Journal: Journal of Biological Chemistry

Article Title: Effective Post-insult Neuroprotection by a Novel Ca2+/ Calmodulin-dependent Protein Kinase II (CaMKII) Inhibitor

doi: 10.1074/jbc.m109.088617

Figure Lengend Snippet: FIGURE 2. CaMKII inhibition reduced excitotoxic neuron death assessed by LDH release. A, CaMKII inhibition by 5 M tatCN21 or 10 M KN93 reduced glutamate-induceddeathalsowhenmeasuredbyLDHassay(**indicatesp 0.001 in Dunnett’s t test ANOVA post hoc analysis). The controls tatRev and KN92 had no effect, whereas the CaMKI and IV inhibitor STO-609 (5 M) increased cell death (p 0.05 in analysis as above). B, glutamate-induced neuron death was significantly reduced by the NMDAR inhibitor D-APV (100 M), and NMDA-induced neuron death was significantly inhibited by tatCN21 (5 M) (** indicates p 0.001 compared with glutamate in Newman-Keuls multiple comparison test after ANOVA; n.s. indicates that there was no differ- ence between these two conditions). C, tatCN21 inhibited NMDA-induced death in an additional independent experiment (p 0.001 in Student’s t test), similar to what was observed for glutamate-induced death in A. D, CaMKII inhibition by 5 and 15 M tatCN21 showed significant neuroprotection (**, p 0.001) that was indistinguishable between the two concentrations (n.s.), whereas 1.5 M tatCN21 also significant neuroprotection but to a much lesser extent (*, p 0.01; all in Newman-Keuls multiple comparison test after ANOVA). Thus, 5 M tatCN21 is sufficient to elicit maximal neuroprotection levels. In all panels, results shown are mean S.E.; N indicates the number of independent experiments; n indicates the number of coverslips.

Article Snippet: Reagents were obtained from Sigma, except for the following: inhibitor peptides and controls (Biomatix, Wilmington, DE, and Global Peptides, Fort Collins, CO); neuron culture supplies, ethidium homodimer 2 (EtDH2), Hoechst 33258, and Lipofectamine 2000 (Invitrogen); KN93, KN92, and STO-609 (Calbiochem); D-APV (Tocris Bioscience, St. Louis, MO); paraformaldehyde (Electron Microscopy Sciences, Hatfield, PA); lactate dehydrogenase assay (LDH) kit (Roche Applied Science); antibodies against MAP2 (Pharmingen); total CaMKII (CB 2; Invitrogen); and phospho-Thr-286 (PhosphoSolutions, Aurora, CO).

Techniques: Inhibition, Comparison

Journal: Advanced Science

Article Title: Decreased RYR2 Cluster Size and Abnormal SR Ca 2+ Release Contribute to Arrhythmogenesis in TMEM43 ‐Related ARVC

doi: 10.1002/advs.202512058

Figure Lengend Snippet: Decreased RYR2 cluster size leads to abnormal SR Ca 2+ release that is causally linked to the arrhythmic phenotypes in ARVC iPSC‐CMs. A,B) Representative Ca 2+ transient tracings from GC and ARVC iPSC‐CMs. Red arrows indicate the irregular arrhythmia‐like Ca 2+ transients. C–G) Bar graphs to compare key Ca 2+ transient parameters between GC and ARVC iPSC‐CMs. n = 79–95 cells in 2 different iPSC lines. H) Representative traces of cytosolic Ca 2+ fluorescence in control (CON), ARVC, and GC iPSC‐CMs in normal Tyrode's solution and exposed to 0 Na + , 0 Ca 2+ solution containing 1 m m tetracaine and 10 m m caffeine. I–L) Bar graphs to compare the Ca 2+ transient amplitude, SR Ca 2+ leak, SR Ca 2+ load, and fractional release between control, ARVC, and GC iPSC‐CMs. n = 87–148 cells in 2 different iPSC lines. M) Bar graph to compare the percentage of cells with irregular Ca 2+ transients between ARVC iPSC‐CMs treated with DMSO (vehicle), KN92, and KN93. n = 98–177 cells in 2 different iPSC lines. N) Bar graph to compare the percentage of cells with arrhythmias between ARVC iPSC‐CMs treated with DMSO, KN92, and KN93. n = 32–37 cells in 2 different iPSC lines. O) Representative Tau‐STED imaging of RYR2 (red) from control, ARVC, and GC iPSC‐CMs. P) Representative threshold binary images of (O). Q) Representative images showing RYR2 clusters in (P) were identified and labeled. R) Bar graph to compare the mean cluster size between control, ARVC, and GC iPSC‐CMs. n = 1973–5343 clusters. Data were collected from 2 different iPSC lines. S) Bar graph to compare the mean cluster density between control, ARVC, and GC iPSC‐CMs. n = 10–23 cells in 2 different iPSC lines. T) The distribution of the number of RYR2 s from clusters over a certain size across increasing cluster size. The vertical dashed line indicates the cluster size of 50. Scale bar, 500 nm. For all panels, data are represented as mean ± SEM. * p < 0.05; *** p < 0.001; **** p < 0.0001, unpaired two‐tailed Student's t ‐test (C–G) or one‐way ANOVA followed by Tukey's HSD post‐hoc test (I–L, R, S).

Article Snippet: KN92 and KN93 were purchased from Selleck (S6507) and Abcam ( Ab120980 ), respectively, and stock solutions were both prepared in 1 m m DMSO.

Techniques: Fluorescence, Control, Imaging, Labeling, Two Tailed Test