Does aberrant architecture of nuclear LINC complex stop muscle cell development?

The linker of nucleoskeleton and cytoskeleton (LINC) complex is a structure that spans the entire nuclear envelope (NE) and integrates the nuclear interior with the cytoskeleton. Lamin A/C together with nesprins that mainly reside along the inner nuclear membrane (INM) and outer nuclear membrane (ONM) are core components of the LINC complex. Integrity of this specific nuclear structure is critical for muscle cell maturation and function. In the present study, we analyzed the ultrastructure of the LINC complex observed in two neonates with severe hypotonia and respiratory distress. Disruption of the LINC complex manifests in a wide separation of the ONM from the INM; the loss of perinuclear space (PNS) and delayed muscle cell maturation were predominating findings. This nuclear phenomenon has never been reported and provides further support for the appearance of a neonatal form of laminopathy.

Is mutation p.Arg168Gly in TPM3 gene responsible for Type 1 fiber hypoplasia and cap structure formation?

Congenital fiber type disproportion with delayed fiber type maturation and the appearance of cap structures were analyzed in a child with p.Arg168Gly mutation in TPM3 gene. Very narrow myotube-like Type 1 fibers with single nuclei decorated by cap structures seem to be a result of a failure in fusion process and mature fiber formation. Repeated mutations in exon 5 of TPM3 gene giving cap structures may be a different consequence of the loss of specific isoform normally operating in the fusion process and sarcomer formation.

LMNA mutations in Polish patients with dilated cardiomyopathy: prevalence, clinical characteristics, and in vitro studies.

BACKGROUND: LMNA mutations are most frequently involved in the pathogenesis of dilated cardiomyopathy with conduction disease. The goal of this study was to identify LMNA mutations, estimate their frequency among Polish dilated cardiomyopathy patients and characterize their effect both in vivo and in vitro. METHODS: Between January, 2008 and June, 2012 two patient populations were screened for the presence of LMNA mutations by direct sequencing: 66 dilated cardiomyopathy patients including 27 heart transplant recipients and 39 dilated cardiomyopathy patients with heart failure referred for heart transplantation evaluation, and 44 consecutive dilated cardiomyopathy patients, referred for a family evaluation and mutation screening. RESULTS: We detected nine non-synonymous mutations including three novel mutations: p.Ser431*, p.Val256Gly and p.Gly400Argfs*11 deletion. There were 25 carriers altogether in nine families. The carriers were mostly characterized by dilated cardiomyopathy and heart failure with conduction system disease and/or complex ventricular arrhythmia, although five were asymptomatic. Among the LMNA mutation carriers, six underwent heart transplantation, fourteen ICD implantation and eight had pacemaker. In addition, we obtained ultrastructural images of cardiomyocytes from the patient carrying p.Thr510Tyrfs*42. Furthermore, because the novel p.Val256Gly mutation was found in a sporadic case, we verified its pathogenicity by expressing the mutation in a cellular model. CONCLUSIONS: In conclusion, in the two referral centre populations, the screening revealed five mutations among 66 heart transplant recipients or patients referred for heart transplantation (7.6%) and four mutations among 44 consecutive dilated cardiomyopathy patients referred for familial evaluation (9.1%). Dilated cardiomyopathy patients with LMNA mutations have poor prognosis, however considerable clinical variability is present among family members.

Morphologic and clinical aspects of Danon disease in a patient with a mutation c.137G > A in the LAMP-2 gene.

BACKGROUND: Danon disease is caused by a primary deficiency of lysosome-associated membrane protein-2 (LAMP-2). MATERIALS AND METHODS: Our study describes a 19-year-old man with isolated hypertrophic cardiomyopathy, in whom we performed DNA analysis and compared results of microscopic analysis of skeletal and cardiac muscles. RESULTS: Sequencing of the LAMP-2 gene revealed a novel point mutation c.137G > A in exon 2, leading to premature stop codon. Ultrastructural analysis of cardiac and skeletal muscles revealed the presence of unusual autophagic vacuoles in both. Although some vacuoles in skeletal muscle reacted strongly with dystrophin, ß-sarcoglycan, and laminin, those in cardiomyocytes showed no immunoreactivity. CONCLUSION: Our immunohistochemical and ultrastructural findings reinforce the claim that in Danon disease the pathomechanism of chaperone-mediated autophagy in cardiomyocytes differs from that in skeletal muscle.

Remodeling of mitochondrial interior in cardiac lipofuscinosis.

Ultrastructural analysis was performed in cardiac ceroidlipofuscinosis to confirm the presence and the nature of storage material. Granular osmophilic deposits characteristic of GROD structures coincidented with particularly aberrant mitochondria. Remodeling of mitochondrial interior with the appearance of several form of abnormal inclusions was never observed in cardiac ceroidlipofuscinosis. The presence of dense osmophilic bodies, glycogen conglomerates, balloon-like and onion-like structures in mitochondrial interior seem to be early events of this storage process.

Did giant mitochondria delay muscle maturation? An uncommon congenital myopathy.

INTRODUCTION: Mitochondria are semi-autonomous organelles that are able to change their shape, size, location, and number inside the living cell. Mitochondrial division is an extremely important process, because cell survival depends on there being an adequate number of mitochondria in each cell. The dynamics of these organelles are particularly important for normal embryonic cell development. We studied morphological and ultrastructural features of muscle-cell immaturity, with the appearance of abnormal giant mitochondria, in a child with an unusual congenital myopathy. METHODS: The biceps brachii and quadriceps femoris muscles were analyzed at the light and electron microscopy levels. RESULTS: Ultrastructural features of muscle-cell immaturity were manifested in the presence of primary and mature myotubes coexisting with abnormal giant mitochondria. CONCLUSIONS: Failure of mitochondrial fusion/fission machinery at an early stage of development may lead to delayed muscle-cell maturation.

[Myopathy in the course of carnitine palmitoyltransferase II deficiency]. / Miopatia w przebiegu niedoboru palmitylotransferazy karnityny II.

Congenital deficiency of carnitine palmitoyltransferase (CPT) II is a disease with an autosomal recessive inheritance of phenotypic variability which depends on age at the onset of symptoms. Three entities associated with deficiency of CPT II are known: the perinatal, the infantile and the adult form. The perinatal disease is the most severe form and is invariably fatal. On the other hand, the adult CPT II clinical phenotype is benign and requires additional external triggers such as high-intensity exercise to provoke myopathic symptoms. We report a case of adult CPT II deficiency presenting with the subtle symptoms of myopathy. A 32-year-old man was admitted to the hospital complaining of muscle pain after exercise. Athletic appearance drew attention, because the patient denied practicing sport. Neurological examination revealed marked tiredness during the single-leg hop test without other abnormalities. Electromyography (EMG) and serum biochemistry were not typical for myopathy. Routine histopathological examination did not reveal any abnormalities of structure of muscle fibers. Diagnosis was established after ultrastructural and biochemical analysis which revealed changes typical for CPT II deficiency.

Genetic and ultrastructural studies in dilated cardiomyopathy patients: a large deletion in the lamin A/C gene is associated with cardiomyocyte nuclear envelope disruption.

Major nuclear envelope abnormalities, such as disruption and/or presence of intranuclear organelles, have rarely been described in cardiomyocytes from dilated cardiomyopathy (DCM) patients. In this study, we screened a series of 25 unrelated DCM patient samples for (a) cardiomyocyte nuclear abnormalities and (b) mutations in LMNA and TMPO as they are two DCM-causing genes that encode proteins involved in maintaining nuclear envelope architecture. Among the 25 heart samples investigated, we identified major cardiomyocyte nuclear abnormalities in 8 patients. Direct sequencing allowed the detection of three heterozygous LMNA mutations (p.D192G, p.Q353K and p.R541S) in three patients. By multiplex ligation-dependant probe amplification (MLPA)/quantitative real-time PCR, we found a heterozygous deletion encompassing exons 3-12 of the LMNA gene in one patient. Immunostaining demonstrated that this deletion led to a decrease in lamin A/C expression in cardiomyocytes from this patient. This LMNA deletion as well as the p.D192G mutation was found in patients displaying major cardiomyocyte nuclear envelope abnormalities, while the p.Q353K and p.R541S mutations were found in patients without specific nuclear envelope abnormalities. None of the DCM patients included in the study carried a mutation in the TMPO gene. Taken together, we found no evidence of a genotype-phenotype relationship between the onset and the severity of DCM, the presence of nuclear abnormalities and the presence or absence of LMNA mutations. We demonstrated that a large deletion in LMNA associated with reduced levels of the protein in the nuclear envelope suggesting a haploinsufficiency mechanism can lead to cardiomyocyte nuclear envelope disruption and thus underlie the pathogenesis of DCM.

Ultrastructural evidence of myocardial capillary remodeling in peripartum cardiomyopathy.

BACKGROUND: The etiology of peripartum cardiomyopathy (PPCM) is not well understood. Potential causal mechanisms include infection, autoimmune disease, and abnormal response to the hemodynamic stress of pregnancy. Recently it was shown that the development of PPCM in an experimental model is associated with the generation of a cleaved antiangiogenic and proapoptotic 16-kDa form of the nursing hormone prolactin, which impairs the cardiac capillary network and its function. The purpose of this study was to evaluate the ultrastructure of the myocardium in a patient with PPCM and in a comparative group of patients and to identify structural characteristics that may predispose to myocardium dysfunction. CASE REPORT: A 28-year-old woman with PPCM had fulminant heart failure leading to the implantation of a biventricular assist device, but no myocarditis was found on histological examination of her myocardial tissue. An ultrastructural study of myocardial tissue taken from three patients (mean age: 22 years, 2 females) with fulminant heart failure not related to PPCM (2 myocarditis, 1 dilated cardiomyopathy) served as a comparison. The ultrastructural analysis revealed the presence of small capillary damage. Vascular lumen occlusion, endothelial cell remodeling, and apoptosis with the appearance of mast cells, plasma cells, and preadipocytes were the most characteristic features of the damaged myocardial tissue with secondary interstitial fibrosis. No vascular pathology of cardiac capillaries was observed in the comparative group. CONCLUSIONS: A myocardial tissue study in PPCM revealed ultrastructural remodeling of small capillaries with the presence of endothelial cell apoptosis and impairment of the microcirculation.

Autophagy in transition from hypertrophic cardiomyopathy to heart failure.

Endomyocardial biopsy of a patient in transition stage from hypertrophic cardiomyopathy to heart failure was investigated. The tissue showed hypertrophy, atrophy of myocytes and an increased amount of fibrosis. In addition, numerous cardiomyocytes revealed ubiquitin positive inclusions. Ultrastructural analysis indicated that cardiomyocytes contained typical autophagic vacuoles including mitochondria, glycogen granules, degraded remnants and myelin structures. The most obvious ultrastructural finding was the presence of amorphous plaques and tubulofilamentous inclusions. Such ultrastructural abnormalities allow us to conclude that degeneration of myocardial cells by autophagy mechanisms leads to cardiomyocyte death, loss and heart failure.

Cardiovascular magnetic resonance findings in a case of Danon disease.

Danon disease is a rare X-linked dominant lysosomal glycogen storage disease that can lead to severe ventricular hypertrophy and heart failure. We report a case of Danon disease with cardiac involvement evaluated with cardiovascular magnetic resonance, including late gadolinium enhancement and perfusion studies.

Obliteration of cardiomyocyte nuclear architecture in a patient with LMNA gene mutation.

OBJECTIVE: The aim of our study was to perform an immunohistochemical and ultrastructural analysis of the nuclear architecture of cardiomyocytes from an end-stage DCM patient with a missense point mutation in the exon 3 of the LMNA gene which is predicted to result in a D192G substitution. METHODS: We studied endomyocardial biopsy samples taken from the right ventricle by immunostaining using antibodies against the lamins A and C and by electron microscopy. The cardiomyocyte ultrastructure was analysed, with particular attention to the nuclear architecture. RESULTS: Thirty percent of cardiomyocyte nuclei from the D192G carrier showed chromatin disorganization and a changed nuclear shape. The most surprising finding was the appearance of sarcoplasmic organelles within the nuclear matrix of well enveloped nuclei. To our knowledge, this intriguing phenomenon was observed for the first time in cardiomyocytes. CONCLUSION: The study documents that D192G mutation in LMNA gene may lead to the disruption of the nuclear wall in cardiomyocytes, thus supporting the mechanical hypothesis of dilated cardiomyopathy development in humans, which might be mutation-specific.

Progeria caused by a rare LMNA mutation p.S143F associated with mild myopathy and atrial fibrillation.

We present a 6-year-old girl with premature aging associated with mild myopathy, displaying muscle weakness, joint contractures and hyporeflexia. Genetic analysis revealed rare heterozygous point mutation in lamin A/C gene, g.428C>T. Cardiological evaluation showed atrial fibrillation, but we did not find signs of coronary heart disease, which is life-threatening cardiovascular complication in progeria. Electron microscopy of the muscle revealed abnormalities in nuclear architecture, i.e. blebbing, thick lamina and peripheral distribution of heterochromatin. As some diagnostic criteria characteristic for classic progeria are not fulfilled, this case could be regarded as atypical progeria associated with myopathy and atrial fibrillation. To our knowledge, this is the second case of such association described in the literature.

Nuclear architecture remodelling in cardiomyocytes with lamin A deficiency.

We analysed the architecture of cardiomyocyte nuclei lacking lamin A activity in three patients with dilated cardiomyopathy. The diagnosis was established on the basis of clinical and electrophysiological examinations, chest radiography and electrocardiography. An ultrastructural study of affected cardiomyocytes showed dramatic alterations in nuclear distribution and organization affecting nuclear shape, lamina structure, chromatin and nuclear interior organization. The most specific hallmark of nuclei with lamin A deficiency was the reorganization of the nuclear interior, the appearance of a various number of mitochondria within the nuclear matrix, and focal or total lack of nuclear membrane.

[Morphological and clinical aspects of Danon disease]. / Morfologiczne i kliniczne aspekty choroby Danona.

BACKGROUND: Danon disease, an X-linked hypertrophic cardiomyopathy, is caused by primary deficiency of lysosome-associated membrane protein (LAMP-2). The pathological hallmark of the disease is the appearance of intracytoplasmic vacuoles containing autophagic material and the absence of LAMP-2 activity in the muscle. AIM: To define the LAMP-2 protein deficiency we investigated cardiac and skeletal muscle of a 19-year-old man with hypertrophic cardiomyopathy without clinically apparent skeletal myopathy or mental impairment, whose mother died suddenly at 46 years of age. METHODS: Clinical, morphological, immunohistochemical and ultrastructural analysis was performed. Paraffin sections of cardiac muscle were stained using routine histochemical methods. Frozen sections of skeletal muscle were stained using histochemical methods as well as using monoclonal antisera against N-terminal of dystrophin and antisera against LAMP-2. Ultrastructural examination of both cardiac and skeletal muscle specimens were performed. RESULTS: Cardiac and skeletal muscle revealed an excessive accumulation of early and late autophagic vacuoles containing various cytoplasmic debris. In immunohistochemical analysis the vacuolar membrane seen in skeletal muscle was decorated with antibody against dystrophin and such vacuoles were negative for LAMP-2. CONCLUSION: Ultrastructural and immunohistochemical analysis of skeletal muscle (less invasive than myocardial biopsy) may be used in diagnosis of Danon disease. Early diagnosis of Danon disease is important for timely cardiac transplantation, the only effective therapeutic option.

Functional and clinical characterization of KCNJ2 mutations associated with LQT7 (Andersen syndrome).

Andersen syndrome (AS) is a rare, inherited disorder characterized by periodic paralysis, long QT (LQT) with ventricular arrhythmias, and skeletal developmental abnormalities. We recently established that AS is caused by mutations in KCNJ2, which encodes the inward rectifier K(+) channel Kir2.1. In this report, we characterized the functional consequences of three novel and seven previously described KCNJ2 mutations using a two-microelectrode voltage-clamp technique and correlated the findings with the clinical phenotype. All mutations resulted in loss of function and dominant-negative suppression of Kir2.1 channel function. In mutation carriers, the frequency of periodic paralysis was 64% and dysmorphic features 78%. LQT was the primary cardiac manifestation, present in 71% of KCNJ2 mutation carriers, with ventricular arrhythmias present in 64%. While arrhythmias were common, none of our subjects suffered sudden cardiac death. To gain insight into the mechanism of arrhythmia susceptibility, we simulated the effect of reduced Kir2.1 using a ventricular myocyte model. A reduction in Kir2.1 prolonged the terminal phase of the cardiac action potential, and in the setting of reduced extracellular K(+), induced Na(+)/Ca(2+) exchanger-dependent delayed afterdepolarizations and spontaneous arrhythmias. These findings suggest that the substrate for arrhythmia susceptibility in AS is distinct from the other forms of inherited LQT syndrome.

Nuclear architecture remodelling in envelopathies.

We performed ultrastructural studies on nuclear abnormalities in muscle from 8 patients with X-linked and autosomal dominant form of Emery-Dreifuss muscular dystrophy (EDMD) and one case with progeroid syndrome. The diagnosis was based on clinical and molecular findings. We detected various degrees of nuclear architecture remodelling ranging from misshapen shape, nuclear disintegration, nuclear chromatin condensation and decondensation, focal chromatin loss to complete nuclear fragmentation. The most interesting finding was the appearance of tubulofilamentous inclusions inside the nuclear matrix of X-linked EDMD patients. All these nuclear aberrations are considered to be structural indicators of nuclear dysfunction evoked by envelope protein deficiency.

Abnormal chaperone-mediated autophagy (CMA) in cardiomyocytes of a boy with Danon disease.

Ultrastructural analysis of the cardiomyocyte structure in Danon disease reveals dramatic accumulation of abnormal late autophagic vacuoles (AVd) suggestive of primary lysosomal defect. Moreover, the accumulation of AVd in cardiomyocytes is consistent with a decreased rate of autophagic to lysosomal trafficking. These results suggest that the loss of the LAMP-2 protein strongly inhibits uptake of proteins into lysosomes for degeneration. The significant reduction of chaperone-mediated autophagy (CMA) activity in the affected cardiomyocytes induces a dramatic increase in the number and size of AVd and a severe reduction of myocardial contractility.

Myelin composition of spinal cord in a model of amyotrophic lateral sclerosis (ALS) in SOD1G93A transgenic rats.

We present the results of biochemical and electron microscopic (EM) examinations of the spinal cord myelin from SOD1G93A transgenic Sprague Dawley rats in the early and late symptom-free period of the disease (60 and 93 days of life) and after four-leg paralysis has occurred (120 days of life). Biochemical and ultrastructural changes of myelin started already in the symptom-free period and become most pronounced in the paralyzed animals. Biochemical examinations indicated a decrease of lipids, phospholipids, cholesterol and cerebrosides. The pattern of particular phospholipids was in the normal range. A progressive decrease of the percentages of proteolipid, DM-20 and Wolfgram proteins was evident. Myelin basic proteins I and II were less affected. In EM,massive myelin disorganization was observed.

Lipid raft disease? A new severe congenital myopathy.

We report a 5-year-old girl with a unique neuromuscular disorder manifested by early onset of the disease, delayed motor development, joint contractures, dysmorphy, cobbler's chest, generalized muscle hypoplasia and weakness. Morphological examination revealed muscle cell immaturity and the appearance of multilamellar myelin-like structures within and outside the sarcolemma. Overexpression of aberrant lipids on the surface of affected muscle cells may suggest some failure in lipid raft formation.