Spatial and Functional Crosstalk between the Mitochondrial Na+-Ca2+ Exchanger NCLX and the Sarcoplasmic Reticulum Ca2+ Pump SERCA in Cardiomyocytes

Abstract

The mitochondrial Na+-Ca2+ exchanger, NCLX, was reported to supply Ca2+ to sarcoplasmic reticulum (SR)/endoplasmic reticulum, thereby modulating various cellular functions such as the rhythmicity of cardiomyocytes, and cellular Ca2+ signaling upon antigen receptor stimulation and chemotaxis in B lymphocytes; however, there is little information on the spatial relationships of NCLX with SR Ca2+ handling proteins, and their physiological impact. Here we examined the issue, focusing on the interaction of NCLX with an SR Ca2+ pump SERCA in cardiomyocytes. A bimolecular fluorescence complementation assay using HEK293 cells revealed that the exogenously expressed NCLX was localized in close proximity to four exogenously expressed SERCA isoforms. Immunofluorescence analyses of isolated ventricular myocytes showed that the NCLX was localized to the edges of the mitochondria, forming a striped pattern. The co-localization coefficients in the super-resolution images were higher for NCLX–SERCA2, than for NCLX–ryanodine receptor and NCLX–Na+/K+ ATPase α-1 subunit, confirming the close localization of endogenous NCLX and SERCA2 in cardiomyocytes. The mathematical model implemented with the spatial and functional coupling of NCLX and SERCA well reproduced the NCLX inhibition-mediated modulations of SR Ca2+ reuptake in HL-1 cardiomyocytes. Taken together, these results indicated that NCLX and SERCA are spatially and functionally coupled in cardiomyocytes.

Mitochondrial sodium/calcium exchanger (NCLX) regulates basal and starvation‐induced autophagy through calcium signaling

“…Indeed, NCLX KD has been extensively associated with decreased cellular Ca 2+ entry and, consequently, low Ca 2+ levels in the ER. 10,11,[55][56][57] To test whether the changes in intracellular Ca 2+ homeostasis could lead to defective autophagy, we preincubated cells for 1 h with the cell-permeant Ca 2+ chelator BAPTA-AM before incubation with HBSS. As expected, pre-incubation with BAPTA-AM prevented FIP200 puncta formation by HBSS (Figure 6B).…”

“… 9,11,53,54 In KD cells, the half‐life decay was strikingly higher (Figure S5A), indicating that other Ca 2+ clearance pathways are strongly affected by decreased NCLX expression, and further linking reduced NCLX activity to impaired cytoplasmic Ca 2+ signals. Indeed, NCLX KD has been extensively associated with decreased cellular Ca 2+ entry and, consequently, low Ca 2+ levels in the ER 10,11,55–57 …”

Mitochondrial Sodium/Calcium Exchanger (NCLX) Regulates Basal and Starvation-Induced Autophagy Through Calcium Signaling

“…In KD cells, the half-life decay was strikingly higher (Fig S5A), indicating that other Ca 2+ clearance pathways are strongly affected by decreased NCLX expression, and further linking reduced NCLX activity to impaired cytoplasmic Ca 2+ signals. Indeed, NCLX KD has been extensively associated with decreased cellular Ca 2+ entry and, consequently, low Ca 2+ levels in the ER (Takeuchi et al, 2013; Parnis et al, 2013; Ben-Kasus Nissim et al, 2017; Emrich et al, 2022; Takeuchi and Matsuoka, 2022).…”

Case report: high-dose carvedilol as a potential key drug for arrhythmias in histiocytoid cardiomyopathy

“…The Ca 2+ concentration in cardiomyocytes is regulated by some exchanger and receptors on cytomembrane, sarcoplasmic reticulum, and mitochondria ( Figure 4 ). 13 , 14 The abnormal mitochondria in cardiomyocytes in histiocytoid cardiomyopathy and resulting in abnormal regulation of intracellular Ca 2+ concentration may be associated with their refractory ventricular tachycardias.…”

“… Ca 2+ regulation in cardiomyocytes. The figure is modified from Takeuchi et al 13 NCX, Na + –Ca 2+ exchanger on cytomembrane; DHPR, dihydropyridine receptor; NCLX, mitochondrial Na + –Ca 2+ exchanger; MCU, mitochondrial Ca 2+ uniporter; SERCA, sarco-endoplasmic reticulum Ca 2+ -ATPase; RyR2, ryanodine receptor 2; BAR, beta-adrenergic receptor. …”