The heart beats approximately 100,000 times per day in humans, imposing substantial energetic demands on cardiac muscle. Adenosine triphosphate (ATP) is an essential energy source for normal function of cardiac muscle during each beat, as it powers ion transport, intracellular Ca²⁺ handling, and actin-myosin cross-bridge cycling. Despite this, the impact of excitation-contraction coupling on the intracellular ATP concentration ([ATP]_i) in myocytes is poorly understood. Here, we conducted real-time measurements of [ATP]_i in ventricular myocytes using a genetically encoded ATP fluorescent reporter. Our data reveal rapid beat-to-beat variations in [ATP]_i. Notably, diastolic [ATP]_i was <1 mM, which is eightfold to 10-fold lower than previously estimated. Accordingly, ATP-sensitive K⁺ (K_ATP) channels were active at physiological [ATP]_i. Cells exhibited two distinct types of ATP fluctuations during an action potential: net increases (Mode 1) or decreases (Mode 2) in [ATP]_i. Mode 1 [ATP]_i increases necessitated Ca²⁺ entry and release from the sarcoplasmic reticulum (SR) and were associated with increases in mitochondrial Ca²⁺. By contrast, decreases in mitochondrial Ca²⁺ accompanied Mode 2 [ATP]_i decreases. Down-regulation of the protein mitofusin 2 reduced the magnitude of [ATP]_i fluctuations, indicating that SR-mitochondrial coupling plays a crucial role in the dynamic control of ATP levels. Activation of β-adrenergic receptors decreased [ATP]_i, underscoring the energetic impact of this signaling pathway. Finally, our work suggests that cross-bridge cycling is the largest consumer of ATP in a ventricular myocyte during an action potential. These findings provide insights into the energetic demands of EC coupling and highlight the dynamic nature of ATP concentrations in cardiac muscle.

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