Cardiomyopathy in a c.1528G>C Hadha mouse is associated with cardiac tissue lipotoxicity and altered cardiolipin species

Fig. 1. FAO enzyme expression, and function in LCHADD hearts. A: mRNA levels of Hadha, Hadhb, Acadvl (N = 4 per group) and (B) protein levels of TFPα, TFPβ and VLCAD (pooled samples, N = 3 per group) are comparable in the cardiac tissue of LCHADD and WT mice. C: LCHADD mice have reduced long-chain fatty acid (LCFA) oxidation in the heart (WT male N = 7, WT female N = 8, LCHADD male N = 5, LCHADD Female N = 11). Data presented as mean ± SD with individual data points. Statistics: (A) 2-way ANOVA (genotype & sex) or (C) t test. ns (not significant), ∗∗∗∗ (P < 0.0001).

Fig. 2. Cardiac cardiolipin profiles in 12-month-old LCHADD mice differ from WT. A, B: Representative mass spectra of cardiolipin species in WT (blue trace) and LCHADD (orange trace) in male (A) and female (B) cardiac tissue illustrating alterations in cardiolipin species due to the LCHADD genotype. C: Relative abundance of individual cardiolipin (CL) species in the heart identified by their ratio of carbons to double bonds. Species with side chains predominant in oleate (OA), linoleate (LA), arachidonate (AA), or docosahexaenoate (DHA) are indicated. Species that exhibit significant differences of P < 0.005 between WT and LCHADD mice are collectively marked with #. D–F: Sex differences can be seen in total cardiolipin (sum of 19 species) (D), total monolysocardiolipin (MLCL) (sum of 9 species) (E), and MLCL/CL ratio (F). G: Oxidized CL showing species 72:7 and 72:8 (tetralinoleoyl), which have 3- and 4-linoleoyl side chains, respectively. For (C–G), N for each genotype: females N = 6, male N = 6, combined N = 12. Bar graphs represent mean ± SD with individual data points. Statistics: individual cardiolipin species (C) were analyzed by multiple t test with a two-stage false discovery…

Fig. 3. Differences in cardiolipin synthesis and remodeling pathway in LCHADD mice. A: Diagram of CL synthesis and remodeling pathways highlighting key enzymes, including MLCL-AT. MLCL-AT is encoded by Hadha and may be affected by the c.1528G>C mutation in LCHADD mice. B: mRNA levels of Crls1 show increased expression in 12-month-old mice of both sexes in LCHADD mice compared to WT (N = 4 per group). C: Western blot of CS and TFPα (MLCL-AT) in individual 11-month-old male and female mice of both genotypes (N = 3 per group). D, E: Densitometric analysis, quantifying CS (D) and TFPα (MLCL-AT) (E) levels relative to GAPDH. Data are presented as mean ± SD with individual data points. Statistics: 2-way ANOVA (genotype & sex) followed by post-hoc Sidak test. ns (not significant), ∗∗ (P < 0.01).

Fig. 4. Concentrations of lipids in serum and cardiac tissue. A: Heart triglyceride levels were higher in LCHADD compared to WT mice in both males and females (male WT N = 4, all other groups N = 5). B: Representative images of Oil Red O staining of WT and LCHADD heart tissue demonstrate greater lipid droplet accumulation (stained in red) in LCHADD, in both sexes. All images are at equal magnification, yellow bar = 50 μm. C: Serum concentrations of oleic (C18:1), linoleic (C18:2), arachidonic (C20:4), and docosahexaenoic (C22:6) acids were not different between WT and LCHADD mice (N = 8 per group). D: In contrast, cardiac tissue oleic acid and linoleic acid concentration was higher in LCHADD compared to WT mice (N = 8 per group). Data presented as mean ± SD with individual data points. Statistics: 2-way ANOVA with genotype and sex or species as the two factors. Post-hoc Sidak test identified differences between WT and LCHADD are denoted by asterisks: ∗(P < 0.05), ∗∗(P < 0.01).

Fig. 5. Mitochondrial copy number and dynamics in LCHADD and WT mice. A: Relative mitochondrial DNA (mtDNA) copy number in the hearts of 12-month-old LCHADD and WT mice were not different (N = 5 per group). B: Representative Western blots of indicated proteins in 12- to 15-month-old male and female LCHADD and WT mice hearts (N = 3 per group) illustrating alterations in S-OPA1/L-OPA1 ratio. C–F: Densitometric analysis of Western blot showing mitofusin-1 (MFN1), dynamin-related protein-1 (DRP1) and the ratio of phosphorylated (pDRP1) to total DRP1, and optic atrophy-1 (OPA1) as the ratio of short to long isoforms (S-OPA1 and L-OPA1). Relative levels to GAPDH. Data are presented as mean ± SD. Statistical significance indicated by asterisks: ∗ (P < 0.05), ∗∗ (P < 0.01).

Fig. 6. TEM of LCHADD and WT cells show various states of LCHADD cardiac mitochondrial health. A, B: LCHADD and WT show healthy mitochondria. C–E: However, LCHADD mitochondria also show mitochondria with disorganized cristae (C), swollen (D), and swollen with cristolysis (E). Swollen mitochondria are marked with arrows. F: Cristolysis is only seen in LCHADD mitochondria.