BIN1 knockdown rescues systolic dysfunction in aging male mouse hearts.

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 CaV1.2 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.

The Piezo channel is a mechano-sensitive complex component in the mammalian inner ear hair cell.

The inner ear is the hub where hair cells (HCs) transduce sound, gravity, and head acceleration stimuli to the brain. Hearing and balance rely on mechanosensation, the fastest sensory signals transmitted to the brain. The mechanoelectrical transducer (MET) channel is the entryway for the sound-balance-brain interface, but the channel-complex composition is not entirely known. Here, we report that the mouse utilizes Piezo1 (Pz1) and Piezo2 (Pz2) isoforms as MET-complex components. The Pz channels, expressed in HC stereocilia, and cell lines are co-localized and co-assembled with MET complex partners. Mice expressing non-functional Pz1 and Pz2 at the ROSA26 locus have impaired auditory and vestibular traits that can only be explained if the Pzs are integral to the MET complex. We suggest that Pz subunits constitute part of the MET complex and that interactions with other MET complex components yield functional MET units to generate HC MET currents.

Guidelines on antibody use in physiology research.

Antibodies are one of the most used reagents in scientific laboratories and are critical components for a multitude of experiments in physiology research. Over the past decade, concerns about many biological methods, including those that use antibodies, have arisen as several laboratories were unable to reproduce the scientific data obtained in other laboratories. The lack of reproducibility could be largely attributed to inadequate reporting of detailed methods, no or limited verification by authors, and the production and use of unvalidated antibodies. The goal of this guideline article is to review best practices concerning commonly used techniques involving antibodies, including immunoblotting, immunohistochemistry, and flow cytometry. Awareness and integration of best practices will increase the rigor and reproducibility of these techniques and elevate the quality of physiology research.

Clinical, Echocardiographic, and Longitudinal Characteristics Associated With Heart Failure With Improved Ejection Fraction.

Heart failure with improved ejection fraction (HFimpEF) has better outcomes than HF with reduced EF (HFrEF). However, factors contributing to HFimpEF remain unclear. This study aimed to evaluate clinical and longitudinal characteristics associated with subsequent HFimpEF. This was a single-center retrospective HFrEF cohort study. Data were collected from 2014 to 2022. Patients with HFrEF were identified using International Classification of Diseases codes, echocardiographic data, and natriuretic peptide levels. The main end points were HFimpEF (defined as EF >40% at ≥3 months with ≥10% increase) and mortality. Cox proportional hazards and mixed effects models were used for analyses. The study included 1,307 patients with HFrEF with a median follow-up of 16.3 months (interquartile range 8.0 to 30.6). The median age was 65 years; 68% were male whereas 57% were White. On follow-up, 38.7% (n = 506) developed HFimpEF, whereas 61.3% (n = 801) had persistent HFrEF. A multivariate Cox regression model identified gender, race, co-morbidities, echocardiographic, and natriuretic peptide as significant covariates of HFimpEF (p <0.05). The HFimpEF group had better survival compared with the persistent HFrEF group (p <0.001). Echocardiographic and laboratory trajectories differed between groups. In this HFrEF cohort, 38.7% transitioned to HFimpEF and approximately 50% met the definition within the first 12 months. In a HFimpEF model, gender, co-morbidities, echocardiographic parameters, and natriuretic peptide were associated with subsequent HFimpEF. The model has the potential to identify patients at risk of subsequent persistent or improved HFrEF, thus informing the design and implementation of targeted quality-of-care improvement interventions.

Clinical, Echocardiographic, and Longitudinal Characteristics Associated with Heart Failure with Improved Ejection Fraction.

Background: Heart failure (HF) with improved ejection fraction (HFimpEF) has better outcomes than HF with reduced ejection fraction (HFrEF). However, factors contributing to HFimpEF remain unclear. This study aimed to evaluate clinical and longitudinal characteristics associated with subsequent HFimpEF. Methods: This was a single-center retrospective HFrEF cohort study. Data were collected from 2014 to 2022. Patients with HFrEF were identified using ICD codes, echocardiographic data, and natriuretic peptide levels. The main endpoints were HFimpEF (defined as ejection fraction >40% at ≥3 months with ≥10% increase) and mortality. Cox proportional hazards and mixed effects models were used for analyses. Results: The study included 1307 HFrEF patients with a median follow-up of 16.3 months (IQR 8.0-30.6). The median age was 65 years; 68% were male while 57% were white. On follow-up, 39% (n=506) developed HFimpEF, while 61% (n=801) had persistent HFrEF. A multivariate Cox regression model identified sex, race comorbidities, echocardiographic, and natriuretic peptide as significant covariates of HFimpEF ( p <0.05). The HFimpEF group had better survival compared to the persistent HFrEF group ( p <0.001). Echocardiographic and laboratory trajectories differed between groups. Conclusion: In this HFrEF cohort, 39% transitioned to HFimpEF and approximately 50% met the definition within the first 12 months. In a HFimpEF model, sex, comorbidities, echocardiographic parameters, and natriuretic peptide were associated with subsequent HFimpEF. The model has the potential to identify patients at risk of subsequent persistent or improved HFrEF, thus informing the design and implementation of targeted quality-of-care improvement interventions.

The Piezo channel is central to the mechano-sensitive channel complex in the mammalian inner ear.

The inner ear is the hub where hair cells transduce sound, gravity, and head acceleration stimuli carried by neural codes to the brain. Of all the senses, hearing and balance, which rely on mechanosensation, are the fastest sensory signals transmitted to the central nervous system. The mechanoelectrical transducer (MET) channel in hair cells is the entryway for the sound-balance-brain interface, but the channel's composition has eluded biologists due to its complexity. Here, we report that the mouse utilizes Piezo1 (Pz1) and Piezo2 (Pz2) isoforms as central components of the MET complex. The Pz channel subunits are expressed in hair-cell stereocilia, are co-localized and co-assembled, and are essential components of the MET complex in vitro and in situ, including integration with the transmembrane channel (Tmc1/2) protein. Mice expressing non-functional Pz1 and Pz2, but not functional Pz1 at the ROSA26 locus under the control of hair-cell promoters, have impaired auditory and vestibular traits that can only be explained if Pz channel multimers are integral to the MET complex. We affirm that Pz protein subunits constitute MET channels and that functional interactions with components of the MET complex yield current properties resembling hair-cell MET currents. Our results demonstrate Pz is a MET channel component central to interacting with MET complex proteins. Results account for the MET channel pore and complex.

Chronic Diclofenac Exposure Increases Mitochondrial Oxidative Stress, Inflammatory Mediators, and Cardiac Dysfunction.

PURPOSE: Nonsteroidal anti-inflammatory drugs (NSAIDs) are among one of the most commonly prescribed medications for pain and inflammation. Diclofenac (DIC) is a commonly prescribed NSAID that is known to increase the risk of cardiovascular diseases. However, the mechanisms underlying its cardiotoxic effects remain largely unknown. In this study, we tested the hypothesis that chronic exposure to DIC increases oxidative stress, which ultimately impairs cardiovascular function. METHODS AND RESULTS: Mice were treated with DIC for 4 weeks and subsequently subjected to in vivo and in vitro functional assessments. Chronic DIC exposure resulted in not only systolic but also diastolic dysfunction. DIC treatment, however, did not alter blood pressure or electrocardiographic recordings. Importantly, treatment with DIC significantly increased inflammatory cytokines and chemokines as well as cardiac fibroblast activation and proliferation. There was increased reactive oxygen species (ROS) production in cardiomyocytes from DIC-treated mice, which may contribute to the more depolarized mitochondrial membrane potential and reduced energy production, leading to a significant decrease in sarcoplasmic reticulum (SR) Ca2+ load, Ca2+ transients, and sarcomere shortening. Using unbiased metabolomic analyses, we demonstrated significant alterations in oxylipin profiles towards inflammatory features in chronic DIC treatment. CONCLUSIONS: Together, chronic treatment with DIC resulted in severe cardiotoxicity, which was mediated, in part, by an increase in mitochondrial oxidative stress.

Disruption of mitochondria-sarcoplasmic reticulum microdomain connectomics contributes to sinus node dysfunction in heart failure.

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 Ca2+ 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 Ca2+ 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.

Disruption of protein quality control of the human ether-à-go-go related gene K+ channel results in profound long QT syndrome.

BACKGROUND: Long QT syndrome (LQTS) is a hereditary disease that predisposes patients to life-threatening cardiac arrhythmias and sudden cardiac death. Our previous study of the human ether-à-go-go related gene (hERG)-encoded K+ channel (Kv11.1) supports an association between hERG and RING finger protein 207 (RNF207) variants in aggravating the onset and severity of LQTS, specifically T613M hERG (hERGT613M) and RNF207 frameshift (RNF207G603fs) mutations. However, the underlying mechanistic underpinning remains unknown. OBJECTIVE: The purpose of the present study was to test the role of RNF207 in the function of hERG-encoded K+ channel subunits. METHODS: Whole-cell patch-clamp experiments were performed in human embryonic kidney (HEK 293) cells and human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) together with immunofluorescent confocal and high resolution microscopy, auto-ubiquitinylation assays, and co-immunoprecipitation experiments to test the functional interactions between hERG and RNF207. RESULTS: Here, we demonstrated that RNF207 serves as an E3 ubiquitin ligase and targets misfolded hERGT613M proteins for degradation. RNF207G603fs exhibits decreased activity and hinders the normal degradation pathway; this increases the levels of hERGT613M subunits and their dominant-negative effect on the wild-type subunits, ultimately resulting in decreased current density. Similar findings are shown for hERGA614V, a known dominant-negative mutant subunit. Finally, the presence of RNF207G603fs with hERGT613M results in significantly prolonged action potential durations and reduced hERG current in human-induced pluripotent stem cell-derived cardiomyocytes. CONCLUSION: Our study establishes RNF207 as an interacting protein serving as a ubiquitin ligase for hERG-encoded K+ channel subunits. Normal function of RNF207 is critical for the quality control of hERG subunits and consequently cardiac repolarization. Moreover, our study provides evidence for protein quality control as a new paradigm in life-threatening cardiac arrhythmias in patients with LQTS.

Selectin-targeting glycosaminoglycan-peptide conjugate limits neutrophil-mediated cardiac reperfusion injury.

AIMS: One of the hallmarks of myocardial infarction (MI) is excessive inflammation. During an inflammatory insult, damaged endothelial cells shed their glycocalyx, a carbohydrate-rich layer on the cell surface which provides a regulatory interface to immune cell adhesion. Selectin-mediated neutrophilia occurs as a result of endothelial injury and inflammation. We recently designed a novel selectin-targeting glycocalyx mimetic (termed DS-IkL) capable of binding inflamed endothelial cells. This study examines the capacity of DS-IkL to limit neutrophil binding and platelet activation on inflamed endothelial cells, as well as the cardioprotective effects of DS-IkL after acute myocardial infarction. METHODS AND RESULTS: In vitro, DS-IkL diminished neutrophil interactions with both recombinant selectin and inflamed endothelial cells, and limited platelet activation on inflamed endothelial cells. Our data demonstrated that DS-IkL localized to regions of vascular inflammation in vivo after 45 min of left anterior descending coronary artery ligation-induced MI. Further, findings from this study show DS-IkL treatment had short- and long-term cardioprotective effects after ischaemia/reperfusion of the left anterior descending coronary artery. Mice treated with DS-IkL immediately after ischaemia/reperfusion and 24 h later exhibited reduced neutrophil extravasation, macrophage accumulation, fibroblast and endothelial cell proliferation, and fibrosis compared to saline controls. CONCLUSIONS: Our findings suggest that DS-IkL has great therapeutic potential after MI by limiting reperfusion injury induced by the immune response.

The Critical Roles of Proteostasis and Endoplasmic Reticulum Stress in Atrial Fibrillation.

Atrial fibrillation (AF) remains the most common arrhythmia seen clinically. The incidence of AF is increasing due to the aging population. AF is associated with a significant increase in morbidity and mortality, yet current treatment paradigms have proven largely inadequate. Therefore, there is an urgent need to develop new effective therapeutic strategies for AF. The endoplasmic reticulum (ER) in the heart plays critical roles in the regulation of excitation-contraction coupling and cardiac function. Perturbation in the ER homeostasis due to intrinsic and extrinsic factors, such as inflammation, oxidative stress, and ischemia, leads to ER stress that has been linked to multiple conditions including diabetes mellitus, neurodegeneration, cancer, heart disease, and cardiac arrhythmias. Recent studies have documented the critical roles of ER stress in the pathophysiological basis of AF. Using an animal model of chronic pressure overload, we demonstrate a significant increase in ER stress in atrial tissues. Moreover, we demonstrate that treatment with a small molecule inhibitor to inhibit the soluble epoxide hydrolase enzyme in the arachidonic acid metabolism significantly reduces ER stress as well as atrial electrical and structural remodeling. The current review article will attempt to provide a perspective on our recent understandings and current knowledge gaps on the critical roles of proteostasis and ER stress in AF progression.

Different arrhythmia-associated calmodulin mutations have distinct effects on cardiac SK channel regulation.

Calmodulin (CaM) plays a critical role in intracellular signaling and regulation of Ca2+-dependent proteins and ion channels. Mutations in CaM cause life-threatening cardiac arrhythmias. Among the known CaM targets, small-conductance Ca2+-activated K+ (SK) channels are unique, since they are gated solely by beat-to-beat changes in intracellular Ca2+. However, the molecular mechanisms of how CaM mutations may affect the function of SK channels remain incompletely understood. To address the structural and functional effects of these mutations, we introduced prototypical human CaM mutations in human induced pluripotent stem cell-derived cardiomyocyte-like cells (hiPSC-CMs). Using structural modeling and molecular dynamics simulation, we demonstrate that human calmodulinopathy-associated CaM mutations disrupt cardiac SK channel function via distinct mechanisms. CaMD96V and CaMD130G mutants reduce SK currents through a dominant-negative fashion. By contrast, specific mutations replacing phenylalanine with leucine result in conformational changes that affect helix packing in the C-lobe, which disengage the interactions between apo-CaM and the CaM-binding domain of SK channels. Distinct mutant CaMs may result in a significant reduction in the activation of the SK channels, leading to a decrease in the key Ca2+-dependent repolarization currents these channels mediate. The findings in this study may be generalizable to other interactions of mutant CaMs with Ca2+-dependent proteins within cardiac myocytes.

AKAP5 complex facilitates purinergic modulation of vascular L-type Ca2+ channel CaV1.2.

The L-type Ca2+ channel CaV1.2 is essential for arterial myocyte excitability, gene expression and contraction. Elevations in extracellular glucose (hyperglycemia) potentiate vascular L-type Ca2+ channel via PKA, but the underlying mechanisms are unclear. Here, we find that cAMP synthesis in response to elevated glucose and the selective P2Y11 agonist NF546 is blocked by disruption of A-kinase anchoring protein 5 (AKAP5) function in arterial myocytes. Glucose and NF546-induced potentiation of L-type Ca2+ channels, vasoconstriction and decreased blood flow are prevented in AKAP5 null arterial myocytes/arteries. These responses are nucleated via the AKAP5-dependent clustering of P2Y11/ P2Y11-like receptors, AC5, PKA and CaV1.2 into nanocomplexes at the plasma membrane of human and mouse arterial myocytes. Hence, data reveal an AKAP5 signaling module that regulates L-type Ca2+ channel activity and vascular reactivity upon elevated glucose. This AKAP5-anchored nanocomplex may contribute to vascular complications during diabetic hyperglycemia.

NODAL inhibition promotes differentiation of pacemaker-like cardiomyocytes from human induced pluripotent stem cells.

Directed cardiomyogenesis from human induced pluripotent stem cells (hiPSCs) has been greatly improved in the last decade but directed differentiation to pacemaking cardiomyocytes (CMs) remains incompletely understood. In this study, we demonstrated that inhibition of NODAL signaling by a specific NODAL inhibitor (SB431542) in the cardiac mesoderm differentiation stage downregulated PITX2c, a transcription factor that is known to inhibit the formation of the sinoatrial node in the left atrium during cardiac development. The resulting hiPSC-CMs were smaller in cell size, expressed higher pro-pacemaking transcription factors, TBX3 and TBX18, and exhibited pacemaking-like electrophysiological characteristics compared to control hiPSC-CMs differentiated from established Wnt-based protocol. The pacemaker-like subtype increased up to 2.4-fold in hiPSC-CMs differentiated with the addition of SB431542 relative to the control. Hence, Nodal inhibition in the cardiac mesoderm stage promoted pacemaker-like CM differentiation from hiPSCs. Improving the yield of human pacemaker-like CMs is a critical first step in the development of functional human cell-based biopacemakers.

Suppression of inflammation and fibrosis using soluble epoxide hydrolase inhibitors enhances cardiac stem cell-based therapy.

Stem cell replacement offers a great potential for cardiac regenerative therapy. However, one of the critical barriers to stem cell therapy is a significant loss of transplanted stem cells from ischemia and inflammation in the host environment. Here, we tested the hypothesis that inhibition of the soluble epoxide hydrolase (sEH) enzyme using sEH inhibitors (sEHIs) to decrease inflammation and fibrosis in the host myocardium may increase the survival of the transplanted human induced pluripotent stem cell derived-cardiomyocytes (hiPSC-CMs) in a murine postmyocardial infarction model. A specific sEHI (1-trifluoromethoxyphenyl-3-(1-propionylpiperidine-4-yl)urea [TPPU]) and CRISPR/Cas9 gene editing were used to test the hypothesis. TPPU results in a significant increase in the retention of transplanted cells compared with cell treatment alone. The increase in the retention of hiPSC-CMs translates into an improvement in the fractional shortening and a decrease in adverse remodeling. Mechanistically, we demonstrate a significant decrease in oxidative stress and apoptosis not only in transplanted hiPSC-CMs but also in the host environment. CRISPR/Cas9-mediated gene silencing of the sEH enzyme reduces cleaved caspase-3 in hiPSC-CMs challenged with angiotensin II, suggesting that knockdown of the sEH enzyme protects the hiPSC-CMs from undergoing apoptosis. Our findings demonstrate that suppression of inflammation and fibrosis using an sEHI represents a promising adjuvant to cardiac stem cell-based therapy. Very little is known regarding the role of this class of compounds in stem cell-based therapy. There is consequently an enormous opportunity to uncover a potentially powerful class of compounds, which may be used effectively in the clinical setting.

Adenylyl cyclase 5-generated cAMP controls cerebral vascular reactivity during diabetic hyperglycemia.

Elevated blood glucose (hyperglycemia) is a hallmark metabolic abnormality in diabetes. Hyperglycemia is associated with protein kinase A (PKA)-mediated stimulation of L-type Ca2+ channels in arterial myocytes resulting in increased vasoconstriction. However, the mechanisms by which glucose activates PKA remain unclear. Here, we showed that elevating extracellular glucose stimulates cAMP production in arterial myocytes, and that this was specifically dependent on adenylyl cyclase 5 (AC5) activity. Super-resolution imaging suggested nanometer proximity between subpopulations of AC5 and the L-type Ca2+ channel pore-forming subunit CaV1.2. In vitro, in silico, ex vivo and in vivo experiments revealed that this close association is critical for stimulation of L-type Ca2+ channels in arterial myocytes and increased myogenic tone upon acute hyperglycemia. This pathway supported the increase in L-type Ca2+ channel activity and myogenic tone in two animal models of diabetes. Our collective findings demonstrate a unique role for AC5 in PKA-dependent modulation of L-type Ca2+ channel activity and vascular reactivity during acute hyperglycemia and diabetes.

Highly efficient transfection of human induced pluripotent stem cells using magnetic nanoparticles.

PURPOSE: The delivery of transgenes into human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes (hiPSC-CMs) represents an important tool in cardiac regeneration with potential for clinical applications. Gene transfection is more difficult, however, for hiPSCs and hiPSC-CMs than for somatic cells. Despite improvements in transfection and transduction, the efficiency, cytotoxicity, safety, and cost of these methods remain unsatisfactory. The objective of this study is to examine gene transfection in hiPSCs and hiPSC-CMs using magnetic nanoparticles (NPs). METHODS: Magnetic NPs are unique transfection reagents that form complexes with nucleic acids by ionic interaction. The particles, loaded with nucleic acids, can be guided by a magnetic field to allow their concentration onto the surface of the cell membrane. Subsequent uptake of the loaded particles by the cells allows for high efficiency transfection of the cells with nucleic acids. We developed a new method using magnetic NPs to transfect hiPSCs and hiPSC-CMs. HiPSCs and hiPSC-CMs were cultured and analyzed using confocal microscopy, flow cytometry, and patch clamp recordings to quantify the transfection efficiency and cellular function. RESULTS: We compared the transfection efficiency of hiPSCs with that of human embryonic kidney (HEK 293) cells. We observed that the average efficiency in hiPSCs was 43%±2% compared to 62%±4% in HEK 293 cells. Further analysis of the transfected hiPSCs showed that the differentiation of hiPSCs to hiPSC-CMs was not altered by NPs. Finally, robust transfection of hiPSC-CMs with an efficiency of 18%±2% was obtained. CONCLUSION: The difficult-to-transfect hiPSCs and hiPSC-CMs were efficiently transfected using magnetic NPs. Our study offers a novel approach for transfection of hiPSCs and hiPSC-CMs without the need for viral vector generation.

Coupling of SK channels, L-type Ca2+ channels, and ryanodine receptors in cardiomyocytes.

Small-conductance Ca2+-activated K+ (SK) channels regulate the excitability of cardiomyocytes by integrating intracellular Ca2+ and membrane potentials on a beat-to-beat basis. The inextricable interplay between activation of SK channels and Ca2+ dynamics suggests the pathology of one begets another. Yet, the exact mechanistic underpinning for the activation of cardiac SK channels remains unaddressed. Here, we investigated the intracellular Ca2+ microdomains necessary for SK channel activation. SK currents coupled with Ca2+ influx via L-type Ca2+ channels (LTCCs) continued to be elicited after application of caffeine, ryanodine or thapsigargin to deplete SR Ca2+ store, suggesting that LTCCs provide the immediate Ca2+ microdomain for the activation of SK channels in cardiomyocytes. Super-resolution imaging of SK2, Cav1.2 Ca2+ channel, and ryanodine receptor 2 (RyR2) was performed to quantify the nearest neighbor distances (NND) and localized the three molecules within hundreds of nanometers. The distribution of NND between SK2 and RyR2 as well as SK2 and Cav1.2 was bimodal, suggesting a spatial relationship between the channels. The activation mechanism revealed by our study paved the way for the understanding of the roles of SK channels on the feedback mechanism to regulate the activities of LTCCs and RyR2 to influence local and global Ca2+ signaling.