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

Abstract

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 Ryanodine receptors (RyR) within the junctional sarcoplasmic reticulum (jSR). 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. Junctophilin-2 (JPH2) enables close association between T-tubules and the jSR 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 (mutPG1JPH2) caused T-tubule remodeling and dyad loss, showing that an interaction between LTCC and JPH2 is crucial for T-tubule stabilization. mut PG1JPH2 caused asynchronous Ca 2+ -release with impaired excitation-contraction (EC) coupling after β-adrenergic stimulation. The disturbed Ca 2+ regulation in mut PG1JPH2 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 CIRC in cardiomyocytes.

The Physiology and Pathophysiology of T-Tubules in the Heart

“…While EM data have historically indicated that the dyadic cleft measures only ≈12 nm across ( Takeshima et al, 2000 ), recent data have suggested that the true dimensions may be even more narrow (<10 nm), and that artifactual expansion of the cleft could have occurred in earlier work as a result of sample fixation procedures ( Rog-Zielinska et al, 2021a ). Consistent dyadic dimensions are ensured by junctophilin-2 (JPH2) which stabilizes the membranes, but also functionally interacts with both RyRs and LTCCs ( Jiang et al, 2016 ; Munro et al, 2016 ; Reynolds et al, 2016 ; Gross et al, 2021 ). Insight into the precise positioning of these dyadic proteins has been made possible by new advances in super-resolution microscopy, including STED ( Wagner et al, 2012 ), 3d STORM ( Shen et al, 2019 ), and DNA-PAINT techniques ( Jayasinghe et al, 2018 ; Sheard et al, 2019 ).…”

“…JPH2 is also reported to modulate LTCC activity, which has important implications for L-type current in HFrEF, where JPH2 expression is lowered ( Jiang et al, 2016 ). Interestingly, recent data from Gross et al (2021) indicate that the “Joining Region” of JPH2 may exert direct effects on the localization of LTCC in t-tubules, indicating that not only the presence of JPH2 but specifically JPH2-LTCC binding is necessary for maintenance of dyadic integrity and Ca 2+ homeostasis.…”

Striated Preferentially Expressed Protein Kinase (SPEG) in Muscle Development, Function, and Disease

“…JPH2 is the cardiac isoform of the junctophilins family that stabilizes the cardiac dyads between T-tubule and junctional SR membranes, which is critical for proper intracellular calcium signaling [ 100 ]. JPH2 knockdown leads to T-tubule disruption in developing myocardium and in the adult mouse heart [ 101 , 102 ].…”

Electrophysiological Remodeling: Cardiac T-Tubules and ß-Adrenoceptors

“…This all occurs against the backdrop of highly specialized, cardiac sub-domains. JPH-2 is understood to control the structure of the calcium dyad by linking ion channel function to control calcium-induced calcium release [ 27 ], as well as being involved in the maintenance of normal tubular morphology [ 28 ]. BIN-1 is also crucial for the maintenance of ion fluxes in healthy ventricular cardiomyocytes [ 29 ].…”

“…The action of LTCC induces the release of calcium from the RYR2 in a process known as calcium-induced calcium release (CICR). JPH-2 is intrinsically important to the process, as it is involved in the direct link between the LTCC and RYR2 to maintain the process [ 27 ]. This sort of complex should be considered similar to the general structural and molecular compartmentation of βAR by membrane microdomains within the TATS.…”