Abstract 801 Genetic Basis of Disease I Platform, Monday, 5/3

Objective: We sought to determine the pathophysiology and molecular basis of stress-induced death in VLCAD deficiency.

Background: Very-long-chain acyl-CoA dehydrogenase deficiency causes lethal cardiomyopathy in children. Patients with this defect are often asymptomatic unless stressed. Environmental stresses, fasting, exercise, and viral illnesses trigger crises in VLCAD deficient humans. The most common phenotypes are cardiomyopathy, metabolic crisis, Reye-like syndrome and sudden death. The pathophysiologic and molecular elements underlying stress-induced illness in this disease are generally unclear or unknown. For this reason a mouse model of VLCAD deficiency that could be systematically studied was generated.

Method: A portion of the mouse VLCAD gene between a BamHI and a XhoI sites was targeted and replaced by the neomycin resistance gene. The targeted linearized vector was electroporated into 129/SV embryonic stem cells. ES cells positive for homologous recombination were injected into C57BL/6 blastocysts to generate chymeric mice. F1 animals were bred with the expected 25% homozygous, 25 % wild type and 50 % heterozygous mice. Two months old homozygous-deleted and heterozygous-deficient mice for VLCAD were monitored in the cold at 4-degree Celsius. The mice were caged individually and fasted with free access to water. Rectal temperature and blood glucose were recorded at specific time intervals after cold exposure, until one of the following occurred: temperature decrease to 25 degree Celsius, blood glucose decrease to 25 mg/dl, sudden death, or a time limit period of 12 hours. Survival was compared among the three genotypes. Tissues from heart, skeletal muscle and brown adipose were collected simultaneously for histologic assessment, protein and RNA analysis.

Results: The VLCAD deficient mouse is asymptomatic unless stressed. The two months old homozygous and heterzygous-deficient mice die in the cold room with an LD/50 of 4 hours and 7 hours respectively. Their comparative survival was 0 % versus 100 % survival of the wild type mice when fasted at 4 degree Celsius. Hypoglycemia, hypothermia, and fatty infiltration of the heart and the liver accompany the cold-induced death. The most severe phenotype is observed in the homozygous-deleted mice, with the heterozygous-deficient animals showing an intermediate response. Levels of uncoupling protein-1 (UCP-1) mRNA in the brown adipose tissue are not affected in the dying mice. However UCP-1 mRNA expression is aberrant in the cardiomyocyte and the skeletal muscle of the homozygous and heterozygous deleted mice.

Conclusion: We generated a mouse model of VLCAD deficiency in which the phenotypes are modulated by physiologic stresses. Cold-induced death is associated with aberrant UCP-1 mRNA expression in the heart and the skeletal muscle. VLCAD function and the integrity of fatty acid β-oxidation in the heart and skeletal muscle are crucial for survival in the cold.