mTOR-mediated calcium transients affect cardiac function in ex vivo ischemia-reperfusion injury

Abstract

The mechanistic target of rapamycin (mTOR) is a key mediator of energy metabolism, cell growth, and survival. While previous studies using transgenic mice with cardiac-specific overexpression of mTOR (mTOR-Tg) demonstrated the protective effects of cardiac mTOR against ischemia-reperfusion (I/R) injury in both ex vivo and in vivo models, the mechanisms underlying the role of cardiac mTOR in cardiac function following I/R injury are not well-understood. Torin1, a pharmacological inhibitor of mTOR complex (mTORC) 1 and mTORC2, significantly decreased functional recovery of LV developed pressure in ex vivo I/R models (p < 0.05). To confirm the role of mTOR complexes in I/R injury, we generated cardiac-specific mTOR-knockout (CKO) mice. In contrast to the effects of Torin1, CKO hearts recovered better after I/R injury than control hearts (p < 0.05). Interestingly, the CKO hearts had exhibited irregular contractions during the reperfusion phase. Calcium is a major factor in Excitation-Contraction (EC) coupling via Sarcoplasmic Reticulum (SR) calcium release. Calcium is also key in opening the mitochondrial permeability transition pore (mPTP) and cell death following I/R injury. Caffeine-induced SR calcium release in isolated CMs showed that total SR calcium content was lower in CKO than in control CMs. Western blotting showed that a significant amount of mTOR localizes to the SR/mitochondria and that GSK3-β phosphorylation, a key factor in SR calcium mobilization, was decreased. These findings suggest that cardiac mTOR located to the SR/mitochondria plays a vital role in EC coupling and cell survival in I/R injury.

Keywords: calcium; cardiomyocyte; ischemia-reperfusion; mTOR.

FIGURE 1

Torin1 significantly decreases %LVDP of wild‐type mice and decreases downstream signaling of mTORC1 and mTORC2. Wild‐type C57BL/6J mice were subjected to ex vivo Langendorff and treated with or without 100 nM Torin1. (a) Representative tracings from the Torin1 experiments. (b) Change in left ventricular developed pressure at baseline and throughout all 40 min of reperfusion. (c) Quantification of the %LVDP recovery of wild‐type mice treated with or without Torin1. The LVDP at baseline and reperfusion at 40 min of reperfusion were compared to calculate the %LVDP. N = 7 (WT) and 8 (WT + Torin1). p < 0.05 by Student's t test. (d) Immunoblot showing signal transduction and phosphorylation of downstream targets (p‐Akt and p‐S6) of mTORC1 and mTORC2 in hearts treated with Torin1. Bar graphs represent the mean densitometric analysis of mTOR and p‐S6. mTOR was normalized to GAPDH. p‐S6 was normalized to total S6. P‐values are displayed on graphs as determined by Student's t test. N = 3 for all groups.

FIGURE 2

Characterization of CKO mice. (a) Representative pictures of whole hearts isolated from Con and CKO mice. Scale bars =2 mm. (b) Left. Representative immunoblot showing a small decrease in mTOR expression in whole hearts and a significant decrease in the expression of mTOR in isolated CMs and in the whole heart compared to control. Right. Densitometric quantification of the amount of mTOR in whole heart and isolated CMs. N = 6 in each group. (c) Representative M‐mode traces from baseline echocardiography analysis of control and CKO mice. CKO mice had normal contractions that were similar to controls. (d) Left. CKO mice LVIDd was comparable to controls. Right. %FS was similar between control and CKO mice. N = 7 in each group.

FIGURE 3

CKO mice have better %LVDP recovery after ex vivo I/R. (a) Left. Representative tracing showing LVDP throughout the Langendorff experiment. Right. Quantification of LVDP at baseline and after every 10 min of reperfusion. (b) Quantification of the %LVDP recovery for control vs. CKO at 40 min of reperfusion. (c) Activity of creatine kinase (CK) in the effluent collected during the reperfusion period. To compare enzyme activities immediately after ex vivo I/R injury between Con and CKO hearts, effluents from either Con or CKO hearts were collected after 40‐min reperfusion. *p < 0.05. All other p‐values displayed on graphs. N = 7 for each group.

FIGURE 4

CKO hearts have irregular contractility following I/R. (a) Left. Representative tracing showing the difference in peak sizes between the control and CKO hearts taken over a 5‐s period. Right. Representative tracing showing the increased variance of contractions CKO hearts have after I/R injury. Representative tracings were taken over a 1‐min period during the last 10 min of reperfusion. (b) Left. Representative diagram displaying how to calculate the variance. Dashed line indicates the average LVDP across the entire interval. Δ and Δ′ indicate the change from the average LVDP for each individual peak. The average variance of peak contractions was then calculated with the formula described in Materials and Methods. Right. Quantification of the average variance of contractions. N = 7 for each group. P‐values listed on graph as determined by Student's t test.

FIGURE 5

Cardiomyocytes isolated from CKO mice exhibit weaker contractions and smaller calcium transients. (a) Representative traces of sarcomere length shortening and Ca²⁺ transients in CMs isolated from Con or CKO mice. (b) Quantification of the sarcomere length and calcium transient tracings. The following parameters were quantified for each group: time to peak shortening, time to 90% relaxation, peak shortening, and calcium transient ratio. N = 3 independent experiments, 8–12 cells total for both groups. P‐values are all displayed on graphs.

FIGURE 6

CKO CMs have lower relative SR calcium content. (a) Representative images from caffeine experiments. Upper. Representative tracing of sarcomere length from Con and CKO CMs stimulated with caffeine. Lower. Representative tracing of Ca²⁺ transients from Con and CKO CMs stimulated with caffeine. (b) Quantification of the peaks resulting from caffeine stimulation. The following parameters were quantified for each group: peak shortening and calcium transient ratio. N = 12 cells analyzed from 3 (Con) and 4 (CKO) mice. P‐values are displayed on graphs as determined by Student's t test.

FIGURE 7

EC Coupling proteins are unchanged in CKO hearts. (a) Representative immunoblot from control and CKO hearts that underwent ultracentrifugation to obtain subcellular fractions. VDAC was used as a loading control for the SR/mito fraction while GAPDH was used as the loading control for the cytosolic fraction. (b) Quantification of the amount of p‐RYR, p‐PLN, and SERCA in controls and CKO hearts. N = 6 for each group. P‐values are shown on graphs.

FIGURE 8

mTOR localizes to the SR/mitochondria (mito) and p‐GSK‐3β is activated in CKO CMs at mitochondrial associated membranes. (a) Representative immunoblot confirming mTOR localizes to both the SR/mito and the cytosol. (b) Densitometric quantification of the amount of mTOR in the cytosol and SR/mito fractions. mTOR was normalized to GAPDH in the cytosol fraction or VDAC in the SR/mito fraction. (c) Densitometric quantification of the amount of p‐GSK‐3β in the cytosolic and SR/mito fractions. p‐GSK‐3β was normalized to total GSK‐3β. N = 6 (Con and CKO cytosolic fractions) and 5 (Con and CKO SR/mito fractions). P‐values are presented on graphs.