Dysregulated Zn2+ homeostasis impairs cardiac type-2 ryanodine receptor and mitsugumin 23 functions, leading to sarcoplasmic reticulum Ca2+ leakage

Figure 1.. Cytosolic Zn2+ increased RyR2 activity in the presence of 1 mm Mg2+. A, representative single RyR2 channel traces with Ca2+ as the permeant ion. Bilayers were voltage-clamped at 0 mV. O and C show the fully open and closed states, respectively. In the presence of activating levels of cytosolic Ca2+ (5 μm), the addition of 1 mm Mg2+ reduced RyR2 activity. In the continued presence of 1 mm Mg2+, the subsequent addition of Zn2+ to the cytosolic face of the channel increased channel open probability. B, scatter plot showing the mean channel open probability of RyR2. Individual data points are shown by ○. Data are expressed as the mean ± S.D.; n = 3–5. Significant difference between treatments was assessed by a one-way ANOVA; (F(5,18) = 7.276, p = 0.0007) followed by a Bonferroni post hoc test. * (p < 0.05) and *** (p < 0.001) indicate significance when compared with Mg2+-treated control.

Figure 2.. Under diastolic conditions, pathophysiological levels of cytosolic free Zn2+ resulted in increased RyR2 activity. A, representative single RyR2 channel traces. Bilayers were voltage-clamped at 0 mV, and Ca2+ was the permeant ion. Open and closed states of RyR2 are indicated by O and C. As expected, RyR2 activity was reduced when the cytosolic Ca2+ were lowered from 5 μm to 100 nm and reduced further after the addition of 1 mm Mg2+. The addition of 2 nm Zn2+ to the cis chamber significantly increased channel activity. B, mean open probability of RyR2. Individual data points are shown by ○. Data are shown as the mean ± S.D.; n = 3–5. The difference between treatments was assessed by a one-way ANOVA followed by a Bonferroni post hoc test. F(5,21) = 8.133; p = 0.0002. * (p < 0.05) and *** (p < 0.001) indicate significance when compared with Mg2+-treated channels in the presence of 100 nm Ca2+. C, scatter plot showing the mean channel open time. Data are shown as the mean ± S.D.; n = 3–5. Individual data points are shown by ○. One-way ANOVA followed by a Bonferroni test was used to test difference between treatments (F(5,26) = 8.501, p < 0.0001). * indicates significance (p ≤ 0.05)…

Figure 3.. Native cardiac MG23 channels displayed distinct gating properties from RyR2. A, the left panel shows representative Ca2+ current fluctuations mediated by MG23, recorded at various holding potentials. The traces were chosen to show current amplitudes clearly. The right panel shows a current-voltage relationship for MG23 using Ca2+ as a permeant ion. Data are shown as the mean ± S.D. (n = 5). B, representative current fluctuations through both MG23 and RyR2 channel gating in the same bilayer under control conditions with Ca2+ as a permeant ion at a holding potential of 0 mV. Cytosolic free Ca2+ was 5 μm. MG23 (OMG23), RyR2 (ORyR2) open, RyR2 + MG23 (ORyR2+MG23) open and closed (C) states are indicated. The addition of 1 mm BAPTA to the cis chamber lowers Ca2+ to <4 nm. This Ca2+ concentration was sub-activating for RyR2 but had no effect on MG23. C, the top panel shows representative Ca2+ current fluctuations mediated through RyR2 under control conditions with Ca2+ as the permeant ion. Cytosolic-free Ca2+ was 5 μm. The addition of 10 mm caffeine to the cis chamber resulted in activation of RyR2 as expected. The lower panel shows a scatter plot of RyR2-channel open probability under…

Figure 4.. Pathophysiological concentrations of Zn2+ altered both the expression and activity of MG23. A, the upper panel representative Western blot shows MG23 protein expression in H9C2 cells exposed to hypoxia. All lanes were loaded with 10 μg of total protein. Western blots were probed with antibodies specific for MG23 or with β-actin as a loading control. Size markers are indicated in kDa. The lower panel scatter plot shows relative density of MG23 under labeled conditions. Band densities were normalized to β-actin loading controls in each lane and are expressed relative to control as % percentage change. The gray dashed line denotes control values. Data are displayed as the mean ± S.D. (n = 4). * denotes a significant difference (p < 0.05) to control. B, scatter plot showing ZnAF-2 DA fluorescence intensity from H9C2 cells exposed to ischemia buffer for indicated time points. For each time point ZnAF-2 DA fluorescence was normalized to DAPI nuclear staining fluorescence intensity for each corresponding well and expressed as percentage change from control. Control values are indicated by the gray dotted line. Data are displayed as the mean ± S.D. (n = 6) from two individual cell…

Figure 5.. Zn2+ modulated the activity of both MG23 and RyR2 but did not result in RyR2 sub-conductance gating. A, representative Ca2+ current fluctuations when both MG23 and RyR2 channels were gating in the same bilayer (left trace) and after the subsequent addition of 1 nm Zn2+ to the cis chamber (right trace). Cytosolic free Ca2+ was 5 μm. MG23 (OMG23), RyR2 (ORyR2) open, RyR2 + MG23 (ORyR2+MG23) open and closed (C) states are indicated. The corresponding all-points-amplitude histogram is shown underneath the respective single-channel recording. Recordings were made using Ca2+ as the permeant ion and at a holding potential of 0 mV. B, representative Ca2+ current fluctuations through RyR2 channels only (left trace) and after the subsequent addition of 1 nm cytosolic Zn2+ (right trace). Cytosolic free Ca2+ was 5 μm. Corresponding all-points-amplitude histograms are shown underneath the respective single-channel recording. C, left panel, Western blot analysis showing the phosphorylation of RyR2 at site serine 2809 after incubation of cardiac HSR vesicles with either 1 unit/μg of protein of the PKA active subunit, 0.1 nm Zn2+, or 1 nm Zn2+. In all experiments 1 mm Mg2+ and 1 mm ATP were…

Figure 6.. Zn2+ potentiated RyR2 activity in Mg23 knock-out mice. A, representative Ca2+ current fluctuations from bilayer experiments using cardiac SR vesicles isolated from wild-type and Mg23-knock-out mice. Functional MG23 channel gating was observed in wild-type experiments but not in Mg23 knock-out experiments. Open (O) and closed (C) states are indicated. Holding potential was 0 mV, and Ca2+ was used as the permeant ion. Cytosolic free Ca2+ was 5 μm. B, scatter plot showing noise analysis data from wild-type experiments where only MG23-mediated Ca2+ currents were observed. Mean average current is shown after incremental addition of Zn2+ to the cis chamber. C, representative Ca2+ current fluctuations from bilayer experiments using cardiac SR vesicles isolated from Mg23 knock-out mice showing functional RyR2 channel gating in the absence and presence of 1 nm Zn2+ added to the cis chamber. The lower panel scatter plot shows RyR2 channel open probability under these conditions. D, paired all-points-amplitude histograms under conditions described in C. Data shown are the mean ± S.D., n = 3, with * and ** indicating significant differences (p < 0.05 and p < 0.01), respectively, from…