Genome-scale modelling and stable isotope tracing reveal dynamic metabolic reprogramming in a human cardiac hypertrophy model

Pathological cardiac hypertrophy is associated with metabolic remodelling; however the temporal dynamics of this remodelling remain poorly defined in human cardiomyocyte systems. Here, we combined transcriptomic profiling, genome-scale metabolic modelling and stable isotope-resolved metabolomics (SIRM) to map the metabolic consequences of endothelin-1 (ET-1) stimulation in human iPSC-derived cardiomyocytes (hiPSC-CMs). Integration of RNA sequencing data obtained after 8 and 48 hours of ET-1 treatment with the Human2.0 genome-scale metabolic model predicted time-dependent remodelling, characterised by progressive increases in glycolytic flux, enhanced glucose carbon entry into the TCA cycle, and a decrease in fatty acid β-oxidation. Computational flux predictions were independently confirmed by extracellular flux analysis and 13C-substrate tracing, with concordance between modelled and measured fluxes. Extracellular flux analysis showed increased glycolytic capacity and mitochondrial respiration after 48 hours, with greater sensitivity to inhibition of mitochondrial pyruvate transport. Glucose tracing revealed increased 13C enrichment of glycolytic, pentose phosphate pathway, serine-glycine and TCA cycle metabolites, consistent with altered glucose-derived carbon allocation. Increased m+3 labelling of selected TCA-related metabolites was compatible with pyruvate anaplerosis, although pathway-specific fluxes were not resolved. In three-dimensional engineered heart tissue (EHTs) treated with ET-1 for 7 days, increased hypertrophic marker expression was accompanied by a blunted inotropic response, and metabolomics showed similar changes in metabolite abundance and glucose-derived labelling to 2D cultures. These findings support hiPSC-CMs and EHTs as complementary platforms for studying early metabolic remodelling in pathological cardiac hypertrophy and provide an integrated framework linking model predictions to functional and isotope-tracing measurements.