ACAD9 treatment with bezafibrate and nicotinamide riboside temporarily stabilizes cardiomyopathy and lactic acidosis

Abstract

Pathogenic ACAD9 variants cause complex I deficiency. Patients presenting in infancy unresponsive to riboflavin have high mortality. A six-month-old infant presented with riboflavin unresponsive lactic acidosis and life-threatening cardiomyopathy. Treatment with high dose bezafibrate and nicotinamide riboside resulted in marked clinical improvement including reduced lactate and NT-pro-brain type natriuretic peptide levels, with stabilized echocardiographic measures. After a long stable period, the child succumbed from cardiac failure with infection at 10.5 months. Therapy was well tolerated. Peak bezafibrate levels exceeded its EC₅₀. The clinical improvement with this treatment illustrates its potential, but weak PPAR agonist activity of bezafibrate limited its efficacy.

Keywords: ACAD9 disorder; Bezafibrate; Cardiomyopathy; Mitochondrial disease; Nicotinamide riboside; Treatment; complex I deficiency.

Figure 1:. Lactate and NT-pro-basic natriuretic peptide levels in a patient with ACAD9 disorder

A. Lactate levels in mmol/L in a patient with ACAD9 disorder. The onset of treatment is shown and the time of an adenoviral gastroenteritis and of a parainfluenza respiratory infection shown. B. Levels of NT-pro-basic natriuretic peptide in pg/mL in this patient with ACAD9 disorder. The bar indicates the first hospital admission with the area in red prior to treatment and in green on treatment with bezafibrate and nicotinamide riboside.

Figure 2:. Basic parameters of the cardiac function on serial echocardiogram evaluations

The ejection fraction (A) and the fractional shortening (B) are shown. The ventricular mass in gram/body surface areâ^2.7 is shown (C). The bar indicates the first hospital admission with the area in red prior to treatment and in green on treatment with bezafibrate and nicotinamide riboside.

Figure 3:. Analysis of mitochondrial function in fibroblasts

(A) The activity and assembly of the respiratory chain enzyme complexes (I, II, IV, and V) were analyzed by blue-native PAGE followed by in-gel activity staining. The activities of all enzyme complexes including complex I were normal with normal assembly of complex V noted. (B) The assembly of complex I is followed on non-denaturing gel after western blotting and identification with an antibody against NDUFS2. Normal fibroblasts show a large amount of fully assembled complex I at 1000 KDa with a small band at 230 kDa. The cells of the patient with ACAD9 show reduced amount of the holocomplex, and an added band of incompletely assembled complex I at 460 kDa. Huh-7cells treated with chloramphenicol are shown as a positive control. (C) Western blot analysis of the ACAD9 protein using SDS-PAGE was performed in fibroblast homogenates followed by detection using enhanced chemiluminescence (ECL). The amount of patient ACAD9 protein was 52% of the average control amount (n=3). Citrate synthase (Abcam: ab129095) was used as the loading control. D. Proteomics analysis after correction of mitochondrial abundance shows a large significant decrease in ACAD9 abundance. The subunits of complex I (red) are decreased. E. Quantification of the abundance of the mitochondrial complexes shows a decrease in complex I, but not the other complexes. The p-value and the relative % of the complex in the patient compared to the controls are provided in sequential rows above the figure. * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001, ns = non-significant. CI = complex I, CII = complex II, CIII = complex III, CIV = complex IV, CV = complex V, mtLSU = large subunit of the mitoribosome, mtSSU = small subunit of the mitoribosome.

Figure 4:. Proteomics analyses of peripheral blood mononuclear cells during treatment with bezafibrate and nicotinamide riboside.

(A) Volcano plot from quantitative proteomics data depicting reduced levels of Complex I subunits (blue) in mitochondrial proteins (gray). (B) Relative abundance of mitochondrial Complex I proteins shows decreased levels on Day 0 compared to controls. Following treatment, the abundance of Complex I increases over time compared to Day 0, except on Day 62. (C) Relative abundance of mitochondrial proteins shows increased level in the overall level of mitochondrial proteins up to Day 62 when compared to Day 0.