Translating cardiovascular ion channel and Ca²⁺ signaling mechanisms into therapeutic insights

Abstract

Cardiovascular diseases remain the leading cause of mortality worldwide, driven by complex, multi-scale mechanisms that span molecular, cellular, and organ-level dysfunction. Effective therapeutic strategies therefore require integrative approaches that link fundamental biology to translational applications. The 8th UC Davis CardioVascular Symposium gathered experts in ion channel biophysics, Ca²⁺ signaling, arrhythmia mechanisms, and cardiovascular physiology to discuss recent advances and define emerging priorities. This white paper synthesizes the key themes and consensus points that emerged, highlighting progress in two core domains: (1) advances in cardiovascular electrophysiology and arrhythmia mechanisms, and (2) spatiotemporal dynamics of Ca²⁺ signaling in cardiac and vascular function and remodeling. We also identify conceptual and technical challenges that must be addressed to accelerate therapeutic discovery and emphasize cross-cutting opportunities where experimental and computational approaches can converge. By integrating ion channel biology and Ca²⁺ signaling mechanisms across scales, this work outlines new directions for advancing cardiovascular research and treatment.

Graphical Abstract:

The white paper integrates mechanistic discoveries across ion channel biology, Ca²⁺ signaling, and multiscale cardiovascular physiology to highlight new opportunities for accelerating research and guiding next-generation therapies.

1. Introduction

Cardiovascular diseases remain the leading cause of death worldwide, reflecting the enormous complexity and multi-scale nature of the underlying pathophysiology (Ahmad et al., 2024). Given the elaborate interplay of molecular and cellular dysfunction, structural remodeling, and organ-level failure, meaningful therapeutic approaches to combat cardiovascular diseases require integrative approaches. The 8^th UC Davis CardioVascular Symposium convened experts in ion channel biophysics, Ca²⁺ signaling, arrhythmia mechanisms, and cardiovascular physiology and pathobiology to discuss recent advances and define emerging priorities for research and translation. Our primary goal was to catalyze interdisciplinary discussion and collaboration. We aimed to identify promising areas where new experimental models and computational tools can converge to yield mechanistic insights, enhance fundamental understanding, and transform and improve the treatment of cardiovascular diseases. This white paper summarizes key themes and consensus points that emerged during the symposium, highlights current research frontiers, and outlines the conceptual and technical challenges that must be addressed to accelerate therapeutic discovery. The work presented here is organized around two core domains: advances in cardiovascular electrophysiology and new insights into the spatiotemporal dynamics associated with the coordinated Ca²⁺ signaling in cardiac and vascular function and remodeling. Finally, we address the unresolved challenges and cross-cutting opportunities for translational innovations. The bold attempt to synthesize discussion of ion channels and Ca²⁺ signaling mechanisms across scales and systems frames a valuable perspective for generating new research questions.

2. Cardiovascular Electrophysiology

Cardiovascular electrophysiology encompasses the electrical activity that drives cardiac contraction and vascular smooth muscle function, with disruptions directly linked to arrhythmias, conduction disorders, and altered vascular reactivity. As electrical signaling involves coordinated activity of multiple ion channels, therapeutic approaches targeting specific electrophysiological components require careful consideration. This section emphasizes the importance of ion channels, regulatory signals, and their impact on the broader electrophysiological landscape within cardiac and vascular tissues in health and disease.

2.1: Cardiac electrophysiology and function

Ion channels in cardiomyocytes orchestrate the cardiac action potential through precisely coordinated activation of distinct channel families. Voltage-gated Na⁺ channels (Na_V1.5) initiate rapid depolarization, while voltage-gated L-type Ca²⁺ channels (LTCCs; Ca_V1.2) sustain the plateau phase and trigger excitation-contraction coupling by initiating Ca²⁺ release from the sarcoplasmic reticulum (SR). Multiple K⁺ channel subtypes, including delayed rectifier channels (KCNQ1/KCNH2) and inward rectifier channels (KCNJ2), govern repolarization and establish the resting membrane potential. Gap junction channels (connexins) provide electrical coupling between cardiomyocytes, enabling synchronized propagation throughout the myocardium. Disruption of these channels through genetic mutations, disease, or pharmacological interference can lead to arrhythmias and sudden cardiac death, making them critical targets for therapeutic interventions. Below we provide specific relevant examples of roles played by cardiac ion channels in health and disease and their promise as therapeutic targets. While ventricular arrhythmias were a primary focus of the symposium, analogous ion channel remodeling also underlies atrial arrhythmias, including atrial fibrillation. Many of the mechanistic themes described here for monogenic perturbations provide mechanistic clarity and precision interventions, but these concepts also apply to acquired cardiac diseases, such as heart failure and atrial dilation.

2.1.1: Treating arrhythmias by targeting ion channels: the road to precision medicine

Dissecting how regulation of ion channels influence the pathogenesis of cardiac arrhythmias represents a major hurdle for treating patients with genetic cardiomyopathies and/or acquired conditions such as ischemic heart disease (Liu et al., 2009; Al-Khatib et al., 2018). In particular, the delay between the first clinical manifestations of the disease and diagnosis has deleterious consequences on patient outcomes. This is further complicated by the absence of high throughput, translational models that effectively predict the onset of arrhythmogenic events and/or if patients will be responsive to the selected treatment (Priori et al., 2013; Zeppenfeld et al., 2022). Indeed, pharmacological interventions, like β-blockers, are frequently aimed at counteracting the downstream effects of ion channel dysfunction but often fail to protect patients from recurring episodes of life-threatening arrhythmias (Priori et al., 2002; Mazzanti et al., 2022). In addition, most anti-arrhythmic drugs carry an intrinsic pro-arrhythmic risk that limits their application to clinical practice (Tisdale et al., 2020). This consideration is also relevant to the treatment of atrial fibrillation, which, while associated with significant morbidity, is not typically life-threatening; therefore, therapeutic strategies must be designed carefully to avoid unintended effects on ventricular rhythm. Targeted approaches directed toward atrial-selective currents including the ultrarapid delayed rectifier (KCNA5), acetylcholine-activated (KCNJ3/KCNJ5), two-pore domain (KCNK3), and small-conductance Ca²⁺-activated (KCNN2) K⁺ channels are anticipated to provide improved therapeutic potential. However, the translation of preclinical findings into clinical efficacy has thus far been inconsistent, as recently summarized (Wiedmann & Schmidt, 2024). These limitations reflect the complexity of ion channel regulation, from molecular to whole-organ levels, and underscore the challenge of selectively targeting events to specifically prevent the “avalanche” effect of ion channel dysfunction and restore membrane stability with limited (adverse) effects on other ion channels. For example, in patients with a clinical diagnosis of Catecholaminergic Polymorphic Ventricular Tachycardia (CPVT), β-blockers (especially non-selective ones, like nadolol and propranolol) are the recommended first-line therapy (Ackerman et al., 2017; Mazzanti et al., 2022; Zeppenfeld et al., 2022), but in ~30% of patients, implantable cardioverter-defibrillator devices are necessary to improve patients’ outcomes (Mazzanti et al., 2022). The class Ic anti-arrhythmic flecainide (a Na⁺ channel blocker) has emerged as an effective adjunctive therapy in CPVT, directly inhibiting RyR2-mediated Ca²⁺ release and suppressing delayed afterdepolarizations, thereby reducing exercise-induced ventricular arrhythmias (Kannankeril et al., 2017). These studies clearly underscore the disconnect between the underlying cause of the disease and the current treatment strategies, emphasizing the need for more targeted, mechanism-based therapeutic approaches.

Gene therapies represent a promising alternative strategy that could overcome the limitations of pharmacological agents (Bains et al., 2025). In the last 10 years, a growing number of phase I-III clinical trials have been conducted to test the safety and efficacy of gene therapies for a variety of disorders, including cardiac arrhythmias (Alliance for Regenerative Medicine, 2020). The field of gene therapy has suffered major setbacks, mostly related to immunogenicity of vector delivery, lack of efficacy, and serious adverse effects (Lapteva et al., 2020). However, the ever-growing field of vector delivery and novel gene editing strategies, especially with the advent of CRISPR-Cas9 systems, promises to overcome some of these limitations, and has led to almost 30 FDA-approved gene-based drug products (Raguram et al., 2022; U.S. Food and Drug Administration, 2025). The broad toolkit of gene editing technologies, offering diverse and adaptable approaches while maintaining target specificity, is particularly appealing for treating cardiac arrhythmias, where ion channel dysregulation can be addressed from multiple angles (Grisorio et al., 2024; Bains et al., 2025) (Fig. 1A). As recently reviewed (Grisorio et al., 2024; Bains et al., 2025), arrhythmia substrates could be modified by: genetically correcting the mutated channel, either by silencing (Bongianino et al., 2017) or direct editing (Pan et al., 2018), increasing the expression of functional channels (Denegri et al., 2012; Wang et al., 2025), modulating accessory/ancillary proteins (Yu et al., 2022), and directly counteracting the effect of the mutant in a “variant-independent” approach (Dotzler et al., 2021; D Santiago Castillo, 2023). In the context of CPVT, for example, there are multiple mechanisms that lead to arrhythmias (Liu et al., 2009; Baltogiannis et al., 2019; Ni et al., 2024), all of them involving abnormal Ca²⁺ handling (Shannon et al., 2000; Sobie et al., 2006; Priori & Chen, 2011). Currently proposed gene therapy strategies aim to correct Ca²⁺ dysregulation by silencing the mutated RYR2 allele (Bongianino et al., 2017) and/or rebalancing Ca²⁺ handling by counteracting the effects of mutant RYR2 with the overexpression of CASQ2 (D Santiago Castillo, 2023). In a similar, “variant-independent” approach, Brugada syndrome caused by SCN5A mutation G1746R could be treated by overexpressing an ancillary protein involved in the trafficking of the channel (Dotzler et al., 2021). Comparable strategies have also been explored in preclinical porcine models of atrial fibrillation, focusing on the suppression of KCNH2 and KCNK3 (Amit et al., 2010; Soucek et al., 2012; Schmidt et al., 2019). While this rapidly evolving field holds great promise, with encouraging results from preclinical models (Bains et al., 2025), several key questions remain unresolved, some broadly relevant to gene therapy and others specific to arrhythmia. Critical aspects such as delivery methods, biodistribution, manufacturing of biological agents, and the determination of an effective therapeutic dose must still be clarified, as each can significantly influence treatment outcomes. Importantly, clinical trials exploring the safety and efficacy of gene therapy for cardiac arrhythmias are still in the early stages and definitive data on efficacy and/or potential new limitations have yet to be determined (https://clinicaltrials.gov/study/NCT07148089, 2025). Nevertheless, the ability to selectively target specific steps in ion channel dysregulation, coupled with a relatively low risk of adverse effects, presents a compelling opportunity to advance precision medicine.

Figure 1: Molecular mechanisms of ion channel dysfunction and multi-scale approaches for translation.

Top: Ion channel dysfunction arises from diverse mechanisms, including genetic mutations, altered trafficking, impaired Ca²⁺ handling, disrupted ubiquitin-mediated protein turnover, aberrant protein–protein interactions, and pathological cell–cell crosstalk. These molecular pathways highlight the complex etiology of ion channelopathies and opportunities for targeted interventions, which include pharmacological agents, antisense oligonucleotides, bioengineered proteins, and viral gene delivery. Bottom: Experimental and modeling frameworks operate across multiple levels of biological organization. At the ion channel level, genetic mutations and regulatory mechanisms (including drug interaction) alter gating and conductance properties. Ion channel scale (left): Cryogenic-electon-microscopy–style channel structure and Markov-state scheme illustrating transitions between C = Closed, O = Open, and B = Blocked states. Example single-channel current trace shows channel gating behavior. At the cellular level, studies in native vascular smooth muscle cells (VSMCs), cardiomyocytes, and human induced pluripotent stem cell–derived cells (hiPSCs) capture disease-relevant electrophysiological behaviors. Cell scale (middle): Representative whole-cell electrophysiological traces (voltage recordings). Schematics depict native VSMCs, native cardiomyocytes, and hiPSCs. Overlapping cells indicate population-based and high-throughput experimental and analysis approaches. At the tissue and organ levels, integration of cellular data into computational models connects molecular defects to clinical outcomes and informs personalized medicine. Tissue/organ scale (right): Models of cardiac and vascular tissue, electrocardiogram traces, and blood flow measurements. Together, this multi-scale approach bridges mechanistic insights with translational applications for precision therapies in cardiovascular disease.

In addition to their limited selectivity, a major challenge of pharmacological treatments for cardiac arrhythmias is the difficulty in accurately predicting individual patient responses and potential adverse effects. As a result, computational models and in vitro platforms using human pluripotent stem cell-derived cardiac cells, such as cardiomyocytes (hPSC-CMs), are gaining increasing attention (Casini et al., 2017; Wu et al., 2024; Lopez-Munoz et al., 2025). These high-throughput systems offer promising tools not only for forecasting patient outcomes and identifying side effects, but also for generating new hypotheses about disease pathogenesis—all while preserving strong translational relevance (Zhu et al., 2019; Mangold et al., 2021; Mangold et al., 2022). HPSC-CMs are capable of recapitulating many of the phenotypic features observed in patients, making them a powerful model for studying cardiovascular diseases such as genetic cardiomyopathies and channelopathies (Groen et al., 2024; Wu et al., 2024; Lopez-Munoz et al., 2025). Their adaptability to complementary platforms, such as high-throughput drug screening and CRISPR-based gene editing, further enhances their value as a versatile and translationally relevant tool. As an example, hPSC-CMs have been used in the Comprehensive In Vitro Proarrhythmia Assay (CiPA) initiative, started in 2013, with the aim of identifying the pro-arrhythmogenic potential of newly developed and/or already commercially available compounds (Blinova et al., 2018). Moreover, an increasing number of clinical trials are incorporating patient-derived hPSCs to predict individual responses to investigational treatments. This approach enhances the likelihood of trial success and aids in identifying molecular signatures associated with treatment responsiveness, offering deeper insights into disease mechanisms and enabling more refined patient stratification (Casini et al., 2017; Wu et al., 2024). hPSC-CMs are also extremely useful in characterizing effects of genetic mutations and interventions in a human-relevant context. For example, hPSC-CMs have been used to describe the relationship between Na_V1.5 trafficking and microtubule-associated proteins EB1 (end-binding protein 1) and CLASP1 (cytoplasmic linker associated protein 2), leading to potential new target approaches (Marchal et al., 2021; Nasilli et al., 2023; Nasilli et al., 2024). Despite the many advantages of hPSC-CM technology, their developmental immaturity remains a major limitation, as they more closely resemble fetal rather than adult ventricular cardiomyocytes, hindering clinical translation (Veerman et al., 2015; Tan & Ye, 2018; Karbassi et al., 2020). Advances in computational modeling and artificial intelligence are increasingly valuable for bridging hPSC-CM data with clinical outcomes (Kernik et al., 2019; Gibbs et al., 2023; Reilly et al., 2024; Gibbs & Boyle, 2025). By integrating multi-omics data and comparing key parameters between hPSC-CMs and adult cardiomyocytes, these in silico tools can identify limitations, predict outcomes, and guide refinement of hPSC-CM models, enhancing their translational relevance and reproducibility (Kernik et al., 2019; Morotti et al., 2021; Moreno & Silva, 2023; Reilly et al., 2024) (Fig. 1B).

2.1.2: KCNJ2 and arrhythmia

KCNJ2 is the gene encoding Kir2.1, which is the major component of the inward K⁺ rectifier current (I_K1)(Reilly & Eckhardt, 2021) that is crucial in maintaining membrane stability and complete repolarization during phase 3 of the cardiac action potential (AP). This channel is characterized by a large inward component at membrane voltages negative to the K⁺ equilibrium potential, and a smaller “inward rectifying” outward component that is responsible for membrane stability at physiological voltages (Mascher & Peper, 1969). By reestablishing the resting membrane potential, I_K1 is fundamental in controlling excitability by allowing Na⁺ channels to recover from inactivation (Nichols et al., 1996). Unsurprisingly, mutations modifying the function of Kir2.1, have been strongly associated with arrhythmogenic disorders (Plaster et al., 2001; Priori et al., 2005; Xia et al., 2005; Van Ert et al., 2017). Importantly, due to its role in excitability, KCNJ2 is also expressed in other excitable tissues, such as neurons and skeletal muscle, and patients with loss-of-function (LOF) mutations (i.e., Andersen-Tawil syndrome, ATS) often present with symptoms associated with neurological and skeletal muscle abnormalities (Tristani-Firouzi & Etheridge, 2010). Mutations that cause a gain-of-function phenotype, like short QT syndrome 3 and atrial fibrillation (Priori et al., 2005; Xia et al., 2005), are extremely rare and characterized by a very short AP duration which strongly abbreviates the refractory period of cardiomyocytes, sustaining the formation and stabilization of reentry-maintaining rotors (Noujaim et al., 2007). ATS patients present clinically with polymorphic VT and bidirectional ventricular VT (BiVT), a trait that is characteristic of CPVT, and led to the classification of KNCJ2 mutations as a risk factor for CPVT (Tristani-Firouzi & Etheridge, 2010; Zeppenfeld et al., 2022). Because a major cause of arrhythmias in CPVT patients is an imbalance in Ca²⁺ regulation due to RYR2/CASQ2 mutations, it has been hypothesized that Ca²⁺ dysregulation might also play a role in ATS and other patients carrying KCNJ2 LOF mutations, as a defect in I_K1 directly affects membrane stability, making Ca²⁺-dependent triggered activity more likely (Maruyama et al., 2011; Myles et al., 2015; Reilly et al., 2020). During adrenergic stimulation, the outward component of I_K1 is enhanced, but is either blunted or decreased in KCNJ2 LOF, diminishing repolarization reserve and potentially initiating arrhythmias via phase 3 early afterdepolarizations (EADs) (Vega et al., 2009; Kalscheur et al., 2014; Reilly et al., 2020). Those phase 3 EADs are characterized by a negative take off potential (below I_CaL activation range, closer to I_Na reactivation, between −60 and −30 mV) and a shorter coupling time from the stimulus (Reilly et al., 2020), which differs from phase 2 EADs that are mainly driven by I_CaL reactivation. Importantly, formation of phase 3 EADs seems to be caused by cardiomyocyte Ca²⁺ overload (Morotti et al., 2016) and delayed afterdepolarizations (DADs), mimicking the condition in CPVT; along with AP prolongation, this behavior typifies long QT syndromes. These alterations have important clinical implications as treatments indicated for CPVT (e.g. β-blockers) might be ineffective in preventing malignant VT in KCNJ2 LOF patients (Reilly et al., 2020; Reilly & Eckhardt, 2021). As with other ion channels, I_K1 activation is regulated by specific molecules in its microdomain. One key regulator is phosphatidylinositol 4,5-bisphosphate (PIP₂), a lipid component of the cell membrane that regulates Kir2.1 ionic conductance upon binding (Hansen et al., 2011). Mutations in the PIP₂ binding pockets are often associated with KCNJ2 LOF phenotypes (Lopes et al., 2002; Munawar et al., 2025). In a recent study, a combination of computational analysis and high-throughput functional characterization led to a better understanding of the binding of Kir2.1 and PIP₂, as well as the identification of arrhythmia mechanisms that could inform therapeutic targeting (Munawar et al., 2025). Indeed, integration of computational modeling with other high-throughput systems represents a promising future path towards patient-centric drug development.

2.1.3: SCN5A/Na_V1.5 dysfunction and therapeutic strategies

Beyond K⁺ channels, the past couple of decades have revealed the need for context-specific interventions in modulating Na⁺ channel activity. From the early success of PVC suppression in a context of acquired heart disease, through negative outcomes in patients post-myocardial infarction, to successful arrhythmia suppression in patients with arrhythmogenic cardiomyopathy, direct targeting of Na_V1.5 has demonstrated inconsistent antiarrhythmic efficacy with a significant risk of pro-arrhythmia (Cardiac Arrhythmia Pilot Study [CAPS] Investigators, 1988, Anon, 1989; Gaine et al., 2025). Even in inherited forms of SCN5A gain-of-function channelopathy, Na⁺ channel blockade had variable success, which was dependent on a specific mutation (Zhu et al., 2019).

The Na_V1.5 complexes with various auxiliary Na_Vβ subunits, which further complicates our understanding of the channel’s function and its pharmacological response to therapies. For instance, coupling of Na_V1.5 with either Na_Vβ1 or Na_Vβ3 differentially modulates the effect of lidocaine and ranolazine on Na_V1.5 (Zhu et al., 2021). This biophysical landscape is further complicated by the various Na_V isoforms present in the heart, which may interact with each other directly (Tarasov et al., 2023; King et al., 2024; Dias et al., 2025). It is yet unclear how these isoforms contribute to integrated Na⁺ current, both biophysically and pharmacologically. For instance, Na_V1.5 is known for its ability to change its biophysical properties and exhibit cooperativity when engaged in homomeric interaction (Clatot et al., 2017; Tarasov et al., 2025). Since the probability of these interactions should increase with multiplying cluster size, as is evident when Na_Vβ3 is co-expressed (Salvage et al., 2020), modulation of clustering via augmentation of adhesion of Na_Vβ subunits may be a viable therapeutic avenue. To that end, recent efforts have focused on developing antagonist and agonist peptides that target Na_Vβ1 and modulate its adhesion, thereby impacting intercalated disc structure and function, including the activity of associated Na_V1.5 channels (Veeraraghavan et al., 2018; Williams et al., 2024). Furthermore, Na_V1.5 homomeric interactions within the channel clusters could also impact the pharmacological response to Na_V1.5 blockers, where interacting channels are less sensitive to block by lidocaine relative to single, non-interacting channels (Tarasov et al., 2025). Recent efforts have also identified 14-3-3 as a potential modulator responsible for the biophysical coupling, which may also impact the response of Na_V1.5 to antiarrhythmic drugs (Zheng & Deschenes, 2023).

Another means of impacting Na_V1.5 clustering is through modulation of surface channel expression. Recent efforts have revealed that targeting the CLASP2-EB1 interaction, along with glycogen synthase kinase 3β (GSK3β) inhibition, can facilitate I_Na selectively at the intercalated disc (Marchal et al., 2021). This observation parallels findings obtained with other Na_V isoforms where GSK3β inhibition increases Na_V1.2 and Na_V1.6 surface expression through direct channel interaction and post-translation modification of the C-terminus (James et al., 2015; Baumgartner et al., 2025). However, other reports have suggested detrimental effects of acute GSK3β inhibition in human hearts (Li et al., 2022). Hence, targeting GSK3β requires more in-depth analysis of efficacy in the setting of various pathological states, as well as safety pharmacology assessments both in health and disease.

Na⁺ channels are also known to be regulated by calmodulin and intracellular fibroblast growth factors (FGFs)/fibroblast growth factor homologous factors (FHFs), with mutations in these proteins affecting channel function that may, in part, underlie arrhythmias (Hennessey et al., 2013; Yin et al., 2014; Tarasov et al., 2023; Selimi et al., 2025). Although three different genes encode for calmodulin, the protein is conserved in mammalian hearts. On the other hand, FGF/FHF exhibits diversity in isoform expression (Hennessey et al., 2013). For this reason, Na_V1.5 is differentially regulated depending on the interacting FGF/FHF isoform (Angsutararux et al., 2023). Notably, there appears to be cross-talk between calmodulin and FHF in regulation of Na_V1.5, where FGF13 or FHF1A tune arrhythmogenic late I_Na in calmodulin binding-deficient channels (Abrams et al., 2020; Chakouri et al., 2022). Using the FGF/FHF interaction site on Na_V1.5, recent efforts have generated an engineered peptide modulator that binds Na⁺ channels, and thus reduces the arrhythmogenic late I_Na (Mahling et al., 2025) that is elevated in heart failure and diabetes (Hegyi et al., 2019; Hegyi et al., 2021; Hegyi et al., 2022a; Hegyi et al., 2022b). These findings highlight that targeting Na⁺ channel function through interacting proteins has therapeutic potential for the management of various disorders and disease states.

Outside of focusing on the ion channels and their interacting partners, disease-modifying strategies may also help restore ion channel function in pathological conditions. One such example is targeting the consequences of inflammation. Inflammation-mediated impairment in cell-cell interaction or communication results in Na_V1.5 de-clustering, that compromises cardiac excitability (George et al., 2017; Raisch et al., 2018; Mezache et al., 2020). Strategies that restore cell-cell interaction through preservation of gap junctions at intercalated discs with a Cx43 mimetic molecule, α carboxyl terminus 1 peptide, restore Na_V1.5 clustering, thereby reducing arrhythmias during inflammatory insult (O'Quinn et al., 2011; Rhett et al., 2011; Jiang et al., 2019; Mezache et al., 2023; Laurita et al., 2024). In line with employing disease-modifying strategies, GSK3β inhibition rescued the disease phenotype and restored balance in arrhythmogenic cardiomyopathy (Asimaki et al., 2014). Notably, this strategy also improved cell-cell communication and cardiac function while reducing arrhythmia burden (Chelko et al., 2016).

2.2: Vascular electrophysiology and function

Vascular electrophysiology is fundamental to understanding how blood vessels regulate myogenic tone, diameter, and blood flow. The electrophysiological properties of cells within the vascular wall, including vascular smooth muscle cells (VSMCs), endothelial cells, and pericytes, are primarily governed by a complex array of ion channels and signaling cascades. These systems are critical for maintaining vascular homeostasis and responding to physiological demands. They also enable (mal)adaptation to pathological conditions, with direct implications for cardiovascular disease treatment and drug development. Ion channels playing central roles in vascular cells include voltage-gated Ca²⁺ channels (e.g. LTCCs), K⁺ channels, Cl⁻ channels, and transient receptor potential (TRP) channels, among others. Each cell type in the vascular wall expresses a distinct profile of ion channels that contribute to their specific physiological roles (Tykocki et al., 2017; Hariharan et al., 2020a; Jackson, 2022). The various cellular components are tightly interconnected. Vascular cells can establish both homocellular and heterocellular connections via gap junctions with adjacent cells in arteries, arterioles and capillaries (Wu et al., 2006; Jackson, 2022). These connexin-mediated junctions facilitate the direct transcellular propagation of membrane potential changes and ionic currents initiated by chemical and electrical signals, thereby modulating contractile responses in mural cells and contributing to the coordination of vasomotor tone (Jackson, 2022).

2.2.1: Ion channels in Vascular Smooth Muscle, Endothelial Cells, and Pericytes

VSMCs are pivotal in controlling vessel tone and diameter, thereby regulating systemic blood pressure and regional blood flow. The integrated activity of various ion channels in VSMCs facilitates control of membrane potential and intracellular Ca²⁺ concentration ([Ca²⁺]_i) (Tykocki et al., 2017). Voltage-gated Ca²⁺ channels, especially LTCCs, are crucial for mediating VSMC contraction (Ghosh et al., 2017). Depolarization leads to Ca_V1.2 L-type Ca²⁺ channel opening, allowing Ca²⁺ influx. The rise in intracellular Ca²⁺ binds calmodulin to form the Ca²⁺-calmodulin complex, which activates myosin light chain kinase (MLCK). MLCK phosphorylates myosin light chain 20 (MLC20), increasing actin-myosin crossbridge cycling and force development. Concurrently, reduced MLC phosphatase activity maintains MLC20 phosphorylation, sustaining VSMC contraction. LTCCs are primary targets for antihypertensive agents such as nifedipine. K⁺ channels, particularly large-conductance Ca²⁺-activated K⁺ channels (BK_Ca), ATP-sensitive K⁺ channels (K_ATP), and voltage-gated K⁺ channels (K_V), also play a crucial role in modulating VSMC membrane potential (Jackson, 2017). Their activation promotes hyperpolarization and vasodilation by reducing LTCC opening and thus Ca²⁺ influx. TRP channels contribute to mechanosensation and Ca²⁺ signaling in response to stretch, pressure, and other stimuli (Earley & Brayden, 2015).

Tissue-specific regulation of LTCC Ca_V1.2 channels

Given the importance of LTCC Ca_V1.2 channels in excitation-contraction coupling in both cardiac and vascular smooth muscle, their regulation is the subject of intense scrutiny. It is well established that cardiac and VSMCs express different pore-forming α1_C splice variants and β subunits required for functional LTCC Ca_V1.2 channels (Navedo & Santana, 2013; Ghosh et al., 2017; Hu et al., 2017). In addition, Ca_V1.2 channels in cardiac and vascular smooth muscle are known to exist within highly organized and distinctive signaling complexes (Dixon, 2022; Pereira da Silva et al., 2022). These signaling complexes play a crucial role in regulating channel activity and tissue function by protein kinase A (PKA), protein kinase C (PKC), and other signaling pathways (Fig. 2).

Figure 2: Divergent regulation of L-type Ca²⁺ channels in cardiac vs. vascular muscle.

In cardiomyocytes (left), β-adrenergic receptor (β-AR) stimulation activates PKA, leading to phosphorylation of the small GTPase Rad, which relieves its inhibitory effect on Ca_V1.2 channels and enhances Ca²⁺ influx. In contrast, angiotensin II type 1 receptor (AT1R) activation triggers phospholipase C-dependent hydrolysis of PIP₂ and subsequent endocytosis of Ca_V1.2 channels, reducing Ca²⁺ entry. In vascular smooth muscle cells (right), P2Y₁₁ receptor activation engages PKA signaling to potentiate Ca_V1.2 activity through phosphorylation of the α_1C subunit at serine 1928 (S1928). Additionally, AT1R stimulation promotes PKC-dependent phosphorylation of α_1C at S1928, enhancing Ca_V1.2 clustering, activity, and cooperative gating. These distinct regulatory mechanisms highlight the tissue-specific modulation of Ca_V1.2 channels in cardiac versus vascular muscle, with divergent implications for physiological contractility and pathological remodeling.

Notably, PKA modulation of Ca_V1.2 represents a key regulatory mechanism in health and disease. Early work identified the α1_C serine 1928 amino acid as a readily phosphorylatable site in response to β-adrenoceptor and PKA signaling in cardiac Ca_V1.2 channels (De Jongh et al., 1996). While this result pointed to a potential role for α1_C serine 1928 phosphorylation in β-adrenoceptor/PKA regulation of Ca_V1.2 channels and cardiac function, subsequent studies found that the phosphorylation of this site or 20+ other potential PKA substrate sites were not necessary (Ganesan et al., 2006; Lemke et al., 2008; Katchman et al., 2017). Rather, it was found that the small GTPase Rad acts as a brake on cardiac Ca_V1.2 activity, which is relieved after β-adrenoceptor/PKA phosphorylation of the Rad associated with the channel (Manning et al., 2013; Liu et al., 2020; Papa et al., 2021; Papa et al., 2022). The current model suggests that the phosphorylated Rad then moves away from the Ca_V1.2 complex, resulting in increased Ca_V1.2 channel activity.

The regulation of vascular Ca_V1.2 by PKA is more controversial, with studies suggesting that the kinase inhibits, potentiates, or has no effect on channel function (Keef et al., 2001; Ghosh et al., 2017). Moreover, specific mechanisms by which PKA affects vascular Ca_V1.2 activity are unclear. Recent studies, however, have shown that elevated extracellular glucose, which mimics diabetic hyperglycemia, potentiates Ca_V1.2 activity through a PKA-dependent mechanism (Navedo et al., 2010b; Nystoriak et al., 2017; Prada et al., 2019; Syed et al., 2019; Prada et al., 2020; Martin-Aragon Baudel et al., 2022). Importantly, the hyperglycemia-induced PKA regulation of vascular Ca_V1.2 channels required α1_C phosphorylation at serine 1928 (Nystoriak et al., 2017; Prada et al., 2019; Martin-Aragon Baudel et al., 2022). The importance of α1_C phosphorylation at serine 1928 in vascular, but not cardiac Ca_V1.2 channels, reflects the tissue-specific and context-dependent regulation of the Ca_V1.2 channel.

Another striking example of the tissue-specific regulation of Ca_V1.2 channels relates to the modulation of Ca_V1.2 activity by angiotensin II signaling in cardiac versus vascular cells. In cardiac myocytes, angiotensin II led to a depletion of the membrane phospholipid PIP₂, resulting in Ca_V1.2 channel internalization and reduced Ca²⁺ current (Voelker et al., 2023). This effect was correlated with a reduction in the amplitude of Ca²⁺ transients (Voelker et al., 2023). These results suggest a potential protective mechanism that prevents cardiomyocyte Ca²⁺ overload during pathological conditions. Conversely, in VSMCs, angiotensin II promoted PKC-mediated α1_C phosphorylation at serine 1928, resulting in enhanced clustering, channel activity, and cooperative gating. The changes in vascular Ca_V1.2 biophysical properties drive pathological vasoconstriction and hypertension (Navedo et al., 2010a; Flores-Tamez et al., 2024).

The contrasting results in LTCC CaV1.2 regulation in cardiac and vascular muscle highlight how evolution has fine-tuned the same ion channel to respond to identical stimuli (e.g. PKA, PKC signaling) through distinct molecular mechanisms and functional outcomes. Accordingly, data emphasize significant tissue-specific and context-dependent regulation of CaV1.2 channels by PKA and PKC. These differences have profound physiological and pathological implications, which could be exploited for the development of new therapeutic interventions.

Endothelial Cells

Endothelial cells lining blood vessels play crucial roles in sensing hemodynamic forces and releasing vasoactive substances such as nitric oxide (NO), prostacyclin, and endothelin-1 (Jackson, 2022). Their unique electrophysiological profile enables rapid responses to mechanical and chemical stimuli. Inward rectifier K⁺ channels (K_ir) and intermediate-conductance Ca²⁺-activated K⁺ channels (IK_Ca) maintain endothelial membrane potential and facilitate hyperpolarization, which transmits to adjacent VSMCs via myoendothelial gap junctions (Jackson, 2016). Importantly, this endothelial hyperpolarization-driven vasodilatory process is a core feature of endothelium-derived hyperpolarization (EDH/EDHF), which is a major regulator of tone in small resistance arteries (Feletou & Vanhoutte, 1988; Garland et al., 1995). TRP channels, particularly TRPV4, TRPC1 and TRPA1, contribute to endothelial Ca²⁺ signaling essential for NO production and vasodilatory responses (Thakore & Earley, 2019). The mechanosensitive polycystins PKD1 and PKD2, which form a cation-permeable complex in endothelial cells, have been implicated in flow-dependent Ca²⁺ signaling and vascular tone regulation (AbouAlaiwi et al., 2009; Hamzaoui et al., 2022). Likewise, chloride channels such as TMEM16A (ANO1) contribute to endothelial electrophysiology and vasomotor control through their effects on membrane potential and Ca²⁺-dependent signaling pathways, and their dysregulation has been linked to endothelial dysfunction in hypertension (Ma et al., 2017; Al-Hosni et al., 2024). Together, these channels complement K⁺ and TRP conductances in integrating mechanical and chemical cues that govern endothelial function and vascular tone.

Pericytes

Pericytes are mural cells embedded within capillary basement membranes that regulate capillary diameter and blood-brain barrier integrity (Hartmann et al., 2022). Despite having a distinctive morphology and expressing different markers than VSMCs, there are similarities regarding their contractile machinery. K⁺ channels dominate pericyte ion channel expression, with Kir6.1 accounting for nearly half of total ion channel pore-forming gene expression (Hariharan et al., 2020b). Indeed, pericyte K_ATP channels contribute to long-range electrical signaling from capillaries to arterioles in response to neuronal signals to promote spatiotemporally precise energy distribution in the brain (Isaacs et al., 2024). K⁺ currents regulating pericyte resting membrane potential are primarily mediated by Kir2 family, BK_Ca and Kv1 channels (Sancho et al., 2024). Ca²⁺ signaling in these cells involves voltage-gated Ca²⁺ channels and TRP channels (Hariharan et al., 2020b), though the involvement of voltage-gated Ca²⁺ channels decreases with distance from upstream arterioles, highlighting functional heterogeneity among pericytes (Klug et al., 2023). TRPC3 channel activation has recently been shown to be critical for proximal pericyte depolarization and pressure-induced contraction (Ferris et al., 2025).

2.2.2: Sex-specific differences in vascular electrophysiology

Accumulating evidence points to significant sex-specific differences in the electrophysiological properties and functional responses of vascular cells. Sex-based biological differences influence the expression, distribution, and functional activity of ion channels in vascular cells. These differences lead to sex-specific variation in vascular tone, reactivity, and disease susceptibility with important therapeutic implications. Moreover, they have profound implications for sex-specific susceptibilities to cardiovascular diseases and for developing targeted therapeutic interventions.

Differential sensitivity to Ca²⁺ channel blockers in males and females

Female VSMCs demonstrate enhanced sensitivity to Ca²⁺ channel blockers compared to males, resulting in greater vasodilation and more significant blood pressure drops in response to drugs like nifedipine (Kloner et al., 1996; Ueno & Sato, 2012). Computational modeling has confirmed and predicted this enhanced sensitivity (Hernandez-Hernandez et al., 2024). Accordingly, multiple contributing factors have been suggested, including higher baseline L-type Ca²⁺ channel activity in females providing greater substrate for inhibition (O'Dwyer et al., 2020), estrogen modulation of channel expression and function (Vega-Vela et al., 2017), and sex differences in VSMC membrane lipid composition affecting drug pharmacokinetics (Arosio et al., 2022; Varghese et al., 2025). These findings have immediate clinical relevance, underscoring the necessity of considering sex as a biological variable in antihypertensive drug development and dosing regimens. Recognizing enhanced female responsiveness to Ca²⁺ channel blockers could guide more precise dosing strategies and reduce adverse effects.

Angiotensin II type 1 receptor activation differs between sexes

Emerging research highlights sex-specific differences in angiotensin II type 1 receptor (AT1R) activation (Nwia et al., 2023). Angiotensin II, a key effector of the renin-angiotensin-aldosterone system, exerts potent vasoconstrictive effects primarily through the AT1R expressed on VSMCs. Activation of AT1R triggers G protein-coupled receptor signaling cascades that elevate intracellular Ca²⁺ levels and promote vasoconstriction.

Male VSMCs typically exhibit stronger contractile responses to angiotensin II, potentially due to higher AT1R expression or enhanced G protein signaling efficacy (Loukotová et al., 2002). In contrast, females show attenuated responses to angiotensin II, possibly because of estrogen-mediated downregulation of AT1R expression (Nickenig et al., 2000), or an increase in the expression of estrogen receptors (Ma et al., 2010). Together with the protective role of estrogen and a higher NO production in females (Viegas et al., 2012), these factors may damp downstream signaling pathways, including reduced phospholipase C activation and diminished inositol-1,4,5-triphosphate (IP₃)-mediated Ca²⁺ release.

AT₂Rs, which generally oppose AT₁R-mediated responses, are also more highly expressed in female than in age-matched males and are considered key players in lowering BP in females (Sandberg & Ji, 2008; Mishra et al., 2016). Sex hormones may augment these functional differences, since testosterone tends to enhance AT1R activity, whereas estrogen exerts suppressive effects (Chinnathambi et al., 2014). These mechanisms likely contribute to the higher prevalence of hypertension and vascular remodeling in males and the relative protection observed in premenopausal females. However, declining estrogen levels with aging can shift this balance, altering AT1R/AT2R signaling and increasing cardiovascular risk in postmenopausal women (Costa et al., 2016; Nicholson et al., 2017).

Integrated perspective on sex-specific vascular regulation

The interplay between ion channel activity, endothelial signaling and hormonal regulation creates dynamic sex-specific control of vascular tone. Estrogen enhances endothelial NO production, augments K⁺ channel activity, and reduces intracellular Ca²⁺ levels in VSMCs, collectively favoring vasodilation (Miller & Duckles, 2008). Female cerebral arteries also exhibit higher BK_Ca channel β1 subunit expression, increasing Ca²⁺ sensitivity and facilitating vasodilation (Li & Qiu, 2015), which may partly underlie the sex-based differences in stroke outcomes. When integrated with sex-dependent signaling described above, these ion channels and hormone-driven mechanisms may help explain why males exhibit greater vascular contractility and higher hypertension prevalence. Meanwhile, females remain protected during premenopause, which may have strong (Nickenig et al., 2000) implications for age-specific therapeutic strategies.

Computational approaches have been helpful in revealing how seemingly disparate sex-specific differences integrate at the cellular and tissue levels. The Hernandez-Hernandez model demonstrated that sex-specific ion channel expression profiles, with females showing higher K_V2.1 expression and longer activation time constants versus male K_V1.5 dominance, combine with hormonal influences to create fundamentally different cellular excitability patterns (Hernandez-Hernandez et al., 2024). Their simulations revealed that these molecular differences amplify when cells are coupled in tissue, predicting enhanced female sensitivity to Ca²⁺ channel blockers that match clinical observations. Future computational models integrating estrogen's multi-target vascular effects (ion channel modulation, AT1R downregulation, enhanced NO signaling) with testosterone's AT1R-promoting actions could predict comprehensive vascular responses across different hormonal states. This will be particularly important during the critical transition from premenopausal protection to postmenopausal risk. The findings stemming from concerted efforts between computational modelers and bench scientists may spur further insights into how vascular smooth muscle regulation helps fine-tune blood flow, and how this fundamental process is controlled and organized in different organs and systems.

2.2.3: Vascular physiology in action: neurovascular coupling and K⁺ signaling

Brain vascular networks must detect and respond to neural activity to deliver oxygen and nutrients on-demand through neurovascular coupling (NVC) (Iadecola, 2017). This process is mediated by the sophisticated ion channel machinery of endothelial and mural cells that serves to sense neural activity and convert stimuli into changes in intracellular Ca²⁺ levels and/or membrane potential. Here, we briefly focus on the evolving understanding of brain capillary ion channels and their impact on NVC, findings from which could ultimately be broadly applicable to many tissues such as cardiac and skeletal muscle (Garcia & Longden, 2020; Longden et al., 2023; Longden & Lederer, 2024).

Capillary endothelial cells express Kir2.1 and K_ATP channels as key hyperpolarizing conductances responding to neuron-derived factors like K⁺ and receptor agonists (Longden et al., 2017; Sancho et al., 2022). Cation channels, including TRPV4, TRPA1, and mechanosensitive Piezo1 channels, modulate membrane potential and initiate Ca²⁺ signaling that facilitates NO production, and potentially other signaling molecules (Harraz et al., 2018; Longden et al., 2021; Thakore et al., 2021; Harraz et al., 2022). Another major player in endothelial cells is the IP₃ receptor (IP₃R) (Longden et al., 2021). Further control of NVC comes from capillary pericytes, which express influx cationic channels such as LTCCs, TRPC3, and Orai (Klug et al., 2023; Phillips et al., 2023; Ferris et al., 2025). Yet, the expression profile of these ion channels is not homogenous across the capillary network, adding complexity to the generalization of the findings of different studies. Several K⁺ channels are present in pericytes, such as K_ATP, Kir2, voltage-activated Kv, and BK_Ca channels (Hariharan et al., 2022; Sancho et al., 2022; Sancho et al., 2024). The Ca²⁺-activated Cl⁻ channel TMEM16A has been additionally described in ensheathing pericytes to play a depolarizing role that promotes contraction (Korte et al., 2022). These findings together underscore the exponential growth in the identification of functional ion channels in brain capillaries over the past decade. We anticipate this will continue with not only the discovery of new ion channels in these compartments of the vasculature, but also with the identification of key regulatory pathways that dictate ion channel function and their influence on blood flow regulation.

Computational modeling studies focused on capillary ion channel conductances and their control may provide crucial insight into the involvement of ion channels in the regulation of NVC. Indeed, a model by Moshkforoush and colleagues (Moshkforoush et al., 2020) suggests a key role for the Kir2.1 channel as a sensor of neuronal activity and an amplifier of retrograde electrical signaling in the brain vasculature. Furthermore, Djurich and Secomb analyzed K⁺ diffusion in silico and predicted that astrocyte endfoot geometry does not prevent extracellular K⁺ in the perivascular space from rising to levels sufficient to cause vasodilation (Djurich & Secomb, 2024). These results support experimental evidence that K⁺ diffusion is a key NVC signaling mechanism and indicate that perivascular K⁺ could increase along timescales that match the rapid dilations characteristic of neurovascular coupling (Djurich & Secomb, 2024). While these modeling studies provide useful insights that can inform further experimentation, a more comprehensive description of ion channel conductances in endothelial cells and pericytes is much needed to gain further insight into the regulation of NVC.

These principles are likely to be important in other vascular beds and not limited to the brain microcirculation. For instance, a similar set of mediators to those proposed to be important for NVC have been implicated in control of skeletal muscle vasculature, including K⁺ ions, NO, epoxyeicosatrienoic acids (EETs), prostaglandins, and adenosine (Ko et al., 1990; Symons et al., 1991; Dirnagl et al., 1993; Hellsten et al., 1998; Stamler & Meissner, 2001; Clifford & Hellsten, 2004; Lacroix et al., 2015; Echagarruga et al., 2020; Longden & Lederer, 2024). Similar studies are warranted to determine whether these are global control mechanisms that are broadly applicable (e.g., in kidney, liver, heart, etc.). It will also be important to elucidate specific molecular details of control and how these differ between distinct vascular beds. This knowledge could reveal important opportunities for specific therapeutic interventions selectively targeting a vascular bed of interest.

Despite the many advances in vascular biology, there are multiple areas for further development. As noted above, there is a pressing experimental need to identify the mediators of vascular and neurovascular communication. We now know that multiple signaling molecules mediate vascular and neurovascular communication with high redundancy, ensuring fidelity (Ko et al., 1990; Dirnagl et al., 1993; Lacroix et al., 2015; Echagarruga et al., 2020). However, a deeper understanding of the spatiotemporal organization of these distinct signaling mechanisms (e.g. ionic (K+), gaseous (NO), and lipid-based EETs and prostanoids) is crucial for developing targeted therapeutic interventions for heart failure, hypertension, stroke, vascular dementia, and other vascular and neurovascular disorders. New computational models should also be developed to integrate these signaling pathways, as they may help refine our understanding of the vasculature.

2.3: Targeting ubiquitin pathways for ion channel regulation

A novel approach for cardiovascular ion channel regulation, particularly in the setting of trafficking-deficient ion channelopathies, is modulation of deubiquitination (Fig. 3). Reduced surface density of ion channels, resulting from abnormalities in trafficking or plasma membrane stability, is a common mechanism behind the loss of function seen in many disease-causing ion channel variants. Hence, stabilization of protein expression represents an attractive agnostic approach that could be used for various pathologies. To achieve selective removal of ubiquitin from target proteins without disrupting others, engineered deubiquitinases (enDUBs) have been developed with a nanobody fused to the catalytic domain of a deubiquitinase in order to rescue KCNQ1 mutation associated with long QT (Kanner et al., 2020). This work has led to the development of enDUBs with preferences for hydrolyzing different polyubiquitin linkages (Shanmugam et al., 2024). Specifically, targeting the K11 chain promoted endoplasmic reticulum (ER) retention/degradation, enhanced endocytosis, and reduced recycling. In contrast, targeting K29/K33 chains impacted ER retention/degradation, targeting K63 chain affected endocytosis and recycling, and targeting K48 chains impacted forward trafficking. Hence, linkage-selective enDUBs could not only help elucidate the expression deficit caused by distinct ion channel disease mutations, but they can be harnessed for precision medicine. Here, the most efficacious polyubiquitin linkage type can be selected for targeted therapy in a particular scenario of ion channel disease mutations. As this technology remains at a proof-of-concept stage, key translational challenges such as in vivo delivery, tissue specificity, and minimizing off-target deubiquitination will need to be addressed before clinical implementation. Nevertheless, this approach highlights a promising conceptual advance in correcting ion-channel trafficking defects.

Figure 3: Targeting ubiquitin pathways for ion channel regulation.

Surface expression of cardiac ion channels is tightly controlled by specific polyubiquitin chains that differentially regulate forward trafficking, endocytosis/recycling, and degradation of membrane ion channels. Targeting K48-linked ubiquitination primarily affects forward trafficking to the plasma membrane, whereas targeting K11- and K63-linked chains affects endocytosis, recycling, and retention/degradation.

Ion channelopathies encompass a remarkable diversity of inherited and acquired cardiovascular disorders arising from molecular defects in gating kinetics, impaired trafficking, and altered protein stability. Beyond inherited channelopathies primarily reflecting genetic mutations, acquired channelopathies often result from environmental stressors, ischemic injury, inflammation, or metabolic disturbances, further complicating therapeutic interventions. This mechanistic diversity underscores the critical need for precision therapeutic approaches: identifying and correcting specific molecular and cellular mechanisms represents an essential paradigm shift toward personalized therapies that can effectively mitigate both inherited and acquired ion channel diseases. Beyond cardiac applications, this approach holds promise for vascular pathologies where ion channel trafficking defects contribute to altered vascular reactivity and diseases such as hypertension. An initial focus could be on voltage-gated Ca2+ channels and K+ channels, which are critical for vascular smooth muscle function and endothelial regulation of vascular reactivity. The repurposing of existing drugs, the use of multichannel blockers, and synergistic drug combinations may also have therapeutic potential. In the context of atrial fibrillation, a phase II trial investigating the multichannel blockade of INa, late INa, ICaL, and IKr by HBI-3000 is ongoing, while the completed phase II HARMONY trial evaluating ranolazine (a late INa blocker) combined with reduced dose dronedarone (a multichannel blocker) demonstrated synergistic reduced atrial fibrillation burden and a favorable safety profile (Reiffel et al., 2015; Saljic et al., 2023; Wiedmann & Schmidt, 2024).

3. Ca²⁺ signaling dynamics in cardiac and vascular cells

It has been more than 40 years since the discovery of the basic mechanisms governing cardiac and vascular smooth muscle cell contraction (Bohr, 1973; Fabiato, 1983; Somlyo & Himpens, 1989). However, the tenets of these mechanisms still stand. In cardiomyocytes, depolarization during the AP opens LTCCs in the surface sarcolemma and t-tubules, and the resulting Ca²⁺ influx triggers RyR2-mediated Ca²⁺ release and contraction. In VSMCs, Ca²⁺ influx through LTCCs is fundamental for contraction (Golenhofen et al., 1973; Belardinelli et al., 1979; Karaki & Weiss, 1984; Knot & Nelson, 1998), with contributions (depending on context) from other plasma membrane ion channels and SR Ca²⁺ release via IP₃R (Tykocki et al., 2017). In contrast, the role of Ca²⁺ in triggering capillary contraction by pericytes is only now beginning to emerge (Gonzales et al., 2020). Here, the mechanisms appear to be broadly similar to VSMCs, with recent work showing that increases in capillary pressure trigger pericyte depolarization, Ca²⁺ elevation, contraction, and directed blood flow (Klug et al., 2023).

3.1: Cardiac Ca²⁺ dynamics

Over the years, there has been growing appreciation that muscle cell Ca²⁺ homeostasis relies on intricate, nanoscale interactions between participating channels. New applications of super-resolution imaging technologies have revealed that LTCCs exhibit clustering behavior that allows their coupled gating in both VSMCs and cardiomyocytes (Del Villar et al., 2021; Martin-Aragon Baudel et al., 2022). In cardiomyocytes, RyR2 release events called Ca²⁺ sparks rely on interaction with nearby channels (Hou et al., 2023), which may occur spontaneously or in response to LTCC currents. Interestingly, Ca²⁺ sparks are generated by RyR2s within the same cluster but can also propagate to trigger nearby RyR2 clusters (Hou et al., 2023; Hurley et al., 2023). When this propagation exceeds a few microns, an arrhythmogenic Ca²⁺ wave occurs, as Ca²⁺-induced Ca²⁺ release propagates across the cell. Removal of this released Ca²⁺ by the Na⁺-Ca²⁺ exchanger induces an inward current, causing delayed afterdepolarizations. Recent advances in electron microscopy have shown that cross-talk between LTCC and RyR2 clusters is truly nanoscale in nature, as the dyadic cleft between t-tubules and SR membranes can measure as little as 6-7 nm across (Rog-Zielinska et al., 2021) and a single junction is 50-200 nm in diameter.

Ongoing work continues to provide new understanding of the vast web of regulatory factors that modulate Ca²⁺ homeostasis in cardiomyocytes and VSMCs. At the smallest scale, the function of individual LTCCs, RyRs, and IP₃Rs is critically determined by posttranslational modifications, including phosphorylation, oxidation, nitrosylation, and O-GlcNAcylation. However, accumulating data have indicated that these channels are also dependent on plasticity of the channel’s physical arrangements. Here, the channels’ partner proteins appear to play key roles, since Junctophilin-2 (JPH2) and Bridging Integrator-1 (BIN1) have been shown to not only control dyadic structure, but to bind and position LTCCs and RyRs (Jones et al., 2018; Dixon, 2022). Evidence from both cardiomyocytes and VSMCs indicates that posttranslational modifications also control channel positioning. Indeed, acute phosphorylation during β-adrenoceptor stimulation leads to greater clustering of LTCCs (Del Villar et al., 2021) and rearrangement of RyRs into a corner-to-corner arrangement of the channels (Asghari et al., 2020). In cardiomyocytes, this repositioning likely favors LTCC-RyR2 crosstalk. However, with more prolonged phosphorylation, as occurs during heart failure, RyR2s clusters disperse (Kolstad et al., 2018; Sheard et al., 2022; Shen et al., 2022). This dispersion not only reduces the overall fidelity of Ca²⁺-induced Ca²⁺ release but also disfavors the generation of Ca²⁺ waves (Shen et al., 2022; Hou et al., 2023).

Mutations in these central Ca²⁺ handling proteins also have established links to disease. Pathological enhancement of diastolic SR Ca²⁺ leak through RyRs underlies CPVT, caused by point mutations in RYR2, calsequestrin and calmodulin. CPVT predisposes to delayed afterdepolarizations, triggered beats, and premature ventricular contractions, and can be mimicked by maladaptive Ca²⁺/calmodulin-dependent protein kinase II (CaMKII) activation as seen in heart failure. Mutations of α1c, the pore-forming subunit of the LTCC Ca_V1.2, are implicated in arrhythmogenesis and vascular pathology. Gain-of-function mutations in CACNA1C are linked to Timothy syndrome (long QT 8), a severe multisystem disorder associated with severe cardiac and neurological defects (Splawski et al., 2004; Bauer et al., 2021; Liu et al., 2023). These mutations can alter activation, voltage-dependent inactivation and/or Ca²⁺-dependent inactivation parameters of the channel, changing the Ca_V1.2 ‘window current’ (Weiss et al., 2010; Harvey & Hell, 2013; Limpitikul & Dick, 2025). Given that Ca_V1.2 is a major determinant of AP duration, these changes can promote AP prolongation and proarrhythmic EADs and DADs (Weiss et al., 2010). Notably, the list of mutations associated with Timothy syndrome continues to grow, and recent reports demonstrate the mechanistic diversity of mutant Ca_V1.2 channels (Bamgboye et al., 2022a). While some mutations have multiple effects on channel gating, others have more selective effects on Ca²⁺- or voltage-dependent inactivation. This has significant implications for treatment, as loss of Ca_V1.2 inactivation reduces the efficacy of Ca²⁺ channel blockers that are often state-dependent (Bamgboye et al., 2022b). Thus, new strategies to target Ca_V1.2 warrant further investigation, such as antisense oligonucleotides. Suppressed expression of Timothy-syndrome associated CACNA1C exon 8A variant with antisense oligonucleotides for that disease-causing allele was recently shown to attenuate Ca²⁺ handling deficits in mutant human neurons (Chen et al., 2024). Whether this approach can work in the cardiovascular system is yet to be explored. The new genetically encoded enhancer of Ca_V1.2 published by Morfin et al. also provides a new avenue to upregulate Ca_V1.2 function and potentially increase excitation-contraction coupling in disease (Del Rivero Morfin et al., 2024). While the above discussion has linked mutations in Ca²⁺ handling channels to the generation of afterdepolarizations, proarrhythmic instability in Ca²⁺ cycling can also manifest as beat-to-beat alternation in Ca²⁺ release and membrane voltage. Known as Ca²⁺-driven alternans, these events can arise from impaired RyR2 refractoriness, reduced SR Ca²⁺ refilling, and perturbed LTCC–RyR2 coupling. Resulting spatially discordant action potential duration alternans can favor unidirectional block and reentry (Sato et al., 2006). Clinically, T-wave alternans provides a noninvasive indicator of such Ca²⁺ cycling instability and serves as a marker of heightened ventricular arrhythmia risk. Modeling and sensitivity analyses indicate that interventions targeting one mechanism can inadvertently worsen the other (Herrera et al., 2025). Indeed, reduced LTCC current suppresses delayed afterdepolarizations but promotes alternans, whereas increased LTCC diminishes alternans yet facilitates Ca²⁺ overload and delayed afterdepolarizations. Likewise, enhanced RyR activation favors delayed afterdepolarizations but can attenuate alternans. These antagonistic interactions suggest that the net outcome of Ca²⁺-modulating therapy depends on the dominant arrhythmia mechanism and disease context.

Despite these advances in our understanding, it remains challenging to link these small-scale changes in the location and function of Ca²⁺-handling proteins into function and dysfunction at larger scales. To address this issue at the cellular level, new mathematical approaches have coupled cellular ultrastructure, electrophysiology, and spatially-detailed Ca²⁺ homeostasis (Loucks et al., 2018; Zhang et al., 2023a; Zhang et al., 2023b; Zhang et al., 2024) (Fig. 4). At the tissue scale, modeling has been used to predict whole-organ contractility and arrhythmia susceptibility based on cellular-level changes in Ca²⁺ handling and electrophysiology across time, space, and tissue layers, with inclusion of the effects of hypertrophy (Chang & Trayanova, 2016; Timmermann et al., 2017; Longobardi et al., 2022; Khalilimeybodi et al., 2023). These approaches have shown us that myocyte Ca²⁺ homeostasis can be adapted to meet physiological demand. For example, VSMCs undergo major structural and functional adaptations during pregnancy. This includes reduced LTCC activity to inhibit VSMC contractility and help accommodate the increased blood volume and ensure uteroplacental perfusion (Hu et al., 2024). Similarly, in cardiomyocytes, dyads have a non-uniform arrangement across the cell, which creates an opportunity to augment dyadic density in times of need, increasing and accelerating overall Ca²⁺ release (Ruud et al., 2024). On the other hand, dyadic loss and channel dysfunction are well-established causes of impaired Ca²⁺ release during diseases such as heart failure and atrial fibrillation (Li et al., 2014; Jones et al., 2018; Dixon, 2022).

Figure 4: Spatial and functional organization of Ca²⁺ microdomains across cardiovascular cell types.

Top panel: schematic representation of Ca²⁺ signal (spatial and temporal) hierarchy that drive contraction and transcriptional regulation. Localized, short-lived events such as Ca²⁺ sparks, sparklets, and puffs occur at nanometer and millisecond scales, whereas global Ca²⁺ transients, nuclear Ca²⁺ signals, and contraction reflect progressively larger and slower phenomena. The central “Ca²⁺ signaling” node emphasizes that these distinct forms of Ca²⁺ activity are interconnected. The schematic in the top right symbolizes systems-level or network modeling of Ca²⁺ signaling processes. The feedback arrow from “Contraction” to “Ca²⁺ signaling” denotes mechanochemical coupling, whereby cellular contraction modulates Ca²⁺ homeostasis. Bottom left (Cardiac): Representative results from a human atrial tissue-level simulation of the Zhang et al., 2023a model. Space time maps display subcellular Ca²⁺ sparks, mini-waves, and large propagating Ca²⁺ waves (bottom) illustrating how local release events integrate into global Ca²⁺ dynamics. Traces (middle) show simulated membrane potentials (V_m) and intracellular Ca²⁺ transients ([Ca²⁺]_i) recorded in two cells (Cell 1 and Cell 2) during tissue-level action potential propagation (top). Bottom right (Vascular): representative vascular Ca²⁺ signals. Ca²⁺ imaging of VSMCs in intact arteries under increasing glucose (D-glu) concentrations reveals enhanced intracellular Ca²⁺ concentration. Schematic of VSMC–endothelial cell interactions underscore cross-cellular Ca²⁺ signaling that should be taken into consideration for more accurate mathematical models. Experimental and simulated Ca²⁺ transients from wild-type (wt) and AC5^−/− arteries demonstrate glucose-dependent PKA regulation of intracellular Ca²⁺ concentration (figure adapted from (Syed et al., 2019) with permission). Imaging of IP₃R- and TRPV4-mediated Ca²⁺ signals in endothelial cells obtained using the en face preparation in mesenteric arteries, along with 3D renderings of local Ca²⁺ domains. These data highlight the spatial complexity of vascular Ca²⁺ regulation, in this case in endothelial cells. Together, these data emphasize how local Ca²⁺ events act as building blocks for higher-order signaling that governs contractility, excitability, and transcriptional responses in the cardiovascular system.

An interesting, perennial question that arises in this work is how variability in Ca²⁺ homeostasis between cells, due to cell-to-cell differences in the expression of Ca²⁺-handling proteins and posttranslational modifications, affects whole-organ electrophysiology. Indeed, while isolated cardiomyocytes exhibit marked differences in AP propagation and configuration (Simitev et al., 2023), these effects are mitigated in situ by tight electrical coupling of neighboring cells via gap junctions, which evens out electrical differences between cells. However, during cardiovascular disease, gap junction function may be impaired (Sedovy et al., 2023; Araujo et al., 2025), and intercellular differences in Ca²⁺ handling proteins then serve as a substrate for uncoordinated electrical activity and contractility (Simitev et al., 2023). Such integrative approaches are critical as we seek to improve treatment approaches for cardiac disease, screening for new potential therapies that have a real chance of success.

3.2: Cardiac hypertrophy and remodeling

Cardiac hypertrophy is a form of structural remodeling of the heart in response to various stressors and stimuli, and is largely attributed to an increase in cardiomyocyte size. Adaptive hypertrophy refers to cardiac growth, typically associated with exercise and pregnancy, that is proportional to ventricular geometry and reversible. For example, pregnancy-induced hypertrophic growth usually regresses over several months following delivery (Mesa et al., 1999). During adaptative hypertrophy, cardiac function is preserved, and cardiac metabolism is balanced. In contrast, maladaptive hypertrophy, often known as pathological hypertrophy, refers to cardiac growth that is disproportional to ventricular geometry and is typically irreversible. Molecular signatures of maladaptive hypertrophy include the reactivation of the “fetal gene program” in adult ventricular cardiomyocytes, and well-established upregulation of β-myosin heavy chain (β-MHC) and atrial natriuretic peptide (ANP) (Calderone et al., 1995). During this process, the heart undergoes a metabolic shift from fatty acid metabolism to glycolytic metabolism, resulting in less efficient energy use (Gibb & Hill, 2018). Maladaptive hypertrophy is also associated with disrupted sarcomere and mitochondrial organization, impaired Ca²⁺ handling, and altered ionic currents. As hypertrophy advances into heart failure, the ventricular chambers become dilated and cardiomyocyte survival declines through multiple cell death pathways, including programmed apoptosis, necrosis, and pyroptosis (van Empel & De Windt, 2004; Xiao et al., 2025). Progressive myocyte loss leads to fibrotic replacement, creating electrical heterogeneity and increasing the risk of cardiac arrhythmias (Hill & Olson, 2008).

Pathological hypertrophy can be promoted via various cues including neurohumoral activation, chronic pressure or volume overload, direct myocardial injury, metabolic stress, or genetic mutations affecting major sarcomeric proteins. To reverse pathological hypertrophy, the timing of the intervention is a critical consideration. Early interventions that correct neurohumoral imbalances (e.g., with β-blockers and angiotensin converting enzyme ACE inhibitors), reduce pressure overload (e.g., through hypertension control), restore coronary perfusion following myocardial infarction, or re-establish a healthy immunoregulatory microenvironment, may stall the progression of hypertrophic remodeling and maintain relatively normal cardiac function. However, effective therapies for genetic forms of hypertrophic cardiomyopathy remain limited to mavacamten and aficamten, which are selective cardiac myosin inhibitors indicated for obstructive hypertrophic cardiomyopathy (Spertus et al., 2021; Maron et al., 2024).

Many molecular mechanisms have been implicated in hypertrophic remodeling, including phosphoinositide 3-kinase (PI3K)-Akt signaling, mechanosensitive ion channels, and signal transduction through cytoskeletal networks (Heineke & Molkentin, 2006). Among these, altered intracellular Ca²⁺ homeostasis is considered a key trigger that activates multiple pro-hypertrophic pathways (Heineke & Molkentin, 2006; Ko et al., 2022). On one hand, elevated Ca²⁺ levels activate calcineurin, a phosphatase commonly upregulated in hypertrophic hearts. Activated calcineurin dephosphorylates nuclear factor of activated T-cells (NFAT), facilitating its translocation to the nucleus and the subsequent activation of pro-hypertrophic transcriptional gene programs (Molkentin et al., 1998; Rossow et al., 2004). In addition, CaMKII not only directly modifies the activity of Ca²⁺-handling apparatus (LTCC, RyR2), multiple ion channels and myofilament properties, but also promotes the transcriptional activation of hypertrophic gene programs (Anderson et al., 2011). CaMKII enhances the activity of myocyte enhancer factor-2 (MEF2), a transcription factor driving the expression of fetal genes such as ANP and β-MHC. Intriguingly, CaMKII can phosphorylate calcineurin at the Serine 197 residue, blocking the Ca²⁺/calmodulin binding site and partially inactivating calcineurin. This negative regulation of calcineurin by CaMKII provides fine-tuned control of the NFAT-mediated hypertrophic pathway (MacDonnell et al., 2009). There is also evidence that CaMKII activity can be locally regulated by cytoskeletal proteins such as βIV-spectrin, which plays a role in maintaining membrane integrity under mechanical stress and organizing ion channel microdomains (Hund et al., 2010). βIV-spectrin directly binds to CaMKII, anchoring it within specific subcellular domains. In murine models, disruption of spectrin-CaMKII interaction domains ameliorates pressure overload-induced pathological hypertrophy (Unudurthi et al., 2018). This positions the spectrin-CaMKII interaction as a novel regulatory target for stress-induced cardiac remodeling. Some evidence also suggests that CaMKII activity intersects with inflammatory signaling pathways, including downstream signals of the Interleukin-1 receptor (IL-1R) and signal transducer and activator of transcription 3 (STAT3), contributing to arrhythmogenesis and remodeling (Suetomi et al., 2018; Unudurthi et al., 2018; Heijman et al., 2020). However, the directionality and mechanistic hierarchy among these molecules require further investigation.

Hormonal fluctuations during pregnancy provide a unique physiological context for studying cardiac hypertrophy. Estrogen and progesterone levels rise substantially throughout gestation and are known to influence vascular tone, fluid retention, and myocardial gene expression. These hormones modulate Ca²⁺ handling, myocardial contractility, and hypertrophic signaling pathways. Pregnancy-induced hypertrophy is typically reversible, in contrast to pathological forms, and is accompanied by balanced growth of cardiomyocytes and vasculature. Pregnancy hormones have a vasodilatory effect on basal VSMC active mechanics, as seen in acute vasodilation. Hormonal effects on cellular active mechanics partially reverse after the stimulus is removed. Mechanics and hormones interact during pregnancy and lead to changes in cell mechanical behavior and internal cytoskeletal structure. More information about cellular-scale memory and timescale is needed to adequately model both short- and long-term arterial mechanics during pregnancy. This proposed model offers insights into mechanisms that support adaptive remodeling and may help identify molecular brakes on hypertrophy that are lost in disease.

A comprehensive understanding of cardiac hypertrophy and vascular dysfunction requires integration across multiple cell types as well as temporal and spatial scales. Acute signaling events, such as Ca2+ transients or kinase activation, operate on the order of milliseconds to minutes, while transcriptional reprogramming and extracellular matrix remodeling unfold over days to weeks. Multi-temporal scale models (Frank et al., 2018; Yoshida & Holmes, 2021; Eggertsen & Saucerman, 2023; Kaissar & Yoshida, 2024; Khorasani et al., 2025) can bridge these dynamics, capturing feedback loops between electrical activity, mechanical stress, and gene expression. Such integrative frameworks are essential to predict the transition from compensatory to decompensated hypertrophy and for identifying optimal therapeutic windows in patients with evolving cardiac disease.

3.3: Vascular smooth muscle Ca²⁺ dynamics

Ca²⁺ signaling in VSMCs is a sophisticated biological control system governing fundamental cardiovascular processes (Hill-Eubanks et al., 2011; Amberg & Navedo, 2013; Ottolini et al., 2019). This essential second messenger orchestrates vessel diameter changes, directly influencing vascular resistance, blood flow distribution, and tissue perfusion throughout the body, with effects that vary between biological sexes (Asuncion-Alvarez et al., 2024). Ca²⁺ signaling events translate diverse mechanical and chemical stimuli into precise contractile control. Thus, in VSMCs, Ca²⁺ signals arise from a complex network of specialized proteins and molecular processes that serve to maintain vascular homeostasis. Within smooth muscle, Ca²⁺ signals demonstrate remarkable diversity in their spatial distribution and functional roles. These roles range from propagating Ca²⁺ waves that traverse entire cells to highly localized events including junctional Ca²⁺ transients triggered by sympathetic nerve stimulation, Ca²⁺ sparks from RyR clusters, Ca²⁺ puffs generated by IP₃R activation, and LTCC and TRPV4 sparklets that create persistent domains of elevated Ca²⁺ (Hill-Eubanks et al., 2011; Amberg & Navedo, 2013; Ottolini et al., 2019) (Fig. 4). Rather than functioning as simple on-off switches, these Ca²⁺ events operate as complex information processing networks where spatial organization, temporal dynamics, and amplitude determine both the magnitude of the reactivity and specific cellular responses activated.

Ca²⁺ microdomains and signal integration

The concept of Ca²⁺ microdomains fundamentally reshapes our understanding of excitation-contraction coupling in VSMCs. While global [Ca²⁺]_i increases invariably lead to contraction, specific subcellular Ca²⁺ signals can produce opposing effects, a paradox highlighting the critical importance of spatial compartmentalization. For instance, Ca²⁺ sparks can trigger vasodilation despite their high local Ca²⁺ concentrations, while other localized signals promote contraction (Amberg & Navedo, 2013; Ottolini et al., 2019). This functional diversity arises from the precise spatial relationships between Ca²⁺ sources and their downstream effectors (e.g. LTCC, BK_Ca. channels and RyR).

Microdomain formation depends critically on cellular architecture, with SR regions positioned within 10-20 nm of the plasma membrane creating specialized signaling domains where local Ca²⁺ concentrations reach micromolar levels while global Ca²⁺ remains unchanged (Biwer & Isakson, 2017; Dixon & Trimmer, 2023). This arrangement enables selective activation of nearby ion channels without triggering cell-wide responses, demonstrating how similar Ca²⁺ signals can produce opposite physiological outcomes based on their molecular environment and vascular bed. Intriguingly, the precise spatial organization creates computational challenges, as mathematical models should incorporate stochastic channel behavior and exact subcellular geometry to accurately predict microdomain function. Model assumptions may be incomplete, as it is known that ion channels could cluster in response to different physiological and pathological stimuli (Dixon et al., 2022), which is not yet captured in any of the mathematical models. Moreover, traditional models focusing on global Ca²⁺ changes may not fully capture the sophisticated regulatory control enabled by this spatial compartmentalization.

The integration of multiple Ca²⁺ microdomains involves complex temporal and spatial summation mechanisms where cellular context and coupling partners determine whether signals promote contraction or relaxation (Karlin, 2015). For example, in cerebral arteries, Ca²⁺ sparks preferentially activate BK_Ca channels, producing spontaneous transient outward currents that lead to hyperpolarization and vasodilation (Nelson et al., 1995). Conversely, in portal vein, similar Ca²⁺ sparks couple to Ca²⁺-activated chloride channels, generating depolarization and promoting contraction (Wang et al., 1992; Saleh & Greenwood, 2005). This differential coupling exemplifies the sophisticated regulatory control enabled by spatial compartmentalization. Moreover, mathematical models incorporating vessel-specific channel expression profiles and spatial coupling arrangements may provide insight as to why identical Ca²⁺ signals produce opposite physiological outcomes.

Multi-scale analysis of Ca²⁺ dynamics

Understanding Ca²⁺ signaling requires analyzing organization across multiple scales, from individual channel clusters to coordinated tissue responses. At the channel cluster level, advances in TIRF microscopy reveal that LTCC and TRPV4 channels segregate into distinct low- and high-activity sites rather than operating randomly across the plasma membrane (Navedo et al., 2005; Sonkusare et al., 2012; Navedo & Santana, 2013; Mercado et al., 2014). High-activity sparklet sites result from coordinated channel opening, requiring A-kinase anchoring protein 150 (AKAP150)-anchored protein kinase activity (PKC and PKA) (Navedo et al., 2006; Navedo et al., 2008; Navedo et al., 2010a; Sonkusare et al., 2014; Nystoriak et al., 2017). Underlying the high-activity LTCC sparklet sites is a dynamic rearrangement of Ca_V1.2 channels into superclusters (Martin-Aragon Baudel et al., 2022; Flores-Tamez et al., 2024). A similar superclustering of TRPV4 channels may underlie the high-activity TRPV4 sparklet sites (Mercado et al., 2014; Sato et al., 2019). These high-activity sites have profound functional consequences, contributing significantly to myogenic tone in resistance arteries and proving necessary for angiotensin II-induced contractions (Amberg et al., 2007; Dixon et al., 2022). The sustained Ca²⁺ microdomains they generate activate specific transcriptional pathways, particularly the Ca²⁺/calcineurin/NFAT cascade, linking acute Ca²⁺ signals to long-term cellular adaptations with important implications for physiological and pathophysiological vascular remodeling. Mathematical models are proving essential for understanding how these localized channel clusters translate into tissue-level responses (Sato et al., 2018; Sato et al., 2019), as the emergent properties of coordinated channel activity cannot be predicted from individual channel behavior alone.

RyR clusters create analogous functional microdomains for Ca²⁺ release (Pritchard et al., 2018; Pritchard et al., 2019). The efficiency with which Ca²⁺ sparks couple to nearby BK channels determines the magnitude of smooth muscle relaxation, providing a key mechanism for vasodilator action (Jaggar et al., 1998). At the whole-cell scale, the transition from local microdomains to integrated dynamics involves complex mechanisms creating spatial and functional compartmentalization within individual cells. Multiple Ca²⁺ clearance systems, including Na⁺/Ca²⁺ exchanger, plasma membrane Ca²⁺ ATPase, sarcoplasmic reticulum Ca²⁺ ATPase (SERCA), and mitochondrial uptake, work in concert to shape signal propagation and termination (Hill-Eubanks et al., 2011; Amberg & Navedo, 2013; Ottolini et al., 2019). Ca²⁺ waves represent the most dramatic manifestation of this integration, arising from sequential Ca²⁺ release events creating regenerative wavefronts that influence contraction through direct contribution to global [Ca²⁺]_i and indirect modulation of plasma membrane Ca²⁺-sensitive ion channels (Jackson, 2016). Computational modeling of these multi-compartment systems will become crucial for understanding how local Ca²⁺ events integrate to produce coordinated cellular responses.

The complexity revealed by current research demands new experimental and theoretical approaches to fully understand Ca2+ signal integration. Three critical areas require immediate attention. First, visualization of cryptic Ca2+ signals remains a technical challenge. For example, Ca2+ puffs generated by IP3R and novel signals associated with store-operated Ca2+ entry await direct assessment and delineation. These signals likely play important roles in vascular regulation but remain hidden from current imaging techniques. Second, understanding the relationship between local and global Ca2+ dynamics requires simultaneous multi-scale measurements. New imaging technologies must capture events ranging from single-channel openings to tissue-wide Ca2+ waves, ideally within intact cardiac and vascular preparations that preserve physiological geometry and complex cell-cell interactions. Third, computational models integrating multiple spatial and temporal scales are essential for predicting emergent properties of the Ca2+ signaling network, particularly in cellular micro- and nanodomains. Only through combined experimental and computational approaches can we hope to better understand how alterations in specific components in discrete subcellular regions contribute to cardiac and vascular dysfunction.

3.4: Pulmonary vasculature Ca²⁺ signaling

The pulmonary vasculature is structurally and functionally distinct from the systemic vasculature. Pulmonary arteries (PAs) receive blood from the right ventricle at low pressure (10-15 mm Hg) with reduced oxygen content, comprising a high-flow vascular bed that accommodates the entire blood supply despite smaller total volume compared to systemic arteries (Suresh & Shimoda, 2016; Sundjaja & Bordoni, 2024). Gas exchange occurs in pulmonary capillaries, after which oxygenated blood is delivered to the left atrium by pulmonary veins (PVs) (Sundjaja & Bordoni, 2024).

Ca²⁺ signaling mechanisms in pulmonary arteries and capillaries

Research on pulmonary vascular Ca²⁺ signaling has focused primarily on PAs and capillaries, particularly in pulmonary hypertension (PH), lung injury, and edema contexts (Thorneloe et al., 2012; Balakrishna et al., 2014; Goldenberg et al., 2015; Suresh et al., 2015; Scheraga et al., 2017; Kuebler et al., 2020; Rajan et al., 2021; Haywood et al., 2022; Sonkusare & Laubach, 2022; Kuppusamy et al., 2023; Li et al., 2025). While various Ca²⁺ signaling pathways are active under physiological conditions, their specific roles in regulating pulmonary arterial pressure (PAP), blood flow, endothelial barrier integrity, and gas exchange remain insufficiently understood.

Pulmonary VSMCs express diverse ion channels, including TRP channels, mechanosensitive Piezo channels, and voltage-gated ion channels (Zhang et al., 2007; Firth et al., 2011; Xia et al., 2013; Goldenberg et al., 2015; Chen et al., 2022; Knoepp et al., 2025; Li et al., 2025), although their precise physiological role is not well understood. In pathological states, dysregulation of these channels contributes to vascular dysfunction. Excessive PA constriction and remodeling are central to elevated PAP in PH, with studies using knockout models and primary cell cultures demonstrating critical roles of smooth muscle TRP and Piezo channels in PA constriction and remodeling (Yang et al., 2012; Goldenberg et al., 2015; Jain et al., 2021; Liao et al., 2021; Masson et al., 2023). The broader implications of ion channel activity and Ca²⁺ signaling in PH pathogenesis have been reviewed elsewhere (Lambert et al., 2018; Ottolini & Sonkusare, 2021; Li et al., 2025).

Endothelium-dependent dilation of PAs, mediated by endothelial TRP channels, Pannexin 1 (an ATP efflux pathway), and purinergic receptors, maintains low resting PAP (Daneva et al., 2021a; Daneva et al., 2021b). As high-flow vessels that experience significantly increased flow during exercise, PAs require effective flow-induced signaling mechanisms that remain largely undefined. While Piezo1 and TRP channels are expressed in PA endothelium similar to systemic arteries (Daneva et al., 2021a; Swain & Liddle, 2021; Wang et al., 2021), their specific roles in flow-mediated responses are not yet well characterized.

Endothelial dysfunction contributes significantly to pulmonary vascular disease development. Abnormal endothelial Piezo1 and TRP channel activity promotes endothelial dysfunction, proliferation, and vascular remodeling in PH (Suresh et al., 2015; Suresh et al., 2018; Daneva et al., 2021a; Wang et al., 2021). The alveolo-capillary barrier, essential for efficient gas exchange, becomes disrupted in disease states, causing fluid accumulation and lung injury. Evidence suggests that endothelial TRP channels, Piezo channels, pannexins, and purinergic receptors contribute critically to barrier dysfunction and lung injury pathogenesis (Thorneloe et al., 2012; Friedrich et al., 2019; Haywood et al., 2021; Swain & Liddle, 2021; Haywood et al., 2022; Kuppusamy et al., 2023).

Ca²⁺ signaling in pulmonary vein myocytes

Despite being the only veins carrying oxygenated blood (aside from umbilical veins), Ca²⁺ signaling mechanisms in PVs remain understudied. Research has focused on large extrapulmonary PVs, while small intrapulmonary veins receive little attention. Large PVs contain myocardial sleeves exhibiting ryanodine RyR-mediated Ca²⁺ signaling and spontaneous contractions (Roux et al., 2004; Jones et al., 2008; Pasqualin et al., 2018). Small PVs also contain cardiomyocyte-like cells expressing cardiac troponin C with spontaneous RyR-mediated contractions (Rietdorf et al., 2014; Chen et al., 2025; Varas et al., 2025). Given dynamic intraluminal pressures throughout the cardiac cycle, pressure likely modulates RyR activity to regulate PV blood flow and prevent backflow into lungs. RyR activity is regulated by voltage-gated Ca²⁺ channels, β-adrenoceptor signaling, protein kinases, and NO (Lehnart & Marks, 2007; Ather et al., 2013). Understanding these regulatory mechanisms in PVs could provide novel insights into PV function, representing a significant gap in cardiopulmonary physiology.

PV Ca²⁺ signaling in disease

PV dysfunction in pulmonary vascular disorders remains largely unexplored. Pulmonary veno-occlusive disease (PVOD), characterized by small PV obstruction leading to elevated PA pressure and PH, is difficult to diagnose with poorly understood pathological mechanisms (Lechartier et al., 2024). Given the critical role of RyR signaling in PV myocytes, its involvement in PVOD pathogenesis warrants investigation, particularly since excessive RyR activation contributes to cardiac hypertrophy and heart failure remodeling (Alvarado et al., 2019). In addition, lung edema represents a major cause of heart failure morbidity and mortality. Elevated pulmonary venous pressure increases capillary hydrostatic pressure and permeability, causing lung fluid accumulation. Current therapies do not specifically target pulmonary edema from elevated PV pressure. Whether PV signaling abnormalities contribute to increased capillary pressure in heart failure remains unclear. Investigating Ca²⁺ signaling in small PVs may reveal new pathogenic mechanisms and therapeutic targets for both heart failure-related pulmonary edema and other forms of lung injury. A comprehensive understanding of PV signaling under physiological and pathological conditions is essential for advancing cardiopulmonary biology.

A crucial and still underexplored frontier in vascular physiology is understanding how Ca2+ signaling pathways integrate and respond to the diverse signals emanating from heterogenous cellular environments. Vascular smooth muscle cells of small resistance vessels exist within complex tissue environments where other cell types generate distinct chemical, mechanical, and electrical cues. Each of these neighboring cell types can modulate vascular Ca2+ handling through paracrine factors, metabolites, mechanical forces, or other modes of direct cell-cell communication. The spatial proximity and temporal coincidence of these signals create intricate local environments whereby Ca2+ microdomains serve as integrative hubs. These hubs likely translate highly localized information into vascular responses tailored to unique metabolic, inflammatory, or mechanical demands of tissues. Dissecting how microdomain signals decode and prioritize competing inputs (from myriad vasoactive metabolites to inflammatory mediators and mechanical stimuli like shear stress) is critical for understanding how blood flow is precisely matched to regional tissue requirements. This could be particularly relevant to pathophysiological states. Future work employing advanced imaging techniques, high-resolution spatial multi-omics, and multiscale computational models will be crucial for understanding how Ca2+ signals function within these heterogeneous contexts. This may open new avenues for precisely targeted therapies for hypertension, stroke, diabetic vascular complications, and other cardiovascular diseases.

3.5: Therapeutic targeting of Ca²⁺ remodeling

As Ca²⁺ dysregulation is a common feature of many cardiovascular pathologies, interventions aimed at modulating aberrant Ca²⁺ handling represent a promising therapeutic avenue. However, despite encouraging results from treatments such as cardiomyocyte SERCA gene therapy (Kawase et al., 2008; Lyon et al., 2011) and RyR stabilizers (Wehrens et al., 2004; Lehnart et al., 2006) in preclinical studies, clinical success with Ca²⁺-targeted therapies has so far been limited. In particular, the large placebo-controlled CUPID 2 (Calcium upregulation by percutaneous administration of gene therapy in patients with cardiac disease) trial reported that SERCA2a gene transfer failed to improve clinical outcomes in patients with advanced heart failure despite encouraging earlier-phase results (Greenberg et al., 2016). The lack of benefit might have been due to the failure to deliver the transgene at sufficiently high levels (Lyon et al., 2020). Limited success of therapy targeting abnormal Ca²⁺ handling may be partly due to late intervention, which occurs at a time point when cardiac remodeling has progressed beyond disrupted Ca²⁺ cycling to include significant structural abnormalities that require additional therapeutic targeting. While initial changes emerge at the subcellular level, they manifest at the cellular and tissue levels, where the loss of local control leads to impaired excitation–contraction coupling. Indeed, because structure and function are intricately linked across scales (e.g., between subcellular RyR2 cluster organization and local Ca²⁺ release, cellular t-tubule density and the synchrony of the Ca²⁺ transient, or tissue-level fibrosis and contractility), effective therapy for Ca²⁺ dysfunction will likely need to address structural remodeling in parallel with functional interventions.

Interestingly, many proteins and signaling molecules that are altered in the disease state interface with both cellular structure and function making them attractive therapeutic targets. Among these, CaMKII and reactive oxygen species stand out due to their broad influence on Ca²⁺-handling and cytoskeletal proteins, fibrotic signaling, and cellular metabolism (Anderson et al., 2011; Dubois-Deruy et al., 2020; Reyes Gaido et al., 2023). However, other downstream effectors may represent promising targets for therapeutic intervention. For example, BIN1 and JPH2 are important regulators of the t-tubule network and respectively modulate LTCC trafficking to the t-tubule (Hong et al., 2010) and RyR2 clustering and activity (van Oort et al., 2011; Munro et al., 2016). Similarly, in addition to supporting BIN1-mediated LTCC trafficking (Hong et al., 2010) and localization of Na_V1.5 to the intercalated disc (Marchal et al., 2021), microtubules, and specifically microtubule detyrosination, has been implicated in diastolic dysfunction and the pathogenesis of heart failure by increasing cellular stiffness and impairing contractility (Chen et al., 2018). Recent work by Fakuade et al. highlighted the dual role of the myofilament protein cardiac troponin C as both a key structural component of the contractile apparatus and a cytosolic Ca²⁺ buffer. Lower cardiac troponin C expression in atrial fibrillation was associated with a proarrhythmic reduction in Ca²⁺ buffering, leading to enhanced Ca²⁺-dependent cellular triggered activity (Fakuade et al., 2024). Dantrolene has been demonstrated to stabilize pathological and CPVT-associated RyR2 channels (Do & Knollmann, 2025; El-Harasis et al., 2025), and ongoing efforts aim to identify new compounds that achieve similar effects at lower concentrations and may serve as novel therapeutic candidates (Rebbeck et al., 2022). Similar structure-function organizations exist in the vasculature. For example, the scaffold protein AKAP5 is known to regulate LTCC function in VSMCs as well as TRPV4 channels in endothelial cells. This arrangement could complicate the specific targeting of proteins such as AKAP5 for controlling Ca²⁺ remodeling, particularly during pathological conditions such as hypertension, as they may lead to unintended interference with beneficial Ca²⁺ signaling pathways. For example, such actions could potentially disrupt protective endothelial TRPV4-mediated vasodilation while attempting to modulate hyperactive LTCC activity in VSMCs.

While simultaneous targeting of Ca²⁺ and structural remodeling is likely to be beneficial, it is important to recognize that advanced tissue-level changes such as cardiac fibrosis and chamber dilation may be irreversible by the time symptoms manifest. In such cases, functional compensation for structural remodeling may be necessary, rather than simply reversing Ca²⁺-handling defects. This could involve interventions to enhance excitation-contraction coupling and conduction, reduce mechanical stress, restore metabolism and oxygenation, and limit further progression of fibrosis. A complementary and potentially more effective strategy may be to shift the therapeutic focus toward prevention or earlier intervention rather than a cure. This could include screening individuals at high risk for disease development using genetic testing for pathogenic mutations, developing and implementing novel assays for circulating biomarkers (Hong et al., 2012; Li et al., 2024), or targeting upstream mediators such as reactive oxygen species and CaMKII with antioxidant therapies, peptides, small molecules, or nucleotides as aberrant signaling begins to emerge (Reyes Gaido et al., 2023; Yan et al., 2023).

Importantly, any therapeutic intervention must carefully account for both intercellular and inter-subject heterogeneity. The heart exhibits significant chamber, transmural and apico-basal differences in cellular composition, t-tubule density, electrical activity, Ca²⁺-cycling, gene expression, β-adrenergic responses, and mechanical strain; many of which are heterogeneously disrupted in disease (Lathers et al., 1986; Caldwell et al., 2014; Frisk et al., 2014; Parks et al., 2014; Yue et al., 2017; Pitoulis et al., 2020; Anto Michel et al., 2022; Koenig et al., 2022; Caldwell et al., 2023; Martinez-Navarro et al., 2025; Smith et al., 2025). Similarly, the vasculature displays remarkable heterogeneity across vessel types, with differences in Ca²⁺-handling proteins, mechanotransduction mechanisms, and responses to vasoactive agents between arteries and veins, large conduit vessels and resistance arterioles, and between different vascular beds. These cardiac and vascular regional variations are further modulated by factors such as age, sex, and hormonal or reproductive state, all of which can influence disease progression and therapeutic outcomes (Ambrosi et al., 2013; Machuki et al., 2019; Maslov et al., 2019; Caldwell et al., 2023; Zhang et al., 2024; Smith et al., 2025). As such, standardized treatment approaches may overlook important biological nuances across diverse patient populations, highlighting the need for personalized or regionally-targeted strategies in both drug development and clinical practice. However, due to the complexity of these factors, substantial future research is needed.

Sex-based differences in ion channels, Ca2+ handling, structural, and mechanical properties of the cardiovascular system significantly impact responses to physiological stress, pharmacological interventions, and disease. This rather recent realization has highlighted the necessity of integrating sex-specific mechanistic and computational models into preclinical and translational research, to more accurately predict human responses, translate across sexes (Hellgren et al., 2023; Shetty et al., 2025), and mitigate sex-based disparities in cardiovascular outcomes. Indeed, recognizing that females may respond more robustly to Ca2+ channel blockers like nifedipine can guide more precise anti-hypertensive dosing strategies and reduce the risk of adverse effects. Estrogen replacement therapy in postmenopausal women has potential cardiovascular benefits, but its effects on ion channel function and long-term outcomes require further investigation. In addition, novel ion channel modulators should be tested for sex-specific efficacy and safety profiles to optimize treatment across diverse populations. Despite clear evidence for sex-specific differences, significant challenges remain in rigorously incorporating sex as a biological variable. These challenges include historical underrepresentation of female subjects in preclinical and clinical studies, insufficient reporting of hormonal and reproductive states, and a lack of standardized protocols for sex-specific data collection and analysis. Additionally, biological complexity, such as interactions between hormonal cycles, aging, and coexisting comorbidities, further complicates data interpretation and modeling efforts. Future research should prioritize 1) mapping the expression and function of ion channels in male vs. female cardiovascular cells at single cell resolution with cardiac region and vascular bed specificity, 2) investigate how sex chromosomes and sex hormones distinctly influence cardiovascular electrophysiology, and 3) exploit computational models and methods to understand sex-linked differences in how cardiovascular electrophysiology integrates to differentially control cardiac and vascular function. Addressing these challenges requires concerted efforts to systematically incorporate balanced cohorts and transparent reporting practices.

4. Challenges, open questions, and conclusions

Despite significant advances in our mechanistic understanding of ion channel dysfunction and Ca²⁺ signaling dysregulation, several critical challenges continue to impede translation of these findings into effective, patient-specific therapies. A central challenge is the integration of high-throughput experimental data with predictive computational models. Mechanistic studies focus on isolated scales, such as single-channel biophysics, subcellular to cellular Ca²⁺ dynamics, or whole-organ electrophysiology, but capturing emergent behaviors like arrhythmia onset, structural remodeling, or therapy responsiveness requires models that bridge disparate spatiotemporal scales and adapt to biological variability (Bhagirath et al., 2024). These integrative frameworks could truly advance precision medicine for the millions of patients affected by arrhythmias, heart failure, and vascular disease, by combining structural modeling (Ngo et al., 2025), dynamically incorporating changes in ion channel trafficking and expression (Heijman et al., 2023; Meier et al., 2023), and accounting for progressive remodeling processes, including fibrosis, metabolic dysfunction, and inflammation. Workflows integrating high-content imaging, multiomics, and stem cell-derived cardiomyocyte functional assays can provide rich experimental datasets for systematic incorporation into mechanistic models. Community-wide efforts are needed to develop shared frameworks, standards, modular, shareable computational models and repositories. Such resources will promote reproducible and comparable simulations, allow integration of diverse datasets, and adaptation to patient-specific scenarios.

Abbreviations

ACE

Angiotensin Converting Enzyme

AKAP150

A-Kinase Anchoring Protein 150

AKAP5

A-Kinase Anchoring Protein 5

ANP

Atrial Natriuretic Peptide

AP

Action Potential

AT1R

Angiotensin II Type 1 Receptor

AT2R

Angiotensin II Type 2 Receptor

β-MHC

β-Myosin Heavy Chain

BK_Ca

Large-conductance Ca²⁺-activated K⁺ channel

BIN1

Bridging Integrator-1

CaMKII

Ca²⁺/calmodulin-dependent protein kinase II

CiPA

Comprehensive in Vitro Proarrhythmia Assay

CLASP1

Cytoplasmic Linker Associated Protein 2

CPVT

Catecholaminergic Polymorphic Ventricular Tachycardia

CRISPR

Clustered Regularly Interspaced Short Palindromic Repeats

DAD

Delayed Afterdepolarizations

EAD

Early Afterdepolarizations

EB1

End-binding Protein 1

EDH/EDHF

Endothelium-derived hyperpolarization

EET

Epoxyeicosatrienoic acid

enDUB

Engineered deubiquitinase

ER

Endoplasmic Reticulum

FHF

Fibroblast Growth Factor Homologous Factor

FGF

Fibroblast Growth Factor

GSK3β

Glycogen Synthase Kinase 3β

hPSC-CM

human Pluripotent Stem Cell-derived Cardiomyocyte

I _CaL

L-type Ca²⁺ Current

I _K1

Inward Rectifier K⁺ Current

I _Kr

Rapidly activating Delayed Rectifier K⁺ Current

IL-1R

Interleukin-1 receptor

I _Na

Voltage-gated Na⁺ Current

IP₃

Inositol-1,4,5-triphosphate

IP₃R

IP₃ Receptor

JPH2

Junctophilin-2

LOF

Loss Of Function

LTCC

L-type Ca²⁺ Channel

K_ATP

ATP-sensitive K⁺ channel

K_V

Voltage-gated K⁺ channel

MEF2

Myocyte Enhancer Factor-2

MLCK

Myosin Light Chain Kinase

MLC20

Myosin Light Chain 20

NFAT

Nuclear Factor of Activated T-cells

NO

Nitric Oxide

NVC

NeuroVascular Coupling

PI3K

Phosphoinositide 3-kinase

PIP₂

Phosphatidylinositol 4,5-bisphosphate

PKA

Protein Kinase A

PKC

Protein Kinase C

PA

Pulmonary Artery

PAP

Pulmonary Arterial Pressure

PH

Pulmonary Hypertension

PV

Pulmonary Vein

PVC

Premature Ventricular Contraction

PVOD

Pulmonary Veno-Occlusive Disease

RyR

Ryanodine Receptor

SERCA

Sarcoplasmic Reticulum Ca²⁺ ATPase

SR

Sarcoplasmic Reticulum

STAT3

Signal Transducer and Activator of Transcription 3

TRP

Transient Receptor Potential

VSMC

Vascular Smooth Muscle Cell

WT

Wild Type