Cardioprotection by post-conditioning with exogenous triiodothyronine in isolated perfused rat hearts and isolated adult rat cardiomyocytes

Abstract

Ischemic post-conditioning (iPoCo) by coronary re-occlusion/reperfusion during immediate reperfusion after prolonged myocardial ischemia reduces infarct size. Mechanical manipulation of culprit lesions, however, carries the risk of coronary microembolization which may obscure iPoCo's cardioprotection. Pharmacological post-conditioning with exogenous triiodothyronine (T3) could serve as an alternative conditioning strategy. Similar to iPoCo, T3 may activate cardioprotective prosurvival pathways. We aimed to study T3's impact on infarct size and its underlying signal transduction. Hearts were isolated from male Lewis rats (200-380 g), buffer-perfused and subjected to 30 min/120 min global zero-flow ischemia/reperfusion (I/R). In additional hearts, either iPoCo (2 × 30 s/30 s I/R) was performed or T3 (100-500 µg/L) infused at reperfusion. Infarct size was demarcated with triphenyl tetrazolium chloride staining and calculated as percent of ventricular mass. Infarct size was reduced with iPoCo to 16 ± 7% vs. 36 ± 4% with I/R only. The maximum infarct size reduction was observed with 300 µg/L T3 (14 ± 2%). T3 increased the phosphorylation of protein kinase B and mitogen extracellular-regulated-kinase 1/2, both key enzymes of the reperfusion injury salvage kinase (RISK) pathway. Pharmacological RISK blockade (RISK-BL) during reperfusion abrogated T3's cardioprotection (35 ± 10%). Adult ventricular cardiomyocytes were isolated from buffer-perfused rat hearts and exposed to 30 min/5 min hypoxia/reoxygenation (H/R); reoxygenation was initiated without or with T3, respectively, and without or with RISK-BL, respectively. Maximal preservation of viability was observed with 500 µg/L T3 after H/R (27 ± 4% of all cells vs. 5 ± 3% in time-matched controls). Again, RISK-BL abrogated protection (11 ± 3%). Mitochondria were isolated at early reperfusion from buffer-perfused rat hearts without or with iPoCo or 300 µg/L T3, respectively, at reperfusion. T3 improved mitochondrial function (i.e.: increased respiration, adenosine triphosphate production, calcium retention capacity, and decreased reactive oxygen species formation) to a similar extent as iPoCo. T3 at reperfusion reduces infarct size by activation of the RISK pathway. T3's protection is a cardiomyocyte phenomenon and targets mitochondria.

Keywords: Cardioprotection; Ischemia/reperfusion; Ischemic conditioning; Post-conditioning; Triiodothyronine.

Fig. 1

Experimental groups and protocols. ERK-BL mitogen extracellular-regulated-kinase phosphorylation blockade, H/R hypoxia and reoxygenation, iPoCo ischemic post-conditioning, I/R ischemia and reperfusion, PI3K-BL phosphatidylinositol(4,5)-bisphosphate-3-kinase blockade, RISK-BL reperfusion injury salvage kinase pathway blockade, SAFE-BL survival activating factor enhancement pathway blockade, T3 triiodothyronine, TC time control, TMP time-matched perfusion, TTC infarct size demarcation by triphenyl tetrazolium chloride staining

Fig. 2

Impact of T3 at reperfusion on infarct size in isolated buffer-perfused rat hearts. Data are presented as means ± standard deviations. For each group, representative triphenyl tetrazolium chloride-stained heart slices are displayed; areas enclosed by yellow lines indicate infarcted tissue. ERK-BL mitogen extracellular-regulated-kinase phosphorylation blockade, iPoCo ischemic post-conditioning, I/R 30 min ischemia and 120 min reperfusion, PI3K-BL phosphatidylinositol(4,5)-bisphosphate-3-kinase blockade, RISK-BL reperfusion injury salvage kinase pathway blockade, SAFE-BL survival-activating factor enhancement pathway blockade, T3 triiodothyronine at reperfusion, a T3 at reperfusion—given in increasing concentrations; *p < 0.001 vs. I/R; ^†p < 0.01 vs. I/R + T3 (100 µg/L); one-way ANOVA with Fisher’s least significant differences post-hoc tests. b T3 at reperfusion—given under RISK or SAFE blockade; I/R and I/R + T3 (300 µg/L): groups are identical to those depicted in Fig. 1a; *p < 0.001 vs. I/R + T3 (300 µg/L); ^#p < 0.01 vs. I/R + T3 (300 µg/L) + SAFE-BL; one-way ANOVA with Fisher’s least significant differences post-hoc tests

Fig. 3

Phosphorylation of AKT1/2/3_Ser473, ERK1/2_{Thr202-Tyr204/Thr185-Tyr187}, GSK-3β_Ser9 and STAT3_tyr705 in isolated buffer-perfused rat hearts. Data are presented as means ± standard deviations. iPoCo ischemic post-conditioning, I/R ischemia and reperfusion, T3 triiodothyronine at reperfusion. a Phosphorylation of protein kinase B (AKT) 1/2/3. Top; middle; bottom: fluorescence signal intensity of phosphorylated AKT1/2/3_Ser473 (green); fluorescence signal intensity of total AKT1/2/3 (red); fluorescence signal intensity of phosphorylated protein was normalized to the respective total AKT1/2/3. *p < 0.01 vs. I/R; ^#p < 0.01 vs. I/R + iPoCo; one-way ANOVA with Fisher’s least significant differences post-hoc tests. b Phosphorylation of mitogen extracellular-regulated-kinase (ERK) 1/2. Top; middle; bottom: fluorescence signal intensity of phosphorylated ERK1/2_{Thr202-Tyr204/Thr185-Tyr187} (green); fluorescence signal intensity of total ERK1/2 (red); fluorescence signal intensity of phosphorylated protein was normalized to the respective total ERK1/2. *p < 0.01 vs. I/R; one-way ANOVA with Fisher’s least significant differences post-hoc tests. c Phosphorylation of glycogen synthase kinase kinase (GSK)-3β. Data are presented as median (vertical line) with interquartile range (from 25 to 75%), horizontal line represents median. Top; middle; bottom: fluorescence signal intensity of phosphorylated GSK-3β_Ser9 (green); fluorescence signal intensity of total GSK-3β (red); fluorescence signal intensity of phosphorylated protein was normalized to the respective total GSK-3β.*p < 0.05 vs. I/R; Kruskal–Wallis ANOVA on ranks with Tukey’s multiple comparisons procedures. d Phosphorylation of signal transducer and activator of transcription (STAT) 3 in myocardial biopsies. Top; middle; bottom: fluorescence signal intensity of phosphorylated STAT3_tyr705 (green); fluorescence signal intensity of total STAT3 (red); fluorescence signal intensity of phosphorylated protein was normalized to the respective total STAT3

Fig. 4

Impact of T3 at reoxygenation on cardiomyocytes viability. Data are presented as means ± standard deviations. ERK-BL mitogen extracellular-regulated-kinase blockade, H/R hypoxia/reoxygenation, PI3K-BL blockade of phosphatidylinositol(4,5)-bisphosphate-3-kinase, RISK-BL reperfusion injury salvage kinase pathway blockade, SAFE-BL signal transducer and activator of transcription pathway blockade, T3 incubation with 100, 200, 300 or 500 µg/L triiodothyronine 5 min before and during reoxygenation, TC time control. a T3 at reoxygenation—incubation with increasing concentrations. Cardiomyocytes were isolated from n = 7 hearts. *p < 0.01 vs. H/R and H/R + T3 (100 µg/L); (*) p = 0.054 vs. H/R; ^#p < 0.01 vs. H/R + T3 (200 µg/L); ^†p = 0.01 vs. H/R + T3 (300 µg/L); one-way ANOVA for repeated measures with Fisher’s least significant differences post-hoc tests. b T3 at reoxygenation—incubation under PI3K-BL, ERK-BL, RISK-BL or SAFE-BL. Cardiomyocytes were isolated from n = 9 hearts. *p < 0.01 vs. H/R; ^#p < 0.01 vs. H/R + T3 (500 µg/L); ^#p = 0.015; ^†p < 0.01 vs. H/R + T3 (500 µg/L) + RISK-BL; ^‡p = 0.022 vs. H/R + T3 (500 µg/L) + SAFE-BL; one-way ANOVA for repeated measures with Fisher’s least significant differences post-hoc tests

Fig. 5

Functional parameters of mitochondria incubated with NaOH or T3, respectively, isolated from naïve rat hearts. Mitochondria were isolated from n = 5 hearts per group (for the respective measurements, see single data points). Data are presented as means ± standard deviations. ADP adenosine diphosphate, ASC ascorbate, ATP adenosine triphosphate, CRC calcium retention capacity, NaOH incubation with buffer and addition of NaOH, ROS reactive oxygen species, T3 incubation with buffer and addition of 300 µg/L triiodothyronine, TMPD tetramethylphenylenediamine

Fig. 6

Functional parameters of mitochondria incubated with NaOH or T3, respectively, after isolation from buffer-perfused rat hearts subjected to 30 min ischemia and 10 min reperfusion. Mitochondria were isolated from n = 5 hearts per group (for the respective measurements, see single data points) hearts. Data are presented as means ± standard deviations. ADP adenosine diphosphate, ASC ascorbate, ATP adenosine triphosphate, CRC calcium retention capacity, NaOH incubation with buffer and addition of NaOH, ROS reactive oxygen species, T3 incubation with buffer and addition of 300 µg/L triiodothyronine, TMPD tetramethylphenylenediamine

Fig. 7

Functional parameters of mitochondria from buffer-perfused rat hearts subjected to I/R or TMP without or with iPoCo or T3, respectively, at reperfusion. Mitochondria were isolated from n = 7–8 hearts per group (for the respective measurements, see single data points). Data are presented as means ± standard deviations. ADP adenosine diphosphate, ASC ascorbate, ATP adenosine triphosphate, CRC calcium retention capacity, iPoCo ischemic post-conditioning, I/R 30 min ischemia and 120 min reperfusion with Krebs–Henseleit buffer, ROS reactive oxygen species, T3 300 µg/L triiodothyronine given at reperfusion or at the corresponding time point in TMP experiments, TMP time-matched perfusion without I/R, TMPD tetramethylphenylenediamine; *p < 0.01 vs. all other groups; one-way ANOVA with Fisher’s least significant differences post-hoc tests