Chronic Akt Activation Attenuated Lipopolysaccharide-Induced Cardiac Dysfunction via Akt/GSK3β-Dependent Inhibition of Apoptosis and ER Stress

Abstract

Sepsis is characterized by systematic inflammation and contributes to cardiac dysfunction. This study was designed to examine the effect of Akt activation on LPS-induced cardiac anomalies and underlying mechanism(s) involved. Mechanical and intracellular Ca²⁺ properties were examined in myocardium from wild-type and transgenic mice with cardiac-specific chronic Akt overexpression following LPS (4 mg/kg, i.p.) challenge. Akt signaling cascade (Akt, PTEN, GSK3β), stress signal (ERK, JNK, p38), apoptotic markers (BAX, caspase-3/-9), ER stress markers (GRP78, GADD153, eIF2α), inflammatory markers (TNFα, IL-1β, IL-6) and autophagic markers (Beclin-1, LC3B, Atg7 and p62) were evaluated. Our results revealed that LPS induced marked decrease in ejection fraction, fractional shortening, cardiomyocyte contractile capacity with dampened intracellular Ca²⁺ release and clearance, elevated ROS generation and decreased GSH/GSSG ratio, increased ERK, JNK, p38, GRP78, GADD153, eIF2α, BAX, caspase-3 and - 9, downregulated Bcl-2, the effects of which were significantly attenuated or obliterated by Akt activation. Akt activation itself did not affect cardiac contractile and intracellular Ca²⁺ properties, ROS production, oxidative stress, apoptosis and ER stress. In addition, LPS upregulated levels of Beclin-1, LC3B and Atg7, while suppressing p62 accumulation. Akt activation did not affect Beclin-1, LC3B, Atg7 and p62 in the presence or absence of LPS. Akt overexpression promoted phosphorylation of Akt and GSK3β. In vitro study using the GSK3β inhibitor SB216763 mimicked the response elicited by chronic Akt activation. Taken together, these data showed that Akt activation ameliorated LPS-induced cardiac contractile and intracellular Ca²⁺ anomalies through inhibition of apoptosis and ER stress, possibly involving an Akt/GSK3β-dependent mechanism.

1. INTRODUCTION

Sepsis is a devastating medical problem, representing a main cause of high mortality in critically ill patients [1–3]. The most severe sequelae for patients with sepsis are considered to be septic shock, leading to multiple organ and system failure. Cardiovascular in particular cardiac anomalies develop in nearly 40% of patients with sepsis, accounting for ~ 15% of mortality as a result of sepsis [4]. Patients with septic shock display ventricular dilation, decreased myocardial ejection fraction and contractility [5, 6]. It is well known that lipopolysaccharide (LPS) from the Gram-negative bacteria triggers sepsis en route to systemic organ failure including compromised cardiovascular function [7]. A number of mechanisms have been put forwards in the etiology of cardiovascular complications in sepsis including oxidative stress, inducible nitric oxide synthase (iNOS), activation of stress signals including mitogen-activated protein kinase (MAPK), and the stress-activated protein kinase/Jun-amino-terminal kinase (SAPK/JNK) [8–11]. Nonetheless, the precise nature behind initiation and progression of cardiac anomalies in sepsis remains elusive, thus making effective therapeutic remedy rather challenging [3].

The phosphoinositide 3 kinase and protein kinase B (PI3K/Akt) signaling pathway plays an essential role in the regulation of cell proliferation, survival and inflammatory processes [12]. Earlier findings from our group revealed that cardiac-specific overexpression of active mutant of Akt rescues endoplasmic reticulum (ER) stress-induced cardiac contractile dysfunction via an Akt-glycogen synthase kinase 3β (GSK3β)-mediated mechanism [13, 14]. Inhibition of GSK3β has also been demonstrated to suppress the proinflammatory response and lessen organ injury in endotoxemia as well as to mediate protection against endotoxin shock [15, 16]. Along the same line, insulin therapy is found beneficial for LPS-induced multiple organ injury through a GSK3β- mediated mechanism independently of any effects on blood glucose levels [17, 18]. Furthermore, Kapoor and colleagues also revealed a role for Akt-GSK3β in peroxisome proliferator-activated receptor-β/δ-offered protection against endotoxemia-induced injury [19]. Despite accumulating evidence for an essential role of GSK3β in endotoxemia [15–20], the precise role of Akt in septic shock-induced myocardial anomalies remains elusive. To this end, our present study was designed to test the hypothesis that the Akt-GSK3β signaling plays an essential role in endotoxin LPS-elicited cardiac dysfunction. Mechanical and intracellular Ca²⁺ properties were examined in hearts from wild-type (WT) and cardiac-specific overexpression of the active mutant of Akt (Akt overexpression, AOE) mice following LPS challenge. Akt signaling (Akt, PTEN and GSK3β), oxidative stress signaling (ERK, JNK and p38), apoptotic protein markers (Bax, caspase-3/-9), endoplasmic reticulum (ER) stress markers (GRP78, GADD153 and eIF2α), proinflammatory markers (TNFα, IL-1β and IL-6) and the autophagic markers (Beclin-1, LC3B, Atg7, and p62) were evaluated.

2. MATERIALS AND METHODS

2.1. Experimental animals and LPS treatment

All animal procedures were approved by the Institutional Animal Use and Care Committee at the University of Wyoming (Laramie, WY). Mice overexpressing the hemagglutinin-tagged Akt (MyAkt) with src myristoylation signal under the direction of the murine α-myosin heavy chain (MHC) promoter were kindly provided by Dr. Anthony Rosenzweig (Harvard Medical School, Boston, MA). The cDNA encoding My- Akt was subcloned downstream of the 5.5-kb murine α-MHC promoter and was used to generate cardiac-specific Akt transgenic mice through oocyte injection. My-Akt transgenic mice were genotyped by PCR. Adult (8–12 week-old) Akt transgenic and wild-type (WT) mice were used. Mice were maintained on a 12:12-h light-dark illumination cycle and were allowed food and water ad libitum. On the day of experimentation, both AOE and WT mice were injected intraperitoneally with 4 mg/kg Escherichia Coli O55:B5 LPS dissolved in sterile saline or an equivalent volume of pathogen-free saline for control. Six hours following LPS injection, mice were sacrificed for experimentation [21, 22].

2.2. Echocardiographic assessment

Cardiac geometry and function were evaluated in anaesthetized ((ketamine 80 mg/kg and xylazine 12 mg/kg, i.p.) mice using the 2-D guided Mmode echocardiography (Sonos 5500, Phillips Medical System, Andover, MA) equipped with a 15–16 MHz linear transducer. Left ventricular (LV) anterior and posterior wall dimensions during diastole and systole were recorded from 3 cycles using M-mode echocardiography using methods adopted by the American Society of Echocardiography. Fractional shortening was calculated from LV end–diastolic (LVEDD) and LV end–systolic (LVESD) diameters using the equation (LVEDD-LVESD)/LVEDD. Echocardiographic LV mass was calculated using the equation of [(LVEDD+septal wall thickness+posterior wall thickness)³−LVEDD³]×1.055, where 1.055 mg/mm³ is the density of myocardium. Ejection faction was calculated from LV enddiastolic and -systolic diameters using the equation of (LVEDD³-LVESD³)/LVEDD³ × 100. Heart rates were averaged over 10 cycles [23].

2.3. Langendorff-perfused heart function

Langendorff-perfused heart function was assessed using the ADInstruments PowerLab system. In brief, mice were anesthetized (ketamine 80 mg/kg and xylazine 12 mg/kg, i.p.) and hearts were perfused with KHB containing 7 mM glucose, 0.4 mM oleate, 1% bovine serum albumin and a low level of insulin (10 µU/ml). The perfusion was initiated in the retrograde mode through the cannulated aorta. Hearts were perfused at a constant aortic pressure of 4 mmHg at baseline for 60 min. A fluid-filled latex balloon connected to a solid-state pressure transducer was inserted into the left ventricle to measure the pressure. Left ventricular developed pressure (LVDP), the first derivative of LVDP, namely, the maximum rate of left ventricular pressure development (+ dP/dt) and the maximum rate of left ventricular pressure decline (− dP/dt) were recorded using a digital acquisition system at a balloon volume which resulted in a baseline LV end-diastolic pressure of 5 mmHg [24].

2.4. Isolation of murine cardiomyocytes and in vitro drug treatment

Cardiomyocytes were isolated as described [25]. In brief, mice were anesthetized using ketamine and xylazine (ketamine 80 mg/kg and xylazine 12 mg/kg, i.p.). Hearts were rapidly removed and perfused with oxygenated (5% CO₂ /95% O₂ ) Krebs-Henseleit bicarbonate (KHB) buffer containing (in mM) 118 NaCl, 4.7 KCl, 1.25CaCl₂, 1.2 MgSO₄, 1.2 KH₂PO₄, 25 NaHCO₃, 10 HEPES, 11.1 glucose. Hearts were then perfused with a Ca²⁺-free KHB containing liberase blendzyme 4 (Hoffmann-La Roche Inc, Indianapolis, IN) for 20 min. After perfusion, left ventricles were removed and minced to disperse cardiomyocytes in a Ca²⁺-free KHB buffer. Extracellular Ca²⁺ was added incrementally back to 1.2 mM. Myocyte yield was ~ 70% which was unaffected by LPS or Akt activation. Only rod-shaped myocytes with clear edges were selected for mechanical and intracellular Ca²⁺ studies. Cells were used within 6 hrs of isolation. To directly assess the role of oxidative stress, ER stress and stress signaling in LPS-induced cardiomyocyte dysfunction, murine cardiomyocytes were incubated with or without LPS (4 µg/ml) and/or the GSK3β inhibitor SB216763 (10 µM) at 37°C for 2 hrs [14, 26].

2.5. Cell shortening /relengthening

Mechanical properties of cardiomyocytes were assessed using a SoftEdge MyoCam system (IonOptix Corporation, Milton, MA). In brief, cardiomyocytes were placed in a chamber mounted on the stage of an inverted microscope (Olympus, IX-70) and superfused at 25°C with a buffer containing (in mM) : 131 NaCl, 4 KCl, 1 CaCl₂, 1 MgCl₂, 10 Glucose and 10 HEPES, at pH 7.4. The cells were field stimulated with suprathreshold voltage at a frequency of 0.5 Hz, 3 ms duration, using a pair of platinum wires placed on opposite sides of the chamber and connected to an electrical stimulator (FHC Inc, Brunswick, NE). The myocyte being studied was displayed on a computer monitor using an IonOptix MyoCam camera. An IonOptix software was used to capture changes in cell length during shortening and relengthening. Cell shortening and relengthening were assessed using the following indices: peak shortening (PS), maximal velocities of cell shortening and relengthening (±dL/dt), time-to-PS (TPS), and time-to-90% relengthening (TR₉₀) [21].

2.6. Intracellular Ca²⁺ transient

Cardomyocytes were loaded with fura-2/AM (0.5µM) for 10 min and fluorescence measurements were recorded with dual-excitation fluorescence photo multiplier system (Ionoptix). Cardiomyocytes were placed on an Olympus IX-70 inverted microscope and imaged through a Fluor 40× oil objective. Cells were exposed to light emitted by a 75-W lamp and passed through either 360 nm or a 380 nm filter, while being stimulated to contract at 0.5Hz. Fluorescence emissions were detected between 480 nm and 520 nm by a photo multiplier tube after first illuminating the cells at a 360nm 0.5s, then at 380 nm for the duration of the recording protocol (333Hz sampling rate). The 360 nm excitation scan can be repeated at the end of the protocol and qualitative changes in intracellular Ca²⁺ concentration were inferred from the ratio of fura-2 fluorescence intensity at two wavelengths (360/380). Fluorescence decay time was assessed as an indication of intracellular Ca²⁺ clearing. Single exponential curve fit was applied to calculate the intracellular decay Ca²⁺ constant [27].

2.7. Generation of intracellular reactive oxygen species (ROS)

Production of ROS was evaluated by changes in the fluorescence intensity resulted from oxidation of the intracellular fluoroprobe 5-(and -6)-chloromethyl-2’,7’-dichlorodihydrofluorescein diacetate (CM-H₂DCFDA, Molecular Probes, Eugene, OR). In brief, cardiomyocytes were incubated with 25 µM CMH₂DCF- DA for 30 min at 37°C. The cells were then rinsed and the fluorescence intensity was measured using a fluorescent micro-plate reader at the excitation and emission wavelengths of 480nm and 530 nm respectively (Molecular Devices, Sunnyvale, CA). Untreated cells without fluorescence were used to determine the background fluorescence. The final results were expressed as the ratio of the fluorescent intensity to respective protein content [27].

2.8. Glutathione and glutathione disulfide (GSH/GSSG) assay

The ratio of GSH/GSSG was used as an indicator of oxidative stress. Myocardial tissues from WT and AOE mice treated with or without LPS were homogenized in 4 volumes (w/v) of 1% picric acid. Acid homogenates were centrifuged at 13500 × g (30 min) and supernatant fractions were collected and were used to assay total GSH and GSSG by the standard recycling method. Half of each sample was used for GSH. Samples for GSSG determination were incubated at room temperature with 2 µl 4- vinyl pyridine (4-vp) per 100 µl sample for 1h after vigorous votexing. Incubation with 4-vp conjugates any GSH present in the sample thus only GSSG is recycled to GSH without potential interference by GSH. The GSSG (as GSH×2) was subtracted from the total GSH to determine actual GSH level and GSH/GSSG level [27].

2.9. Western blot analysis

Expression of the stress signaling molecules ERK, JNK and p38, the ER stress proteins GRP78, GADD153, eIF2α, the autophagy markers Beclin-1, LC3B, Atg7 and p62 as well as the proinflammatory markers TNFα, IL-1β and IL-6, the Akt signals Akt, PTEN, GSK3β, as well as the apoptotic markers Bax, Bcl-2, caspase-3 and -9 were assessed. In brief, left ventricular tissue was sonicated in a lysis buffer containing (in mM): TRIS 10, NaCl 150, EDTA 5, 1%Triton X-100 and protease inhibitor cocktail followed by centrifugation at 12,000g for 10 min. Equal amount (30 µg) protein was separated using 7–15% SDS-polyacrylamide gels in minigel apparatus (Mini-PROTEAN II, Bio-Rad, Hercules, CA), and were transferred to nitrocellulose membranes (0.2 µm pore size, Bio-Rad). Membranes were blocked for 1 hr in 5% nonfat milk before being rinsed in TBS-T. The membranes were incubated overnight at 4°C with primary antibodies (1:1,000) against protein of interest (Santa Cruz Biotechnology, Santa Cruz, CA or Cell Signaling technology, Beverly, MA). After incubation with primary antibodies, blots were incubated with horseradish peroxidase-linked secondary antibodies (1:5,000) for 60 min at room temperature. Immunoreactive bands were detected using Super Signal West Dura Extended Duration Substrate (Pierce, Milwaukee, WI). The intensity of bands was measured with a scanning densitometer (Model GS-800, Bio-Rad) coupled with a Bio-Rad personal computer analysis software. GAPDH was used as the loading control [21, 27]. GAPDH was chosen over β-actin as the loading control marker since the later has been shown to be affected by malnutrition state such as cachexia [28].

2.10. Statistical analysis

Data were Mean±SEM. Difference was assessed using one-way analysis of variance (ANOVA) followed by a Tukey’s post hoc test. A p value < 0.05 was considered statistically significant.

3. RESULTS

3.1. General biometric and echocardiographic properties of WT and AOE mice

LPS treatment did not affect body liver, or kidney weights in either WT or AOE mice. AOE transgene itself did not affect body or organ weights. LPS significantly increased LVESD while reduced fractional shortening and ejection fraction (p < 0.05 vs. LPS-untreated groups), the effect of which was ablated by Akt activation. Akt transgene itself did not affect ESD, fractional shortening and ejection fraction (Table 1).

Table 1.

General characteristics of WT and AOE mice treated with or without LPS (4 mg/kg. i.p., 6 hrs)

	WT	WT-LPS	AOE	AOE-LPS
Body Weight (g)	24.5 ± 0.7	24.4 ± 0.4	242 ± 1.8	23.5 ± 0.7
Heart Weight (mg)	114 ± 4	114 ± 4	121 ± 26	118 ± 8
Heart/Body Weight (mg/g)	5.64 ± 0.21	5.31 ± 0.18	5.49 ± 0.31	5.41 ± 0.25
Liver weight (g)	0.95 ± 0.15	0.98 ± 0.07	0.97 ± 0.04	1.01 ± 0.09
Kidney weight(g)	0.29 ± 0.03	0.29 ± 0.01	0 27 ± 0.01	0.28 ± 0.01
Heart Rate (bpm)	489 ± 27	466 ± 36	460 ± 40	470 ± 22
Wall Thickness (mm)	0.92 ± 0.02	0.87 ± 0.05	0.95 ± 0.09	0.88 ± 0.06
LVEDD (mm)	2.29 ± 0.17	2.33 ± 0.14	2.39 ± 0.14	2.26 ± 0.09
LVESD (mm)	1.09 ± 0.08	1.39 ± 0.13*	1.17 ± 006	0.93 ± 0.07
Normalized LV mass (mg/g)	2 41 ± 0.20	2.45 ± 0.27	2.55 ± 0.48	2.39 ± 0.24
Fractional Shortening (%)	52.4 ± 1.9	36.2 ± 3.5*	50.7 ± 2.2	52.5 ± 2.8#
Ejection Fraction (%)	88.9 ± 1.4	72.8 ± 4.3*	90.2 ± 1.8	88.2 ± 2.6#

LV: left ventricular; EDD: end diastolic diameter; ESD: end systolic diameter; Mean ± SEM, n = 6–7 mice per group,

^*p < 0.05 vs. WT group,

^#p < 0.05 vs. WT-LPS group.

3.2. Langendorff-perfused heart function of WT and AOE mice

The Langendorff perfused heart assessment revealed that LPS challenge significantly decreased LVDP, the maximal rate of LV pressure development (+ dP/dt) and the maximal rate of LV pressure decline (−dP/dt) in WT group (p < 0.05 vs. LPS untreated hearts), the effect of which was abrogated by Akt activation. Chronic Akt activation did not elicit any notable effects on Langendorff perfused heart function (Fig. 1)

Fig. 1.

Effect of Akt overexpression (AOE) on LPS-induced changes in whole heart function using the Langendorff perfusion system. WT and AOE mice received LPS (4 mg/kg, i.p., 6 hrs) prior to assessment of heart function. A: Left ventricular developed pressure (LVDP); B: + dP/dt; and C: - dP/dt. Mean ± SEM, n = 5–6 mice per group, *p < 0.05 vs. WT group, ^#p < 0.05 vs. WTLPS group.

3.3. Mechanical and intracellular Ca²⁺ properties of cardiomyocytes

Mechanical properties revealed unchanged resting cell length regardless of LPS exposure or Akt activation (p > 0.05). LPS challenge reduced peak shortening and maximal velocity of shortening/ relengthening (± dL/dt), prolonged TR₉₀ associated with comparable TPS (p < 0.05 vs. LPS untreated groups), the effects of which were ablated by Akt transgene. Akt transgene itself did not exert any notable effect on cell mechanics (Fig. 2). To understand the mechanism(s) behind Akt activation-offered cardiac protection, Fura-2 fluorescence was monitored to evaluate intracellular Ca²⁺ handling. Cardiomyocytes displayed significantly reduced intracellular Ca²⁺ release in response to electrical stimuli (ΔFFI) and prolonged intracellular Ca²⁺ decay (single or bi-exponential curve fitting, p < 0.05 vs. LPS untreated groups) along with unchanged resting intracellular Ca²⁺ (resting FFI) following LPS treatment, the effect of which was reconciled by Akt activation. Akt transgene itself did not alter intracellular Ca²⁺ homeostasis (Fig. 3).

Fig. 2.

Effect of Akt overexpression (AOE) on LPS-induced changes in cardiomyocyte contractile properties. WT and AOE mice received LPS (4 mg/kg, i.p., 6 hrs) prior to assessment of mechanical function. A: Resting cell length; B: Peak shortening (% of resting cell length); C: Maximal velocity of shortening (+ dL/dt); D: Maximal velocity of relengthening (− dL/dt); E: Time-to-peak shortening (TPS); and F: Time-to-90% relengthening (TR₉₀). Mean ± SEM, n = 90 – 100 cells from 3 mice per group, *p < 0.05 vs. WT group, ^# p < 0.05 vs. WT-LPS group.

Fig. 3.

Effect of Akt overexpression (AOE) on LPS-induced changes in intracellular Ca²⁺ handling measured using Fura-2. WT and AOE mice received LPS (4 mg/kg, i.p., 6 hrs) prior to assessment of intracellular Ca²⁺ properties in cardiomyocytes. A: Baseline Fura-2 fluorescence intensity (FFI); B: Change in FFI (ΔFFI) in response to electrical stimuli; C: Single exponential intracellular Ca²⁺ decay rate; and D: Bi-exponential intracellular Ca²⁺ decay rate. Mean ± SEM, n = 80 – 90 cells from 3 mice per group, *p < 0.05 vs. WT group, ^#p < 0.05 vs. WT-LPS group.

3.4. Effect of Akt activation on LPS-induced ROS production and oxidative stress

To examine the potential mechanism behind Akt-elicited protection against LPS-induced cardiac mechanical anomalies, ROS was evaluated using DCF fluorescence in cardiomyocytes, and GSH/GSSG assay in myocardial tissues from WT and Akt transgenic mice. Results shown in Fig. 4A–D indicate that ROS production was significantly enhanced whereas GSH levels and GSH/GSSG ratio was overtly reduced following LPS treatment (p < 0.05 vs. LPS untreated groups). Consistent with its mechanical responses, Akt activation obliterated LPS-induced increase in ROS production and drop in GSH and GSH/GSSG ratio. Akt activation exerted little effects on ROS production and oxidative stress (GSH levels and GSH/GSSG ratio).

Fig. 4.

ROS production, oxidative stress as well as changes in phosphorylation of Akt (Ser473 or Thr308), GSK3β, and PTEN in cardiomyocytes from WT and AOE mice treated with or without LPS (4 mg/kg, i.p., 6 hrs). A: ROS production measured by DCF fluorescence; B: Glutathione (GSH) level; C: Oxidized glutathione (GSSG) level; D: GSH/GSSG ratio; E: pAkt (Ser473) level; F: pAkt (Thr308) level; G: pGSK3β level; and H: pPTEN level. Insets: Representative gel blots depicting proteins of interest (GAPDH as loading control). Mean ± SEM, n = 5–6 mice per group, *p < 0.05 vs. WT group, ^# p < 0.05 vs. WT-LPS group.

3.5. Effects of Akt activation on LPS-induced ER stress response

As depicted in Fig. 5, Western blot analysis demonstrated a robust increase in the ER stress protein markers including GRP78, Gadd153, peIF2α, as well as phosphorylation of the stress signaling p38, ERK, and JNK (p < 0.05 vs. LPS untreated groups), the effects were ablated by Akt activation. Akt activation itself did not affect ER stress or stress signaling activation.

Fig. 5.

Effect of Akt overexpression (AOE) on LPS-induced ER stress. WT and AOE mice received (4 mg/kg, i.p., 6 hrs) prior to assessment of ER stress. A: pERK level; B: pJNK level; C: pp38 level; D: GRP78 level; E: Gadd153; and F: peIF2α level. Insets: Representative gel blots of stress signaling and ER stress proteins (GAPDH used as loading control). Mean ± SEM, n = 5–8 per group; *p < 0.05 vs. WT group, ^#p < 0.05 vs. WT-LPS group.

3.6. Effects of Akt activation on LPS-induced changes on Akt signal pathways

Western blot analysis revealed significantly increased phosphorylation of Akt (Ser⁴⁷³ and Thr³⁰⁸) and GSK3β in myocardium from WT and AOE mouse hearts following LPS treatment (p < 0.05 vs. LPS untreated groups). Akt activation exerted notable increase in the phosphorylation of Akt (Ser⁴⁷³ and Thr³⁰⁸) without notable effect on GSK3β phosphorylation. Examination of the Akt negative regular PTEN phosphorylation revealed little change in WT or AOE myocardium, regardless of LPS challenge (Fig. 4E–H).

3.7. Effects of Akt activation on LPS-induced changes on proinflammatory cytokines

To detect the effect of inflammation on Akt-induced cardioprotection against LPS, levels of the proinflammatory cytokines such as TNFα, IL-1β and IL-6 were evaluated. As shown in Fig. 6AD, LPS significantly upregulated expression of TNFα, IL-1β and IL- 6 as well as phosphorylation of the NFΚB inhibitory protein IΚBα (p < 0.05 vs. LPS untreated groups). Although Akt activation did not affect levels (or phosphorylation) of these proinflammatory cytokines, it mitigated LPS-induced changes in these cytokines.

Fig. 6.

Effect of Akt overexpression (AOE) on LPS-induced changes in phosphorylation of IΚBα, as well as levels of TNFα, IL-1β, IL-6, Bax, caspase-3, -9 and Bcl-2. WT and AOE mice received LPS (4mg/kg, i.p., or 6 hrs) prior to determination of these proteins. A: phosphorylation of IΚBα; B: TNFα level; C: IL-1β level; D: IL-6 level; E: Bax level; F: Bcl-2 level; G: Cleaved caspase-3 level; and H: Cleaved caspase-9 level. Insets: Representative gel blots of proteins of interest (GAPDH used as loading control). Mean ± SEM, n = 5–8 per group; *p < 0.05 vs. WT group, ^#p < 0.05 vs. WT-LPS group.

3.8. Effects of Akt activation on LPS-induced changes on apoptotic markers

To evaluate the possible role of apoptosis in Akt activation-offered cardioprotection against LPS treatment, the apoptotic proteins Bax, caspase-3, -9 and Bcl-2 were examined. As shown in Fig. 6E–H, LPS challenge significantly upregulated expression of Bax, Caspase-3 and -9, while reducing the level of Bcl-2 (p < 0.05 vs. LPS untreated groups), the effects of which were ablated by Akt activation. Akt activation itself did not affect these apoptotic markers.

3.9. Effects of Akt activation on LPS-induced autophagic response

To explore if autophagy contributes to Akt activation-offered cardioprotection against LPS treatment, levels of autophagic markers including Beclin-1, Atg7, Atg5 and LC3B were evaluated. Results shown in Fig. 7 depicted that LPS significantly increased levels of Beclin-1, Atg7 and the LC3B II/I ratio while suppressing p62 accumulation (p < 0.05 vs. LPS untreated groups), the effects of which were unaffected by Akt activation (with the exception of p62). Akt activation itself did not alter levels of these autophagic markers.

Fig. 7.

Effect of Akt overexpression (AOE) on LPS-induced changes in autophagic markers. WT and AOE mice received LPS (4mg/kg, i.p.) for 6 hrs prior to assessment of autophagy. A: Belin- 1 level; B: LC3-I level; C: LC3-II level; D: LC3-II to LC3-I ratio; E: Atg7 level; and F: p62 level; Insets: Representative gel blots of autophagy proteins and GAPDH (used as loading control). Mean ± SEM, n = 5–8 per group; *p < 0.05 vs. WT group, ^#p < 0.05 vs. WT-LPS group.

3.10. Effect of Akt activation and GSK3β inhibition on LPS-induced changes in mechanical and intracellular Ca²⁺ properties in cardiomyocytes in vitro

Our in vitro data revealed that LPS exposure significantly decreased peak shortening and maximal velocity of shortening/relengthening (± dL/dt), prolonged TR₉₀ without affecting TPS (p < 0.05 vs. LPS untreated group), in a manner reminiscent of in vivo LPS treatment. Although Akt activation and GSK3β inhibition using SB216763 failed to exert notable effects themselves on cardiomyocyte mechanics, they effectively ablated LPS-induced changes in cardiomyocyte mechanical and intracellular Ca²⁺ properties. Neither Akt activation nor SB216763 affected cardiomyocyte mechanics and intracellular Ca²⁺ homeostasis (Fig. 8 & Fig. 9).

Fig. 8.

Effect of SB216763 (10 µM) on cardiomyocyte contractile properties in cardiomyocytes from WT and AOE mice following LPS exposure (4 µg/ml, 2 hrs). A: Resting cell length; B: Peak shortening (% of resting cell length); C: Maximal velocity of shortening (+ dL/dt); D: Maximal velocity of relengthening (− dL/dt); E: Time-to-peak shortening (TPS); and F: Time-to- 90% relengthening (TR₉₀). Mean ± SEM, n = 90 – 100 cells from 3 mice per group, *p < 0.05 vs. WT group, ^# p < 0.05 vs. WT-LPS group.

Fig. 9.

Effect of SB216763 (10 µM) on intracellular Ca²⁺ properties measured using Fura-2 in cardiomyocytes from WT and AOE mice after LPS exposure (4 µg/ml, 2 hrs). A: Baseline fura-2 fluorescence intensity (FFI); B: Change in FFI (ΔFFI) in response to electrical stimuli; C: Single exponential intracellular Ca²⁺ decay rate; and D: Bi-exponential intracellular Ca²⁺ decay rate. Mean ± SEM, n = 80 – 90 cells from 3 mice per group, *p < 0.05 vs. WT group, ^#p < 0.05 vs. WT-LPS group.

3.11. Effect of Akt activation and SB216763 on LPS-induced ER stress in vitro

As shown in Fig. 10A–C, Western blot analysis revealed a robust increase in ER stress manifested by GRP78, Gadd153 and peIF2α in cardiomyocytes from WT mouse hearts (p < 0.05 vs. LPS untreated group). Chronic Akt activation and SB216763 significantly attenuated LPS-induced changes in these ER stress markers without eliciting any notable effect by themselves.

Fig. 10.

Effect of SB216763 (10 µM) on ER stress proteins and proinflammatory cytokines in cardiomyocytes from WT and AOE mice following LPS exposure (4 µg/ml, 2 hrs). A: Gadd153; B: GRP78; C: peIF2α; D: ROS production; E: phosphorylation of IΚBα; F: TNFα level; G: IL-1β level; and H: IL-6 level. Insets: Representative gel blots of proteins of interest (GAPDH as loading control). Mean ± SEM, n = 5 – 8 isolations per group; *p < 0.05 vs. WT group, ^#p < 0.05 vs. WT-LPS group.

3.12. Effect of SB216763 on proinflammatory cytokines following LPS treatment in vitro

As shown in Fig. 10E–H, LPS significantly induced expression of pIΚBα, TNFα, IL-1β, and IL- 6 in cardiomyocytes (p < 0.05 vs. LPS untreated groups), the effect of which was attenuated or ablated by chronic Akt activation or SB216763. Neither Akt activation nor SB216763 affected the levels of these proinflammatory cytokines.

3.13. Effect of SB216763 on apoptotic proteins following LPS treatment in vitro

As shown in Fig. 11A–D, LPS significantly induced expression of Bax, Caspase-3 and -9, while it reduced Bcl-2 expression in cardiomyocytes (p < 0.05 vs. LPS untreated group), the effects of which were ablated by Akt overexpression or SB216763. Neither Akt activation nor SB216763 alone affected levels of these apoptotic proteins.

Fig. 11.

Effect of SB216763 (10 µM) on the levels of apoptotic proteins Bax, caspase-3, -9 and Bcl-2 in cardiomyocytes from WT and AOE mice following LPS exposure (4 µg/ml, 2 hrs). A: Bax levels; B: Bcl-2 levels; C: Cleaved caspase-3 levels; D: Cleaved caspase-9 levels. Insets: Representative gel blots depicting levels of Bax, Bcl-2, Cleaved caspase-3, Cleaved caspase-9 and GAPDH (loading control). Mean ± SEM, n = 5 – 8 isolations per group; * p < 0.05 vs. WT group, ^#p < 0.05 vs. WT-LPS group. E: Schematic diagram depicting LPS-induced cardiac contractile dysfunction, intracellular Ca²⁺ mishandling, oxidative stress, ER stress, apoptosis and inflammation in vivo (left panel) and in vitro (right panel). Activation of Akt rescues against LPS-induced anomalies possibly through a mechanism involving alleviation of pro-inflammatory cytokines and stress signaling activation. As a result, Akt activation ameliorates LPS-induced oxidative stress, ER stress and apoptosis en route to cardioprotection.

4. DISCUSSION

The salient finding of our study showed that chronic Akt activation alleviates cardiac contractile and intracellular Ca²⁺ defect, ROS accumulation, oxidative stress, ER stress and apoptosis as well as downregulates proinflammatory cytokines following LPS challenge. These findings implicated a protective effect of Akt against septic shock, implicating the therapeutic potential of Akt in the management of cardiac anomalies in sepsis. These findings are supportive to the notion for a role of the Akt downstream signaling molecule GSK3β in endotoxemia organ injury [15–20]. Previous reports have demonstrated that chronic Akt activation, with time, may promote cardiac hypertrophy, fibrosis and pronounced cardiac injury in response to ischemia/perfusion challenge [29–32]. Similar finding was noted by our group in old Akt transgenic mice [33]. However, in this study, 8–12 week-old mice were employed and the results failed to reveal overt changes in cardiac geometry and contractile function, suggesting that endogenous overactivation of Akt may not be innately harmful at a younger age.

Ample clinical and experimental evidence has consolidated cardiac contractile anomalies in sepsis [21, 26]. Echocardiographic and cardiomyocyte mechanical observations from our present study revealed enlarged left ventricular end systolic diameter, decreased ejection faction, fractional shortening, peak shortening, and maximal velocity of shortening/relengthening, as well as prolonged relaxation in association with minimal changes in LV diastolic diameter following LPS challenge. This is substantiated by the Langendorff perfused heart system where LPS decreased LVDP, maximum rate of LV pressure development (+ dP/dt) and decline (− dP/dt). These findings favor the presence of mixed systolic and diastolic dysfunction in response to LPS challenge. The inconsistent findings in the systolic versus diastolic parameters may be due to difference in levels of assessment (in vivo echocardiographic, ex vivo perfused heart and isolated cardiomyocytes). Interestingly, our study revealed that Akt activation attenuated or mitigated LPS-induced myocardial dysfunction. Further observation displayed reduced intracellular Ca²⁺ release in response to electrical stimuli (ΔFFI) and prolonged intracellular Ca²⁺ decay with unchanged resting intracellular Ca²⁺ (resting FFI) in cardiomyocytes from LPS-challenged WT mice, the effect of which was reconciled by Akt activation. These findings depicted a role of intracellular Ca²⁺ handling in LPS- and Akt activation-induced changes in myocardial contractile function. Our examination of the signaling molecules related to Akt signaling including PTEN and GSK3β revealed little role in the Akt negative regulator PTEN in LPS- and Akt activation-induced myocardial responsiveness. However, reduced phosphorylation of GSK3β was noted following LPS treatment, the effect of which was reversed by Akt overactivation. GSK3β is a highly conserved protein kinase ubiquitously expressed in nearly all mammalian tissues [34, 35]. GSK3β is involved in a multitude of biological process such as cell cycle control, cell differentiation, cell motility, adhesion and inflammation [36]. PI3K/Akt inactivates GSK3β (phosphorylation) to benefit cell survival and suppress inflammation [20, 37]. As our data indicated, reduced inactivation of GSK3β may be involved in LPS-induced cardiac anomalies, in line with a recent report where PI3K protects against sepsis through GSK3β phosphorylation in a murine model of cardiac-specific overexpression of PI3K p110α [38]. Accumulating evidence has revealed proven benefit of GSK3β inhibition in endotoxemia-associated organ injury [15–20].

Our results indicated that chronic Akt activation alleviated ROS production, oxidative and ER stress as depicted by ERK, p38, JNK, GRP78, Gadd153, peIF2α, and GSH/GSSG ratio. Moreover, chronic Akt activation inhibited proinflammatory and proapoptotic molecules such as TNFα, IL-1β, IL-6, Bax, caspase-3, and casepase-9. These findings suggested a beneficial role of Akt in sepsis-induced cellular injuries including oxidative damage, ER stress, inflammation and apoptosis. Meanwhile, our data further indicated activated NF-κB following LPS challenge as manifested by elevated IκBα phosphorylation, the effect of which was nullified by Akt activation. These findings favor a role of cytokine and inflammation in PI3K/Akt-elicited cytoprotection, consistent with the notion for a role of PI3K/Akt in the regulation of host immune homeostasis and sepsis [39–41]. PI3K/Akt may neutralize the deleterious action of NF-κB to alleviate organ damage in sepsis [42, 43]. Akt is capable of attenuating inflammation through inactivating NF κB by way of suppression of mitogen-activated kinase (p38) and c-jun N-terminal kinase (JNK) [44, 45]. NF-κB inhibitor was found to significantly attenuate TNFα-induced cytotoxicity along with preserved PI3K/Akt signaling [46]. Interleukin-1 (IL-1) was initially described as the first “endogenous pyrogen” with an essential role in myocardial dysfunction and infarct healing [47]. The pro-inflammatory cytokines such as TNFα, IL-1β and IL-6 have been shown to trigger heart anomalies through induction of apoptosis, impairment of bone marrow function, and interruption of endothelial cell function [48]. As shown in our study, changes in proinflammatory cytokines (TNFα, IL-1β and IL-6) and stress signaling molecules coincided with phosphorylation of IκBα in response to LPS challenge and Akt activation. These data support a role of NF-κB signaling in Akt activation-induced protection against LPS-induced cell stress and inflammation in the heart.

ER stress refers to unfolded protein response (UPR), or an imbalance between delivery of unfolded proteins to the ER and the ER capacity for cellular folding [49]. Three ER integral membrane proteins namely PERK (RNA-activated protein kinase-like ER kinase), IRE (inositolrequiring kinase) and ATF6 (activating transcription factor) comprise the main pathways for ER stress [50]. ER stress plays a rather pivotal role in diabetes mellitus, neurodegenerative diseases, obesity, trauma and ischemic heart diseases [51–54]. It is believed that sepsis perturbs the ER, leading to a situation of ER stress [55]. In our in vivo study, ER stress markers including GRP78, Gadd153 and peIF2α were markedly elevated in response to LPS challenge, the effects of which were attenuated or obliterated by chronic Akt activation. Similar results were found from in vitro study where SB216763 (an inhibitor of GSK3β) inhibits LPS-induced ER stress and contractile dysfunction in cardiomyocytes, consistent with our previous report that ER stress contributes to cardiac dysfunction [14]. Although ROS may be necessary for normal cellular function and survival, oxidative stress develops when the balance between ROS production and clearance is interrupted [56]. LPS induces superoxide production to promote oxidative stress [57]. Findings from our study revealed that LPS prompted accumulation of ROS and oxidative stress (reduced GSH/GSSG ratio) in conjunction with impaired cardiac contraction and intracellular Ca²⁺ handling. Our result revealed that Akt may exert its beneficial role in sepsis through alleviating LPS-induced ROS production and oxidative stress.

Apoptosis plays important role in a wide variety of cardiovascular diseases including ischemia/reperfusion injury, atherosclerosis, alcoholic cardiomyopathy, diabetic cardiomyopathy and sepsis-associated cardiac dysfunction [58–60]. Our results demonstrated that Akt activation ablated sepsis-elicited activation of stress signaling (ERK, JNK and p38) and apoptosis (Bax, Bcl-2, Caspase-3 and -9), in line with the changes in ROS accumulation and the proinflammatory cytokines. This is consistent with the notion that endotoxin is capable of inducing proapoptotic markers such as Bax and caspase-3 in the heart to contribute to heart defect [61, 62]. Activation of MAPK stress signaling has been widely documented in septic heart contributing to cell death and myocardial dysfunction [21, 26]. In our study, we also found that LPS upregulated the levels of Bax, caspase-3 and -9 while downregulating the anti-apoptotic protein Bcl-2. Although Akt activation did not exert any effect on these apoptotic proteins, it ablated LPS-induced changes in these apoptosis markers. In vitro evidence further revealed that SB216763 mimicked the effect of Akt activation upon LPS challenge. Data from our study revealed increased proinflammatory factors such as TNFα, IL-1β and IL-6 along with ROS accumulation and ER stress following LPS challenge. Chronic Akt activation alleviated ROS production, ER stress, and apoptosis in response to LPS challenge. Adequate ER reaction and ROS production are the biologically protective responses although excessive ER stress and ROS production are deemed deleterious. Excessive ER stress and oxidative stress may induce apoptosis [63]. Many studies have demonstrated that PI3K/Akt may suppress apoptosis in macrophages, lymphocytes, neutrophils to protect the host against sepsis [64, 65]. In particular, cardiac-specific PI3K p110α may suppress apoptosis in sepsis [38]. These findings are in all favor of a pivotal role of Akt in attenuating LPS-induced myocardial damage through inhibition of apoptosis.

Autophagy is a conserved cellular mechanism through which mammalian cells degrade and recycle damaged macromolecules and organelles [66, 67]. A role of autophagy has been reported in an array of cardiovascular diseases including obesity, sepsis, aging, alcoholism and ischemic injury [68–74]. Autophagy usually starts with an “induction phase” which is initiated by Beclin-1 as an internal stimulus followed by a second “formation phase” involving Atg proteins such as Atg7 and autophagosome membrane specific protein light chain 3 (LC3) or Atg8 [75]. Sequestosome-1 (SQSTM, also known as p62) is an adaptor for autophagosome formation. The higher expression of p62 usually means hindering of fusion of autophagosome and lysosome [76, 77]. Data from our study suggest that LPS facilitates levels of autophagy as depicted by higher levels of Beclin-1, Atg7, LC3B, and LC3B II-to LC3B I ratio, as well as decreased p62 in WT mice. Akt activation failed to attenuate or reverse LPS-induced responses in autophagy signaling molecules, suggesting a lesser role of autophagy in Akt-induced cardioprotection against sepsis.

In conclusion, data from our study revealed that chronic Akt activation rescues against LPS-induced cardiac contractile and intracellular Ca²⁺ anomalies as well as cell death through an Akt/GSK3β-dependent mechanism involving interplay between activation of proinflammatory cytokines and stress signaling (Fig.11E). Akt activation alleviates stress signaling activation, oxidative stress, ER stress, apoptosis and inflammation in sepsis. These results may implicate the possible therapeutic potential of Akt cascade in the management of cardiac dysfunction in sepsis. Nonetheless, although LPS infusion/injection has been widely employed for sepsis research to mimics many of the initial clinical features of sepsis including increases in proinflammatory cytokines such as TNF-α and interleukin-1, it suffers from limitations such as lack of bacteremia compared with sepsis resulted from intestinal leakage (e.g., cecal ligation and puncture or colon ascendens stent peritonitis) [78]. To this end, precaution should be taken to apply knowledge obtained from LPS endotoxemia animal model to human sepsis.

Research highlights.

• We examine the effect of Akt activation in sepsis-induced cardiomyopathy;

• Chronic Akt activation alleviates LPS-induced cardiac mechanical defect;

• Akt activation inhibits apoptosis and ER stress in sepsis;