Hacd2 deficiency in mice leads to an early and lethal mitochondrial disease

Abstract

Objective: Mitochondria fuel most animal cells with ATP, ensuring proper energetic metabolism of organs. Early and extensive mitochondrial dysfunction often leads to severe disorders through multiorgan failure. Hacd2 gene encodes an enzyme involved in very long chain fatty acid (C ≥ 18) synthesis, yet its roles in vivo remain poorly understood. Since mitochondria function relies on specific properties of their membranes conferred by a particular phospholipid composition, we investigated if Hacd2 gene participates to mitochondrial integrity.

Methods: We generated two mouse models, the first one leading to a partial knockdown of Hacd2 expression and the second one, to a complete knockout of Hacd2 expression. We performed an in-depth analysis of the associated phenotypes, from whole organism to molecular scale.

Results: Thanks to these models, we show that Hacd2 displays an early and broad expression, and that its deficiency in mice is lethal. Specifically, partial knockdown of Hacd2 expression leads to death within one to four weeks after birth, from a sudden growth arrest followed by cachexia and lethargy. The total knockout of Hacd2 is even more severe, characterized by embryonic lethality around E9.5 following developmental arrest and pronounced cardiovascular malformations. In-depth mechanistic analysis revealed that Hacd2 deficiency causes altered mitochondrial efficiency and ultrastructure, as well as accumulation of oxidized cardiolipin.

Conclusions: Altogether, these data indicate that the Hacd2 gene is essential for energetic metabolism during embryonic and postnatal development, acting through the control of proper mitochondrial organization and function.

Keywords: ELOVL; Fatty acid; Heart development; OXPHOS coupling; Phospholipid; VLCFA.

Figure 1

Hacd2 expression during embryonic development and adulthood. (A–E) Expression pattern of LacZ-expressing cells on whole-mount (A–C) and transverse sections (D,E) of E9.5 Hacd2^KO/+ embryos. (A) At this stage, embryos have 20–22 pairs of somites, they have turned, and two pharyngeal bars and the forelimb are visible. After X-Gal staining, the embryo is uniformly blue, reflecting ubiquitous expression of Hacd2. (B) Focus on the region outlined in (A), showing Hacd2 expression in the embryonic heart and branchial arches. (C) Dorsal view of the embryo showing Hacd2 expression in the neural tube and somites. (D) All cell layers of the embryo express Hacd2, here on a transverse section at the level of the heart. (E) In the heart, staining is visible in the pericardium, myocardium and endocardium (arrowhead). (F) RT-qPCR quantification of Hacd2 mRNA in adult WT mice, normalized to Actin mRNA. (G) RT-qPCR quantification of Hacd2 mRNA in WT pups at postnatal day nine (P9), normalized to Rpl32 mRNA. In (A–E), as is aortic sac; ba1, first branchial arch; ba2, second branchial arch; bc, bulbus cordis; bw, body wall, consisting in this region of pericardium and surface ectoderm; cj, cardiac jelly; cvch, common ventricular chamber of the heart; d, diencephalon; da, dorsal aorta; en, endocardium; fb, forelimb bud; h, heart; m, mesencephalon; my, myocardium; nt, neural tube; p, pericardium; so, somite and t, telencephalon. Scale bars are 500 μm in A and 100 μm in B-E. Individual data are plotted, along with the mean and standard error of the mean.

Figure 2

Hacd2 knockdown leads to hyperlactatemia and lethality. (A) RT-qPCR quantification of Hacd2 mRNA in tissues from WT and Hacd2-KD pups at P14, normalized to Rpl32 mRNA. (B) Body mass of WT and Hacd2-KD pups at P5. (C) Representative picture of WT (left) and Hacd2^flox/flox(Hacd2-KD) littermates at postnatal day 22 (P22). (D) Percent survival of Hacd2-KD pups compared to WT. (E–F) Glycemia (E) and lactatemia (F) in Hacd2-KD growing pups and control littermates at P7. Individual data are plotted, along with the mean and standard error of the mean. Individual data are plotted, along with the mean and standard error of the mean. ∗, P < 0.05; ∗∗, P < 0.01.

Figure 3

Impaired mitochondrial function in kidney and liver of Hacd2-KD mice. (A–C) Seahorse oxygraphy analysis on kidney homogenates. (A) Phosphorylating oxidation rate in the presence of pyruvate, malate, glutamate and succinate (Complex I and II). (B) Non-phosphorylating respiration after the addition of oligomycin. (C) Mitochondrial coupling (Respiratory Control Ratio, i.e. ratio of phosphorylating to non-phosphorylating oxidation rates from A and B). (D) COX activity staining and quantification in kidney cortex. (E–G) Seahorse oxygraphy analysis on liver homogenates. (E) Phosphorylating oxidation rate in the presence of pyruvate, malate, glutamate and succinate (Complex I and II). (F) Non-phosphorylating respiration after the addition of oligomycin. (G) Mitochondrial coupling (Respiratory Control Ratio, i.e. ratio of phosphorylating to non-phosphorylating oxidation rates from E and F). Data correspond to Hacd2-KD pups aged P9 to P14. Individual data are plotted, along with the mean and standard error of the mean. ∗, P < 0.05.

Figure 4

Anomalies of cardiovascular development in E9.5 Hacd2-KO embryos. (A) Representative control and knockout (Hacd2-KO) embryos from E9.5 to E11.5. Note the reduced size of Hacd2-KO embryos, some of them exhibit a poorly defined tail, rudimentary head parts with anterior and posterior neuropores still open. Most Hacd2-KO embryos display a pericardial edema (delimited by white arrows). Erythrocytes are visible in dorsal aorta, but they are seldom seen in head vessels and very often found sedimenting in the pericardial cavity (asterisk). In the most advanced embryos, the beating heart is not compartmentalized, in a U-shaped tubular form that contains no erythrocytes. (B) Representative pictures of the yolk sac surrounding the embryo. At this stage, yolk vascularization in the control is composed of vessels filled with red blood cells. These vessels form a tree-like network (part of which has been outlined by dotted lines) distributed over the entire surface of the sphere. In Hacd2-KO embryos, the early stage of a capillary plexus, formed of unconnected cell islands, is identifiable and no red blood cells are visible. (C) Z-stack in confocal microscopy of blood vessels revealed by labelling of endothelial cells with an anti-CD31 antibody. Vascular projections between somites are poorly visible in Hacd2-KO embryo, whereas they are regular and interconnected in the control. In (A), a is amnion; an, anterior neuropore; ba, branchial arch; da, dorsal aorta; fb, forelimb bud; fv, forebrain vesicle; h, heart; hb, hindlimb bud; ht, heart tube; htr, heart tube remnant; lp, lens placode; lv, lens vesicle; nf, neural folds; ov, otic vesicle; pc, pericardial cavity; so, somites and vv, vitelline vein. In (B), em is embryo; vp, vascular plexus and ysv, yolk sac vessel. In (C), isv is intersomitic vessel; pcv, posterior cardinal vein; so, somite. Scale bars are 500 μm in A and B and 100 μm in C.

Figure 5

Impaired mitochondrial function in Hacd2-KO embryos. (A–C) Seahorse oxygraphy analysis of whole embryos homogenates at E9.5. (A) Phosphorylating oxidation rate in the presence of pyruvate, malate, glutamate and succinate (Complex I and II). (B) Non-phosphorylating respiration after the addition of oligomycin. (C) Mitochondrial coupling (Respiratory Control Ratio, i.e. ratio of phosphorylating to non-phosphorylating oxidation rates from A and B). (D–E) Transverse sections from E9.5 wild-type (WT) and Hacd2-KO embryos were examined by transmission electron microscopy. Images are representative of cells from the myocardium. (D) Ultrastructural organization of WT cells reveals an active rough endoplasmic reticulum and fields of mitochondria with evidence of tubular-shaped invaginations (cristae) of their internal membrane, unambiguously identified on the same plane than the outer membrane. Myocardial cells are identified by the presence of developing sarcomeres, organized between z-lines, adjacent to numerous glycogen rosettes. (E) Myocardial cells from age matched Hacd2-KO embryos display a rather normal global organization, with similar cytoplasm, nucleus and rough endoplasmic reticulum membrane systems. However, many mitochondria appear dilated and are abnormally compartmentalized (arrow heads). g, glycogen rosettes; m, mitochondrion; mf, myofibrils; n, nucleus; ne, nuclear envelope; rer, rough endoplasmic reticulum and z, z-line. Scale bars are 250 nm. (F) Repartition of cardiolipin species in WT and Hacd2-KO embryos according to their proportion in WT. Oxidized cardiolipin species are depicted on a grey background. (G) Relative percentage of oxidized cardiolipins in WT and Hacd2-KO embryos. (H) Heatmap of cardiolipin species in WT and Hacd2-KO embryos according to their repartition in WT. Oxidized cardiolipin species are highlighted in pink. Individual data are plotted, along with the mean and standard error of the mean. ∗, P < 0.05; ∗∗∗, P < 0.001.