Multiple manifestations of coronary microvascular dysfunction in a double-hit mouse model of HFpEF

Heart failure with preserved ejection fraction (HFpEF) is increasingly linked to coronary microvascular dysfunction (CMD), but early, mechanism-resolved phenotyping remains challenging. We developed a multimodal, non-invasive imaging platform to interrogate CMD in a double-hit HFpEF mouse model induced by high-fat diet and chronic nitric oxide synthase inhibition (L-NAME). C57BL/6 mice underwent cine and strain cardiac MRI, dynamic contrast-enhanced MRI with macromolecular (galbumin) and redox-sensitive (3CP) contrast agents, and Doppler-based assessment of coronary flow reserve induced by multiple vasodilators. Ex vivo coronary micro-CT angiography, transmission electron microscopy, coronary endothelial cell transcriptomics, and high-energy phosphate and NAD(H) profiling in heart and blood were performed. Double-hit mice developed an early HFpEF-like phenotype with hypertension, metabolic dysfunction, exercise intolerance, lung congestion, increased natriuretic peptides, impaired diastolic relaxation, longitudinal strain and dobutamine reserve, with preserved systolic function. Coronary tree size and length were maintained, but branching complexity was reduced and ultrastructure revealed endothelial swelling, mitochondrial damage and perivascular/interstitial remodeling. Galbumin DCE-MRI demonstrated increased K_trans, consistent with microvascular barrier failure, whereas 3CP imaging suggested enhanced myocardial reduction capacity. Coronary flow reserve was globally blunted across endothelium-dependent and -independent vasodilators, with elevated basal coronary flow. Endothelial and cardiac transcriptomics indicated inflammatory, fibrotic and redox-stress signatures, while bioenergetic analysis showed ATP and NAD+ depletion with diminished myocardial creatine buffering. This platform sensitively captured early, predominantly functional CMD without overt rarefaction in double-hit HFpEF mice. These results identify impaired vasodilatory reserve, increased endothelial permeability, vascular inflammatory and fibrotic activation, and capillary extracellular matrix deposition as early pathophysiological events.