Activation of Akt rescues endoplasmic reticulum stress-impaired murine cardiac contractile function via glycogen synthase kinase-3β-mediated suppression of mitochondrial permeation pore opening

Abstract

Aims: The present study was designed to examine the impact of chronic Akt activation on endoplasmic reticulum (ER) stress-induced cardiac mechanical anomalies, if any, and the underlying mechanism involved.

Results: Wild-type and transgenic mice with cardiac-specific overexpression of the active mutant of Akt (Myr-Akt) were subjected to the ER stress inducer tunicamycin (1 or 3 mg/kg). ER stress led to compromised echocardiographic (elevated left ventricular end-systolic diameter and reduced fractional shortening) and cardiomyocyte contractile function, intracellular Ca(2+) mishandling, and cell survival in wild-type mice associated with mitochondrial damage. In vitro ER stress induction in murine cardiomyocytes upregulated the ER stress proteins Gadd153, GRP78, and phospho-eIF2α, and promoted reactive oxygen species production, carbonyl formation, apoptosis, mitochondrial membrane potential loss, and mitochondrial permeation pore (mPTP) opening associated with overtly impaired cardiomyocyte contractile and intracellular Ca(2+) properties. Interestingly, these anomalies were mitigated by chronic Akt activation or the ER chaperon tauroursodeoxycholic acid (TUDCA). Treatment with tunicamycin also dephosphorylated Akt and its downstream signal glycogen synthase kinase 3β (GSK3β) (leading to activation of GSK3β), the effect of which was abrogated by Akt activation and TUDCA. The ER stress-induced cardiomyocyte contractile and mitochondrial anomalies were obliterated by the mPTP inhibitor cyclosporin A, GSK3β inhibitor SB216763, and ER stress inhibitor TUDCA.

Innovation: This research reported the direct relationship between ER stress and cardiomyocyte contractile and mitochondrial anomalies for the first time.

Conclusion: Taken together, these data suggest that ER stress may compromise cardiac contractile and intracellular Ca(2+) properties, possibly through the Akt/GSK3β-dependent impairment of mitochondrial integrity.

FIG. 1.

Effect of in vivo tunicamycin (TN) challenge (1 or 3 mg/kg, i.p. for 48 h) on echocardiographic and cardiomyocyte contractile properties from wild-type (WT) and MyAkt mice. (A) Left ventricular end-systolic diameter (LVESD) using echocardiography; (B) left ventricular end-diastolic diameter (LVEDD) using echocardiography; (C) fractional shortening using echocardiography; (D) peak shortening (PS) (% of cell length) in cardiomyocytes; (E) maximal velocity of shortening (+dL/dt); (F) maximal velocity of relengthening (−dL/dt); (G) time-to-peak shortening (TPS); and (H) time-to-90% relengthening (TR₉₀). Mean±SEM, n=6–7 mice and 86–87 cells for panels (A–C) and panels (D–H), respectively, *p<0.05 versus WT group; ^#p<0.05 versus respective WT-TN group.

FIG. 2.

Effect of in vivo TN challenge (1 or 3 mg/kg, i.p. for 48 h) on intracellular Ca²⁺ properties in cardiomyocytes from WT and MyAkt mice. (A) Representative intracellular Ca²⁺ transients in cardiomyocytes from WT mice with or without TN; (B) representative intracellular Ca²⁺ transients in cardiomyocytes from MyAkt mice with or without TN; (C) resting fura-2 fluorescence intensity (FFI); (D) electrically stimulated increase in FFI (ΔFFI); (E) intracellular Ca²⁺ decay rate (single exponential); and (F) intracellular Ca²⁺ decay rate (biexponential). Mean±SEM, n=60 cells per group, *p<0.05 versus WT group; ^#p<0.05 versus respective WT-TN group.

FIG. 3.

Effect of in vivo TN challenge (3 mg/kg, i.p. for 48 h) on endoplasmic reticulum (ER) stress, cell survival, mitochondrial integrity (aconitase activity), and phosphorylation of Akt and glycogen synthase kinase 3β (GSK3β) in WT and MyAkt mice. A cohort of WT mice were injected with the ER stress inhibitor tauroursodeoxycholic acid (TUDCA) (50 mg/kg, i.p.) at the time of TN challenge. (A) Gadd153 expression; (B) GRP78 expression; (C) cell survival; (D) aconitase activity; (E) pAkt-to-Akt ratio; and (F) pGSK3β-to-GSK3β ratio. Insets: Representative gel blots depicting the ER stress proteins Gadd153 and GRP78, as well as pan and phosphorylated Akt and GSK3β using specific antibodies (β-actin was used as the loading control). All protein expressions were normalized to that of β-actin. Mean±SEM, n=5–7 hearts per group, *p<0.05 versus WT group, ^#p<0.05 versus WT-TN group.

FIG. 4.

Effect of TN on cell shortening in isolated cardiomyocytes from WT and MyAkt mice. Murine cardiomyocytes were incubated with TN (3 μg/ml) for 5–6 h in vitro before assessment of mechanical properties. A cohort of WT cardiomyocytes were coincubated with the ER stress inhibitor TUDCA (500 μM) alone with TN. (A) Representative contractile traces in cardiomyocytes from WT mice in the absence or presence of TN or TUDCA; (B) representative contractile traces in cardiomyocytes from MyAkt mice in the absence or presence of TN; (C) resting cell length; (D) PS (% of resting cell length); (E) maximal velocity of shortening (+dL/dt); (F) maximal velocity of relengthening (−dL/dt); (G) TPS; and (H) time-to-90% relengthening (TR₉₀). Mean±SEM, n=80 cells per group, *p<0.05 versus WT group; ^#p<0.05 versus WT-TN group.

FIG. 5.

Effect of TN on intracellular Ca²⁺ properties in cardiomyocytes from WT and MyAkt mice. Murine cardiomyocytes were incubated with TN (3 μg/ml) for 5–6 h in vitro before intracellular Ca²⁺ evaluation. (A) Baseline FFI; (B) electrically stimulated increase in FFI (ΔFFI); (C) intracellular Ca²⁺ decay rate (single exponential); and (D) intracellular Ca²⁺ decay rate (biexponential). Mean±SEM, n=52–54 cells per group, *p<0.05 versus WT group; ^#p<0.05 versus WT-TN group.

FIG. 6.

Effect of TN on ER stress in cardiomyocytes from WT and MyAkt mice. Freshly isolated murine cardiomyocytes were incubated with TN (3 μg/ml) for 5–6 h in vitro before assessment of ER stress. A cohort of WT cardiomyocytes were coincubated with the ER stress inhibitor TUDCA (500 μM) alone with TN. (A) Representative gel blots depicting the ER stress proteins Gadd153, GRP78, and peIF2α using specific antibodies (β-actin was used as the loading control); (B) Gadd153 expression; (C) GRP78 expression; and (D) peIF2α expression. All protein expression was normalized to that of β-actin. Mean±SEM, n=5–7 isolations per group, *p<0.05 versus WT group; ^#p<0.05 versus WT-TN group.

FIG. 7.

Effect of TN on reactive oxygen species (ROS) and cell survival in cardiomyocytes from WT and MyAkt mice. Murine cardiomyocytes were incubated with TN (3 μg/ml) for 5–6 h in vitro before examination. A cohort of WT cardiomyocytes were coincubated with the ER stress inhibitor TUDCA (500 μM), the antioxidant catalase-polyethylene glycol (PEG, 15,000 IU/ml), or the mitochondrial permeation pore (mPTP) inhibitor cyclosporin A (200 nM) at the same time of TN exposure. (A). Representative DCF fluorescent images depicting cardiomyocytes from WT and MyAkt mice treated with TN or TN-treated WT cells in the presence of the ER stress inhibitor TUDCA (500 μM). A cohort of WT cardiomyocytes were coincubated with H₂O₂ (100 μM, positive control) for 5 h in the absence or presence of the antioxidant catalase-PEG; (B) ROS production; and (C) MTT cell survival. Mean±SEM, n=5–8 isolations per group, *p<0.05 versus WT group; ^#p<0.05 versus WT-TN group. (To see this illustration in color the reader is referred to the web version of this article at www.liebertonline.com/ars).

FIG. 8.

Effect of TN on protein carbonyl formation and apoptosis (caspase-3 activity, caspase-8, pro-caspase-9, and caspase-12 expression) in cardiomyocytes from adult WT and MyAkt mice. For all panels except panel (B), murine cardiomyocytes were incubated with TN (3 μg/ml) for 5–6 h in vitro before examination. For panel (B), TN was injected (3 mg/kg, i.p. for 48 h) in vivo along with the ER stress inhibitor TUDCA (50 mg/kg, i.p.). (A) Carbonyl formation; (B) protein carbonyl expression evaluated by Western blot analysis; (C) caspase-3 activity measured by a colorimetric assay; (D) caspase-8 expression; (E) pro-caspase-9 expression; and (F) cleaved caspase-12 expression; Insets: Representative gel blots depicting expression of carbonyl, caspase-8, pro-caspase-9, and cleaved caspase-12 using specific antibodies. All protein expression was normalized to that of the loading control β-actin. Mean±SEM, n=5–8 isolations per group, *p<0.05 versus WT group; ^#p<0.05 versus WT-TN group.

FIG. 9.

Effect of TN on mitochondrial integrity using mitochondrial membrane potential and mitochondrial permeability transition pore opening (mPTP) in cardiomyocytes from adult WT and MyAkt mice. Murine cardiomyocytes were incubated with TN (3 μg/ml) for 5–6 h in vitro before assessment of mitochondrial function. A cohort of WT cardiomyocytes were coincubated with the mPTP inhibitor cyclosporin A (200 nM), the GSK3β inhibitor SB216763 (10 μM), or the ER stress inhibitor TUDCA (500 μM) along with TN exposure. (A) Representative JC-1 fluorochrome images depicting mitochondrial membrane potential in the above-mentioned cardiomyocyte groups. The mitochondrial uncoupler carbonyl cyanide m-chlorophenylhydrazone (CCCP) (10 μM) was used as a positive control; (B) pooled data of mitochondrial membrane potential (ratio of the red fluorescence obtained at 590 nm to the green fluorescence at 530 nm); and (C) mPTP opening evaluated by NAD⁺, a marker for mitochondrial permeability transition pore opening; Mean±SEM, n=6 isolations per group, *p<0.05 versus WT group; ^#p<0.05 versus WT-TN group. (To see this illustration in color the reader is referred to the web version of this article at www.liebertonline.com/ars).

FIG. 10.

Effect of the mPTP inhibitor cyclosporine A, the GSK3β inhibitor SB216763 and the ER stress inhibitor TUDCA on TN-induced cardiomyocyte contractile dysfunction. Freshly isolated murine cardiomyocytes were incubated with TN (3 μg/ml) for 5–6 h in vitro in the absence or presence of cyclosporine A (200 nM), SB216763 (10 μM), or TUDCA (500 μM) before assessment of mechanical properties. (A) Resting cell length; (B) PS (% of resting cell length); (C) maximal velocity of shortening (+dL/dt); (D) maximal velocity of relengthening (−dL/dt); (E) TPS; and (F) time-to-90% relengthening (TR₉₀). Mean±SEM, n=52–53 cells per group, *p<0.05 versus control (without drug treatment) group; ^#p<0.05 versus TN group.

FIG. 11.

Schematic diagram depicting ER stress-induced cellular events leading to mitochondrial damage, intracellular Ca²⁺ dysregulation, and contractile dysfunction in the heart as well as how chronic Akt activation may rescue the heart from ER stress-induced cardiac anomalies. TUDCA (an ER chaperon to inhibit ER stress). Please note that intracellular Ca²⁺ overload and dysregulation may occur immediately after induction of ER stress and/or as the final instigator to trigger cardiac contractile dysfunction after onset of mitochondrial damage and ROS production. Also note that ROS production and mitochondrial damage may happen in a reciprocal manner, leading to a vicious cycle for ROS accumulation.