Abstract
Mitochondrial trifunctional protein deficiency, due to mutations in hydratase subunit A (HADHA), results in sudden infant death syndrome with no cure. To reveal the disease etiology, we generated stem cell-derived cardiomyocytes from HADHA-deficient hiPSCs and accelerated their maturation via an engineered microRNA maturation cocktail that upregulated the epigenetic regulator, HOPX. Here we report, matured HADHA mutant cardiomyocytes treated with an endogenous mixture of fatty acids manifest the disease phenotype: defective calcium dynamics and repolarization kinetics which results in a pro-arrhythmic state. Single cell RNA-seq reveals a cardiomyocyte developmental intermediate, based on metabolic gene expression. This intermediate gives rise to mature-like cardiomyocytes in control cells but, mutant cells transition to a pathological state with reduced fatty acid beta-oxidation, reduced mitochondrial proton gradient, disrupted cristae structure and defective cardiolipin remodeling. This study reveals that HADHA (tri-functional protein alpha), a monolysocardiolipin acyltransferase-like enzyme, is required for fatty acid beta-oxidation and cardiolipin remodeling, essential for functional mitochondria in human cardiomyocytes.
Highlights
In this study, we analyze mitochondrial trifunctional protein deficiency by generating stem cell derived cardiomyocytes from HADHA-deficient human induced pluripotent stem cells and accelerate their maturation by our engineered microRNA maturation cocktail
We utilized the human induced pluripotent stem cells (hiPSCs) line HEL87.1, which was derived from a patient carrying the founder point mutation most common in mitochondrial trifunctional protein disorder, HADHA c.1528G>C (Supplemental Fig. 1C, D)[25]
CL is important for mitochondrial architecture, it has been shown to function in organizing the cristae and electron transport chain (ETC) higher order structure, important for ETC activity, and acts as a proton trap on the outer leaflet of the inner mitochondrial membrane[84]
Summary
We analyze mitochondrial trifunctional protein deficiency by generating stem cell derived cardiomyocytes from HADHA-deficient human induced pluripotent stem cells (hiPSCs) and accelerate their maturation by our engineered microRNA maturation cocktail. The mutations found in the HADHAMut line resulted in a predicted early stop codon on both alleles (Supplemental Fig. 1H). The expressed HADHAMut protein skips the expression of exons 1–3, 60 amino acids, generating a truncated ClpP/crotonase domain, which likely compromises the mitochondrial localization and protein folding of the enzyme pocket resulting in the inability to stabilize enolate anion intermediates during FAO (Supplemental Fig. 1J). Using a monolayer directed differentiation protocols[26,27] we generated human induced pluripotent stem cell derived cardiomyocytes (hiPSC-CMs) from the WT and hiPSC lines with HADHA mutations. Analyzing each miR’s predicted targets affecting glucose and/or fatty acid metabolism, cell growth and hypertrophy and cell cycle, six miRs were chosen to assess for their CM maturation potency: three upregulated miRs (miR-452, −208b33 and −378e34) and three downregulated miRs (miR-122, −200a, and −205) (Fig. 2a)
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