Fueling the heartbeat: Dynamic regulation of intracellular ATP during excitation-contraction coupling in ventricular myocytes

Abstract

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.

Keywords: calcium; electrometabolic coupling; mitochondria; mitofusin 2.

Fig. 1.

Two different modalities of [ATP]_i fluctuations during EC coupling. (A) Cartoon of the structure of iATP, which was created by fusing a circularly permuted superfolder GFP (cpSFGFP) with the epsilon subunit of the F₀F₁ ATP synthase. iATP increases its fluorescence upon binding ATP. (B) Images of a representative ventricular myocyte expressing iATP loaded with the fluorescent Ca²⁺ indicator Rhod-3. Line-scan images of [Ca²⁺]_i (Left column) and [ATP]_i (Center column) in a myocyte with cell-wide Mode 1 (C) or Mode 2 (D) ATP dynamics. The red traces above the [Ca²⁺]_i line scans show the cell-wide time course of [Ca²⁺]_i. The black traces show the time course of cell length. The green traces above the [ATP]_i line scans show the cell-wide time course of [ATP]_i. (E) Representative cell with Mode 1 and 2 sites. The time courses for [Ca²⁺]_i and [ATP]_i in these sites (marked by dashed lines) are shown to the right of each image. The right column shows the same [Ca²⁺]_i and [ATP]_i traces in the left and center column in μM units. White bars in each image are 10 μm long.

Fig. 2.

Oxidative phosphorylation is critical for diastolic and [ATP]_i transients during contraction. (A) Confocal image of an exemplar ventricular myocyte expressing iATP. The plot below the image shows the time course of [ATP]_i in the region within the white circle before and after the application of 1 μM FCCP. The plot underneath shows the time course of [ATP]_i from a different cell before and after the application of 1 μM FCCP and 1 μM oligomycin. The white bar on the image equals 10 μm. The double-headed arrow indicates the change in [ATP]_i in response to inhibition of mitochondrial oxidative phosphorylation, which represents ATP consumption by homeostatic cellular processes. Insets show summary change in [ATP]_i in response to FCCP (N = 4, n = 10) or FCCP and oligomycin (N = 4, n = 11). (B) Line-scan images of [Ca²⁺]_i and [ATP]_i from 2 ventricular myocytes with Mode 1 and 2 sites before and after FCCP application. The arrows indicate the timing of field stimulation.

Fig. 3.

[Ca²⁺]_mito changes in Mode 1 and Mode 2 ventricular myocytes. (A) Two-dimensional and line-scan confocal images of a representative ventricular myocyte expressing iATP loaded with the mitochondrial fluorescent Ca²⁺ indicator dh-Rhod-2. The traces below each line scan show the time course of [ATP]_i and [Ca²⁺]_mito in Mode 1 and 2 sites in this cell. White bars in each image are 10 μm long. (B) Scatter plot of the amplitudes of [ATP]_i and [Ca²⁺]_mito transients in Mode 1 and Mode 2 sites (N = 3/n = 27). All significant values are provided from a nested t test. The mean values ± SEM of all individual values are in black. Panels (C) and (D) show the relationship between [ATP]_i and [Ca²⁺]_mito during the contraction and is plotted as a trajectory. For each trace, the cell begins and ends in a relaxed state at low [ATP]_i and [Ca²⁺]_mito. Thus, the diagrams “begin” and “end” at the intersection of 1.0 in the x- and y- axes. The lines with arrows indicate the trajectory of the relationship. (E) Plot showing the relationship between the amplitude of simultaneously recorded [ATP]_i and [Ca²⁺]_mito signals in individual myocytes. The solid line shows a linear fit to the data with a slope of 0.89 Δ[ATP]_i/Δ[Ca²⁺]_mito. The dashed lines show the 95% CI of the fit.

Fig. 4.

Eliminating SR Ca²⁺ release decreases [Ca²⁺]_mito and [ATP]_i in ventricular myocytes. (A) Time course from line scans of [ATP]_i and [Ca²⁺]_i in Modes 1 and 2 sites before (Left) and after (Right) application of 1 μM thapsigargin. Paired scatter plot of the amplitudes of [ATP]_i (B) and [Ca²⁺]_i (C) transients in Modes 1 and 2 sites (N = 4, n = 10/7, respectively). (D) Time course from line scans of [ATP]_i and [Ca²⁺]_mito in Mode 1 and 2 sites before (Left) and after (Right) application of 1 μM thapsigargin. Scatter plot of the amplitudes of [ATP]_i (E) and [Ca²⁺]_mito (F) transients in Mode 1 and 2 sites (N = 4, n = 22/20, respectively). All significant values are provided from a paired t test.

Fig. 5.

Mitofusin 2 is important for Mode 1 Ca²⁺–ATP relationship in ventricular myocytes. (A) Time course from line scans of [Ca²⁺]_i, [Ca²⁺]_mito and [ATP]_i in Mode 1 and Mode 2 sites recorded from control and Mfn2-shRNA cells. Scatter plots of the spatial spread of [ATP]_i (B) (N = 15, n = 45/34 and N = 3, n = 27/24 of Modes 1 and 2 sites in control and Mfn2 shRNA, respectively) and amplitudes of [ATP]_i (C) and [Ca²⁺]_i (D) transients in control and Mfn2-shRNA expressing myocytes (N = 8, n = 35 and N = 14, n = 34 of control Mode 1 and Mode 2, and N = 3, n = 23 and N = 3, n = 17 Mfn2 shRNA Mode 1 and Mode 2 sites, respectively). Scatter plots of the amplitudes of [ATP]_i (E) and [Ca²⁺]_mito (F) transients in control and Mfn2-shRNA expressing myocytes (N = 4, n = 43/37 of control Mode 1 and Mode 2 sites, and N = 3, n = 15/21 of Mfn2 shRNA Mode 1 and Mode 2 sites, respectively). All significant values are provided from a nested t test. The mean values ± SEM of all individual values are in black. (G) Plot showing the relationship between the amplitude of simultaneously recorded [ATP]_i and [Ca²⁺]_mito signals in control cells and cells expressing Mfn2-shRNA. The solid lines show linear fits to the data with a slope of 0.89 Δ[ATP]_i/Δ[Ca²⁺]_mito in control myocytes and 0.29 Δ[ATP]_i/Δ[Ca²⁺]_mito in Mfn2-shRNA myocytes. The dashed lines show the 95% CI of the fits.

Fig. 6.

Contraction consumes significant ATP during cardiac EC coupling. (A) [Ca²⁺]_i and [ATP]_i transients recorded from the same cell sites before and after the acute (3 min) application of 10 μM blebbistatin (BB). (B) [Ca²⁺]_i and [ATP]_i transients recorded from cell sites in control cells and in cells exposed to 10 μM BB for 30 min. Scatter plots of the amplitude of [ATP]_i (C) and [Ca²⁺]_i (D) transients under control conditions and in the presence of 10 μM BB for 30 min (N = 4, n = 9/9 of control Mode 1 and Mode 2 sites, and N = 4, n = 16/5 of BB Mode 1 and Mode 2 sites, respectively). (E) Amplitude (%) of contraction in control cells (N = 4, n = 14) and cells treated with 10 μM blebbistatin (N = 4, n = 18). All significant values are provided from a nested t test. The mean values ± SEM of all individual values are in black.

Fig. 7.

Active sarcolemmal K_ATP channels in ventricular myocytes. (A) Whole cell K⁺ currents recorded at −100 mV from a holding potential of −80 mV before and after 10 μm glibenclamide. The green Inset and dashed red line show the control current before drug. Scatter plots of current density before and after glibenclamide (B) and for the glibenclamide sensitive current (I_KATP) (C) (N = 4, n = 17). (D) Current clamp recordings of AP-evoked in ventricular myocytes before and after application of glibenclamide. Black arrows note early and delayed after depolarizations (EADs or DADs, respectively) in the glibenclamide-treated cells. (E) Percentage of cells with recorded EADs or DADs. Scatter plots of membrane diastolic potential (MDP) (F) and the time to 90% of the action potential (APD₉₀) (G) before and after glibenclamide (N = 4, n = 14). (H) AP-evoked (1 Hz) [Ca²⁺]_i transients in a ventricular myocyte before and after the application of 10 μM glibenclamide. The black arrows mark when an AP was evoked via field stimulation. The red arrows identified spontaneous Ca²⁺ release events. Scatter plots of diastolic [Ca²⁺]_i (I) and the amplitudes of [Ca²⁺]_i transients (J) under control conditions and in the presence of 10 μM glibenclamide (N = 4, n = 17). (K) Percentage of cells with spontaneous SR Ca²⁺ release events. All significant values are provided from a paired t test.

Fig. 8.

Proposed model for Modes 1 and 2 ATP dynamics in ventricular myocytes during EC coupling. (A) In Mode 1 sites, mitochondria, anchored to the SR by Mfn2, strategically positioned near sites of Ca²⁺ entry and release, efficiently absorb Ca²⁺ through the MCU, leading to ATP production. (B) In Mode 2 sites, mitochondria are likely located farther away from the dyad. Mitochondria in these sites initially experience a decrease in [Ca²⁺]_mito, likely mediated by the NCLX. However, as Ca²⁺ diffuses to these mitochondria and enters via the MCU, this influx surpasses NCLX efflux, leading to an augmentation in [Ca²⁺]_mito that increases ATP synthesis and restores diastolic [ATP]_i levels in the vicinity.