The importance of Zn²⁺ in cardiac function was first reported in 1965, where it was demonstrated that increasing [Zn²⁺] decreased contractility and disrupted intracellular Ca²⁺-handling in rabbit atria [1]. [Zn²⁺] is elevated in ischaemia and rapidly declines in reperfusion [2] and intracellular Zn²⁺ levels and protein expression of Zn²⁺ transporters are altered in the failing heart [3]. The molecular mechanisms linking disrupted Zn²⁺ homeostasis with altered intracellular Ca²⁺-dynamics remain poorly understood. We have shown that Zn²⁺ influences intracellular Ca²⁺ dynamics in cardiomyocytes by modulating the function of the type-2 ryanodine receptor [4,5]. We have also shown that Mitsugumin 23 (MG23), a recently identified cation channel located on the sarcoplasmic reticulum (SR) and nuclear membranes, is regulated by Zn²⁺ [5].

The aim of this research was to investigate the structure-function relationship of Zn²⁺ modulation of MG23.

MG23 channels were enriched from mouse heart by affinity purification and recombinant tagged human MG23 was purified using SMA co-polymers. Site-directed mutagenesis of human MG23 was achieved using PCR. Biophysical characterisation of channel function was carried out using the planar lipid bilayer technique. Cardiomyocytes were isolated from mouse hearts by a Langendorff-free method. Live cell imaging of Zn²⁺ and Ca²⁺-dynamics were measured using 1 µM FluoZin-3 AM or 5μM Fluo-4 AM respectively. SR Ca²⁺-store levels were assessed by addition of 10 mM caffeine. Statistical significance was calculated with student’s t-test or one-way ANOVA with Tukey’s or Dunnett’s post-hoc test (as specified in figure legend). The institutional ethics committee at the University of St. Andrews and Kyoto University approved the study. Work was carried out under project licence P82006EDF.

Our data show that pathophysiological [Zn²⁺] (1 nM) increase both mouse and human MG23 channel activity, suggesting that MG23 modulation by Zn²⁺ is conserved across species (n ≥ 3). To probe Zn²⁺ binding sites of MG23, we constructed a E79Q channel mutant. The activity of the MG23 mutant was not increased by 1 nM Zn²⁺, suggesting this residue is important in Zn²⁺ regulation of channel activity (n = 4).

In isolated cardiomyocytes, we confirm that hypoxia (0.5% O₂) leads to Zn²⁺ dyshomeostasis (n = 2 animals). We also show that cardiomyocytes exposed to hypoxic conditions have increased MG23 protein expression (n ≥ 2 animals) and a significant reduction in the caffeine-induced SR Ca²⁺-release response (n ≥ 20 cells). Under the same conditions, store levels were restored by chelation of Zn²⁺ with TPEN (10 µM).  To investigate the role of MG23 in Zn²⁺-mediated SR Ca²⁺ leak, cardiomyocytes isolated from MG23 knock-out mice were exposed to hypoxic conditions. Our data reveal that SR Ca²⁺ load was not decreased in these cells (n ≥ 20 cells).

These preliminary data suggest MG23 is a Zn²⁺-regulated Ca²⁺ permeable channel and provide the first evidence that MG23 is a key driver in deleterious Ca²⁺ leak in progressive deterioration of cardiac function in pathophysiology.