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Advances in understanding cardiolipin synthesis and pathological effects of dysregulation in cells and animals

Background: Cardiolipin must be remodeled to contain a fatty acyl profile that is appropriate for cell-and tissue-specific requirements. In addition to the four known enzymes that participate in this reaction, wehave identified a fifth,HRASLS1, and found that the partially-purified enzymecan directly esterify monolysocardiolipinto form cardiolipin, while overexpression of the enzyme in cells indirectlypromotedcellular cardiolipin synthesis. Since HRASLS1 is also known to synthesize endocannabinoids, thisprompted us to investigate a potential beneficial role for lipid signalingin cultured lymphoblasts from donors with Barth Syndrome, and to study Taz-KO mice as a model for testing novel therapeutics.
Methods: Hemagluttinin-tagged HRASLS1 was affinitypurifiedfrom HEK293cells for enzyme activity analysis, and cells overexpressing Hrasls1were studied for cardiolipin content and biosynthesis. Oleoylethanolamide(OEA)and linoleoylethanolamide(LEA)were studied for effects on cell growth, mitochondrial morphology, and mitochondrial dynamics regulation inlymphoblast lines from 5 healthy donors and 5 donors with Barth Syndrome.Male Taz-KO mice and wildtype littermates were studied at ages 3, 6 and 12 months for organ and tissue size, exercise tolerance, and metabolic measures to establish differences across the lifespan.

Results: Vmax and Km valueswere calculatedfor monolysocardiolipin esterificationby HRASLS1 using dioleoyl-phosphatidylcholine and distearoyl-phosphatidylcholine. HRASLS1wasnot active with 1-palmitoyl-2-linoleoyl-phosphatidylcholine. Overexpression of Hrasls1 in HEK293 cells increased total cardiolipin levels and the expression of genes involved in cardiolipin synthesis. Together, these data suggested that HRASLS1 may produce a signal that indirectly promotes cardiolipin synthesis, in addition to directly catalyzing the re-esterification of monolysocardiolipin.Testing of lymphoblasts deficient in Taz demonstrated that OEA, but not LEA, could restore a growth deficitin these lines. This was associated with a reduction in the number of large mitochondriaand a reduction to control levels of the mitochondrial fusion activatorOPA1, but surprisingly was not associated with changes in cardiolipin levels or content. Characterization of Taz-KO mice at 3, 6, and 12 months of age demonstratedthat while progressive age-related changes in physiological measures often occur similarly to wildtype mice, measures tended to differ between groups at any given age.

Conclusions: Our results show that HRASLS1 promotes cardiolipin synthesis directly and indirectly. OEA may be one signalingmolecule involved,and testing in Taz-deficient lymphoblasts indicated a beneficial effect, although this effect was independent of cardiolipin content or composition.The Taz-KO mouse model shows promise for usein testing novel therapeuticsacross the lifespan.

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