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.

Ca_V1.2 channels are critical players in cardiac excitation-contraction coupling, yet we do not understand how they are affected by an important therapeutic target of heart failure drugs and regulator of blood pressure, angiotensin II. Signaling through G_q-coupled AT1 receptors, angiotensin II triggers a decrease in PIP₂, a phosphoinositide component of the plasma membrane (PM) and known regulator of many ion channels. PIP₂ depletion suppresses Ca_V1.2 currents in heterologous expression systems but the mechanism of this regulation and whether a similar phenomenon occurs in cardiomyocytes is unknown. Previous studies have shown that Ca_V1.2 currents are also suppressed by angiotensin II. We hypothesized that these two observations are linked and that PIP₂ stabilizes Ca_V1.2 expression at the PM and angiotensin II depresses cardiac excitability by stimulating PIP₂ depletion and destabilization of Ca_V1.2 expression. We tested this hypothesis and report that Ca_V1.2 channels in tsA201 cells are destabilized after AT1 receptor-triggered PIP₂ depletion, leading to their dynamin-dependent endocytosis. Likewise, in cardiomyocytes, angiotensin II decreased t-tubular Ca_V1.2 expression and cluster size by inducing their dynamic removal from the sarcolemma. These effects were abrogated by PIP₂ supplementation. Functional data revealed acute angiotensin II reduced Ca_V1.2 currents and Ca²⁺ transient amplitudes thus diminishing excitation-contraction coupling. Finally, mass spectrometry results indicated whole-heart levels of PIP₂ are decreased by acute angiotensin II treatment. Based on these observations, we propose a model wherein PIP₂ stabilizes Ca_V1.2 membrane lifetimes, and angiotensin II-induced PIP₂ depletion destabilizes sarcolemmal Ca_V1.2, triggering their removal, and the acute reduction of Ca_V1.2 currents and contractility.

Cardiac dysfunction is a hallmark of aging in humans and mice. Here we report that a two-week treatment to restore youthful Bridging Integrator 1 (BIN1) levels in the hearts of 24-month-old mice rejuvenates cardiac function and substantially reverses the aging phenotype. Our data indicate that age-associated overexpression of BIN1 occurs alongside dysregulated endosomal recycling and disrupted trafficking of cardiac Ca_V1.2 and type 2 ryanodine receptors. These deficiencies affect channel function at rest and their upregulation during acute stress. In vivo echocardiography reveals reduced systolic function in old mice. BIN1 knockdown using an adeno-associated virus serotype 9 packaged shRNA-mBIN1 restores the nanoscale distribution and clustering plasticity of ryanodine receptors and recovers Ca²⁺ transient amplitudes and cardiac systolic function toward youthful levels. Enhanced systolic function correlates with increased phosphorylation of the myofilament protein cardiac myosin binding protein-C. These results reveal BIN1 knockdown as a novel therapeutic strategy to rejuvenate the aging myocardium.

A mistake was identified for the paper [1] in the treatment of the radion [2] cross-sections, which resulted in multiple changes.