Rose Ellen Dixon

L‐type Ca _V 1.2 channels are key regulators of cellular function in diverse cell types, controlling processes that range from gene expression to muscle contraction. In arterial myocytes, Ca _V 1.2 channels traffic to the plasma membrane and assemble into clusters that control Ca ²⁺ influx. Changes in the size and number of these Ca _V 1.2 clusters have been linked to altered channel and vascular function in diabetic hyperglycaemia, angiotensin II (Ang II) signalling and hypertension. Yet, the dynamics governing the distribution of vascular Ca _V 1.2 at the plasma membrane remain poorly understood. Here, we found that microtubule‐mediated transport actively shapes Ca _V 1.2 distribution under hyperglycaemia and Ang II signalling. Employing high‐resolution imaging, we tracked live Ca _V 1.2 trafficking in male arterial myocytes using a dual approach: unpassaged cells expressing vascular Ca _V 1.2‐ and freshly isolated cells from a Ca _V 1.2–mClover2 knock‐in mouse, which enabled endogenous channel visualization in native tissue. By recreating diabetic hyperglycaemia (20 mM d ‐glucose) and enhancing Ang II signalling in vitro , we uncovered distinct Ca _V 1.2 trafficking patterns in both cultured and freshly isolated arterial myocytes that were dependent on an intact microtubular network. Importantly, microtubule disruption abolished Ca _V 1.2 dynamics and prevented Ang II‐induced calcium elevations in both isolated myocytes and intact arteries. These findings identify microtubule‐dependent Ca _V 1.2 trafficking as a key mechanism by which hyperglycaemia and Ang II signalling may drive channel reorganization and calcium dysregulation. The results have implications for vascular dysfunction in diabetes and hypertension, and open new potential therapeutic avenues targeting the cellular transport machinery. Moreover, the Ca _V 1.2–mClover2 mouse provides a useful tool for investigating native channel behaviour in living tissues across physiological and pathological contexts. image Key points: Live‐cell total internal reflection fluorescence and spinning‐disk confocal imaging revealed that vascular Ca _V 1.2 channels exhibit distinct perimembrane behaviours in male arterial myocytes, including kiss‐and‐stay, kiss‐and‐run, and homotypic fusion and fission events. Microtubules are essential for vascular Ca _V 1.2 structural dynamics, with nocodazole‐induced microtubule disruption significantly reducing channel mobility, speed and displacement. Hyperglycaemic or angiotensin II‐stimulated environments rapidly increased vascular Ca _V 1.2 clustering at the plasma membrane, and microtubule disruption abolished these pathological elevations in clustering and intracellular Ca ²⁺ in both isolated myocytes and intact arteries. A CRISPR/Cas9‐generated Ca _V 1.2–mClover2 knock‐in mouse enabled endogenous channel visualization in native tissue, providing a transformative tool for investigating Ca _V 1.2 behaviour in physiological and pathological contexts.

Disrupted calcium (Ca ²⁺ ) homeostasis is a hallmark of neurodegenerative diseases, yet the mechanisms driving excessive Ca ²⁺ entry at the neuronal plasma membrane remain poorly understood. Here, we show that α-synuclein pre-formed fibrils trigger a reorganization of voltage-gated calcium (Ca _V ) channels in cultured mouse cortical neurons, increasing their clustering at the soma and dendrites. We find that α-synuclein fibrils promote cyclin-dependent kinase 5–mediated phosphorylation of K _V 2.1 at serine 603, enhancing its scaffolding capacity for Ca _V channels. Disrupting Ca _V –K _V 2.1 coupling with a competitive peptide or pharmacological CDK5 inhibition restores channel proximity to control levels. Functionally, the enhanced Ca _V clustering amplifies depolarization-evoked Ca ²⁺ influx and drives aberrant excitation–transcription coupling, as reflected by elevated expression of the immediate early gene c-Fos. All effects were rescued by disrupting Ca _V –K _V 2.1 interactions or inhibiting Ca _V channel activity. These findings identify nanoscale Ca _V channel remodeling as a mechanistic link between α-synuclein pathology and Ca ²⁺ -dependent transcriptional dysregulation, positioning Parkinson’s disease as a nanostructural channelopathy.

BACKGROUND: Contractile dysfunction, hypertrophy, and cell death during heart failure are linked to altered Ca ²⁺ handling and elevated levels of the hormone AngII (angiotensin II), which signals through G _q (Guanine nucleotide-binding protein alpha subunit q)-coupled AT ₁ Rs (AngII type 1 receptors), initiating hydrolysis of phosphatidylinositol (4,5)-bisphosphate. Chronic elevation of AngII contributes to cardiac pathology, but the mechanisms linking sustained AngII signaling to heart dysfunction remain incompletely understood. Here, we demonstrate that chronic AngII exposure profoundly disrupts cardiac phosphoinositide homeostasis, triggering a cascade of cellular adaptations that ultimately impair cardiac function. METHODS: Mice received 1-week infusions of AngII, bisperoxovanadium (1,10 phenanthroline), both, or saline via osmotic minipumps. We used mass spectrometry, super-resolution microscopy, electrophysiology, confocal imaging, immunoblot, echocardiography, and histology to assess phosphoinositide levels, L-type voltage-gated calcium channel Ca _V 1.2 localization, Ca ²⁺ handling, protein phosphorylation, cardiac function, and fibrosis. RESULTS: Chronic AngII infusion caused widespread phosphoinositide imbalance, reducing phosphatidylinositol, phosphatidylinositol phosphate, phosphatidylinositol bisphosphate, and phosphatidylinositol (3,4,5)-trisphosphate levels. Ca _V 1.2 channels were partially redistributed from t-tubules to endosomal compartments. Despite reduced sarcolemmal channel expression, Ca ²⁺ currents and transients were maintained through enhanced PKA (protein kinase A)–mediated and CaMKII (Ca ²⁺ /calmodulin-dependent protein kinase II)–mediated phosphorylation of Ca ²⁺ -handling proteins. However, this compensation proved insufficient as cardiac function progressively declined, marked by pathological hypertrophy, t-tubule disruption, and diastolic dysfunction. PTEN (phosphatase and tensin homolog) inhibition preserved Akt signaling and protected against cardiac dysfunction and fibrosis without preventing cellular remodeling or altered calcium handling. CONCLUSIONS: These findings reveal a complex interplay between phosphoinositide signaling, ion channel trafficking, and compensatory phospho-regulation in AngII-induced cardiac pathology. We establish phosphatidylinositol (3,4,5)-trisphosphate depletion as a critical link between chronic AngII signaling and cardiac dysfunction. The dissociation between persistent cellular remodeling and preserved organ function with PTEN inhibition reveals that cardioprotection occurs primarily through reduced fibrosis. PTEN inhibition, thus, emerges as a promising therapeutic strategy for heart failure associated with pathological renin-angiotensin system activation, with potential to complement existing therapies by targeting antifibrotic responses.

Lysosomes are essential organelles that regulate cellular homeostasis through complex membrane interactions. Phosphoinositide lipids play critical roles in orchestrating these functions by recruiting specific proteins to organelle membranes. The PIKfyve/Fig4/Vac14 complex regulates PI(3,5)P₂ metabolism, and intriguingly, while loss-of-function mutations cause neurodegeneration, acute PIKfyve inhibition shows therapeutic potential in neurodegenerative disorders. We demonstrate that PIKfyve/Fig4/Vac14 dysfunction triggers a compensatory response where reduced mTORC1 activity leads to ULK1-dependent trafficking of ATG9A and PI4KIIα from the TGN to lysosomes. This increases lysosomal PI(4)P, facilitating cholesterol and phosphatidylserine transport at ER-lysosome contacts to promote membrane repair. Concurrently, elevated lysosomal PI(4)P recruits ORP1L to ER-lysosome-mitochondria three-way contacts, enabling PI(4)P transfer to mitochondria that drives ULK1-dependent fragmentation and increased respiration. These findings reveal a role for PIKfyve/Fig4/Vac14 in coordinating lysosomal repair and mitochondrial homeostasis, offering insights into cellular stress responses.

Bridging integrator 1, initially named box-dependent myc-interacting protein-1 (BIN1), and also known as Amphiphysin 2 is a versatile N-BAR protein that plays essential roles in membrane remodeling, protein trafficking, and cellular organization across multiple tissues. Although extensively studied in cancer and Alzheimer’s disease, BIN1’s critical functions in cardiac physiology and pathology represent an emerging frontier with significant therapeutic implications. This review provides a synopsis of our current understanding of BIN1’s structure-function relationships, with particular emphasis on cardiac-specific isoforms and their roles in heart function. We examine how BIN1’s various domains—including the membrane-curvature forming and sensing BAR domain, phosphoinositide-binding motif, and SH3 protein-protein interaction domains—orchestrate its diverse cellular functions, from t-tubule growth, microfolding, and anchoring to directed protein trafficking and complex assembly. Recent discoveries highlight BIN1’s involvement in cardiac aging and disease, where both deficiency and excess of BIN1 can lead to dysfunction. Notably, BIN1 levels are reduced in heart failure while increasing significantly during cardiac aging, suggesting a bidirectional pathophysiology where both insufficient and excessive BIN1 expression can impair cardiac function. We discuss emerging evidence regarding the role of BIN1 in cardiac pathologies, offering potential therapeutic targets. Understanding BIN1’s membrane-shaping capabilities and its roles in organizing excitation-contraction coupling machinery could yield novel therapeutic strategies for addressing cardiac dysfunction in various disease contexts.

14‐3‐3 proteins are highly conserved proteins that regulate numerous cellular processes mostly through phosphorylation‐dependent protein–protein interactions. In the heart 14‐3‐3 proteins play critical roles in cardiac conduction pathways, excitation–contraction (EC) coupling, development and stress responses. This review summarizes the current understanding of cardiac 14‐3‐3 regulation and function, with particular emphasis on its role in ion channel regulation and β‐adrenergic signalling. We discuss how 14‐3‐3 proteins act through three main mechanisms – masking, clamping, and scaffolding – to regulate target proteins, including Cx43, Ca_V1.2, Na_V1.5, and various potassium channels. The seven mammalian 14‐3‐3 isoforms display distinct but overlapping functions, with tissue‐specific expression patterns and isoform‐specific regulation through phosphorylation and dimerization. Recent work has revealed 14‐3‐3's importance in cardiac development and stress responses, where it generally serves a cardioprotective role. However in some pathological contexts such as ischaemia–reperfusion injury, 14‐3‐3 can be detrimental. We highlight emerging themes in cardiac 14‐3‐3 biology, including its role in prolonging β‐adrenergic signalling. Understanding the complex regulation of cardiac 14‐3‐3 and its numerous targets presents both opportunities and challenges for therapeutic development. image

Contractile dysfunction, hypertrophy, and cell death during heart failure are linked to altered Ca²⁺ handling, and elevated levels of the hormone angiotensin II (AngII), which signals through G_q-coupled AT₁ receptors, initiating hydrolysis of PIP₂. Chronic elevation of AngII contributes to cardiac pathology, but the mechanisms linking sustained AngII signaling to heart dysfunction remain incompletely understood. Here, we demonstrate that chronic AngII exposure profoundly disrupts cardiac phosphoinositide homeostasis, triggering a cascade of cellular adaptations that ultimately impair cardiac function. Using in vivo AngII infusion combined with phospholipid mass spectrometry, super-resolution microscopy, and functional analyses, we show that sustained AngII signaling reduces PI(4,5)P₂ levels and triggers extensive redistribution of Ca_V1.2 channels from t-tubules to various endosomal compartments. Despite this t-tubular channel loss, enhanced sympathetic drive maintains calcium currents and transients through increased channel phosphorylation via PKA and CaMKII pathways. However, this compensation proves insufficient as cardiac function progressively declines, marked by pathological hypertrophy, t-tubule disruption, and diastolic dysfunction. Notably, we identify depletion of PI(3,4,5)P₃ as a critical mediator of AngII-induced cardiac pathology. While preservation of PI(3,4,5)P₃ levels through PTEN inhibition did not prevent cellular remodeling or calcium handling changes, it protected against cardiac dysfunction, suggesting effects primarily through reduction of fibrosis. These findings reveal a complex interplay between phosphoinositide signaling, ion channel trafficking, and sympathetic activation in AngII-induced cardiac pathology. Moreover, they establish maintenance of PI(3,4,5)P₃ as a promising therapeutic strategy for hypertensive heart disease and as a potential protective adjunct therapy during clinical AngII administration. Chronic AngII signaling through AT1R activates G_q-coupled signaling, leading to PI(4,5)P₂ depletion that destabilizes Ca_V1.2 in the plasma membrane (PM), triggering their endocytosis and reduced channel numbers at the PM. The remaining Ca_V1.2 channels and RyR2 undergo compensatory phosphorylation by CaMKII and PKA, triggered by sympathetic activation (β-AR signaling), leading to enhanced calcium-induced calcium release (CICR). Meanwhile, AngII promotes fibroblast-to-myofibroblast transition via M1 macrophage phenotype activation, increasing cardiac fibrosis. PTEN inhibition preserves PIP₃ levels and promotes anti-inflammatory M2 macrophage activation, resulting in reduced fibrosis. These findings reveal a complex interplay between cardiac phosphoinositide signaling, calcium handling, and fibrotic remodeling with chronic AngII. AC, adenylyl cyclase; β-AR, beta-adrenergic receptor; DAG, diacylglycerol; IP₃, inositol trisphosphate; PLC, phospholipase C; PKA, protein kinase A; PTEN, phosphatase and tensin homolog.

The L-type Ca ²⁺ channel (Ca _V 1.2) is essential for cardiac excitation–contraction coupling. To contribute to the inward Ca ²⁺ flux that drives Ca ²⁺ -induced-Ca ²⁺ -release, Ca _V 1.2 channels must be expressed on the sarcolemma; thus the regulatory mechanisms that tune Ca _V 1.2 expression to meet contractile demand are an emerging area of research. A ubiquitously expressed protein called 14-3-3 has been proposed to affect Ca ²⁺ channel trafficking in nonmyocytes; however, whether 14-3-3 has similar effects on Ca _V 1.2 in cardiomyocytes is unknown. 14-3-3 preferentially binds phospho-serine/threonine residues to affect many cellular processes and is known to regulate cardiac ion channels including Na _V 1.5 and the human ether-à-go-go–related gene (hERG) potassium channel. Altered 14-3-3 expression and function have been implicated in cardiac pathologies including hypertrophy. Accordingly, we tested the hypothesis that 14-3-3 interacts with Ca _V 1.2 in a phosphorylation-dependent manner and regulates cardiac Ca _V 1.2 trafficking and recycling. Confocal imaging, proximity ligation assays, superresolution imaging, and coimmunoprecipitation revealed a population of 14-3-3 colocalized and closely associated with Ca _V 1.2. The degree of 14-3-3/Ca _V 1.2 colocalization increased upon stimulation of β -adrenergic receptors with isoproterenol. Notably, only the 14-3-3-associated Ca _V 1.2 population displayed increased cluster size with isoproterenol, revealing a role for 14-3-3 as a nucleation factor that directs Ca _V 1.2 superclustering. Isoproterenol-stimulated augmentation of sarcolemmal Ca _V 1.2 expression, Ca ²⁺ currents, and Ca ²⁺ transients in ventricular myocytes were strengthened by 14-3-3 overexpression and attenuated by 14-3-3 inhibition. These data support a model where 14-3-3 interacts with Ca _V 1.2 in a phosphorylation-dependent manner to promote enhanced trafficking/recycling, clustering, and activity during β -adrenergic stimulation.

The L-type Ca ²⁺ channel (Ca _V 1.2) is essential for cardiac excitation-contraction coupling. To contribute to the inward Ca ²⁺ flux that drives Ca ²⁺ -induced-Ca ²⁺ -release, Ca _V 1.2 channels must be expressed on the sarcolemma; thus the regulatory mechanisms that tune Ca _V 1.2 expression to meet contractile demand are an emerging area of research. A ubiquitously expressed protein called 14-3-3 has been proposed to affect Ca ²⁺ channel trafficking in non-myocytes, however whether 14-3-3 has similar effects on Ca _V 1.2 in cardiomyocytes is unknown. 14-3-3 preferentially binds phospho-serine/threonine residues to affect many cellular processes and is known to regulate cardiac ion channels including Na _V 1.5 and hERG. Altered 14-3-3 expression and function have been implicated in cardiac pathologies including hypertrophy. Accordingly, we tested the hypothesis that 14-3-3 interacts with Ca _V 1.2 in a phosphorylation-dependent manner and regulates cardiac Ca _V 1.2 trafficking and recycling. Confocal imaging, proximity ligation assays, super-resolution imaging, and co-immunoprecipitation revealed a population of 14-3-3 colocalized and closely associated with Ca _V 1.2. The degree of 14-3-3/Ca _V 1.2 colocalization increased upon stimulation of β -adrenergic receptors with isoproterenol. Notably, only the 14-3-3-associated Ca _V 1.2 population displayed increased cluster size with isoproterenol, revealing a role for 14-3-3 as a nucleation factor that directs Ca _V 1.2 super-clustering. 14-3-3 overexpression increased basal Ca _V 1.2 cluster size and Ca ²⁺ currents in ventricular myocytes, with maintained channel responsivity to isoproterenol. In contrast, isoproterenol-stimulated augmentation of sarcolemmal Ca _V 1.2 expression and currents in ventricular myocytes were abrogated by 14-3-3 inhibition. These data support a model where 14-3-3 interacts with Ca _V 1.2 in a phosphorylation-dependent manner to promote enhanced trafficking/recycling, clustering, and activity during β -adrenergic stimulation. Significance Statement: The L-type Ca ²⁺ channel, Ca _V 1.2, plays an essential role in excitation-contraction coupling in the heart and in part regulates the overall strength of contraction during basal and fight- or-flight β -adrenergic signaling conditions. Proteins that modulate the trafficking and/or activity of Ca _V 1.2 are interesting both from a physiological and pathological perspective, since alterations in Ca _V 1.2 can impact action potential duration and cause arrythmias. A small protein called 14-3-3 regulates other ion channels in the heart and other Ca ²⁺ channels, but how it may interact with Ca _V 1.2 in the heart has never been studied. Examining factors that affect Ca _V 1.2 at rest and during β -adrenergic stimulation is crucial for our ability to understand and treat disease and aging conditions where these pathways are altered.