study was to investigate the possible anticancer effect of single or dual targeting of HSP90 and HSP70 and the combination treatment with HSP inhibitors and chemotherapeutic agents in bladder cancer cells. The expression of HSP90 and the anticancer effect of the HSP90 inhibitor 17-N-allylamino-17-demethoxygeldanamycin (17-AAG) coupled with cisplatin, docetaxel, or gemcitabine were examined using immunohistochemistry, quantitative real-time PCR, cell growth, flow cytometry, immunoblots and caspase-3/7 assays. The expression of HSP70 under HSP90 inhibition and the additive effect of HSP70 inhibitor pifithrin-µ (PFT-µ) were examined by the same assays and transmission electron microscopy. HSP90 was highly expressed in bladder cancer tissues and cell lines. 17-AAG enhanced the antiproliferative and apoptotic effects of each chemotherapeutic agent. 17-AAG also suppressed Akt activity but induced the upregulation of HSP70. PFT-µ enhanced the effect of 17-AAG or chemotherapeutic agents; the triple combination of 17-AAG, PFT-µ and a chemotherapeutic agent showed the most significant anticancer effect on the T24 cell line. The combination of 17-AAG and PFT-µ markedly suppressed Akt and Bad activities. With HSP90 suppression, HSP70 overexpression possibly contributes to the avoidance of cell death and HSP70 may be a key molecule for overcoming resistance to the HSP90 inhibitor. The dual targeting of these two chaperones and the combination with conventional anticancer drugs could be a promising therapeutic option for patients with advanced bladder cancer.

Immuno-histochemistry and three-dimensional architecture of the intermediate filaments in Purkinje cells in mammalian hearts.

In mammalian hearts, Purkinje cells varied greatly in morphological appearance in different species, and were divided into three groups. Bovine Purkinje cells corresponding to group I were a large size, and had a few myofibrils and abundant intermediate filaments throughout the cytoplasm. The aim of the present study was to clarify the more detailed distribution and three-dimensional architecture of intermediate filaments in Purkinje cells. The hearts in various mammals including humans were investigated by both immuno-histochemistry and scanning electron microscopy (SEM).Immuno-histochemical studies demonstrated that sheep Purkinje cells in group I had a great number of intermediate filaments of 10 nm positive for desmin antibody. Purkinje cells in group II (humans, monkeys and dogs) and group III (mice) were somewhat larger or smaller in size than myocardial cells, but also showed a strong positive reaction for desmin antibody. The saponin or NaOH treatment of cardiac tissues in sheep and humans enabled us to view intermediate filaments by SEM three-dimensionally. Intermediate filaments in sheep Purkinje cells formed a considerably delicate network, and were distributed throughout the cytoplasm. In contrast, those in human Purkinje cells were lower in density, and were present around the nucleus and between myofibrils. It was concluded that a delicate network of intermediate filaments in Purkinje cells of mammalian hearts acted as the cytoskeleton to maintain intercellular stability.