Protein Expression of Canine and Feline Muscular Dystrophies.

Muscular dystrophies in dogs and cats represent a heterogeneous group of inherited, sometimes congenital, but infrequently diagnosed, progressive neuromuscular disorders. A correct identification and characterization of canine and feline muscular dystrophies could increase diagnostic and treatment strategies for veterinary neurologists and could identify useful animal models for the study of human dystrophies. However, in dogs and cats, diagnosis of muscular dystrophies is challenging due to a nonspecific clinical phenotype and pathological lesions, thus is most likely underestimated. We performed immunofluorescence and Western blot techniques using a wide panel of antibodies against proteins involved in human dystrophies (dystrophin mid-rod and carboxyterminal domain, α, ß, γ, and δ-sarcoglycan, α-dystroglycan, caveolin-3, emerin, merosin, dysferlin, calpain-3, spectrin epitopes), on 9 canine and 3 feline muscle biopsies characterized by myopathic changes. Dystrophin deficiency was detected in 3 dogs and 2 novel canine muscular dystrophies have been identified, characterized by deficiency of caveolin-3 and calpain-3, respectively. In 2 cats, deficiency of ß-SG and carboxyterminal domain of dystrophin in all muscle fibers has been detected. Performing immunofluorescence and Western blot analyses with a wider panel of antibodies allowed a correct identification of muscular dystrophies in dogs and cats and provides a direction for subsequent targeted genetic testing.

Uptake and intracellular distribution of different types of nanoparticles in primary human myoblasts and myotubes.

The use of nanoparticles as drug carriers in the field of skeletal muscle diseases has been poorly addressed and the interaction of nanoparticles with skeletal muscle cells has been investigated almost exclusively on C2C12 murine myoblasts. In this study we investigated the effects poly(lactide-co-glycolide) nanoparticles, mesoporous silica nanoparticles and liposomes, on the viability of primary human myoblasts and analyzed their cellular uptake and intracellular distribution in both primary human myoblasts and myotubes. Our data demonstrate that poly(lactide-co-glycolide) nanoparticles do not negatively affect myoblasts viability, contrarily to mesoporous silica nanoparticles and liposomes that induce a decrease in cell viability at the highest doses and longest incubation time. Poly(lactide-co-glycolide) nanoparticles and mesoporous silica nanoparticles are internalized by endocytosis, poly(lactide-co-glycolide) nanoparticles undergo endosomal escape whereas mesoporous silica nanoparticles always occur within vacuoles. Liposomes were rarely observed within the cells. The uptake of all tested nanoparticles was less prominent in primary human myotubes as compared to myoblasts. Our findings represent the first step toward the characterization of the interaction between nanoparticles and primary human muscle cells and suggest that poly(lactide-co-glycolide) nanoparticles might find an application for drug delivery to skeletal muscle.

Characterization of sarcoplasmic reticulum Ca(2+) ATPase pumps in muscle of patients with myotonic dystrophy and with hypothyroid myopathy.

Sarcoplasmic/endoplasmic reticulum Ca(2+) ATPase (SERCA) pumps play the major role in lowering cytoplasmic calcium concentration in skeletal muscle by catalyzing the ATP-dependent transport of Ca(2+) from the cytosol to the lumen of the sarcoplasmic reticulum (SR). Although SERCA abnormalities have been hypothesized to contribute to the dysregulation of intracellular Ca(2+) homeostasis and signaling in muscle of patients with myotonic dystrophy (DM) and hypothyroid myopathy, the characterization of SERCA pumps remains elusive and their impairment is still unclear. We assessed the activity of SR Ca(2+)-ATPase, expression levels and fiber distribution of SERCA1 and SERCA2, and oligomerization of SERCA1 protein in muscle of patients with DM type 1 and 2, and with hypothyroid myopathy. Our data provide evidence that SR Ca(2+) ATPase activity, protein levels and muscle fiber distribution of total SERCA1 and SERCA2, and SERCA1 oligomerization pattern are similar in patients with both DM1 and DM2, hypothyroid myopathy and in control subjects. We prove that SERCA1b, the neonatal isoform of SERCA1, is expressed at protein level in muscle of patients with DM2 and, in lower amount, of patients with DM1. Our present study demonstrates that SERCA function is not altered in muscle of patients with DM and with hypothyroid myopathy.

Brody syndrome: a clinically heterogeneous entity distinct from Brody disease: a review of literature and a cross-sectional clinical study in 17 patients.

Brody disease is a rare inherited myopathy due to reduced sarcoplasmic reticulum Ca(2+) ATPase (SERCA)1 activity caused by mutations in ATP2A1, which causes delayed muscle relaxation and silent cramps. So far the disease has mostly been diagnosed by measurement of SERCA1 activity. Since mutation analysis became more widely available, it has appeared that not all patients with reduced SERCA1 activity indeed have ATP2A1 mutations, and a distinction between Brody disease (with ATP2A1 mutations) and Brody syndrome (without ATP2A1 mutations) was proposed. We aim to compare the clinical features of patients with Brody disease and those with Brody syndrome and detect clinical features which help to distinguish between the two. In addition, we describe the Brody syndrome phenotype in more detail. We therefore performed a literature review on clinical features of both Brody disease and Brody syndrome and a cross-sectional clinical study consisting of questionnaires, physical examination, and a review of medical files in 17 Brody syndrome patients in our centre. The results showed that Brody disease presents with an onset in the 1st decade, a generalized pattern of muscle stiffness, delayed muscle relaxation after repetitive contraction on physical examination, and autosomal recessive inheritance. Patients with Brody syndrome more often report myalgia and experience a considerable impact on daily life. Future research should focus on the possible mechanisms of reduction of SERCA activity in Brody syndrome and other genetic causes, and on evaluation of treatment options.

Adult-onset muscular dystrophy in a cat associated with a presumptive alteration in trafficking of caveolin-3.

A 10-year-old neutered female domestic longhaired cat was referred for evaluation of forelimb weakness and lameness. There was hypertrophy and firmness of the musculature with no neurological deficits. Moderate increase of creatine kinase activity was present. Muscle biopsy showed rounded atrophic and hypertrophic fibres, an increased number of centrally located myofibre nuclei, scattered rimmed vacuoles and mild perimysial and endomysial fibrosis. Myofibre necrosis with phagocytosis was present in the gluteal muscle. Immunohistochemistry revealed absence of sarcolemmal caveolin-3 in almost all muscle fibres and sarcoplasmic accumulation of the protein in approximately 30% of myofibres. Normal expression of caveolin-3 was detected by immunoblotting, so protein mislocalization in the sarcoplasm due to aberrant trafficking towards the sarcolemma was suspected. This case represents the first example of muscular dystrophy due to a caveolinopathy in animals.

Clinical, morphological and genetic studies in a cohort of 21 patients with myofibrillar myopathy.

The term myofibrillar myopathies (MFM) refers to uncommon neuromuscular disorders that pathologically are characterized by myofibrillar degeneration and ectopic expression of several proteins. MFM are partly caused by mutations in genes that encode mainly Z-disk-related proteins (desmin, alphaB-crystallin, myotilin, ZASP, filamin C and BAG3). We reviewed clinical, light and electron microscopy, immunohistochemistry, immunoblotting and genetic findings of 21 patients with MFM (15 unrelated patients and three pairs of brothers) investigated at our neuromuscular center. MFM patients begin to show symptoms at any age, from juvenile to late adult life and present a different distribution of muscle weakness. Cardiac involvement and peripheral neuropathy are common. Typical histological features include focal areas with reduction/loss of ATPase and oxidative enzyme activity, and amorphous material (eosinophilic on hematoxylin and eosin and dark blue on Engel-Gomori trichrome) in these abnormal fiber areas. Electron microscopy shows disintegration of myofibrils starting from the Z-disk and accumulation of granular and filamentous material among the myofilaments. Immunohistochemical studies demonstrate focal accumulation of desmin, alphaB-crystallin and myotilin in abnormal muscle fibers while immunoblot analysis does not highlight differences in the expression of these proteins also including ZASP protein. Therefore, unlike immunoblot, immunohistochemistry together with light and electron microscopy is a useful diagnostic tool in MFM. Finally three of our 21 patients have missense mutations in the desmin gene, two brothers carry missense mutations in the gene encoding myotilin, one has a missense mutation in alphaB-crystallin, and none harbour pathogenic variations in the genes encoding ZASP and BAG3.

Dystrophin restoration in skeletal, heart and skin arrector pili smooth muscle of mdx mice by ZM2 NP-AON complexes.

Potentially viable therapeutic approaches for Duchenne muscular dystrophy (DMD) are now within reach. Indeed, clinical trials are currently under way. Two crucial aspects still need to be addressed: maximizing therapeutic efficacy and identifying appropriate and sensible outcome measures. Nevertheless, the end point of these trials remains painful muscle biopsy to show and quantify protein restoration in treated boys. In this study we show that PMMA/N-isopropil-acrylamide+ (NIPAM) nanoparticles (ZM2) bind and convey antisense oligoribonucleotides (AONs) very efficiently. Systemic injection of the ZM2-AON complex restored dystrophin protein synthesis in both skeletal and cardiac muscles of mdx mice, allowing protein localization in up to 40% of muscle fibers. The mdx exon 23 skipping level was up to 20%, as measured by the RealTime assay, and dystrophin restoration was confirmed by both reverse transcription-PCR and western blotting. Furthermore, we verified that dystrophin restoration also occurs in the smooth muscle cells of the dorsal skin arrector pili, an easily accessible histological structure, in ZM2-AON-treated mdx mice, with respect to untreated animals. This finding reveals arrector pili smooth muscle to be an appealing biomarker candidate and a novel low-invasive treatment end point. Furthermore, this marker would also be suitable for subsequent monitoring of the therapeutic effects in DMD patients. In addition, we demonstrate herein the expression of other sarcolemma proteins such as alpha-, beta-, gamma- and delta-sarcoglycans in the human skin arrector pili smooth muscle, thereby showing the potential of this muscle as a biomarker for other muscular dystrophies currently or soon to be the object of clinical trials.

Muscular dystrophy with reduced beta-sarcoglycan in a cat.

A partial beta-sarcoglycan (SG) deficiency with retention of other components of the SG complex (SGC) is described in 6-month-old, intact male domestic shorthaired kitten that was referred for evaluation of weakness, reluctance to move and dyspnoea. Neurological deficits were restricted to the neuromuscular system. Muscle biopsy revealed moderate variability in myofibre size, with numerous atrophic rounded fibres, rare myofibre necrosis, regeneration and moderate perimysial and endomysial fibrosis. Immunohistochemistry revealed decreased expression of beta- and gamma-SG and western blotting revealed markedly decreased beta-SG with normal expression of alpha-, gamma- and delta-SG, caveolin-3 and calpain-3. Sarcoglycanopathy has not previously been described in cats. In human and canine sarcoglycanopathies the deficiency in any one of the SGs leads to secondary deficiency of the entire SGC. Such spontaneously arising muscular disease in animals can provide valuable models for equivalent human disorders.

Transcriptional behavior of DMD gene duplications in DMD/BMD males.

DMD gene exons duplications account for up to 5-10 % of Duchenne (DMD) and up to 5-19% of Becker (BMD) muscular dystrophies; as for the more common deletions, the genotype-phenotype correlation and the genetic prognosis are generally based on the "reading frame rule". Nevertheless, the transcriptional profile of duplications, abridging the genomic configuration to the eventual protein effect, has been poorly studied. We describe 26 DMD gene duplications occurring in 33 unrelated patients and detected among a cohort of 194 mutation-positive DMD/BMD patients. We have characterized at the RNA level 16 of them. Four duplications (15%) behave as exception to the reading frame rule. In three BMD cases with out-of-frame mutations, the RNA analysis revealed that exon skipping events occurring in the duplicated region represent the mechanism leading to the frame re-establishment and to the milder phenotype. Differently, in a DMD patient carrying an in-frame duplication the RNA behaviour failed to explain the clinical phenotype which is probably related to post-transcriptional-translational mechanisms. We conclude that defining the RNA profile in DMD gene duplications is mandatory both for establishing the genetic prognosis and for approaching therapeutic trials based on hnRNA modulation.

Eyelid ptosis from sympathetic nerve dysfunction mistaken as myopathy: a simple test to identify this condition.

Acquired isolated unilateral or bilateral blepharoptosis has many aetiologies. When the pupils are normal, a myasthenic syndrome or myopathy has to be ruled out. If the tests for myasthenia gravis are negative, the next step is to perform a muscle biopsy to establish a diagnosis. Muscle examination may show a mitochondrial disorder, non-specific abnormalities or be quite normal. We identified three patients, who had previously undergone various investigations, including a muscle biopsy, whose lid ptosis disappeared using eye drops containing naphazoline nitrate, a sympathomimetic drug, thus suggesting partial Horner's syndrome. We emphasise the usefulness of this simple and cheap test before performing more traumatic and expensive investigations.

The role of muscle biopsy in investigating isolated muscle pain.

OBJECTIVE: To evaluate the muscle biopsy findings from 240 patients who had isolated muscle pain. METHODS: Histopathology, immunohistochemistry for dystrophin, dystrophin-related proteins, major histocompatibility complex type I, and biochemical analysis of glycolytic and mitochondrial respiratory chain enzymes were performed on muscle biopsies. An attempt was made to correlate pathologic data and clinical findings (sex, age, quality and distribution of symptoms, serum CK levels, and EMG recording). RESULTS: We have described five groups of patients based on muscle biopsy findings: 51.6% had heterogeneous myopathic abnormalities; only 19% of them had a specific myopathic picture, i.e., central nuclei myopathy, central core disease, myopathy with tubular aggregates or with trabecular fibers or abnormalities of fiber typing; 20% had signs of respiratory chain dysfunction but only one patient had a probable mitochondrial disease; 7% had a neurogenic pattern; 2.4% had a metabolic myopathy (phosphorylase or phosphofructokinase deficiency); and 19% had normal muscle biopsy. No clear-cut correlation between muscle biopsy and clinical data was observed except for those patients with a metabolic myopathy. CONCLUSIONS: The probability that a patient complaining only of muscle pain and with a normal neurologic examination has a definite muscle pathology is 2%. Only patients with sole exercise-related muscle pain and sCK seven times higher than the normal value are strongly suspected of having a metabolic myopathy. A rigorous selection of patients is needed before performing a muscle biopsy.

Expression of late myogenic differentiation markers in sarcoplasmic masses of patients with myotonic dystrophy.

Sarcoplasmic masses contain disorganized myofibrillar material and are a striking feature of myotonic dystrophy. However their significance is still unclear. Using immunocytochemistry we studied the expression of cytoskeletal proteins (desmin and vimentin), dystrophin, markers of myogenic differentiation (foetal myosin, neural cell adhesion molecule, bcl-2, insulin-like growth factor-I, fibroblast growth factor, retinoblastoma protein and myoD1), cell cycle regulators (Cdk2, p16, p27 and p57) and muscle proteases (ubiquitin, micro and m calpain and cathepsin D) in muscle biopsies from four patients with myotonic dystrophy. Sarcoplasmic masses were strongly positive for desmin, neural cell adhesion molecule, bcl-2, insulin-like growth factor I, retinoblastoma protein and p57, weakly positive for dystrophin and p16 and negative for vimentin, fibroblast growth factor, myoD1, Cdk2 and p27. Immunoreactivity for foetal myosin was detected only in a few fibres (< 1%). Our data suggest that the late myogenic differentiation programme is activated in sarcoplasmic masses although these areas do not reach complete maturation.

Mutant ubiquitin UBB+1 is accumulated in sporadic inclusion-body myositis muscle fibers.

Mutant ubiquitin (UBB+1), a product of "molecular misreading," is toxic to cells because its ubiquitinated form inhibits the proteasome, contributing to accumulation of misfolded proteins and their ensuing toxicity. The authors demonstrate in 10 sporadic inclusion body myositis (s-IBM) muscle biopsies that UBB+1 is accumulated in aggregates containing amyloid-beta and phosphorylated-tau. In s-IBM, UBB+1 may be pathogenic by inhibiting proteasome, thereby promoting accumulation of cytotoxic misfolded amyloid-beta and phosphorylated-tau.

Expression of protein kinase C isoforms and interleukin-1beta in myofibrillar myopathy.

BACKGROUND: The term myofibrillar myopathy refers to a rare and clinically heterogeneous group of muscle disorders. The pathogenesis of this myopathy is not well understood. The morphologic hallmark is myofibrillar destruction with abnormal expression of numerous proteins, most consistently of desmin. METHODS: The authors investigated eight patients with myofibrillar myopathy belonging to four families. They studied the role of different protein kinase C isoforms and of interleukin-1beta, a cytokine that might activate protein kinase C and, in addition, mediate myofibrillar proteolysis. RESULTS: Immunohistochemical analysis showed the expression of alpha, eta, and zeta isoforms of protein kinase C and of interleukin-1beta in abnormal muscle fibers. Immunoblots confirmed the immunohistochemical data and revealed the absence of protein kinase C delta and epsilon in muscle fibers from patients and controls. CONCLUSIONS: These data suggest that protein kinase C and interleukin-1beta may play a role in the pathogenesis of myofibrillar myopathy.