Massimiliano Memo

Investigation of a transgenic mouse model of familial dilated cardiomyopathy

We have investigated a transgenic mouse model of inherited dilated cardiomyopathy that stably expresses the ACTC E361G mutation at around 50% of total actin in the heart. F-actin isolated from ACTC E361G mouse hearts was incorporated into thin filaments with native human tropomyosin and troponin and compared with NTG mouse actin by in vitro motility assay. There was no significant difference in sliding speed, fraction of filaments motile or Ca(2+)-sensitivity (ratio EC(50) E361G/NTG=0.95+/-0.08). The Ca(2+)-sensitivity of force in skinned trabeculae from ACTC E361G mice was slightly higher than NTG (EC(50) E361G/NTG=0.78+/-0.04). The molecular phenotype was revealed when troponin was dephosphorylated; Ca(2+)-sensitivity of E361G-containing thin filaments was now lower than NTG (EC(50) E361G(dPTn)/NTG(dPTn)=2.15+/-0.09). We demonstrated that this was due to uncoupling of Ca(2+)-sensitivity from troponin I phosphorylation by comparing Ca(2+)-sensitivity of phosphorylated and dephosphorylated thin filaments. For NTG actin-containing thin filaments EC(50) native/dPTn=3.0+/-0.3 but for E361G-containing thin filaments EC(50) native/dPTn=1.04+/-0.07.We studied contractility in isolated myocytes and found no significant differences under basal conditions. We measured cardiac performance by cine-MRI, echocardiography and with a conductance catheter over a period of 4 to 18 months and found minimal systematic differences between NTG and ACTC E361G mice under basal conditions. However, the increase in septal thickening, ejection fraction, heart rate and cardiac output following dobutamine treatment was significantly less in ACTC E361G mice compared with NTG. We propose that the ACTC E361G mutation uncouples myofilament Ca(2+)-sensitivity from Troponin I phosphorylation and blunts the response to adrenergic stimulation, leading to a reduced cardiac reserve with consequent contractile dysfunction under stress, leading to dilated cardiomyopathy.

Mutations in repeating structural motifs of tropomyosin cause gain of function in skeletal muscle myopathy patients

The congenital myopathies include a wide spectrum of clinically, histologically and genetically variable neuromuscular disorders many of which are caused by mutations in genes for sarcomeric proteins. Some congenital myopathy patients have a hypercontractile phenotype. Recent functional studies demonstrated that ACTA1 K326N and TPM2 ΔK7 mutations were associated with hypercontractility that could be explained by increased myofibrillar Ca(2+) sensitivity. A recent structure of the complex of actin and tropomyosin in the relaxed state showed that both these mutations are located in the actin-tropomyosin interface. Tropomyosin is an elongated molecule with a 7-fold repeated motif of around 40 amino acids corresponding to the 7 actin monomers it interacts with. Actin binds to tropomyosin electrostatically at two points, through Asp25 and through a cluster of amino acids that includes Lys326, mutated in the gain-of-function mutation. Asp25 interacts with tropomyosin K6, next to K7 that was mutated in the other gain-of-function mutation. We identified four tropomyosin motifs interacting with Asp25 (K6-K7, K48-K49, R90-R91 and R167-K168) and three E-E/D-K/R motifs interacting with Lys326 (E139, E181 and E218), and we predicted that the known skeletal myopathy mutations ΔK7, ΔK49, R91G, ΔE139, K168E and E181K would cause a gain of function. Tests by an in vitro motility assay confirmed that these mutations increased Ca(2+) sensitivity, while mutations not in these motifs (R167H, R244G) decreased Ca(2+) sensitivity. The work reported here explains the molecular mechanism for 6 out of 49 known disease-causing mutations in the TPM2 and TPM3 genes, derived from structural data of the actin-tropomyosin interface.

Abnormal actin binding of aberrant β-tropomyosins is a molecular cause of muscle weakness in TPM2-related nemaline and cap myopathy.

NM (nemaline myopathy) is a rare genetic muscle disorder defined on the basis of muscle weakness and the presence of structural abnormalities in the muscle fibres, i.e. nemaline bodies. The related disorder cap myopathy is defined by cap-like structures located peripherally in the muscle fibres. Both disorders may be caused by mutations in the TPM2 gene encoding β-Tm (tropomyosin). Tm controls muscle contraction by inhibiting actin-myosin interaction in a calcium-sensitive manner. In the present study, we have investigated the pathogenetic mechanisms underlying five disease-causing mutations in Tm. We show that four of the mutations cause changes in affinity for actin, which may cause muscle weakness in these patients, whereas two show defective Ca2+ activation of contractility. We have also mapped the amino acids altered by the mutation to regions important for actin binding and note that two of the mutations cause altered protein conformation, which could account for impaired actin affinity.

Nemaline myopathy with stiffness and hypertonia associated with an ACTA1 mutation

Nemaline myopathy, known to be caused by mutations in 7 genes, including skeletal muscle α-actin ( ACTA1 ),1 is characterized by muscle weakness, hypotonia, and nemaline rods in muscle biopsy. Here we report a patient with nemaline rods but the opposite phenotype of hypercontractility. ### Case report. The John Radcliffe Hospital ethics review board approved the study. The first child of nonconsanguineous European parents presented at 6 weeks of age with an apneic episode thought due to bilateral strangulated inguinal hernias and an umbilical hernia. However, after herniotomy, rigidity and recurrent apneas requiring mechanical ventilation continued. The patient was born by elective Caesarean section at 39 weeks, with no perinatal complications, although the mother, with hindsight, thought there might have been stiffness in the weeks after birth as she had difficulty clothing him. Polyhydramnios had been noted. On examination, he had very stiff and hypertrophic abdominal and proximal limb muscles and elbow and knee contractures. Deep tendon reflexes showed brisk contraction and slow relaxation and percussion myotonia could be elicited. However, EMG in multiple muscles showed no evidence of myotonia. He also had normal nerve conduction studies, MRI, EEG, and cardiac assessment, including echocardiography. Neurometabolic investigations were also normal including serum and CSF lactate, CSF neurotransmitters, urinary catecholamines, carnitine profile, white cell enzymes, and screening for mucopolysaccharidosis. Serum creatine kinase was slightly raised (370 IU, range 24–195). A sleep study showed hypoventilation and carbon dioxide retention. Hyperekplexia, paroxysmal episodic pain disorder, neuromyotonia, and episodic ataxia were considered, though none exactly matched the phenotype because of the underlying stiffness, but relevant testing ( ARX , FRAXA , GLRA1 , KCN1A , POLG , SCN9A , anti GAD, and VGKC antibody studies) was negative. Pharmacologic treatment including phenytoin, carbamazepine, flecainide, baclofen, dantrolene, and acetazolamide was ineffective. He continued having recurrent episodes of stiffness, where he stopped …

K7del is a common TPM2 gene mutation associated with nemaline myopathy and raised myofibre calcium sensitivity

Mutations in the TPM2 gene, which encodes β-tropomyosin, are an established cause of several congenital skeletal myopathies and distal arthrogryposis. We have identified a TPM2 mutation, p.K7del, in five unrelated families with nemaline myopathy and a consistent distinctive clinical phenotype. Patients develop large joint contractures during childhood, followed by slowly progressive skeletal muscle weakness during adulthood. The TPM2 p.K7del mutation results in the loss of a highly conserved lysine residue near the N-terminus of β-tropomyosin, which is predicted to disrupt head-to-tail polymerization of tropomyosin. Recombinant K7del-β-tropomyosin incorporates poorly into sarcomeres in C2C12 myotubes and has a reduced affinity for actin. Two-dimensional gel electrophoresis of patient muscle and primary patient cultured myotubes showed that mutant protein is expressed but incorporates poorly into sarcomeres and likely accumulates in nemaline rods. In vitro studies using recombinant K7del-β-tropomyosin and force measurements from single dissected patient myofibres showed increased myofilament calcium sensitivity. Together these data indicate that p.K7del is a common recurrent TPM2 mutation associated with mild nemaline myopathy. The p.K7del mutation likely disrupts head-to-tail polymerization of tropomyosin, which impairs incorporation into sarcomeres and also affects the equilibrium of the troponin/tropomyosin-dependent calcium switch of muscle. Joint contractures may stem from chronic muscle hypercontraction due to increased myofibrillar calcium sensitivity while declining strength in adulthood likely arises from other mechanisms, such as myofibre decompensation and fatty infiltration. These results suggest that patients may benefit from therapies that reduce skeletal muscle calcium sensitivity, and we highlight late muscle decompensation as an important cause of morbidity.

Skeletal muscle myopathy mutations at the actin tropomyosin interface that cause gain- or loss-of-function

It is well known that the regulation of muscle contraction relies on the ability of tropomyosin to switch between different positions on the actin filament, but it is still not well understood which amino acids are directly involved in the different states of the interaction. Recently the structure of the actin–tropomyosin interface has been determined both in the absence and presence of myosin heads. Interestingly, a number of mutations in tropomyosin that are associated with skeletal muscle myopathy are located within this interface. We first give an overview of the functional effect of mutations on amino acids that are involved in the contact with actin asp25, which represent a pattern repeated seven times along tropomyosin. It is explained how some of these amino acids (R167 and R244) which are thought to be involved in a salt bridge contact with actin in the closed state can produce a loss-of-function when mutated, while other positively charged tropomyosin amino acids positioned on the downstream side of the contact (K7, K49, R91, K168) can produce a gain-of-function when mutated. We then consider mutations of amino acids involved in another salt bridge contact between the two proteins in the closed state, actin K326N (which binds on five different points of tropomyosin) and tropomyosin ∆E139 and E181K, and we report how all of these mutations produce a gain-of-function. These observations can be important to validate the proposed structures and to understand more deeply how mutations affect the function of these proteins and to enable prediction of their outcomes.

C.P.15 K7del is a recurrent TPM2 nemaline myopathy mutation associated with joint contractures and increased calcium sensitivity

Abstract The TPM2 gene encodes beta-tropomyosin (βTm). A range of TPM2 mutations have been associated with several congenital myopathies and distal arthrogryposis. We report a novel dominant TPM2 mutation, K7del, in five unrelated families (de novo occurrence was likely in four families and could be confirmed in two). This mutation removes a highly conserved lysine residue within the N-terminal region that mediates βTm head-to-tail polymerisation. Patients had postnatal large joint contractures and little skeletal muscle weakness until adulthood. Muscle histology showed nemaline bodies and core-like areas, and there was marked fatty infiltration of lower limb muscles by mid-adulthood on muscle MRI. We performed a wide range of studies to investigate the causes of muscle dysfunction. 2D-gel electrophoresis on patient muscle and primary patient cultured myotubes suggested that mutant protein incorporates poorly into sarcomeres and most resides in nemaline rods. Recombinant K7del protein showed a reduced ability to polymerise into long filaments and reduced actin affinity but bound to thin filaments when mixed with wild-type β Tm and caused an in myofibrillar calcium sensitivity. Single dissected patient muscle fibres also showed increased calcium sensitivity of force generation. In summary, K7del is a recurrent TPM2 mutation that causes mild nemaline myopathy with core-like features and postnatal contractures. Mutant K7del protein incorporates poorly into sarcomeres likely due to abnormal head-to-tail polymerisation and reduced actin affinity. Joint contractures may arise from muscle hypercontraction due to increased calcium sensitivity while declining strength in adulthood likely arises from other mechanisms, such as myofibre decompensation and fatty infiltration. These results suggest that patients may benefit from therapies that reduce skeletal muscle calcium sensitivity and we highlight late muscle decompensation as an important cause of morbidity.

C.P.17 A repeating structural motif in tropomyosin that is responsible for multiple gain-of-function skeletal myopathy mutations

Abstract It was recently shown that the beta-tropomyosin ΔK7 mutation caused a gain of function, detected by increased Ca2+-sensitivity in single filament assays and increased Ca2+-sensitivity and maximium force in skinned fibres. Lysine 7 is adjacent to lysine 6 that has been shown to interact with actin Asp25 in the structure of actin-tropomyosin when tropomyosin is in the closed (inactive) state. We hypothesised that the mutation one amino acid from the amino acid that interacts with actin destabilises the closed state and therefore increase the probability of the muscle being in the open (active) state. Tropomyosin has a 7-fold repeated motif of about 40 amino acids that corresponds to the 7 actins that one tropomyosin molecule is in contact with. The contact with actin Asp 25 is repeated at K6, K48, R90, K128, R167, K205 and R244. We therefore asked whether further gain of function mutations might be associated with the other interaction sites. ΔK49 in beta-tropomyosin (second repeat) is reported to be a cap myopathy-causing mutation. We found that in vitro, this mutation increased myofilament Ca2+-sensitivity like ΔK7. R91G in beta-tropomyosin (third period) is a well document mutation associated with distal arthrogryposis that also increases Ca2+-sensitivity. K168E (fifth period) and R245G (sixth period) in gamma-tropomyosin have been reported associated with congenital fibre-type disproportion, but have not been studied in vitro. In summary, disease-related mutations resulting in the loss of positive charge in the amino acid immediately downstream of the basic amino acid that interacts with Asp25 have been noted in 5 of the 7 repeating motifs in tropomyosin. Three of these have been investigated and show an increased Ca2+-sensitivity and the remainder have not yet been tested. We propose that the repeating motif demonstrates one molecular mechanism for gain of function mutations.