Novel TRIM32 mutation in sarcotubular myopathy

Tripartite motif-containing protein 32 (TRIM32) is a member of the TRIM ubiquitin E3 ligases which ubiquitinates different substrates in muscle including sarcomeric proteins. Mutations in TRIM32 are associated with Limb-Girdle Muscular Dystrophy 2H. In a 66 old woman with disto-proximal myopathy, we identified a novel homozygous mutation of TRIM32 gene c.1781G > A (p. Ser594Asn) localised in the c-terminus NHL domain. Mutations of this domain have been also associated to Sarcotubular Myopathy (STM), a form of distal myopathy with peculiar features in muscle biopsy, now considered in the spectrum of LGMD2H. Muscle biopsy revealed severe abnormalities of the myofibrillar network with core like areas, lobulated fibres, whorled fibres and multiple vacuoles. Desmin and Myotilin stainings also pointed to accumulation as in Myofibrillar Myopathy. This report further confirms that STM and LGMD2H represent the same disorder and suggests to consider TRIM32 mutations in the genetic diagnosis of Sarcotubular Myopathy and Myofibrillar Myopathy

Modifications induced by acylphosphatase in the functional properties of heart sarcolemma Na+,K+ pump

AbstractAcylphosphatase purified from cardiac muscle actively hydrolyzes the phosphoenzyme intermediate of heart sarcolemma Na+,K+-ATPase. This effect occurred with acylphosphatase amounts (up to 800 unitsmg membrane protein) that fall within the physiological range and the low value of the apparent Km (0.69 × 10−7 M) indicates a considerable affinity of the enzyme towards this specific substrate. Acylphosphatase addition to purified sarcolemmal vesicles significantly increased the rate of Na+,K+-dependent ATP hydrolysis. Maximal stimulation, observed with 800 unitsmg protein, resulted in an ATPase activity which was about 2-fold over basal value. The same acylphosphatase amounts significantly stimulated, in a similar and to an even greater extent, the rate of ATP driven Na+ transport into sarcolemmal vesicles. These findings lead to suppose that an accelerated hydrolysis of the phosphoenzyme may result in an enhanced activity of heart sarcolemmal Na+,K+ pump, therefore suggesting a potential role of acylphosphatase in the control of this active transport system

Stimulation of cardiac sarcoplasmic reticulum calcium pump by acylphosphatase: relationship to phospholamban phosphorylation.

Abstract Ca2+ transport by cardiac sarcoplasmic reticulum is tightly coupled with the enzymatic activity of Ca2+-dependent ATPase, which forms and decomposes an intermediate phosphoenzyme. Heart sarcoplasmic reticulum Ca2+ pump is regulated by cAMP-dependent protein kinase (PKA) phospholamban phosphorylation, which results in a stimulation of the initial rates of Ca2+ transport and Ca2+ ATPase activity. In the present studies we found that acylphosphatase from heart muscle, used at concentrations within the physiological range, actively hydrolyzes the phosphoenzyme of cardiac sarcoplasmic reticulum Ca2+ pump, with an apparent Km on the order of 10−7 M, suggesting an high affinity of the enzyme for this special substrate. In unphosphorylated vesicles acylphosphatase enhanced the rate of ATP hydrolysis and Ca2+ uptake with a concomitant significant decrease in apparent Km for Ca2+ and ATP. In vesicles whose phospholamban was PKA-phosphorylated, acylphosphatase also stimulated the rate of Ca2+ uptake and ATP hydrolysis but to a lesser extent, and the Km values for Ca2+ and ATP were not significantly different with respect to those found in the absence of acylphosphatase. These findings suggest that acylphosphatase, owing to its hydrolytic effect, accelerates the turnover of the phosphoenzyme intermediate with the consequence of an enhanced activity of Ca2+ pump. It is known that phosphorylation of phospholamban results in an increase of the rate at which the phosphoenzyme is decomposed. Thus, as discussed, a competition between phospholamban and acylphosphatase effect on the phosphoenzyme might be proposed to explain why the stimulation induced by this enzyme is less marked in PKA-phosphorylated than in unphosphorylated heart vesicles

A novel interaction mechanism accounting for different acylphosphatase effects on cardiac and fast twitch skeletal muscle sarcoplasmic reticulum calcium pumps

AbstractIn cardiac and skeletal muscle Ca2+ translocation from cytoplasm into sarcoplasmic reticulum (SR) is accomplished by different Ca2+-ATPases whose functioning involves the formation and decomposition of an acylphosphorylated phosphoenzyme intermediate (EP). In this study we found that acylphosphatase, an enzyme well represented in muscular tissues and which actively hydrolyzes EP, had different effects on heart (SERCA2a) and fast twitch skeletal muscle SR Ca2+-ATPase (SERCA1). With physiological acylphosphatase concentrations SERCA2a exhibited a parallel increase in the rates of both ATP hydrolysis and Ca2+ transport; in contrast, SERCA1 appeared to be uncoupled since the stimulation of ATP hydrolysis matched an inhibition of Ca2+ pump. These different effects probably depend on phospholamban, which is associated with SERCA2a but not SERCA1. Consistent with this view, the present study suggests that acylphosphatase-induced stimulation of SERCA2a, in addition to an enhanced EP hydrolysis, may be due to a displacement of phospholamban, thus to a removal of its inhibitory effect

A novel mutation in the N-terminal acting-binding domain of Filamin C protein causing a distal myofibrillar myopathy

Variants in Filamin C (FLNC) gene may cause either cardiomyopathies or different myopathies. We describe a family affected by a distal myopathy with autosomal dominant inheritance. The onset of the disease was in the third decade with gait impairment due to distal leg weakness. Subsequently, the disease progressed with an involvement of proximal lower limbs and hand muscles. Muscle biopsy, performed in one subject,identified relevant myofibrillar abnormalities. We performed a target gene panel testing for myofibrillar myopathies by NGS approach which identified a novel mutation in exon 3 of FLNC gene (c.A664G:p.M222V), within the N-terminal actin-binding (ABD) domain. This variant has been identified in all affected members of the family, thus supporting its pathogenic role. Differently from previously identified variants, our family showed a predominant leg involvement and myofibrillar aggregates, thus further expanding the spectrum of Filamin C related myopathies