Glutamate drives ‘local Ca2+ release’ in cardiac pacemaker cells

Figures

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  Fig. 1. Glutamate was enriched in the cytoplasm but not in the mitochondria in rat SANPCs. a Representative confocal images of rat iGluSnFR-overexpressing SANPCs which were stained with Mito-tracker or Cyto-tracker. Scale bar = 20 µm. b Quantification of colocalization coefficients between glutamate and Mito-tracker/Cyto-tracker (n = 6). c Glutamate concentrations in mitochondrial and cytoplasmic subcellular components (n = 6). *P < 0.05, calculated by unpaired Student’s t-test.

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  Fig. 2. Colocalization of glutamate accumulation and LCR events in rat SANPCs. a Representative confocal images demonstrating the subcellular colocalization of glutamate accumulation and LCR events. b Line plot profiles of the fluorescence along the white dotted line in a. c Scatterplots showing the Pearson’s correlation coefficient (Rr) between the fluorescent signals of glutamate accumulation and LCR events. d Typical traces of spontaneous Ca2+ transients in SANPCs upon the intracellular injection of vehicle or 5 mM glutamate. e, f Pooled data from d demonstrated that glutamate application significantly increased the LCR number per cycle (e) and the Ca2+ transients rates (f) in SANPCs compared with the vehicle (n = 6 per group). g Representative confocal line-scan images of LCRs in permeabilized SANPCs. h–k The pooled data from g demonstrated that the application of 5 mM glutamate significantly increased LCR number (h), LCR size (i), LCR duration (j) and LCR amplitude (k) in permeabilized SANPCs compared to the vehicle group (n = 8–12 cells per group, cells were isolated from at least 5 rats). *P < 0.05, calculated by unpaired Student’s t-test.

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  Fig. 3. Glutamate triggered LCRs by increasing the oxidation of Ca2+-handling proteins. a, b To assess the oxidation status of RyR2, the free thiol content of immunoprecipitated RyR2 was measured using the anti-DNP antibody. Western blot showing increased oxidation of RyR2 under 5 mM glutamate treatment. a Representative western blot bands. b Pooled data from a. n = 6 per group. c, d To assess the oxidation status of SERCA2a, the free thiol content of immunoprecipitated SERCA2a was measured using the DNP antibody. Western blot showing increased oxidation of SERCA2a under glutamate treatment. c Representative western blot bands. d Pooled data from c. n = 6 per group. e, f To assess the oxidation status of CaMKII, the free thiol content of immunoprecipitated CaMKII was measured using the DNP antibody. Western blot showing increased oxidation of CaMKII under glutamate treatment. e Representative western blot bands. f Pooled data from e. n = 6 per group. g Representative confocal line-scan images of LCRs in permeabilized SANPCs. h–k Pooled data from g demonstrated that oxidation inhibitors (5 mM DTT or 10 μM dantrolene) reversed glutamate-induced increases in LCR number (h), LCR size (i),…

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  Fig. 4. Glutamate-induced mitochondrial ROS generation promoted the oxidation of Ca2+-handling proteins in rat SANPCs. a Measurement of ROS levels in isolated SANPC mitochondria with or without 5 mM glutamate treatment (n = 5 per group). b Measurement of glutamate-induced ROS in SANPC mitochondria treated with vehicle or 10 µM FCCP, 10 µM rotenone or 10 µM HQNO (n = 5 per group). c–h To assess the oxidation status of RyR2, SERCA2a and CaMKII, the free thiol contents of these immunoprecipitated proteins were measured using the DNP antibody. Representative western blot images and semiquantitative analyses of glutamate-induced oxidation of RyR2 (c, d), SERCA2a (e, f) and CaMKII (g, h) treated with vehicle, 10 µM FCCP, 10 µM rotenone or 10 µM HQNO are shown. *P < 0.05, calculated by unpaired Student’s t-test (a) or one-way ANOVA with Dunnett post-hoc test (b, d, f, h).

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  Fig. 5. Glutamate transporters mediated glutamate-induced ROS production and LCR occurrence. a Measurement of 5 mM glutamate-induced ROS levels in rat SANPC mitochondria treated with vehicle or 100 μM inhibitors of glutamate receptors/transporters (n = 5–6 per group). b Representative confocal line-scan images of glutamate-induced LCRs in permeabilized SANPCs treated with vehicle or 100 μM D-AP5, CNQX and DL-TBOA. c–f Pooled data from b demonstrated that only DL-TBOA decreased the LCR number (c), LCR size (d), LCR duration (e) and LCR amplitude (f) compared with the vehicle (n = 4–7 per group, cells were isolated from at least 5 rats). *P < 0.05, calculated by one-way ANOVA with Dunnett post-hoc test.

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  Fig. 6. EAAT1 was abundantly expressed in rat SANPC mitochondria. a Western blot analysis confirmed that EAAT1 was abundantly expressed in SANPC mitochondria. b Pooled data from a (n = 5 per groups). c Immunofluorescence staining with anti-EAAT1 antibody, anti-GRIA3 antibody, anti-GRIN1 antibody and anti-Cytochrome C antibody (mitochondrial marker) in isolated SANPCs (left); line plot profiles of the fluorescence along the white dotted line on the left (right).

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  Fig. 7. EAAT1 depletion attenuated mitochondrial glutamate import and automaticity in mouse SANPCs. a The mitochondrial FAM-glutamate import experiment showed that EAAT1 knockdown (EAAT1 KD) interrupted the time-dependent import of FAM-labeled glutamate into mitochondria (n = 4–6). b EAAT1 KD decreased glutamate-induced ROS production in SANPCs (n = 4–6). c Representative confocal line-scan images of spontaneous Ca2+ transients in SANPCs from the EAAT1 KD and control groups. d, e Pooled data from c (n = 9–10 per group). f Representative field potential recordings of SAN tissues from the EAAT1 KD and control groups. g Pooled data from f (n = 7–8 per group). h Representative surface ECG recordings of anesthetized mice from the EAAT1 KD and control groups. i Pooled data from h (n = 7–8 per group). *P < 0.05, calculated by unpaired Student’s t-test.