Junctophilin-2 tethers T-tubules and recruits functional L-type calcium channels to lipid rafts in adult cardiomyocytes

Abstract Aim In cardiomyocytes, transverse tubules (T-tubules) associate with the sarcoplasmic reticulum (SR), forming junctional membrane complexes (JMCs) where L-type calcium channels (LTCCs) are juxtaposed to Ryanodine receptors (RyR). Junctophilin-2 (JPH2) supports the assembly of JMCs by tethering T-tubules to the SR membrane. T-tubule remodelling in cardiac diseases is associated with downregulation of JPH2 expression suggesting that JPH2 plays a crucial role in T-tubule stability. Furthermore, increasing evidence indicate that JPH2 might additionally act as a modulator of calcium signalling by directly regulating RyR and LTCCs. This study aimed at determining whether JPH2 overexpression restores normal T-tubule structure and LTCC function in cultured cardiomyocytes. Methods and results Rat ventricular myocytes kept in culture for 4 days showed extensive T-tubule remodelling with impaired JPH2 localization and relocation of the scaffolding protein Caveolin3 (Cav3) from the T-tubules to the outer membrane. Overexpression of JPH2 restored T-tubule structure and Cav3 relocation. Depletion of membrane cholesterol by chronic treatment with methyl-β-cyclodextrin (MβCD) countered the stabilizing effect of JPH2 overexpression on T-tubules and Cav3. Super-resolution scanning patch-clamp showed that JPH2 overexpression greatly increased the number of functional LTCCs at the plasma membrane. Treatment with MβCD reduced LTCC open probability and activity. Proximity ligation assays showed that MβCD did not affect JPH2 interaction with RyR and the pore-forming LTCC subunit Cav1.2, but strongly impaired JPH2 association with Cav3 and the accessory LTCC subunit Cavβ2. Conclusions JPH2 promotes T-tubule structural stability and recruits functional LTCCs to the membrane, most likely by directly binding to the channel. Cholesterol is involved in the binding of JPH2 to T-tubules as well as in the modulation of LTCC activity. We propose a model where cholesterol and Cav3 support the assembly of lipid rafts which provide an anchor for JPH2 to form JMCs and a platform for signalling complexes to regulate LTCC activity.

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Interaction of the Joining Region in Junctophilin-2 With the L-Type Ca 2+ Channel Is Pivotal for Cardiac Dyad Assembly and Intracellular Ca 2+ Dynamics

Circulation Research ◽

10.1161/circresaha.119.315715 ◽

2021 ◽

Vol 128 (1) ◽

pp. 92-114

Author(s):

Polina Gross ◽

Jaslyn Johnson ◽

Carlos M. Romero ◽

Deborah M. Eaton ◽

Claire Poulet ◽

...

Keyword(s):

Sarcoplasmic Reticulum ◽

Ventricular Myocytes ◽

Close Association ◽

Ryanodine Receptors ◽

Left Ventricular ◽

Induced Mutations ◽

Adrenergic Stimulation ◽

T Tubules ◽

Junctional Sarcoplasmic Reticulum

Rationale: Ca 2+ -induced Ca 2+ release (CICR) in normal hearts requires close approximation of L-type calcium channels (LTCCs) within the transverse tubules (T-tubules) and RyR (ryanodine receptors) within the junctional sarcoplasmic reticulum. CICR is disrupted in cardiac hypertrophy and heart failure, which is associated with loss of T-tubules and disruption of cardiac dyads. In these conditions, LTCCs are redistributed from the T-tubules to disrupt CICR. The molecular mechanism responsible for LTCCs recruitment to and from the T-tubules is not well known. JPH (junctophilin) 2 enables close association between T-tubules and the junctional sarcoplasmic reticulum to ensure efficient CICR. JPH2 has a so-called joining region that is located near domains that interact with T-tubular plasma membrane, where LTCCs are housed. The idea that this joining region directly interacts with LTCCs and contributes to LTCC recruitment to T-tubules is unknown. Objective: To determine if the joining region in JPH2 recruits LTCCs to T-tubules through direct molecular interaction in cardiomyocytes to enable efficient CICR. Methods and Results: Modified abundance of JPH2 and redistribution of LTCC were studied in left ventricular hypertrophy in vivo and in cultured adult feline and rat ventricular myocytes. Protein-protein interaction studies showed that the joining region in JPH2 interacts with LTCC-α1C subunit and causes LTCCs distribution to the dyads, where they colocalize with RyRs. A JPH2 with induced mutations in the joining region (mut PG1 JPH2) caused T-tubule remodeling and dyad loss, showing that an interaction between LTCC and JPH2 is crucial for T-tubule stabilization. mut PG1 JPH2 caused asynchronous Ca 2+ -release with impaired excitation-contraction coupling after β-adrenergic stimulation. The disturbed Ca 2+ regulation in mut PG1 JPH2 overexpressing myocytes caused calcium/calmodulin-dependent kinase II activation and altered myocyte bioenergetics. Conclusions: The interaction between LTCC and the joining region in JPH2 facilitates dyad assembly and maintains normal CICR in cardiomyocytes.

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CaM kinase augments cardiac L-type Ca2+ current: a cellular mechanism for long Q-T arrhythmias

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.1999.276.6.h2168 ◽

1999 ◽

Vol 276 (6) ◽

pp. H2168-H2178 ◽

Cited By ~ 36

Author(s):

Yuejin Wu ◽

Leigh B. MacMillan ◽

R. Blair McNeill ◽

Roger J. Colbran ◽

Mark E. Anderson

Keyword(s):

Ventricular Myocytes ◽

Ryanodine Receptors ◽

Cam Kinase ◽

Early Afterdepolarizations ◽

Ultrastructural Studies ◽

Intracellular Ca ◽

Dependent Protein ◽

T Tubules ◽

Rabbit Ventricular Myocytes

Early afterdepolarizations (EAD) caused by L-type Ca2+ current ( I Ca,L) are thought to initiate long Q-T arrhythmias, but the role of intracellular Ca2+ in these arrhythmias is controversial. Rabbit ventricular myocytes were stimulated with a prolonged EAD-containing action potential-clamp waveform to investigate the role of Ca2+/calmodulin-dependent protein kinase II (CaM kinase) in I Ca,L during repolarization. I Ca,L was initially augmented, and augmentation was dependent on Ca2+ from the sarcoplasmic reticulum because the augmentation was prevented by ryanodine or thapsigargin. I Ca,Laugmentation was also dependent on CaM kinase, because it was prevented by dialysis with the inhibitor peptide AC3-I and reconstituted by exogenous constitutively active CaM kinase when Ba2+ was substituted for bath Ca2+. Ultrastructural studies confirmed that endogenous CaM kinase, L-type Ca2+ channels, and ryanodine receptors colocalized near T tubules. EAD induction was significantly reduced in current-clamped cells dialyzed with AC3-I (4/15) compared with cells dialyzed with an inactive control peptide (11/15, P = 0.013). These findings support the hypothesis that EADs are facilitated by CaM kinase.

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Local hyperactivation of L-type Ca2+ channels increases spontaneous Ca2+ release activity and cellular hypertrophy in right ventricular myocytes from heart failure rats

Scientific Reports ◽

10.1038/s41598-021-84275-w ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Roman Y. Medvedev ◽

Jose L. Sanchez-Alonso ◽

Catherine A. Mansfield ◽

Aleksandra Judina ◽

Alice J. Francis ◽

...

Keyword(s):

Heart Failure ◽

Structural Changes ◽

Ventricular Myocytes ◽

Cellular Mechanisms ◽

Open Probability ◽

Surface Flattening ◽

Cellular Hypertrophy ◽

T Tubules ◽

Optimized Treatment ◽

Ca2 Channels

AbstractRight ventricle (RV) dysfunction is an independent predictor of patient survival in heart failure (HF). However, the mechanisms of RV progression towards failing are not well understood. We studied cellular mechanisms of RV remodelling in a rat model of left ventricle myocardial infarction (MI)-caused HF. RV myocytes from HF rats show significant cellular hypertrophy accompanied with a disruption of transverse-axial tubular network and surface flattening. Functionally these cells exhibit higher contractility with lower Ca2+ transients. The structural changes in HF RV myocytes correlate with more frequent spontaneous Ca2+ release activity than in control RV myocytes. This is accompanied by hyperactivated L-type Ca2+ channels (LTCCs) located specifically in the T-tubules of HF RV myocytes. The increased open probability of tubular LTCCs and Ca2+ sparks activation is linked to protein kinase A-mediated channel phosphorylation that occurs locally in T-tubules. Thus, our approach revealed that alterations in RV myocytes in heart failure are specifically localized in microdomains. Our findings may indicate the development of compensatory, though potentially arrhythmogenic, RV remodelling in the setting of LV failure. These data will foster better understanding of mechanisms of heart failure and it could promote an optimized treatment of patients.

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Spontaneous Ca2+ sparks and Ca2+ homeostasis in a minimal model of permeabilized ventricular myocytes

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.00293.2010 ◽

2010 ◽

Vol 299 (6) ◽

pp. H1996-H2008 ◽

Cited By ~ 13

Author(s):

Jana M. Hartman ◽

Eric A. Sobie ◽

Gregory D. Smith

Keyword(s):

Dwell Time ◽

Ventricular Myocytes ◽

Molecular Level ◽

Cell Model ◽

Ryanodine Receptors ◽

Low Frequency ◽

Open Probability ◽

Long Duration ◽

Release Flux ◽

Health And Disease

Many issues remain unresolved concerning how local, subcellular Ca2+ signals interact with bulk cellular concentrations to maintain homeostasis in health and disease. To aid in the interpretation of data obtained in quiescent ventricular myocytes, we present here a minimal whole cell model that accounts for both localized (subcellular) and global (cellular) aspects of Ca2+ signaling. Using a minimal formulation of the distribution of local [Ca2+] associated with a large number of Ca2+-release sites, the model simulates both random spontaneous Ca2+ sparks and the changes in myoplasmic and sarcoplasmic reticulum (SR) [Ca2+] that result from the balance between stochastic release and reuptake into the SR. Ca2+-release sites are composed of clusters of two-state ryanodine receptors (RyRs) that exhibit activation by local cytosolic [Ca2+] but no inactivation or regulation by luminal Ca2+. Decreasing RyR open probability in the model causes a decrease in aggregate release flux and an increase in SR [Ca2+], regardless of whether RyR inhibition is mediated by a decrease in RyR open dwell time or an increase in RyR closed dwell time. The same balance of stochastic release and reuptake can be achieved, however, by either high-frequency/short-duration or low-frequency/long-duration Ca2+ sparks. The results are well correlated with recent experimental observations using pharmacological RyR inhibitors and clarify those aspects of the release-reuptake balance that are inherent to the coupling between local and global Ca2+ signals and those aspects that depend on molecular-level details. The model of Ca2+ sparks and homeostasis presented here can be a useful tool for understanding changes in cardiac Ca2+ release resulting from drugs, mutations, or acquired diseases.

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Revealing t-tubules in striated muscle with new optical super-resolution microscopy techniques

European Journal of Translational Myology ◽

10.4081/ejtm.2015.4747 ◽

2014 ◽

Vol 25 (1) ◽

pp. 15 ◽

Cited By ~ 11

Author(s):

Isuru D. Jayasinghe ◽

Alexander H. Clowsley ◽

Michelle Munro ◽

Yufeng Hou ◽

David J. Crossman ◽

...

Keyword(s):

Electron Microscopy ◽

Light Microscopy ◽

Single Molecule ◽

Striated Muscle ◽

Calcium Signalling ◽

Super Resolution ◽

Muscle Cells ◽

Muscle Fibres ◽

T Tubules ◽

Microscopy Techniques

The t-tubular system plays a central role in the synchronisation of calcium signalling and excitation-contraction coupling in most striated muscle cells. Light microscopy has been used for imaging t-tubules for well over 100 years and together with electron microscopy (EM), has revealed the three-dimensional complexities of the t-system topology within cardiomyocytes and skeletal muscle fibres from a range of species. The emerging super-resolution single molecule localisation microscopy (SMLM) techniques are offering a near 10-fold improvement over the resolution of conventional fluorescence light microscopy methods, with the ability to spectrally resolve nanometre scale distributions of multiple molecular targets. In conjunction with the next generation of electron microscopy, SMLM has allowed the visualisation and quantification of intricate t-tubule morphologies within large areas of muscle cells at an unprecedented level of detail. In this paper, we review recent advancements in the t-tubule structural biology with the utility of various microscopy techniques. We outline the technical considerations in adapting SMLM to study t-tubules and its potential to further our understanding of the molecular processes that underlie the sub-micron scale structural alterations observed in a range of muscle pathologies.

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Sarcolemmal distribution of ICa and INCX and Ca2+ autoregulation in mouse ventricular myocytes

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.00117.2017 ◽

2017 ◽

Vol 313 (1) ◽

pp. H190-H199 ◽

Cited By ~ 6

Author(s):

Hanne C. Gadeberg ◽

Cherrie H. T. Kong ◽

Simon M. Bryant ◽

Andrew F. James ◽

Clive H. Orchard

Keyword(s):

Steady State ◽

Sarcoplasmic Reticulum ◽

Cardiac Myocytes ◽

Ventricular Myocytes ◽

Surface Membrane ◽

Ryanodine Receptors ◽

Cell Voltage ◽

Intact Cells ◽

T Tubules

The balance of Ca2+ influx and efflux regulates the Ca2+ load of cardiac myocytes, a process known as autoregulation. Previous work has shown that Ca2+ influx, via L-type Ca2+ current ( ICa), and efflux, via the Na+/Ca2+ exchanger (NCX), occur predominantly at t-tubules; however, the role of t-tubules in autoregulation is unknown. Therefore, we investigated the sarcolemmal distribution of ICa and NCX current ( INCX), and autoregulation, in mouse ventricular myocytes using whole cell voltage-clamp and simultaneous Ca2+ measurements in intact and detubulated (DT) cells. In contrast to the rat, INCX was located predominantly at the surface membrane, and the hysteresis between INCX and Ca2+ observed in intact myocytes was preserved after detubulation. Immunostaining showed both NCX and ryanodine receptors (RyRs) at the t-tubules and surface membrane, consistent with colocalization of NCX and RyRs at both sites. Unlike INCX, ICa was found predominantly in the t-tubules. Recovery of the Ca2+ transient amplitude to steady state (autoregulation) after application of 200 µM or 10 mM caffeine was slower in DT cells than in intact cells. However, during application of 200 µM caffeine to increase sarcoplasmic reticulum (SR) Ca2+ release, DT and intact cells recovered at the same rate. It appears likely that this asymmetric response to changes in SR Ca2+ release is a consequence of the distribution of ICa, which is reduced in DT cells and is required to refill the SR after depletion, and NCX, which is little affected by detubulation, remaining available to remove Ca2+ when SR Ca2+ release is increased. NEW & NOTEWORTHY This study shows that in contrast to the rat, mouse ventricular Na+/Ca2+ exchange current density is lower in the t-tubules than in the surface sarcolemma and Ca2+ current is predominantly located in the t-tubules. As a consequence, the t-tubules play a role in recovery (autoregulation) from reduced, but not increased, sarcoplasmic reticulum Ca2+ release.

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Cardiac Alternans Occurs through the Synergy of Voltage- and Calcium-Dependent Mechanisms

Membranes ◽

10.3390/membranes11100794 ◽

2021 ◽

Vol 11 (10) ◽

pp. 794

Author(s):

Minh Tuan Hoang-Trong ◽

Aman Ullah ◽

William Jonathan Lederer ◽

Mohsin Saleet Jafri

Keyword(s):

Sarcoplasmic Reticulum ◽

Calcium Channels ◽

Calcium Release ◽

Ryanodine Receptors ◽

Cellular Level ◽

Open Probability ◽

Calcium Dependent ◽

Cardiac Alternans ◽

Rapid Pacing ◽

Dependent Mechanism

Cardiac alternans is characterized by alternating weak and strong beats of the heart. This signaling at the cellular level may appear as alternating long and short action potentials (APs) that occur in synchrony with alternating large and small calcium transients, respectively. Previous studies have suggested that alternans manifests itself through either a voltage dependent mechanism based upon action potential restitution or as a calcium dependent mechanism based on refractoriness of calcium release. We use a novel model of cardiac excitation-contraction (EC) coupling in the rat ventricular myocyte that includes 20,000 calcium release units (CRU) each with 49 ryanodine receptors (RyR2s) and 7 L-type calcium channels that are all stochastically gated. The model suggests that at the cellular level in the case of alternans produced by rapid pacing, the mechanism requires a synergy of voltage- and calcium-dependent mechanisms. The rapid pacing reduces AP duration and magnitude reducing the number of L-type calcium channels activating individual CRUs during each AP and thus increases the population of CRUs that can be recruited stochastically. Elevated myoplasmic and sarcoplasmic reticulum (SR) calcium, [Ca2+]myo and [Ca2+]SR respectively, increases ryanodine receptor open probability (Po) according to our model used in this simulation and this increased the probability of activating additional CRUs. A CRU that opens in one beat is less likely to open the subsequent beat due to refractoriness caused by incomplete refilling of the junctional sarcoplasmic reticulum (jSR). Furthermore, the model includes estimates of changes in Na+ fluxes and [Na+]i and thus provides insight into how changes in electrical activity, [Na+]i and sodium-calcium exchanger activity can modulate alternans. The model thus tracks critical elements that can account for rate-dependent changes in [Na+]i and [Ca2+]myo and how they contribute to the generation of Ca2+ signaling alternans in the heart.

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