Identification of an endogenous glutamatergic transmitter system controlling excitability and conductivity of atrial cardiomyocytes

Figures

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  Fig. 1. Observation of the vesicles containing glutamate in the rat atrial cardiomyocytes. a Representative TEM images showing vesicles beneath the plasma membrane of atrial cardiomyocytes (yellow arrows). Scale bar, 200 nm. b The immunofluorescence confocal images showing glutamate (red) colocalized with the secretory vesicles marker, CAST (green) in atrial cardiomyocytes. Scale bar, 10 μm. c 3D reconstruction from the z-stack confocal image showing colocalization of glutamate and CAST in the atrial cardiomyocyte. Left, 3D view of the whole cell labeled with glutamate antibody (red) and CAST antibody (green). Right, 2D section view of the x-z coordinates showing the colocalization (white arrows) of glutamate (red) and CAST (green) in the atrial cardiomyocyte.

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  Fig. 2. Rat atrial cardiomyocytes express core elements of the glutamatergic transmitter system. a Heat map showing expression of 34 assayed genes in single atrial cardiomyocytes. Rows are the samples of single atrial cardiomyocytes; columns are the genes. Color denotes the relative expression levels of the genes. b Representative agarose gel image of the semi-qPCR products demonstrats the expression of the glutamatergic transmitter system genes in atrial cardiomyocytes. c Immunofluorescent localization of GLS, GRIA3, GRIN1 and EAAT1 in ANP-expressing rat atrial cardiomyocytes. Scale bar, 25 μm.

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  Fig. 3. Identification of iGluRs-gated currents in the rat atrial cardiomyocytes. a Representative patch-clamp recordings of whole-cell currents elicited by glutamate in rat atrial cardiomyocytes. The holding potential was set at −60 mV. b Pooled data of the glutamate-elicited currents with different concentrations of glutamate. n = 9. c The AMPA or NMDA iGluR-gated currents in atrial cardiomyocytes were blocked by local application of CNQX or AP-5 in the perfusion solution at the holding potential of −60 mV. d Pooled data of the AMPA or NMDA iGluR-gated current densities from c. *P < 0.05, calculated by Student’s t-test, n = 9 per group.

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  Fig. 4. Activation of iGluRs excited the isolated rat atrial cardiomyocytes. a Action potentials were evoked by the increasing injection currents from 5 pA to 125 pA by 5 pA step. The cells were separately treated with iGluR agonist puffs (1 mM glutamate, 1 mM AMPA or 1 mM NMDA, respectively), or the iGluR antagonists (100 μM CNQX, MK-801 or AP-5, respectively), which were delivered through the perfusion solution. Black arrows indicate the minimum threshold current evoking the action potential. b, c Pooled data of the minimum threshold current evoking action potential and the amplitude of the action potentials from a. *P < 0.05, calculated by Student’s t-test, n = 6 per group.

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  Fig. 5. Inhibition of iGluRs attenuated electrical conduction velocity in rat atrial myocardium. a Representative right atrial ECG recordings (shown as the derivative of the electric potential as dV/dt) in anesthetic rats. The iGluR antagonists (CNQX, MK-801, and AP-5) respectively slow the electrical conduction velocity. b Pooled data of electrical conduction velocity from a. *P < 0.05, calculated by Student’s t-test or Kolmogorov–Smirnov test, n = 4−5 per group. c Typical conduction heatmap of isolated rat atria perfused with 100 μM CNQX, MK-801 or AP-5, respectively. The black arrows indicate the conduction direction of the action potential. Scale bar, 4 mm. d Pooled data of electrical conduction velocity from c. *P < 0.05, calculated by Student’s t-test, n = 7−8 per group.

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  Fig. 6. Knockdown of iGluRs decreased the excitability and conductivity in the cultured human iPSC-derived atrial cardiomyocyte monolayers. a Experimental protocol for the MEA analysis in cultured human iPSC-derived atrial cardiomyocyte monolayers. b, c Knockdown efficiency of GRIA3 (b) and GRIN1 (c) in the human iPSC-derived atrial cardiomyocytes at mRNA level. *P < 0.05, calculated by Student’s t-test, n = 3. d, e Knockdown efficiency of GRIA3 (d) and GRIN1 (e) in human iPSC-derived atrial cardiomyocytes at protein level. *P < 0.05, calculated by Student’s t-test, n = 3. f Typical heatmap of the excitatory firing rate in the cultured atrial cardiomyocyte monolayers with GRIA3 or GRIN1 knockdown. g Pooled data of firing rate from f. *P < 0.05, calculated by Student’s t-test, n = 5−7 per group. h Typical conduction heatmap of cultured atrial cardiomyocyte monolayers with GRIA3 or GRIN1 knockdown. i Pooled data of conduction velocity from h. *P < 0.05, calculated by Student’s t-test, n = 5−8 per group.

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  Fig. 7. Blockage of iGluRs prevented AF. a Experimental design for analyzing the effects of the iGluR antagonists on AF. b Representative ECG and right atrial electrograms (RAE) in the heart showing that an atrial 40 Hz pacing protocol successfully induced AF in the vehicle control. However, pre-treatment with the iGluR antagonists, including CNQX, MK-801 or AP-5, prevented the occurrence of the AF under the same burst pacing stimulation. c, d Pooled data of the incidence and duration of AF induced by the 40 Hz burst pacing protocol in atria with or without the pre-treatment of the iGluR antagonists. *P < 0.05, calculated by χ2 test, n = 8−9.

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  Fig. 8. Blockage of iGluRs terminated AF. a Experimental design for analyzing the effects of the iGluR antagonists on treating AF. b Representative ECG and RAE in the heart showing that AF was terminated by the application of CNQX, MK-801, or AP-5 but not the vehicle. c, d Pooled data of the incidence and duration of the burst pacing-induced AF with or without treatment of the iGluR antagonists. *P < 0.05, calculated by χ2 test, n = 8−9.

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  Fig. 9. Expression and functional role of the glutamatergic transmitter system in human atrial cardiomyocytes. a qPCR analysis of the core elements of the glutamatergic transmitter system in human atrial cardiomyocytes illustrating that GLS, GRIA3, GRIN1 and SLC1A3 were highly expressed in human atrial cardiomyocytes at mRNA level. b Western blot analysis of the core elements of the glutamatergic transmitter system illustrating that GLS, GRIA3, GRIN1 and EAAT1 are expressed in human atrial cardiomyocytes at protein level. c Immunofluorescence images of the core elements of the glutamatergic transmitter system in human atrial samples showing the spatial location of GLS, GRIA3, GRIN1, and EAAT1 in the human atrial cardiomyocytes. Scale bar, 50 μm. d Representative patch-clamp recordings of whole-cell currents elicited by the application of 1 mM glutamate in human atrial cardiomyocytes. The holding potential was set at −60 mV. e Pooled data of the glutamate-elicited currents induced by different concentrations of glutamate in isolated human atrial cardiomyocytes. n = 6.