Selective protein kinase C inhibition switches time-dependent glucose cardiotoxicity to cardioprotection

Figures

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  FIGURE 1. Time course of glucose-induced dysregulation of cardiomyocyte contractile function. (A) Illustration of the experimental design used in this figure. (B) Example traces showing cardiomyocyte contractions, initiated by 1 Hz EFS, in the presence of 5 or 20 mM glucose. Cardiomyocytes that contracted outside of the 1 Hz EFS, as demonstrated by # in panel B, or failed to contract were not defined as contractile cells. Therefore, only cardiomyocytes that contracted in time with 1 Hz EFS were counted as contractile cells. (C) Bar chart showing the mean percentage change in contractile cells (*p < 0.05, ***p < 0.001. One Way ANOVA with Holm-Sidak post-test). (D) Bar chart showing the mean number of arrhythmic episodes in each data set. ***p < 0.001 One-Way ANOVA with Holm-Sidak post-test. n = 6 experiments from six animals (>98 cells for each data set).

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  FIGURE 2. High glucose pre-treatment delays activation of the metabolic inhibition-sensitive KATP current and limits calcium overload during metabolic inhibition. (A) Example traces of whole-cell current recorded at 0 mV showing the activation of a metabolically-sensitive outward K+ current. (B) Bar chart showing the mean time to activation of this metabolic inhibition-sensitive current in control conditions (perfused for 15 min with NT prior to SFT-MI) or with 5 min high glucose pre-treatment. Mean time to activation was significantly delayed following 20 mM PT [***P > 0.001, Unpaired T-test, n = 9 (>14) for each group]. (C) Example traces of cell attached recording of sarcolemmal Kir6.2/SUR2A (IKATP) current activation by metabolic inhibition, identified by its single channel conductance. (D) Bar chart showing the mean time to activation of Kir6.2/SUR2A channels with a burst duration longer than 250 ms. [***P > 0.001, Unpaired T-test, n = > 6 (>10) for each group]. (E) Example traces and expanded transients, recorded at the beginning and end of the protocol, from three cells in control conditions (perfused for 15 mins prior to SFT-MI with NT solution) and three cells in a 20 mM glucose…

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  FIGURE 3. A short pre-treatment with elevated glucose imparts a cardioprotection, but longer durations reveal glucose-dependent toxicity. (A) Example video edge detection measurements from a cell in control (5 mM glucose) conditions and a cell following 5 min of pre-treatment with glucose prior to the MI/R protocol. (ii) Expanded traces showing 30 s of contractions from the beginning (1) and end (2) of the traces in panel (A). (B) Time course showing the number of rod-shaped contractile cells from 3 min before the start of the metabolic inhibition in control (no pre-treatment), or following a 5, 10, or 15 min pre-treatment with 20 mM glucose NT. Bar charts showing (C) the mean time to contractile failure in metabolic inhibition, (D) the mean percentage of contractile recovery at the end of the MI/R protocol and (E) the mean percentage cell survival at the end of the MI/R protocol [**P < 0.01, ***P < 0.001, One-Way ANOVA with Holm-Sidak post-test, n = 6 (>131) cells for each group].

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  FIGURE 4. A 5 min pre-treatment with 20 mM glucose imparts a short-lived cardioprotection. (A) Time course showing the percentage of contractile cardiomyocytes throughout the metabolic inhibition protocol following a 5 min high glucose pre-treatment and a variable gap between the pre-treatment and metabolic insult. (B) Mean time to contractile failure, (C) mean percentage contractile recovery and (D) mean percentage of cell survival [**p < 0.01, ***p < 0.001, One-Way ANOVA with Holm-Sidak post-test. n = 6 experiments from six animals (>86 cells for each data set)].

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  FIGURE 5. Protection afforded by high glucose pre-treatment can be mimicked with other glycolytic metabolites. (A) Example time course showing the percentage of contractile cells during the MI/R protocol following 5 min pre-treatment with 20 mM glucose, 15 mM fructose, 10 mM pyruvate, or 20 mM L-glucose. (B) Bar chart showing mean time to contractile failure. All pre-treatments show a significant delay in the time to contractile failure, except for L-glucose, which is a non-metabolised enantiomer [**P < 0.01, ***P < 0.001, One-Way ANOVA with Holm-Sidak post-test, n = 6 (>98 cells) for each group]. Bar charts showing (C) the percentage contractile recovery and (D) cell survival at the end of the MI/R protocol. In each case there is a significant increase in contractile recovery, except for the L-glucose pre-treated group [***P < 0.001, One-Way ANOVA with Holm-Sidak post-test, n = 6 (>102 cells) for each group].

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  FIGURE 6. The high glucose-induced disruption of contractile function in isolated cardiomyocytes can be reversed with selective PKCα/β inhibition and mimicked with a PKCβ activator. (A) Time course showing the loss of contractile cardiomyocytes over a 20 min exposure to 20 mM glucose in the absence (black circles) and presence (white circles, Gö6976, grey circles, LY379196) of PKCα/β inhibitors. (B) Time course showing the number of contractions outside of the 1 Hz EFS in the cardiomyocytes recorded in panel (A). (C) The mean change in contractile cells at the end of the protocol and (D) the mean peak number of arrhythmic evens in each condition (***P < 0.001, **P < 0.01, One Way ANOVA with Holm-Sidak post-test, n = 6 experiments from six animals, >123 cells for each group). (E) Time course showing the loss of contractile function in cardiomyocytes pre-incubated for 15 min with Tat-ψRACK-PKCβ-activating peptide in the absence (light grey) and presence (dark grey) Gö6976 in 5 mM glucose. (F) Time course showing the number of contractions outside of the 1 Hz EFS in the cardiomyocytes recorded in panel (E). (G) The mean change in contractile cells at the end of the protocol and (H) the mean…

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  FIGURE 7. PKCα and/or β-selective inhibitors enhance the protected phenotype imparted by short high glucose pre-treatment. (A) Time course showing the percentage of contractile cardiomyocyte throughout the metabolic inhibition protocol following high glucose pre-treatment with or without PKC inhibitors, as indicated. (B) Bar chart showing the mean time to contractile failure, (C) mean percentage contractile recovery and (D) mean percentage of cell survival following metabolic inhibition in the absence and presence of selective PKC inhibition and with or without pre-treatment [***p < 0.001, One Way ANOVA with Holm-Sidak post-test. n = > 6 animals (>83 cells) for each data set. Each data set is compared to PT in the absence of PKC inhibitor. Control (no PT) data shown for comparison].

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  FIGURE 8. Selective inhibition of PKCα/β with Gö6976 reveals a 20 mM glucose pre-treatment dependent reduction in infarct size. (A) Example images from a control heart, heart following an amended protocol using a 15-min pre-treatment with 20 mM glucose and a heart following 15-min pre-treatment with 20 mM glucose in the presence of 300 nM Gö6976. The experimental protocol is shown above. (B) Bar chart showing the mean percentage infarct by weight of the different groups. Circles show data from individual hearts (**P < 0.01 and ***P < 0.001 Two-way ANOVA with Tukey’s post-test, n = 12, 6, 4, and 5 hearts for control, 20 mM PT, 5 mM glucose with Gö6976 and 20 mM PT with Gö6976 groups). There was no difference in the effectiveness of coronary blockade in the 4 groups measured as the percentage area at risk (41.9 ± 4.5, 43.2 ± 4.5, 39.4 ± 2.3, and 38.9 ± 2.6% (mean ± S.D., n = 12, 6, 4, and 5 hearts for control, 20 mM PT, 5 mM glucose with Gö6976 and 20 mM PT with Gö6976 groups, Two-way ANOVA).