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.

Ageing Causes Ultrastructural Modification to Calcium Release Units and Mitochondria in Cardiomyocytes

“…Data in Figure 1 were also confirmed by images in Figure 2, where we immunolabeled the external membranes (i.e., T-tubule) using WGA (Figure 2A,D) [37] and antibodies against Cav-3 (Figure 2B,E) [38] and JP-2 (Figure 2C,F) [38]. Cav-3 is a protein that plays a key role in T-tubulation, whereas JP-2 is a membrane bridging protein which supports the assembly of junctional membrane complexes by tethering T-tubules to the Double-immunolabeling with primary antibodies against RYR2 and TOM20 revealed that in adult cardiomyocytes (a) the EC coupling apparatus has an ordered transversal disposition that creates a cross-striated pattern (Figure 1A; red labeling) while (b) mitochondria are mainly disposed longitudinally between the myofibrils (Figure 1A; green labeling).…”

“…Data in Figure 1 were also confirmed by images in Figure 2, where we immunolabeled the external membranes (i.e., T-tubule) using WGA (Figure 2A,D) [37] and antibodies against Cav-3 (Figure 2B,E) [38] and JP-2 (Figure 2C,F) [38]. Cav-3 is a protein that plays a key role in T-tubulation, whereas JP-2 is a membrane bridging protein which supports the assembly of junctional membrane complexes by tethering T-tubules to the SR membrane [37,38]: in aged cardiomyocytes the transversal labeling is frequently interrupted by longitudinally oriented fluorescence, indicative of a loos of integrity and organization of the sarcotubular system. By WB we also evaluated the expression levels of either JP-2 or Cav-3 and verified that both were reduced in samples from aged mice (Figure 2G,H), providing a possible molecular mechanism underlying the age-related disarray of the T-tubule network.…”

“…See Supplementary Table S1 for the numeric values used in Figure 3B-D. Data in Figure 1 were also confirmed by images in Figure 2, where we immunolabeled the external membranes (i.e., T-tubule) using WGA (Figure 2A,D) [37] and antibodies against Cav-3 (Figure 2B,E) [38] and JP-2 (Figure 2C,F) [38]. Cav-3 is a protein that plays a key role in T-tubulation, whereas JP-2 is a membrane bridging protein which supports the assembly of junctional membrane complexes by tethering T-tubules to the SR membrane [37,38]: in aged cardiomyocytes the transversal labeling is frequently interrupted by longitudinally oriented fluorescence, indicative of a loos of integrity and organization of the sarcotubular system.…”

“…SR membrane [37,38]: in aged cardiomyocytes the transversal labeling is frequently interrupted by longitudinally oriented fluorescence, indicative of a loos of integrity and organization of the sarcotubular system. By WB we also evaluated the expression levels of either JP-2 or Cav-3 and verified that both were reduced in samples from aged mice (Figure 2G,H), providing a possible molecular mechanism underlying the age-related disarray of the T-tubule network.…”

Mechanisms and Regulation of Cardiac CaV1.2 Trafficking

“…An interesting avenue for future research in those topics is that of the role of the junctional tethering protein junctophilin 2 (JPH2) in targeting Ca V 1.2 to microdomains within the sarcolemma. A recent study reported that JPH2 recruits Ca V 1.2 to lipid rafts on the T-tubules wherein overexpression of JPH2 in cultured rat cardiomyocytes led to increased channel density at the t-tubule and surface membrane that was not accompanied by an increase in total cellular Ca V 1.2 protein, implying JPH2 increased channel trafficking rather than altered channel biosynthesis [ 143 ]. Future studies should also parse out the mechanistic details of trafficking alterations that may explain how failing hearts redistribute Ca V 1.2 from the t-tubular sarcolemma to the surface membrane [ 144 , 145 , 146 ], and others that mediate the enhanced Ca V 1.2 sarcolemmal expression observed with aging [ 147 , 148 , 149 ].…”