Metabolic Myopathies II: Lipid-Related Disorders and Mitochondrial Myopathies

Skeletal muscles not only use glycogen but also lipid as a source of energy. The oxidation of lipids occurs in the mitochondria and involves a series of enzymatic reactions ( Fig. 18.1a, b ). Defects in these pathways and in the pathways of oxidative phosphorylation in the mitochondria lead to a heterogeneous group of muscle disorders.

Disorders of Muscle Lipid Metabolism

The first association of a myopathy with a disorder of lipid metabolism was the report by , who suggested the possibility in 18-year-old identical twin sisters, who from childhood had muscle cramps associated with myoglobinuria, at times occurring some hours after exercise. Carbohydrate metabolism was normal. Attacks could be provoked by prolonged fasting or by a high-fat, low-carbohydrate diet. Muscle biopsy was histologically normal but showed excess lipid droplets on oil red O staining. They postulated a defect in the utilization of long-chain fatty acids. In a prophetic annotation in the same journal, predicted a deficiency of either carnitine or carnitine palmitoyl transferase (CPT) to account for this lipid storage myopathy. were subsequently able to demonstrate carnitine deficiency in the muscle of a 24-year-old woman with weakness all her life and progression from the age of 19 years. Muscle biopsy showed a vacuolar-like myopathy filled with lipid droplets on histochemical staining. In the same year, reported a 29-year-old man with episodic cramps and myoglobinuria of 16 years’ duration but no muscle weakness. Muscle biopsy showed no excess of lipid but a deficiency of the enzyme CPT.

Morphological Aspects

The triglycerides are stored in muscle in the form of lipid droplets ( ). Under the light microscope they are readily revealed by stains for neutral fat such as oil red O or Sudan black. The droplets are more numerous in the type 1 fibres and tend to be concentrated more at the periphery of the fibre. The presence of excess lipid can give a very vacuolated-like appearance with routine histological stains such as haematoxylin and eosin (H&E) and may be more marked in type 1 fibres ( Figs. 18.2–18.4 ), but as lipid droplets are not membrane bound they are not vacuoles. Under the electron microscope, the lipid droplets appear as empty, rounded spaces of uniform size and do not have a limiting membrane. They are located between the myofibrils and under the sarcolemma and are often adjacent to mitochondria. An excess of lipid, as in carnitine deficiency, shows as a striking increase in the number and size of the droplets under light microscopy, as well as with electron microscopy, and may be associated with structural abnormalities in the mitochondria (see Fig. 18.4 ). In cases with CPT II deficiency, however, the amount of lipid may not appear particularly increased, and in other enzyme defects it varies with the metabolic state of the patient.

Fig. 18.2, Biopsy from the quadriceps muscle of a 43-year-old woman with deficiency of adipose triglyceride lipase (ATGL) due to mutations in PNPLA2 . She had muscle fatigue on exercise and slowly progressive distal muscle weakness from age 20. The biopsy demonstrates atrophy, fibrosis and vacuolar appearance of many muscle fibres (a, H&E) and type 1 fibre predominance (b, ATPase pH 4.3). The content of some of the vacuoles shows a granular appearance as in rimmed vacuoles (c, Gomori trichrome), and there is massive lipid storage which gives a vacuolar appearance (d, Sudan black).

Fig. 18.3, Postmortem biopsy of the psoas muscle in a 5-year-old boy with recurrent rhabdomyolysis. The patient died during an acute attack. He was found to be homozygous for a truncating mutation in LIPIN1 . The muscle demonstrated lipid accumulation as seen in transverse (a) and longitudinal (b) sections after staining with Sudan black.

Fig. 18.4, Biopsy from the quadriceps of a 4-year-old child (a, b) and a 21-month-old infant (c, d) with carnitine deficiency. (a) There is a microvacuolar appearance in some fibres caused by lipid accumulation (Gomori trichrome); (b) accumulation of lipid is seen in several fibres (Sudan black); (c) The accumulation of fat between myofibrils seen with the electron microscope; (d) ultrastructurally abnormal mitochondria.

The primary lipid metabolism disorders with most extensive lipid storage are neutral lipid storage disease, lipin-1 deficiency, systemic (primary) carnitine deficiency and multiple acyl-CoA dehydrogenase deficiency. However, mitochondrial diseases (see below) frequently show lipid storage secondary to defective oxidative phosphorylation. Other conditions that may also show lipid storage include inflammatory and drug-induced myopathies. Congenital myasthenic syndromes associated with DOK7 mutations were also reported to be associated with lipid accumulation in several cases ( ).

Clinical Syndromes Associated with Abnormalities in Fatty Acid Metabolism

The clinical features of lipid disorders of muscle fall into two broad groups: those in which muscle symptoms are the predominant abnormality and those in which muscle involvement is part of a more general systemic illness ( ). In patients in whom the muscle involvement is the major or only clinical feature, the presenting symptoms and signs may be proximal or diffuse muscle weakness, or muscle pain, particularly on prolonged exertion, which may be associated with muscle necrosis and myoglobinuria. The muscle symptoms seen in children in whom muscle involvement is part of a systemic illness are predominantly hypotonia and generalized muscle weakness. In some, the symptoms may resolve as the clinical condition improves; in others, the hypotonia and muscle weakness persist, and recovery may take several months.

Neutral Lipid Storage Disease

Neutral lipid storage disease with myopathy may present in childhood or adolescence as slowly progressive muscle weakness. The muscle weakness is usually proximal but may be predominantly distal. Predominantly affected muscles are found in the posterior compartments of the legs, in the shoulders and upper arms and in paraspinal muscles ( ). The disease is sometimes accompanied by cardiomyopathy ( ). Muscle biopsy shows massive lipid accumulation and rimmed vacuoles are frequently encountered (see Fig. 18.2 ). Lipid droplets are also present in leucocytes. Mutations are found in the PNPLA2 gene, which encodes adipose triglyceride lipase (ATGL), which catalyzes the first step in hydrolysis of triacylglycerol ( ) (see Fig. 18.1a ).

Neutral lipid storage disease with ichthyosis is a childhood-onset disease with congenital ichthyosiform erythroderma, mild myopathy and hepatomegaly. Several other manifestations may be present, including mental retardation, microcephaly, hearing loss and cataracts. There is lipid accumulation in various cell types, including muscle fibres and blood leucocytes. Mutations have been identified in ABHD5 encoding the protein CGI-58, which is an activator of ATGL ( ).

Lipin-1 Deficiency

Lipin-1, encoded by the LPIN1 gene, converts phosphatidic acid to diacylglycerol, which is an important step in triglyceride and phospholipid synthesis. Mutations in LPIN1 have been identified as a cause of severe and occasionally lethal recurrent rhabdomyolysis in children ( ). Muscle histology is either normal or shows moderate abnormalities, including lipid accumulation, predominance of type 1 muscle fibres, atrophy of type 2 fibres and, rarely, subsarcolemmal aggregates of mitochondria (see Fig. 18.3 ). If biopsy is performed shortly after an episode of rhabdomyolysis, necrotic fibres and regeneration may be apparent.

Carnitine Deficiency

Carnitine (β-hydroxy-α-trimethylaminobutyric acid) is the indispensable carrier of medium- and long-chain fatty acids across the inner mitochondrial membrane into the mitochondrion, where they undergo β-oxidation. There are two sources of carnitine – diet and synthesis. The synthesis of carnitine (which is dependent on two essential amino acids, lysine and methionine) takes place predominantly in the liver, and carnitine is then transported by the blood to other tissues. The highest concentration of free carnitine is in muscle, followed by liver, with about half the concentration, and the heart, which is lower still. Carnitine is transported across the cell membrane in many tissues by means of the high-affinity plasma membrane sodium-dependent carnitine transporter (OCTN2).

The selective excretion of acylcarnitine provides a route for removal of accumulating intermediates in various metabolic disorders. Excessive loss of these specific acylcarnitines in the urine results in loss of free carnitine and increases the probability of secondary carnitine deficiency. Such deficiency is seen in various disorders of β-oxidation and oxidative phosphorylation in the mitochondria and secondary to various drugs ( ).

Systemic Carnitine Deficiency

Systemic (primary) carnitine deficiency is caused by mutations in the carnitine transporter OCTN2 encoded by the SLC22A5 gene ( ). In addition to the lipid storage myopathy and cardiomyopathy that usually start in childhood, these patients may also have recurrent episodes of acute hepatic encephalopathy, with nausea, vomiting, confusion or coma (reminiscent of Reye syndrome) and, in some, associated hypoglycaemia, and a metabolic acidosis caused by increased levels of lactate and ketoacids.

Of the eight cases reviewed by , six had died from cardiorespiratory failure, five of them before 20 years of age. In two patients, the weakness worsened towards the end of pregnancy or after delivery. The serum carnitine concentration was markedly reduced in all patients tested. The serum creatine kinase (CK) was elevated in some but not in others and electromyography showed a myopathic pattern.

Replacement therapy with oral carnitine is feasible in systemic carnitine deficiency ( ) as well as in secondary carnitine deficiency ( ).

Muscle biopsy shows severe lipid storage and there may also be mitochondrial proliferation and mitochondrial ultrastructural abnormalities (see Fig. 18.4 ).

Carnitine Palmitoyl Transferase Deficiency

Carnitine palmitoyl transferase is an enzyme that catalyzes the reversible reaction of carnitine and long-chain fatty acyl groups. It exists in two forms, CPT I and CPT II. CPT I is located on the inner side of the outer mitochondrial membrane, whereas CPT II is on the inner side of the inner mitochondrial membrane. Deficiency of either CPT I or CPT II can present as attacks of hypoketotic hypoglycaemia, but the most common manifestation of CPT II deficiency is the benign myopathic form ( ).

Myopathy Associated with CPT II Deficiency

The adult myopathic form of CPT II deficiency is characterized by recurrent myoglobinuria, often precipitated by prolonged exercise, fasting or a combination of the two. reviewed 16 cases from the literature and added three of their own, plus two others in which they had studied the biopsies. Most patients recalled recurrent muscle pains since childhood but they did not seem to have cramps during exercise, which act as warning signals in patients with phosphorylase or phosphofructokinase (PFK) deficiency to stop using their muscles. This may explain the earlier onset as well as the greater frequency of myoglobinuria in CPT deficiency than in phosphorylase or PFK deficiency. By the time patients with CPT deficiency experienced muscle stiffness or pain, it was usually too late to avoid myoglobinuria. The myoglobinuria usually followed vigorous exercise of a few hours’ duration, such as a long hike or a football game, and fasting before exercise was recognized by most patients as a precipitating factor. Five of the 21 had renal failure. In about one-third of cases, there was no apparent precipitating cause for at least some of the episodes of myoglobinuria.

During attacks, affected muscles became swollen, tender and weak. Respiratory muscles were often severely involved, and three cases needed assisted ventilation. Between attacks, patients were usually normal and did not show residual weakness. In 19 of the 21 cases the diagnosis was established between 15 and 30 years of age.

Serum CK was normal between attacks, and blood lactate rose normally after ischaemic exercise. Plasma lipids were abnormal in four cases, with increased triglycerides and cholesterol in three and slightly increased triglycerides in one.

Muscle biopsies taken during the quiescent phase between attacks were normal in about two-thirds of the reported cases. When lipid storage was present, this was usually less marked than in carnitine deficiency, and in two reports was only noted in one out of two biopsies on the patients. Areas of necrosis noted in the limb muscle of one patient and the intercostal muscle of another were probably related to a recent episode of myoglobinuria. Liver biopsy in one case showed no lipid storage but some abnormality of mitochondria, and the morphology of leucocytes was normal in two cases. In general, muscle biopsies rarely show significant pathological changes in CPT deficiencies and levels of lipid within fibres often appear normal. If a sample is taken soon after an episode of myoglobinuria, however, necrosis and fibre regeneration will be apparent.

The diagnosis of CPT II deficiency should be considered in any patient with recurrent myoglobinuria, particularly if precipitated by prolonged exercise or fasting. Two clinical criteria are helpful in distinguishing it from phosphorylase or PFK deficiency: (1) there is no intolerance to vigorous exercise of short duration and no ‘second-wind’ phenomenon, and (2) cramps are unusual and contracture is not induced by ischaemic exercise. Long-chain acylcarnitines may be increased in blood (C ₁₆ , C _18:1 , C ₁₈ ), and the CPT II activity can be measured in fibroblasts, muscle tissue or leucocytes. Mutations are found in the CPT2 gene ( ).

Acyl-CoA Dehydrogenase Deficiency

Before the β-oxidation of fatty acids can occur, they have to be converted first to their coenzyme A (CoA) thioesters, which is catalyzed by the acyl-CoA synthetases in cytosol. The fatty acyl-CoA is then reacted with carnitine by CPT I and transported into the mitochondria by carnitine-acylcarnitine translocase. Carnitine is removed by CPT II and fatty acyl-CoA then enters the β-oxidation pathway, where the initial step is oxidation by acyl-CoA dehydrogenases. There are several types of acyl-CoA dehydrogenases (see Fig. 18.1b ). Defects of the acyl-CoA dehydrogenases are the most frequently identified abnormalities of fatty acid oxidation.

Short-Chain Acyl-CoA Dehydrogenase (SCAD) Deficiency

This deficiency has been described in different clinical situations ( ). There is a myopathic form in which the defect is limited to muscle and presents with a slowly progressive muscle weakness and exercise-induced pain ( ) and a systemic form with hepatomegaly and microcephaly ( ). A 16-year-old girl documented by had recurrent myoglobinuria, hypoketotic hypoglycaemia, encephalopathy and an associated cardiomyopathy. Muscle histology may in some cases show lipid storage and also multicore changes with staining for oxidative enzymes ( ). The presence of increased ethylmalonic acid in urine is a major biochemical marker. SCAD deficiency is associated with recessive mutations in the ACADS gene ( ).

Medium-Chain Acyl-CoA Dehydrogenase (MCAD) Deficiency

This may be one of the most commonly inherited metabolic disorders, with an incidence of 1 in 5000 to 10,000 live births. It usually presents in infancy with an episodic illness in which muscle symptoms and signs are not prominent ( ). The clinical presentations include sudden infant death, Reye syndrome and hypoglycaemic episodes. Some cases, however, present in later life, and exercise-induced muscle pain may be a feature; yet others are asymptomatic and are detected only when the disorder is diagnosed in another family member ( ). MCAD deficiency is inherited in an autosomal recessive pattern and is associated with mutations in the ACDM gene ( ). Increased amounts of medium-chain acylcarnitines are present in blood and urine.

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