PurposeTo demonstrate the feasibility of mapping intracellular pH within the in vivo rodent heart. Alterations in cardiac acid-base balance can lead to acute contractile depression and alterations in Ca2+ signaling. The transient reduction in adenosine triphosphate (ATP) consumption and cardiac contractility may be initially beneficial; however, sustained pH changes can be maladaptive, leading to myocardial damage and electrical arrhythmias. MethodsSpectrally selective radiofrequency (RF) pulses were used to excite the HCO3- and CO2 resonances individually while preserving signal from the injected hyperpolarized [1-C-13]pyruvate. The large flip angle pulses were placed within a three-dimensional (3D) imaging acquisition, which exploited CA-mediated label exchange between HCO3- and CO2. Images at 4.5 x 4.5 x 5 mm(3) resolution were obtained in the in vivo rodent heart. The technique was evaluated in healthy rodents scanned at baseline and during high cardiac workload induced by dobutamine infusion. ResultsThe intracellular pH was measured to be 7.150.04 at baseline, and decreased to 6.90 +/- 0.06 following 15 min of continuous -adrenergic stimulation. ConclusionsVolumetric maps of intracellular pH can be obtained following an injection of hyperpolarized [1-C-13]pyruvate. The new method is anticipated to enable assessment of stress-inducible ischemia and potential ventricular arrythmogenic substrates within the ischemic heart. Magn Reson Med 77:1810-1817, 2017. (c) 2016 The Authors Magnetic Resonance in Medicine published by Wiley Periodicals, Inc. on behalf of International Society for Magnetic Resonance in Medicine. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.