Transferability of the classic volumetric myocardial contrast echocardiography formula to constant-flow Langendorff-perfused rabbit hearts

The classic volumetric myocardial contrast echocardiography (MCE) formula estimates absolute myocardial blood flow (MBF) by normalizing myocardial contrast intensity to the left ventricular blood pool. Because Langendorff-perfused isolated hearts lack this physiological reference, we substituted the aortic-root blood pool to evaluate formula transferability and develop a condition-specific calibration. Twenty-four rabbit hearts underwent constant-flow perfusion at 21 mL/min and flash-replenishment MCE during continuous microbubble infusion. Plateau intensity (A), replenishment rate constant (β), and A × β were measured, with segment-matched fluorescent-microsphere-derived MBF as the reference. Of 408 segments, 407 were analyzed. The adapted estimate preserved the basal-to-apical perfusion pattern but showed poor agreement with reference MBF (CCC = 0.080; RMSE = 3.479 mL·min⁻¹·g⁻¹). After adjustment for myocardial level, the adapted estimate did not improve model fit ( P = 0.328), whereas A × β remained associated with reference MBF ( P = 0.005). The final mixed-effects calibration equation was MBF = 2.863 + 0.176I_Basal − 0.906I_Apical + 0.012(A × β). Adding A × β to myocardial level alone changed leave-one-heart-out cross-validation RMSE from 0.510 to 0.508 mL·min⁻¹·g⁻¹ and CCC from 0.648 to 0.653. Predictive performance was driven predominantly by myocardial level, with A × β providing modest additional predictive information. This condition-specific proof-of-concept calibration under standardized constant-flow conditions requires validation in independent preparations and across broader perfusion conditions before wider application.