Hyperglycaemia‐induced reactive oxygen species production in cardiac ventricular myocytes differs among mammals

Figures

• 

  Figure 1. Acute hyperglycaemia (HiGlu)‐induced reactive oxygen species (ROS) production in rat cardiomyocytes A, representative images of rat cardiomyocytes loaded with DCF, and representative of DCF raw image after exposure to HiGlu. Scale bar of 20 µm. B, representative of DCF raw trace of rat cardiomyocytes in response to HiGlu in the absence of an after pretreatment with KN93. The baseline slopes of fluorescence increase are indicated by extended red lines. C, quantification of DCF slope in presence of HiGlu or ThmG ± KN93 or OSMI‐1, normalized to the slope prior to HiGlu or ThmG exposure. GlAnT: combined HiGlu, angiotensin II and Thiamet G. D, HiGlu‐induced ROS is suppressed by TEMPO, but not mito‐TEMPO. E, NADPH oxidase 2 (NOX2) inhibitor GP91ds‐tat blocks HiGlu‐induced ROS. (Data are shown as mean ± standard deviation (SD); number of cells/animals used is shown in the bar; one‐sample t test; **** p < 0.0001).

• 

  Figure 2. Acute hyperglycaemia (HiGlu) and angiotensin II (Ang II)‐induced cytosolic and mitochondrial reactive oxygen species (ROS) production in rabbit cardiomyocytes A, representative images of DCF‐loaded isolated rabbit cardiomyocytes before and after exposure to HiGlu or GLANT (HiGlu + Ang II + ThmG). Scale bar of 20 µm. B, quantification of DCF slope in presence of HiGlu, ThmG, Ang II or in combination (GlAnT). C, increased pacing rate does not affect the ROS level in the cells. D, inhibition of GlAnT‐induced ROS: inhibition of CaMKII (KN93), O‐GlcNAcylation (OSMI‐1), mitochondrial or cytosolic ROS (mito‐TEMPO or TEMPO) failed to suppress ROS increase. Only combined inhibitors could prevent GlAnT‐induced ROS. E, averaged mitoSOX signals of time course with different treatments. (Data are shown as mean ± standard deviation (SD); number of cells/animals used is shown in the bars; one‐sample t test for DCF experiments; one‐way analysis of variance (ANOVA) for mitoSOX measurement; *p < 0.05, **p < 0.01, ***p < 0.001, **** p < 0.0001).

• 

  Figure 3. Acute hyperglycaemia (HiGlu)‐induced reactive oxygen species (ROS) production in human cardiomyocytes A, representative ROS traces with and without HiGlu superperfusion. B, quantification of DCF slope without or with pretreatment of KN93, OSMI‐1, GP91 or mito‐TEMPO normalized to the slope prior to HiGlu exposure. The samples from trauma death are darker with black border. (Data are shown as mean ± standard deviation (SD); number of cells/patients used is shown in the bar; one‐sample t test, * p < 0.05).

• 

  Figure 4. Measurement of HiGlu‐induced cytosolic glucose level increase A, exemplar images of CFP and YFP in rat myocytes expressing FlipGlu700. Scale bar 20 µm. B, selective photo‐bleach of YFP acceptor enhances donor fluorescence by 16%. C, representative trace of time course of FRET change in a saponin‐permeabilized rat myocyte for different glucose levels. D, calibration curve of FlipGlu700 as function of [glucose]. E, representative intact myocyte FRET trace with acute extracellular HiGlu perfusion and final maximal glucose (50 mM). F, HiGlu perfusion significantly increases cytosolic glucose in rat cardiomyocytes. (Data are shown as mean ± standard deviation (SD); number of cells/animals used is shown in the bar; Student's t test; *** p < 0.001).

• 

  Figure 5. Measurement of HiGlu‐induced cytosolic glucose in isolated rabbit myocytes A, representative images of rabbit cardiomyocytes transfected with FlipGlu700. Scale bar 20 µm. B, selective YFP acceptor photo‐bleach results in increased donor fluorescence. C, calibration of FlipGlu700 in rabbit myocytes. D, HiGlu perfusion significantly increases cytosolic glucose in rabbit cardiomyocytes. (Data are shown as mean ± standard deviation (SD); number of cells/animals used is shown in the bar; Student's t test; ****p < 0.0001).

• 

  Figure 6. Redox sensor evaluation of reactive oxygen species (ROS) production in isolated rat myocytes A, representative images of isolated rat cardiomyocytes transfected with four different redox sensors. Scale bar 20 µm. B, averaged traces of cytosolic localized redox sensors responses to HiGlu perfusion. C, averaged traces of mitochondrial localized redox sensors responses to HiGlu perfusion. D, calculated EGSH and H2O2 concentrations of cyto‐Grx and Orp sensors based on H2O2 and DTT in response to HiGlu. E, summarized fluorescence ratio redox levels with HiGlu and GlAnT treatments. Data are shown as mean ± standard deviation (SD); number of cells/animals used is shown in the bar; Student's t test on EGSH; one‐sample t test for normalized sensor data; *p < 0.05, **p < 0.01, ***p < 0.001, **** p < 0.0001).

• 

  Figure 7. Redox sensor evaluation of reactive oxygen species (ROS) production in isolated rabbit myocytes A, representative images of isolated rat cardiomyocytes transfected with four different redox sensors. Scale bar 20 µm. B, averaged traces of cytosolic localized redox sensors’ responses to GlAnT perfusion. C, respectively calculated EGSH and H2O2 concentrations of cyto‐Grx and Orp sensors based on H2O2 and DTT of baseline and in response to HiGlu + ThmG, Ang II and GlAnT. D, summarized redox levels with different treatments. Data are shown as mean ± standard deviation (SD); number of cells/animals used is shown in the bar; one‐sample t test for normalized sensor data; **p < 0.01, ***p < 0.001).

• 

  Figure 8. Pacing cultured cardiomyocytes increased reactive oxygen species (ROS) level, but divergent HiGlu responses in rat and rabbit A, strategy of isolated cardiomyocytes with pacing culture. B, calculated H2O2 levels based on calibration curves show increased baseline and HiGlu response of ROS after pacing culture in rat. C, calculated H2O2 levels based on calibration curves show increased level of ROS after pacing in culture in rabbit. Data are shown as mean ± standard deviation (SD); number of cells/animals used is shown in the bar; one‐sample t test for normalized sensor data; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001).

• 

  Figure 9. Superoxide dismutase (SOD) activity in rat and rabbit cardiomyocytes A, SOD activities remain unchanged with 20 min HiGlu treatment. B, SOD activities measured after 24 h pacing culture. Data are shown as mean ± standard deviation (SD), number of cells/animals used is shown in the bar; two‐way analysis of variance (ANOVA) was used, **P < 0.01).

• 

  Figure A1. Representative traces of DCF, mitoSOX and fluorescent reactive oxygen species (ROS) sensors A, representative trace of DCF with normalized baseline. B, DCF signals with NOX4 inhibitor (GLX351322) pretreated rat cardiomyocytes. C, representative trace of mitoSOX intensity in rabbit cardiomyocytes. D, representative traces of cyto‐Grx, cyto‐Orp, mito‐Grx and mito‐Orp in rat with HiGlu perfusion at 405 nm and 488 nm, respectively.

• 

  Figure A2. Excitation‐contraction coupling remains unchanged under HiGlu stimulus A, traces of sarcomere length, cell length and Ca2+ transient. B–E, quantification of sarcomere length, % shortening, twitch Tpeak and Ca2+ transient amplitude. The mean values are marked as short lines in (B) and (C). F, quantification of DCF slope under 2 Hz and 5 Hz pacing frequency.

• 

  Figure A3. Excitation‐contraction coupling remains unchanged under HiGlu and GlAnT stimuli A, traces of sarcomere length, cell length and Ca2+ transient. B–E, quantification of sarcomere length, % shortening, twitch Tpeak and Ca2+ transient amplitude. The mean values are marked as short lines in figure (D) and (E). F, quantification of DCF slope under 2 Hz w/o HiGlu.