Abstract Body (Do not enter title and authors here): Background: In healthy hearts, fatty acid oxidation (FAO) supplies most ATP. As dilated cardiomyopathy (DCM) progresses to heart failure, a shift to alternative fuels like ketones is often considered as a secondary adaptation. Whether this metabolic remodeling contributes to disease onset remains unclear. We recently identified a nonsense variant in the FA transporter CD36 (rs3211938), uniquely found in ~9% of individuals with African ancestry, associated with increased DCM risk. We investigated its functional and metabolic consequences in human induced pluripotent stem cell cardiomyocytes (hiPSC-CM).
Methods: HiPSCs were CRISPR-edited to generate heterozygous (Het) and homozygous (Hom) rs3211938 lines, then differentiated into cardiomyocytes. These were compared to isogenic iPSC-CMs containing titin truncating variants (TTNtv), a known structural DCM mutation. CD36 expression and plasma ketone levels were assessed in human carriers and validated in human heart tissue and hiPSC-CMs. Contractile force was measured in engineered cardiac rings under different nutrient conditions. Mitochondrial oxygen consumption rate (OCR) and substrate utilization were evaluated via metabolic assays.
Results: CD36 mRNA and protein levels were reduced in Het (-60%) and Hom (-97%) hiPSC-CM, consistent with human heart tissue and carrier plasma, suggesting nonsense-mediated decay. Contractile force analysis revealed substrate-dependent changes. CD36-deficient tissues preserved contractile force in glucose but reduced contractility in palmitate-only media (Het -29%; Hom -55%), with slower contraction and impaired calcium handling; the stronger Hom cellular phenotype aligns with higher DCM risk in homozygous patients. TTNtv showed reduced force regardless of substrate. CD36-deficient hiPSC-CM also exhibited a gene-dose-dependent reduction in FA uptake and impairment of FAO, ATP production, and mitochondrial OCR in palmitate-only media. Glycolysis was increased in Het and Hom lines, while only Hom cells showed enhanced ketone utilization. Correspondingly, plasma ketone levels were subclinically elevated in homozygous rs3211938 carriers, not in TTNtv, revealing a distinct metabolic fingerprint.
Conclusion: The rs3211938 variant in CD36 links impaired FA metabolism to nutrient-dependent contractile dysfunction, defining a distinct DCM subtype separable from typical structural forms. This human cell model captures early pathogenic features relevant to ancestry-specific genetic risk.