Matteo Marini

Increased protein nitration in mitochondrial diseases: evidence for vessel wall involvement.

Mitochondrial diseases (MD) are heterogeneous disorders because of impairment of respiratory chain function leading to oxidative stress. We hypothesized that in MD the vascular endothelium may be affected by increased oxidative/nitrative stress causing a reduction of nitric oxide availability. We therefore, investigated the pathobiology of vasculature in MD patients by assaying the presence of 3-nitrotyrosine in muscle biopsies followed by the proteomic identification of proteins which undergo tyrosine nitration. We then measured the flow-mediated vasodilatation as a proof of altered nitric oxide generation/bioactivity. Here, we show that 3-nitrotyrosine staining is specifically located in the small vessels of muscle tissue and that the reaction is stronger and more evident in a significant percentage of vessels from MD patients as compared with controls. Eleven specific proteins which are nitrated under pathological conditions were identified; most of them are involved in energy metabolism and are located mainly in mitochondria. In MD patients the flow-mediated vasodilatation was reduced whereas baseline arterial diameters, blood flow velocity and endothelium-independent vasodilatation were similar to controls. The present results provide evidence that in MD the vessel wall is a target of increased oxidative/nitrative stress.

Brody disease: insights into biochemical features of SERCA1 and identification of a novel mutation.

Brody disease is an inherited disorder of skeletal muscle function characterized by increasing impairment of relaxation during exercise. The autosomal recessive form can be caused by mutations in the ATP2A1 gene, which encodes for the sarcoplasmic/endoplasmic reticulum Ca2+-ATPase 1 (SERCA1) protein. We studied 2 siblings affected by Brody disease. The patients complained of exercise-induced delay of muscle relaxation and stiffness since childhood and had gene analysis of ATP2A1. Morphologic and biochemical studies were performed on a muscle biopsy from 1 patient. The biopsy showed fiber size variation and increased numbers of fibers with internal nuclei. Ultrastructural examination revealed dilatation of lateral cisternae and proliferation of tubular elements of the sarcoplasmic reticulum. By immunohistochemistry, SERCA1 was expressed in a normal pattern, but sarcoplasmic reticulum Ca2+-ATPase activity was significantly reduced. Immunoblotting after high-resolution 2-dimensional gel electrophoresis showed a significant difference in the amount of SERCA1 protein between the patient and controls. Both patients were found to have 2 previously unreported in-frame deletions in ATP2A1. Because SERCA1 protein has specific biochemical characteristics in our patient, these results underline the importance of a pathologic and biochemical analyses for the diagnosis. In addition, we describe 2 novel mutations in the ATP2A1 gene.

SERCA1 protein expression in muscle of patients with Brody disease and Brody syndrome and in cultured human muscle fibers

Brody disease is an inherited myopathy associated with a defective function of sarcoplasmic/endoplasmic reticulum Ca(2+)-ATPase 1 (SERCA1) protein. Mutations in the ATP2A1 gene have been reported only in some patients. Therefore it has been proposed to distinguish patients with ATP2A1 mutations, Brody disease (BD), from patients without mutations, Brody syndrome (BS). We performed a detailed study of SERCA1 protein expression in muscle of patients with BD and BS, and evaluated the alternative splicing of SERCA1 in primary cultures of normal human muscle and in infant muscle. SERCA1 reactivity was observed in type 2 muscle fibers of patients with and without ATP2A1 mutations and staining intensity was similar in patients and controls. Immunoblot analysis showed a significant reduction of SERCA1 band in muscle of BD patients. In addition we demonstrated that the wild type and mutated protein exhibits similar solubility properties and that RIPA buffer improves the recovery of the wild type and mutated SERCA1 protein. We found that SERCA1b, the SERCA1 neonatal form, is the main protein isoform expressed in cultured human muscle fibers and infant muscle. Finally, we identified two novel heterozygous mutations within exon 3 of the ATP2A1 gene from a previously described patient with BD.

Sarcoidosis and inclusion body myositis

directed against RANKL, has been demonstrated to increase bone mass at both axial and peripheral skeletal sites, as well as in trabecular and cortical areas of bone [8]. It can probably prevent not only the generalized bone loss in RA, but also the joint destruction inhibiting the bone erosion [9]. On the other hand, zoledronic acid is a potent third-generation aminobisphosphonate that is thought to act by inhibiting the osteoclast lifespan. It has been suggested that, added to MTX, it reduces the number of bone erosions in RA [10]. It seems clear that the new challenge in the treatment of RA should be not only to arrest the structural damage, but also to repair the previous erosions. Anti-TNFtherapy, zoledronic acid and denosumab, alone or in combined therapy, are among the potential candidates to achieve that ambitious but, we think, realistic goal.

Muscular dystrophy with reduced β-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.

Polymyositis in solid organ transplant recipients receiving tacrolimus.

Tacrolimus, also known as FK506, is an immunosuppressive agent widely used for the prevention of acute allograft rejection in organ transplantation and for the treatment of immunological diseases. This study reports two male patients who underwent solid organ transplantation (liver and kidney). After transplant, the patients received continuous immunosuppressive therapy with oral tacrolimus and later presented clinical manifestations and laboratory signs of myopathy. Muscle biopsies of both patients clearly documented an inflammatory myopathy with the histological features of polymyositis including CD8+ T cells which invaded healthy muscle fibers and expressed granzyme B and perforin, many CD68+ macrophages and MHC class I antigen upregulation on the surface of most fibers. Because of the temporal association while receiving tacrolimus and since other possible causes for myopathy were excluded, the most likely cause of polymyositis in our patients was tacrolimus toxicity. We suggest that patients on tacrolimus should be carefully monitored for serum CK levels and clinical signs of muscle disease.

Differential regulation of TNF receptors in maternal leukocytes is associated with severe preterm preeclampsia

Abstract We tested the hypothesis that maternal peripheral blood leukocytes contribute to elevated levels of soluble TNF receptors (sTNFR) in preeclampsia (PE) with concomitant intrauterine growth restriction (IUGR). TNFR1 and TNFR2 were evaluated in a cross-sectional study comparing preeclamptic (n = 15) with or without IUGR versus normotensive pregnant women (PREG, n = 30), and non-pregnant controls (Con; n = 20). Plasma levels of sTNFR1 were higher in PE (1675.0 ± 227.1 pg/mL) compared with PREG (1035.0 ± 101.1 pg/mL) and Con (589.3 ± 82.67 pg/mL), with the highest values observed in PE with IUGR (2624.0 ± 421.4 pg/mL; n = 6). Plasma sTNFR2 was higher during pregnancy (PE: 1836.0 ± 198.7 pg/mL; PREG: 1697.0 ± 95.0 pg/mL) compared with Con (598.3 ± 82.7 pg/mL). Urinary levels of sTNFR1 and sTNFR2 were higher in PE and PREG compared with the Con group. Abundance of TNFR1 mRNA in peripheral blood leukocytes was strongly correlated with plasma levels of sTNFR1 in PE. However, TNFR2 mRNA accumulation in leukocytes did not correlate with sTNFR2 plasma levels. The level of sTNFR1 in plasma was correlated with body weight of the newborn (r = −0.56). The data suggest that maternal leukocytes contribute to sTNFR1 levels in plasma in association with decreasing newborn weight and PE with concomitant IUGR.

Calpain 3 deficiency presenting as fibre type disproportion.

Congenital fibre type disproportion (CFTD) is a histological abnormality characterized by small type 1 muscle fibres, normally sized type 2 fibres and absence of other pathological features [1]. Type 1 fibre hypotrophy is observed in various neuromuscular disorders; thus CFTD should refer only to cases of fibre type disproportion (FTD) having the clinical features of a congenital myopathy and when other neuromuscular conditions have been ruled out [1]. A 36-year-old man, the only son of nonconsanguineous parents, had a normal birth and early development. Family history was unremarkable. At 4 years of age he presented with difficulty in climbing stairs and serum creatine kinase was twice the normal value. On clinical examination, proximal limb and spine flexor muscles were weak, whereas facial and neck muscles were spared. Shortening of the Achilles tendon was observed. A biopsy of the left vastus lateralis muscle showed FTD (Figure 1 and Table 1). During the following years, the patient developed elbow and knee contractures and at 9 years of age he underwent tenotomy of both Achilles tendons. At 19 years of age he complained of increased difficulty in walking, but did not need any aid, was unable to rise from the squatting position, but could ride his bicycle and lifted the arms over his shoulders. Contractures were severe at the elbow and knee and very mild in the hands where movements and strength were preserved. The patient had a scoliosis and could only partially flex the trunk. Muscle atrophy was predominant at the proximal muscles of the limbs. Deep tendon reflexes were lost and creatine kinase values were increased to over 10 times the normal value. ECG was normal. A second biopsy from the right vastus lateralis muscle showed dystrophic features, including marked endomysial fibrosis, vacuolar degeneration in a few fibres, increased number of central nuclei and occasional ring fibres. Unexpectedly, there was also a change in the size and number of fibre types as compared with the first muscle biopsy because type 2 fibres were smaller and more numerous than type 1 (Table 1); in addition, most of the type 1 fibres were lobulated (Figure 1). Four years after the second muscle biopsy, the patient was wheelchairbound. ECG and Holter ECG were normal. Immunohistochemistry for dystrophin (mid-rod, amino-terminal and carboxy-terminal domains), a-sarcoglycan (SG), b-SG, g-SG, d-SG, dysferlin, telethonin, caveolin-3, emerin, merosin, titin and collagen VI, done on the second muscle biopsy, was normal, as well as an immunoblot for dysferlin. Immunoblot for calpain 3, however, showed a severe reduction of the 94and 60-kDa protein bands, as compared with control muscles, and the absence of the 30-kDa band (Figure 1). Mitochondria respiratory chain enzyme activities were normal. All the coding exons of the following genes were amplified by PCR and sequenced: Selenoprotein N isoform 1 gene (SEPN1), including the selenocysteine insertion sequence, the SECIS element, located in the 3′ UTR of the gene; skeletal muscle alpha Actin gene (ACTA1); Caveolin-3 gene (CAV3); FukutinRelated Protein gene (FKRP); and lamin A/C gene (LMNA). Finally, the Calpain-3 gene (CAPN3) and the gene encoding the slow chain of a-tropomyosin (TPM3) were studied at transcript level. The analysis of all these genes did not show any mutation. At the present time, the patient, who has a degree in musicology, plays the barrel organ; he moves only from the wheelchair to bed and raises the hands up to the mouth. Foot movements are preserved and contractures are unchanged. The first muscle biopsy from this patient, done when he was 4 years old, fulfilled the histopathological criteria for the diagnosis of FTD. Fifteen years later, remarkably, the morphological pattern had turned into a dystrophic one and the ratio between the diameter of type 1 and type 2 fibres was reversed. The lack of mutations in SEPN1, ACTA1 and TPM3 genes, which have been associated with CTFD, indicates that he is not affected with any of the known genetic causes of CFTD [2]. Data on the histological changes in muscle from patients with FTD are scarce and, to our knowledge, only 11 patients with FTD had two or more muscle biopsies taken at different ages [3–10]. In four of these patients the histological abnormalities were no more evident by the second muscle biopsy [3,4], in two patients were unchanged [5,6], in two patients were

Bortezomib-induced muscle toxicity in multiple myeloma

Multiple myeloma (MM) accounts for ∼13% of all hematologic malignancies. Bortezomib treatment is effective in MM, but can be complicated with neurological side effects. We describe a patient with symptomatic MM who had a reversible metabolic myopathy associated with bortezomib administration and pathologically characterized by excessive storage of lipid droplets together with mitochondrial abnormalities. In a single-center prospective study, 14 out of 24 patients with symptomatic MM were treated with bortezomib and, among these, 7 developed muscular signs and/or symptoms. The myopathy was characterized by a proximal muscle weakness involving lower limbs and was an early complication. Complete resolution of muscle weakness occurred after treatment discontinuation. Conversely, none of the patients who received a treatment without bortezomib developed muscular symptoms. Experimental studies demonstrate that in primary human myoblasts bortezomib at low concentrations leads to excessive storage of lipid droplets together with structural mitochondrial abnormalities, recapitulating the pathologic findings observed in patients muscle. Our data suggest that patients treated with bortezomib should be monitored for muscular signs and/or symptoms and muscle weakness should alert the clinician to the possibility of myopathy. Bortezomib-induced metabolic myopathy is a potentially reversible entity with important implications for management and treatment of patients with MM.

Evidence for caspase-dependent programmed cell death along with repair processes in affected skeletal muscle fibres in patients with mitochondrial disorders

Mitochondrial disorders are heterogeneous multisystemic disorders due to impaired oxidative phosphorylation causing defective mitochondrial energy production. Common histological hallmarks of mitochondrial disorders are RRFs (ragged red fibres), muscle fibres with abnormal focal accumulations of mitochondria. In contrast with the growing understanding of the genetic basis of mitochondrial disorders, the fate of phenotypically affected muscle fibres remains largely unknown. We investigated PCD (programmed cell death) in muscle of 17 patients with mitochondrial respiratory chain dysfunction. We documented that in affected muscle fibres, nuclear chromatin is condensed in lumpy irregular masses and cytochrome c is released into the cytosol to activate, along with Apaf-1 (apoptotic protease-activating factor 1), caspase 9 that, in turn, activates effector caspase 3, caspase 6, and caspase 7, suggesting the execution of the intrinsic apoptotic pathway. Whereas active caspase 3 underwent nuclear translocation, AIF (apoptosis-inducing factor) mainly stayed within mitochondria, into which an up-regulated Bax is relocated. The significant increase in caspase 2, caspase 3 and caspase 6 activity strongly suggest that the cell death programme is caspase-dependent and the activation of caspase 2 together with PUMA (p53 up-regulated modulator of apoptosis) up-regulation point to a role for oxidative stress in triggering the intrinsic pathway. Concurrently, in muscle of patients, the number of satellite cells was significantly increased and myonuclei were detected at different stages of myogenic differentiation, indicating that a reparative programme is ongoing in muscle of patients with mitochondrial disorders. Together, these data suggest that, in patients with mitochondrial disorders, affected muscle fibres are trapped in a mitochondria-regulated caspase-dependent PCD while repairing events take place.