Cardiac-specific VLCAD deficiency induces dilated cardiomyopathy and cold intolerance.

The very long-chain acyl-CoA dehydrogenase (VLCAD) enzyme catalyzes the first step of mitochondrial ß-oxidation. Patients with VLCAD deficiency present with hypoketotic hypoglycemia and cardiomyopathy, which can be exacerbated by fasting and/or cold stress. Global VLCAD knockout mice recapitulate these phenotypes: mice develop cardiomyopathy, and cold exposure leads to rapid hypothermia and death. However, the contribution of different tissues to development of these phenotypes has not been studied. We generated cardiac-specific VLCAD-deficient (cVLCAD(-/-)) mice by Cre-mediated ablation of the VLCAD in cardiomyocytes. By 6 mo of age, cVLCAD(-/-) mice demonstrated increased end-diastolic and end-systolic left ventricular dimensions and decreased fractional shortening. Surprisingly, selective VLCAD gene ablation in cardiomyocytes was sufficient to evoke severe cold intolerance in mice who rapidly developed severe hypothermia, bradycardia, and markedly depressed cardiac function in response to fasting and cold exposure (+5°C). We conclude that cardiac-specific VLCAD deficiency is sufficient to induce cold intolerance and cardiomyopathy and is associated with reduced ATP production. These results provide strong evidence that fatty acid oxidation in myocardium is essential for maintaining normal cardiac function under these stress conditions.

Cardiac and skeletal muscle defects in a mouse model of human Barth syndrome.

Barth syndrome is an X-linked genetic disorder caused by mutations in the tafazzin (taz) gene and characterized by dilated cardiomyopathy, exercise intolerance, chronic fatigue, delayed growth, and neutropenia. Tafazzin is a mitochondrial transacylase required for cardiolipin remodeling. Although tafazzin function has been studied in non-mammalian model organisms, mammalian genetic loss of function approaches have not been used. We examined the consequences of tafazzin knockdown on sarcomeric mitochondria and cardiac function in mice. Tafazzin knockdown resulted in a dramatic decrease of tetralinoleoyl cardiolipin in cardiac and skeletal muscles and accumulation of monolysocardiolipins and cardiolipin molecular species with aberrant acyl groups. Electron microscopy revealed pathological changes in mitochondria, myofibrils, and mitochondrion-associated membranes in skeletal and cardiac muscles. Echocardiography and magnetic resonance imaging revealed severe cardiac abnormalities, including left ventricular dilation, left ventricular mass reduction, and depression of fractional shortening and ejection fraction in tafazzin-deficient mice. Tafazzin knockdown mice provide the first mammalian model system for Barth syndrome in which the pathophysiological relationships between altered content of mitochondrial phospholipids, ultrastructural abnormalities, myocardial and mitochondrial dysfunction, and clinical outcome can be completely investigated.

Reye-like syndrome resulting from novel missense mutations in mitochondrial medium- and short-chain l-3-hydroxy-acyl-CoA dehydrogenase.

Medium- and short-chain l-3-hydroxy-acyl-CoA dehydrogenase (M/SCHAD) deficiency is a recessively inherited disorder of fatty acid oxidation. Currently, only four patients from three families have been reported in the literature. All these patients presented with hypoglycemia associated with hyperinsulinism (HI). This association suggests that there is a role for M/SCHAD in regulating the pancreatic secretion of insulin. We present a fifth patient whose presentation was similar to Reye syndrome, a feature in common with most of the previously recognized disorders of fatty acid oxidation but with no clinical evidence of HI. Sequencing of the HAD1 gene on chromosome 4 revealed compound heterozygosity for two novel missense mutations, 170A>G, resulting in D45G, and 676T>C, resulting in Y214H. The mutant enzymes were expressed and subjected to kinetic analysis. Y214H has no detectable activity, whilst D45G, which resides in the cofactor-binding pocket, has an altered K(m) for NADH (96 microM versus 24 microM for the wild-type). This represents the first kinetic M/SCHAD mutant, which explains the high residual activity in skin fibroblasts. The lack of obvious HI in this patient may be related to the high residual activity and indicates that HI associated with M/SCHAD deficiency may only be present with complete deficiency. The spectrum of M/SCHAD phenotype should be broadened to include acute liver disease.

Tissue carnitine homeostasis in very-long-chain acyl-CoA dehydrogenase-deficient mice.

Deficiency of very-long-chain acyl-CoA dehydrogenase (VLCAD) is the most common long-chain fatty acid oxidation defect and presents with heterogeneous clinical manifestations. Accumulation of long-chain acylcarnitines and deficiency of free carnitine have often been proposed to play an important role in disease pathogenesis. The VLCAD-deficient mouse exhibits similar clinical and biochemical phenotypes to those observed in humans and, therefore, represents an excellent model to study VLCAD deficiency. We measured carnitine and acylcarnitine profiles in liver, skeletal muscle (SkM), bile, and blood from VLCAD knock-out mice and controls under nonstressed and various stress conditions. Carnitine and acylcarnitines were extracted from body fluids with methanol and from tissues with acetonitrile, respectively, and were analyzed as their butyl esters using electrospray ionization tandem mass spectrometry. Fasting combined with a cold challenge for 8 h significantly induced liver long-chain acylcarnitine and free carnitine production. Acylcarnitines in SkM predominantly accumulated during exercise with a concomitant decrease of free carnitine. Changes in blood free carnitine did not correlate with carnitine homeostasis in liver and SkM. Our results demonstrate different tissue-specific long-chain acylcarnitine profiles in response to various stressors, which may be of importance with respect to the heterogeneous clinical manifestations of VLCAD deficiency in humans. Furthermore, we conclude that carnitine biosynthesis in the liver seems sufficiently active to maintain liver carnitine levels during increased demand. Our data suggest that carnitine supplementation in long-chain beta-oxidation defects may not be required, and blood carnitine concentrations do not reflect tissue carnitine homeostasis.