The sinoatrial node (SAN), the leading pacemaker region, generates electrical impulses that propagate throughout the heart. SAN dysfunction with bradyarrhythmia is well documented in heart failure (HF). However, the underlying mechanisms are not completely understood. Mitochondria are critical to cellular processes that determine the life or death of the cell. The release of Ca ²⁺ from the ryanodine receptors 2 (RyR2) on the sarcoplasmic reticulum (SR) at mitochondria–SR microdomains serves as the critical communication to match energy production to meet metabolic demands. Therefore, we tested the hypothesis that alterations in the mitochondria–SR connectomics contribute to SAN dysfunction in HF. We took advantage of a mouse model of chronic pressure overload–induced HF by transverse aortic constriction (TAC) and a SAN-specific CRISPR-Cas9–mediated knockdown of mitofusin-2 ( Mfn2 ), the mitochondria–SR tethering GTPase protein. TAC mice exhibited impaired cardiac function with HF, cardiac fibrosis, and profound SAN dysfunction. Ultrastructural imaging using electron microscope (EM) tomography revealed abnormal mitochondrial structure with increased mitochondria–SR distance. The expression of Mfn2 was significantly down-regulated and showed reduced colocalization with RyR2 in HF SAN cells. Indeed, SAN-specific Mfn2 knockdown led to alterations in the mitochondria–SR microdomains and SAN dysfunction. Finally, disruptions in the mitochondria–SR microdomains resulted in abnormal mitochondrial Ca ²⁺ handling, alterations in localized protein kinase A (PKA) activity, and impaired mitochondrial function in HF SAN cells. The current study provides insights into the role of mitochondria–SR microdomains in SAN automaticity and possible therapeutic targets for SAN dysfunction in HF patients.

Small-conductance Ca ²⁺ -activated K ⁺ channels (SK, K _Ca 2) are gated solely by intracellular microdomain Ca ²⁺ . The channel has emerged as a therapeutic target for cardiac arrhythmias. Calmodulin (CaM) interacts with the CaM binding domain (CaMBD) of the SK channels, serving as the obligatory Ca ²⁺ sensor to gate the channels. In heterologous expression systems, phosphatidylinositol 4,5-bisphosphate (PIP2) coordinates with CaM in regulating SK channels. However, the roles and mechanisms of PIP2 in regulating SK channels in cardiomyocytes remain unknown. Here, optogenetics, magnetic nanoparticles, combined with Rosetta structural modeling, and molecular dynamics (MD) simulations revealed the atomistic mechanisms of how PIP2 works in concert with Ca ²⁺ -CaM in the SK channel activation. Our computational study affords evidence for the critical role of the amino acid residue R395 in the S6 transmembrane segment, which is localized in propinquity to the intracellular hydrophobic gate. This residue forms a salt bridge with residue E398 in the S6 transmembrane segment from the adjacent subunit. Both R395 and E398 are conserved in all known isoforms of SK channels. Our findings suggest that the binding of PIP2 to R395 residue disrupts the R395:E398 salt bridge, increasing the flexibility of the transmembrane segment S6 and the activation of the channel. Importantly, our findings serve as a platform for testing of structural-based drug designs for therapeutic inhibitors and activators of the SK channel family. The study is timely since inhibitors of SK channels are currently in clinical trials to treat atrial arrhythmias.

Background/Objectives: Ischemic heart disease remains a leading cause of morbidity and mortality worldwide, accompanied by a major decline in local myocardial pH. However, the mechanisms of pH regulation and the homeostasis of H⁺ neutralizing buffers, such as HCO₃⁻, in cardiomyocytes remain incompletely understood. We identified a solute carrier, Slc26a6, in mouse and human hearts playing key roles in the regulation of cardiac pH, excitability, and contractility. Slc26a6 is an acid loader, so we hypothesized that ablation of Slc26a6 may protect the heart from ischemia/reperfusion (I/R) injury. Methods: The I/R model was generated using wild type (WT) and Slc26a6 knockout (Slc26a6^−/−) mice. Multidisciplinary in vivo, in vitro, and ex vivo approaches were used, including echocardiography, electrophysiology, hemodynamic monitoring, fluorescence microscopy, histochemistry, and cellular Ca²⁺ transients, sarcoplasmic reticulum Ca²⁺ load, and sarcomere shortening were recorded. Results: Troponin I level was lower in Slc26a6^−/− I/R mice. Slc26a6^−/− mice showed better systolic and diastolic function, reduced collagen deposition, and reduced infarct size compared to that of WT mice. Cellular experiments in measurement of sarcomere shortening, Ca²⁺ transients, and sarcoplasmic reticulum Ca²⁺ load in cardiomyocytes from the infarct zone supported the in vivo findings, demonstrating better single cell function in Slc26a6^−/− compared to WT mice. Ex vivo pH_i measurement showed elevated pH_i in Slc26a6^−/− mouse heart. Conclusions: Ablation of Slc26a6 protects the heart from I/R injury, suggesting the importance of Cl⁻/HCO₃⁻ exchange in cardiac pH regulation and I/R injury. The elevated pH_i in Slc26a6^−/− mouse heart may counterbalance the effects of the myocardium acidosis resulting from ischemia.