The discovery of allosteric modulators is an emerging paradigm in drug discovery, and signal transduction is a subtle and dynamic process that is challenging to characterize. We developed a time-correlated single photon counting (TCSPC) imaging approach to investigate the structural mechanisms for small-molecule activation of the cardiac sarcoplasmic reticulum Ca²⁺-ATPase (SERCA2a), a pharmacologically important pump that transports Ca²⁺ at the expense of ATP hydrolysis. We first tested whether the dissociation of SERCA2a from its regulatory protein phospholamban (PLN) is required for small-molecule activation. We found that CDN1163, a validated SERCA2a activator, does not have significant effects on the stability of the SERCA2a–PLN complex. TCSPC imaging experiments using the non-hydrolyzable ATP analog AMP-PCP showed ATP is an allosteric modulator of SERCA2a, increasing the fraction of catalytically competent structures at physiologically relevant Ca²⁺ concentrations. Unlike ATP, CDN1163 alone has no significant effects on the Ca²⁺-dependent shifts in the structural populations of SERCA2a, and it does not increase the pump’s affinity for Ca²⁺ ions. However, we found that CDN1163 enhances the ATP-mediated modulatory effects to increase the population of catalytically competent SERCA2a structures. Importantly, this structural shift occurs within the physiological window of Ca²⁺ concentrations at which SERCA2a operates. We demonstrated that ATP is both a substrate and modulator of SERCA2a and showed that CDN1163 and ATP act synergistically to populate SERCA2a structures that are primed for phosphorylation. This study provides novel insights into the structural mechanisms for SERCA2a activation by its substrate and a synthetic allosteric modulator.

Obesity is a major risk factor for atrial fibrillation (AF), the most common serious cardiac arrhythmia, but the molecular mechanisms underlying obesity-induced AF remain unclear. In this study, we subjected mice to a chronic high-fat diet and acute sympathetic activation to investigate how obesity promotes AF. Surface electrocardiography revealed that obesity and sympathetic activation synergize during intracardiac tachypacing to induce AF. At the cellular level, this combination facilitated delayed afterdepolarizations in atrial myocytes, implicating altered Ca²⁺ dynamics. Interestingly, obesity did not affect the expression of key atrial Ca²⁺-handling proteins, including the cardiac sarcoplasmic reticulum Ca²⁺-ATPase (SERCA2a). However, obesity increases the proportion of inhibitory phospholamban (PLN) monomers and decreases PLN phosphorylation, suggesting reduced SERCA2a activity. Paradoxically, Ca²⁺ reuptake in atrial myocytes from obese mice was similar to that achieved by potent small-molecule SERCA2a activators. We found that adrenergic stimulation increased Ca²⁺ transient amplitude without altering Ca²⁺ reuptake in myocytes from obese mice. Transcriptomic analysis revealed that a high-fat diet upregulated neuronatin, a protein involved in obesity that enhances SERCA2-mediated Ca²⁺ reuptake in neurons. We propose that obesity enables SERCA2a activation independently of PLN regulation, while adrenergic stimulation triggers arrhythmogenic Ca²⁺-induced Ca²⁺ release, promoting AF. In conclusion, this study demonstrates that obesity causes a paradoxical dysregulation of SERCA2a in atrial myocytes, with increased activity despite higher levels of inhibitory PLN monomers and reduced PLN phosphorylation. These findings offer new insights into the cellular mechanisms of obesity-induced AF and suggest potential therapeutic targets.

Background: The Na⁺/K⁺-ATPase (NKA) maintains electrochemical gradients by exporting Na+ and importing K⁺ at the expense of ATP hydrolysis. Although NKA inhibition is a well-established strategy for increasing cardiac contractility, existing inhibitors such as cardiotonic steroids (CTS) are limited by serious adverse effects. N106 is a small molecule previously shown to enhance cardiac lusitropy by promoting SERCA2a SUMOylation and, intriguingly, also exerts positive inotropic effects, suggesting additional mechanisms of action. Methods: To test whether N106 directly modulates NKA, we combined ATPase activity assays with molecular docking and microsecond-scale molecular dynamics simulations. Results: Biochemical measurements showed that N106 partially inhibits NKA, achieving ~80% maximal inhibition with an IC50 of 7 ± 1 µM, while leaving the pump’s apparent affinity for Na⁺, K⁺, and ATP unchanged. Computational analyses suggest that N106 binds within the canonical CTS-binding pocket but undergoes intermittent unbinding events, consistent with the partial inhibition observed experimentally. Conclusions: These findings identify N106 as a first-in-class dual modulator of cardiac ion pumps, partially inhibiting NKA while previously shown to activate SERCA2a through enhanced SUMOylation. This combined mechanism likely underlies its positive inotropic and lusitropic effects and positions the N106 scaffold as a promising lead for developing next-generation dual-target therapeutics for heart failure.

P-type ATPases share a conserved catalytic architecture but have different sensitivities to small-molecule inhibitors. We previously identified several statins as inhibitors of the sarco/endoplasmic reticulum Ca²⁺-ATPase (SERCA), but the extent to which this inhibitory activity reflects broader pharmacological cross-reactivity among P-type ATPases remains unknown. Here, we compared the effects of seven structurally diverse statin lactones, which share a common δ-lactone moiety but contain distinct core scaffolds, including hexahydronaphthalene-based and aromatic heterocyclic ring systems, on SERCA and the Na⁺/K⁺-ATPase (NKA). The structurally related lovastatin and simvastatin produced similar concentration-dependent inhibitory profiles toward SERCA and NKA, despite the otherwise distinct pharmacological sensitivities of these pumps. In contrast, atorvastatin, rosuvastatin, and pitavastatin showed more divergent inhibitory profiles, generally with greater inhibition of SERCA than NKA at higher concentrations. Pravastatin and fluvastatin, however, had minimal effects on either ATPase over the concentration range examined. These findings build on our previous identification of selected statins as SERCA inhibitors by demonstrating that their activity can extend to NKA. Overall, the results reveal substantial structure-dependent variation in the effects of statin lactones on these P-type ATPases, ranging from convergent inhibition to differential inhibition and minimal activity.