Oxidative activation of Ca²⁺/calmodulin-activated kinase II mediates ER stress-induced cardiac dysfunction and apoptosis

Abstract

Endoplasmic reticulum (ER) stress elicits oxidative stress and intracellular Ca²⁺ derangement via activation of Ca²⁺/calmodulin-dependent protein kinase II (CaMKII). This study was designed to examine the role of CaMKII in ER stress-induced cardiac dysfunction and apoptosis as well as the effect of antioxidant catalase. Wild-type FVB and transgenic mice with cardiac-specific overexpression of catalase were challenged with the ER stress inducer tunicamycin (3 mg/kg ip for 48 h). Presence of ER stress was verified using the ER stress protein markers immunoglobulin binding protein (BiP) and C/EBP homologous protein (CHOP), the effect of which was unaffected by catalase overexpression. Echocardiographic assessment revealed that tunicamycin elicited cardiac remodeling (enlarged end-systolic diameter without affecting diastolic and ventricular wall thickness), depressed fractional shortening, ejection fraction, and cardiomyocyte contractile capacity, intracellular Ca²⁺ mishandling, accumulation of reactive oxygen species (superoxide production and NADPH oxidase p47phox level), CaMKII oxidation, and apoptosis (evidenced by Bax, Bcl-2/Bax ratio, and TUNEL staining), the effects of which were obliterated by catalase. Interestingly, tunicamycin-induced cardiomyocyte mechanical anomalies and cell death were ablated by the CaMKII inhibitor KN93, in a manner reminiscent of catalase. These data favored a permissive role of oxidative stress and CaMKII activation in ER stress-induced cardiac dysfunction and cell death. Our data further revealed the therapeutic potential of antioxidant or CaMKII inhibition in cardiac pathological conditions associated with ER stress. This research shows for the first time that contractile dysfunction caused by ER stress is a result of the oxidative activation of the CaMKII pathway.

the endoplasmic reticulum (ER) is the organelle responsible for protein and lipid synthesis, Ca²⁺ storage, and toxin degradation. The ER is essential to the maintenance of cardiac homeostasis as it regulates intracellular Ca²⁺ cycling and excitation-contraction coupling (27). Periods of starvation, increased protein synthesis, and accumulation of misfolded proteins lead to a phenomenon called ER stress. ER stress stimulates the unfolded protein response (UPR), a complex cell survival mechanism to reduce protein synthesis and promote molecular chaperone production to properly fold new proteins (19). Prolonged activation of UPR or ER stress that surpasses the ability of UPR to neutralize the stress leads to deleterious effects. ER stress has been implicated in cardiac diseases including obesity and diabetes-related cardiomyopathy, ischemia-reperfusion, and dilated cardiomyopathy (7, 12, 21, 41). In the heart, prolonged ER stress often results in apoptosis, disruption of intracellular Ca²⁺ homeostasis, cardiac contractile dysfunction, and the production of reactive oxygen species (ROS) (29, 40). Although a causative link between ER stress and cardiac contractile dysfunction has been established (10, 19), the precise mechanisms of action remain elusive.

Ca²⁺/calmodulin-activated protein kinase II (CaMKII) is a serine/threonine protein kinase activated in response to changes in intracellular Ca²⁺ while its inhibition has shown some promise in the treatment of heart disease (4, 6). It was reported that CaMKII may be oxidized by ROS at methionine residues 281/282 following its activation by Ca²⁺/calmodulin, leading to a sustained activation even after the removal of free Ca²⁺ (14). This ROS-dependent CaMKII activation is believed to be the primary mechanism responsible for ANG II-induced cardiac dysfunction and apoptosis as well as cardiac rupture after myocardial infarction (14, 22, 30). Changes in CaMKII activity may have profound effects on intracellular Ca²⁺ handling while a number of signaling targets have been identified in response to CaMKII activation including ryanodine receptor and phospholamban (36, 38).

Given that ER stress increases intracellular Ca²⁺ levels and stimulates ROS production, we hypothesized that ER stress increases ROS leading to CaMKII activation, apoptosis, and cardiac mechanical dysfunction. To this end, this study was designed to examine the impact of oxidative stress inhibition on ER stress-induced cardiac anomalies with a focus on CaMKII. ER stress was induced by tunicamycin in wild-type and transgenic mice with cardiac overexpression of the antioxidant enzyme catalase. Tunicamycin is a potent inhibitor of N-glycosylation, triggering ER stress. Animals subjected to tunicamycin develop ER stress in many body tissues, including the heart. Cardiac phenotypic changes in response to tunicamycin challenge may be represented as depressed systolic function such as fractional shortening (44, 45). The myocardial consequence of ER stress have been examined in depth by our group and others although the cell signaling mechanism downstream of ER stress in ER stress-initiated cardiac anomalies have not been evaluated. To this end, the present study was designed to evaluate the role of ROS in ER stress-induced cardiac contractile dysfunction and apoptosis. Cardiac contractile function was examined using echocardiography and cell shortening. Apoptosis was assessed using TUNEL staining and protein expression of apoptosis-regulatory proteins Bax, Bcl-2, and Fas. Superoxide (O₂⁻), the key enzyme responsible for O₂⁻ generation NADPH oxidase, and oxidation of CaMKII were monitored. To evaluate if CaMKII was necessary for ER stress-induced cardiac contractile dysfunction and apoptosis, tunicamycin-induced cardiac dysfunction and apoptosis were evaluated in the absence or presence of the specific CaMKII inhibitor KN93.

MATERIALS AND METHODS

Experimental animals.

The experimental procedures used here were approved by the University of Wyoming Institutional Animal Use and Care Committee (Laramie, WY). Adult male cardiac-specific catalase overexpression mice were used as described previously (11, 17, 23, 31). A primer pair derived from the MHC promoter and rat catalase cDNA was used for identification of the catalase transgene, with the reverse sequence of 5′-AATATCGTGGGTGACCTCAA-3′ and the forward sequence of 5′-CAGATGAAGCAGTGGAAGGA-3′. The FVB littermates were used as the wild-type mice. Mice were housed in a 12:12-h light/dark cycle and given free access to food and water. Tunicamycin or equal volume of DMSO was injected into 3 mo-old wild-type FVB or catalase mice (3 mg/kg ip) (45) and were used 48 h later.

Echocardiographic assessment.

Cardiac geometry and function were evaluated in anesthetized (ketamine 80 mg/kg and xylazine 12 mg/kg ip) mice using a 2-D guided M-mode echocardiography (Sonos 5500) equipped with a 15- to 6-MHz linear transducer. Fractional shortening was calculated from left ventricular (LV) end-diastolic diameter (LVEDD) and LV end-systolic diameter (LVESD) using the following equation: (LVEDD − LVESD)/LVEDD. Ejection fraction was calculated using the following equation: [(EDD³ − ESD³)/EDD³] × 100 (43).

Cardiomyocyte isolation and in vitro ER stress induction.

Hearts were rapidly removed from anesthetized (ketamine 80 mg/kg and xylazine 12 mg/kg ip) mice and mounted onto a temperature-controlled (37°C) Langendorff system. After perfusion with a modified Tyrode's solution (Ca²⁺ free) for 2 min, the heart was digested with a Ca²⁺-free Krebs-Henseleit (KHB) buffer containing liberase blendzyme 4 (Hoffmann-La Roche, Indianapolis, IN) for 15 min. The modified Tyrode's solution (pH 7.4) contained the following (in mM): 135 NaCl, 4.0 KCl, 1.0 MgCl₂, 10 HEPES, 0.33 NaH₂PO₄, 10 glucose, and 10 butanedione monoxime. The solution was gassed with 5% CO₂-95% O₂. The digested heart was removed from the cannula, and left ventricle was cut into small pieces in the modified Tyrode's solution. Tissue pieces were gently agitated, and the pellet of cells was resuspended. Extracellular Ca²⁺ was added incrementally back to 1.20 mM over 30 min. A yield of at least 50–60% viable rod-shaped cells with clear sarcomere striations was achieved. To induce ER stress, cardiomyocytes were incubated with tunicamycin (3 μg/ml, 3 h) (45) in the absence or presence of the CaMKII inhibitor KN93 (0.5 μM, 30 min pretreatment) at 37°C (45).

Cell shortening and intracellular Ca²⁺ measurement.

Mechanical properties of myocytes were assessed using an IonOptix soft-edge system (IonOptix, Milton, MA) (45). Myocytes were placed in a chamber mounted on the stage of an Olympus IX-70 microscope and superfused (∼2 ml/min at 25°C) with a buffer containing (in mM) 131 NaCl, 4 KCl, 1 CaCl₂, 1 MgCl₂, 10 glucose, and 10 HEPES. Myocytes were field stimulated at 0.5 Hz and 3-ms duration. Cell shortening and relengthening were assessed using the following indexes: peak shortening (PS), time to PS (TPS), time to 90% relengthening (TR₉₀), and maximal velocities of shortening/relengthening (±dL/dt). For intracellular Ca²⁺ handling, cardiomyocytes were loaded with fura-2/AM (0.5 μM) for 10 min, and fluorescence measurements were recorded with a dual-excitation fluorescence photomultiplier system (IonOptix). Loaded myocytes were placed on an Olympus IX-70 inverted microscope and observed using a Fluor ×40 objective. Cells were exposed to light emitted by a 75-W lamp and passed through either a 360- or 380-nm filter while being stimulated to contract at 0.5 Hz. Fluorescence emissions were detected between 480–520 nm by a photomultiplier tube after first illuminating the cells at 360 nm for 0.5 s, then at 380 nm for the duration of the recording. The 360-nm excitation scan was repeated at the end of the protocol and qualitative changes in intracellular Ca²⁺ level were inferred from the ratio of the fura-2 fluorescence intensity (FFI) at the two wavelengths (360/380 nm). Fluorescence decay time was also calculated as an indication of the intracellular Ca²⁺ clearing rate.

Western blot analysis.

Protein was prepared as previously described (45). Briefly, heart tissue was homogenized in RIPA lysis buffer (Millipore, Billerica, MA), sonicated, and centrifuged at 12,000 g for 20 min at 4°C. Protein concentrations of supernatants were measured using the Bradford assay. Samples containing equal protein concentrations were separated on a 7%, 10%, or 12% SDS-polyacrylamide gel in a mini-gel apparatus (Mini-PROTEAN II, Bio-Rad, Hercules, CA). Membranes were blocked with 5% milk in TBS-Tween and were incubated overnight at 4°C with anti-BiP (1:1,000), anti-CHOP (1:1,000), anti-p47phox (1:1,000), anti-Bax (1:1,000), anti-Bcl2 (1:1,000), and anti-FAS (1:1,000) from Santa Cruz Biotechnology (Santa Cruz, CA); anti-GAPDH (1:2,000) and anti-α-tubulin (1:2,000) from Cell Signaling (Beverly, MA); and anti-OxCaMKII (1:500) from Millipore. Blots were washed and incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies and detected by enzymatic chemiluminescence by a Bio-Rad Calibrated Densitometer (32).

Detection of O₂⁻ production.

ROS levels were determined as described (18). Fresh heart sections were frozen in OCT embedding compound. Using a Leica cryostat, 30-μm transverse sections were cut and incubated with dihydroethidium (DHE, 3 μM, 30 min) at room temperature in the dark. Sections were washed twice with PBS and then fixed in 4% paraformaldehyde for 10 min at 4°C. Sections were washed twice with PBS, mounted, and stored in the dark. Images were acquired using a Zeiss 710 laser scanning confocal microscope. The fluorescence intensity per image was quantified using ImageJ analysis software (NIH, Bethesda, MD).

TUNEL staining.

Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) staining of DNA strand breaks was performed using a fluorescence detection kit (Roche Applied Science, Indianapolis, IN) as previously described (43). Fresh frozen heart sections were cut using a Leica cryostat to produce 5-μm tissue sections. Tissue sections were fixed with 4% paraformaldehyde for 20 min and permeabilized in 0.1% Triton X-100 in 0.1% sodium citrate for 2 min at 4°C. Fifty microliters of a reaction mixture containing terminal deoxynucleotidyl transferase (TdT), fluorescein-dUTP was added to each section and incubated in a humidified chamber for 60 min at 37°C. Sections were washed three times with PBS and counterstained with 4′,6′-diamidino-2-phenylindole (DAPI, 5 μg/ml) for 1 min. Slides were mounted with Prolong Gold mounting medium (Invitrogen, Carlsbad, CA), and five images per tissue section were obtained using an Olympus BX51 microscope equipped with an Olympus MagnaFire SP digital camera and ImagePro image analysis software as previously described (24).

MTT assay for cell viability and mitochondrial membrane potential.

3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was performed as previously described (45). Isolated cardiomyocytes incubated with or without tunicamycin and with or without KN93 pretreatment were plated in a microtiter plate at 3 × 10⁵ cells/ml. MTT was added to each well for a final concentration of 0.5 mg/ml and incubated at 37°C for 2 h. DMSO (150 μl) was added to each well to dissolve the formazan crystals. Absorbance was measured at 540 nm using a SpectraMax 190 spectrophotometer. Mitochondrial membrane potential was detected as described previously (9, 44, 45). Myocytes incubated with or without tunicamycin and/or KN93 were incubated with JC-1 (5 μM) at 37°C in the dark for 10 min. Cells were washed three times with HEPES-saline buffer, and fluorescence was measured with a SpectraMax Gemini XS plate reader.

Statistical analysis.

Data are presented as means ± SE. Statistical significance was estimated by one-way ANOVA followed by Tukey's post hoc analysis.

RESULTS

Catalase overexpression prevents ER stress-induced cardiac dysfunction.

The experimental model of ER stress was confirmed using the ER stress protein markers immunoglobulin binding protein (BiP) and C/EBP homologous protein (CHOP). Tunicamycin challenge significantly upregulated levels of both BiP and CHOP. Catalase overexpression did not affect ER stress in the presence or absence of tunicamycin challenge (Fig. 1, A and B). Echocardiographic evaluation revealed that ER stress induction increased LVESD as well as suppressed fractional shortening and ejection fraction without affecting LV wall thickness and LVEDD, depicting the presence of systolic dysfunction following ER stress challenge. Although catalase overexpression did not elicit any overt effect on echocardiographic properties, it effectively ablated ER stress-induced cardiac remodeling and contractile defect (Fig. 1, C–H). Analysis of cardiomyocyte contractile function revealed that ER stress depressed PS and ±dL/dt without affecting TPS and TR₉₀. Catalase overexpression reversed tunicamycin-induced cardiomyocyte contractile anomalies without eliciting any effect by itself. Interestingly, catalase overexpression further increased −dL/dt while it shortened TR₉₀ under ER stress (Fig. 2).

Fig. 1.

Effect of cardiac-specific overexpression of catalase on tunicamycin (3 mg/kg ip for 48 h)-induced endoplasmic reticulum (ER) stress and changes in echocardiographic indexes. A: BiP expression. B: CHOP expression. Insets in A and B: representative gel blots depicting expression of BiP, CHOP, and α-tubulin (loading control). C: representative echocardiographic images from FVB and catalase mice treated with or without tunicamycin. D: left ventricular (LV) wall thickness. E: LV end-diastolic diameter (LVEDD). F: LV end-systolic diameter (LVESD). G: fractional shortening. H: ejection fraction. Values are means ± SE; n = 5–7 mice/group. *P < 0.05 vs. FVB. #P < 0.05 vs. tunicamycin group.

Fig. 2.

Effect of cardiac-specific overexpression of catalase on tunicamycin (3 mg/kg ip for 48 h)-induced changes in cardiomyocyte contractile properties. A: representative cell shortening traces. B: peak shortening amplitude (normalized to cell length). C: maximum velocity of shortening (+dL/dt). D: maximum velocity of relengthening (−dL/dt). E: time to shortening (TPS). F: time to 90% relengthening (TR₉₀). Values are means ± SE; n = 141–150 cells per group, *P < 0.05 vs. FVB. #P < 0.05 vs. tunicamycin group.

ER stress-induced apoptosis is prevented by catalase overexpression.

ER stress is known to promote myocardial apoptosis (28). To evaluate the effect of catalase on ER stress-induced apoptosis, levels of the proapoptotic proteins Fas and Bax as well as the antiapoptotic protein Bcl-2 were examined in myocardium from FVB and catalase mice with or without tunicamycin challenge. Our data revealed that tunicamycin upregulated levels of Bax and decreased the Bcl-2-to-Bax ratio without affecting the levels of Fas and Bcl-2. Although catalase overexpression did not exert any overt effect on these apoptotic proteins, it mitigated ER stress-induced changes in Bax and Bcl-2-to-Bax ratio with little effect on Fas and Bcl-2 (Fig. 3). Assessment of apoptosis using TUNEL staining indicated that tunicamycin significantly increased the number of TUNEL-positive nuclei, the effect of which was prevented by catalase, corroborating the immunoblot data. Catalase overexpression did not exert any notable effect using TUNEL staining (Fig. 4).

Fig. 3.

Effect of cardiac-specific overexpression of catalase on tunicamycin (TN, 3 mg/kg ip for 48 h)-induced changes in apoptotic proteins Bax, Bcl-2, and Fas. A: Fas expression. B: Bax expression. C: Bcl-2 expression. D: Bax-to-Bcl-2 ratio. Inset: gel blots of Fas, Bax, Bcl-2, and GAPDH (loading control). TN, tunicamycin. Values are means ± SE; n = 3 hearts per group. *P < 0.05 vs. FVB. #P < 0.05 vs. tunicamycin group.

Fig. 4.

TUNEL staining of myocardium from FVB and cardiac-specific catalase overexpression mice with or without tunicamycin (3 mg/kg ip for 48 h) challenge. A: representative images of TUNEL staining (left column) and DAPI nuclear labeling (right column). Arrows are pointing to apoptotic nuclei. B: quantitative analysis of TUNEL positive nuclei per 1,000 nuclei. Values are means ± SE; n = 3–4 hearts per group. *P < 0.05 vs. FVB. #P < 0.05 vs. tunicamycin group.

ER stress induces superoxide production, upregulates NADPH p47phox, and promotes oxidative activation of CaMKII.

DHE staining and Western blot analyses were performed in myocardial tissues to evaluate O₂⁻ production and the O₂⁻ generation enzyme NADPH oxidase (p47phox subunit), respectively, in FVB and catalase mice with or without tunicamycin challenge. Our data revealed that tunicamycin enhanced O₂⁻ production and upregulated p47phox expression, the effect of which was negated by catalase in a manner consistent with mechanical and apoptotic data. To examine the possible downstream target for oxidative stress, oxidized CaMKII [a key player for oxidative stress-induced apoptosis (14)] was evaluated in myocardium using an antibody against oxidized methionine residues 281–282 of CaMKII. Our result indicated upregulated oxidation of CaMKII in the heart following tunicamycin treatment, the effect of which was prevented by catalase. Catalase overexpression itself did not exert effect on O₂⁻ production and oxidation of CaMKII (Fig. 5).

Fig. 5.

Effect of cardiac-specific overexpression of catalase on tunicamycin (3 mg/kg ip for 48 h)-induced changes in superoxide production, NADPH oxidase p47phox level, and oxidation of Ca²⁺/calmodulin-activated kinase II (CaMKII). A: representative images of DHE-stained heart sections. B: pooled data summarizing DHE fluorescence intensity. C: representative gel blots depicting p47phox, oxidized CaMKII, and GAPDH (loading control). D: p47phox expression. E: levels of oxidized CaMKII assessed using Western blot analysis. Values are means ± SE; n = 3–4 hearts per group. *P < 0.05 vs. FVB. #P < 0.05 vs. tunicamycin group.

CaMKII inhibition prevents ER stress-induced cardiac contractile dysfunction.

Given the possible role of oxidation of CaMKII in ER stress-induced cardiac anomalies, ER stress-induced cardiomyocyte contractile dysfunction was examined in the presence of the specific CaMKII inhibitor KN93. Our data shown in Fig. 6 demonstrated that KN93 incubation ablated ER stress-elicited cardiomyocyte mechanical defects, including decreased PS and ±dL/dt and prolonged TR₉₀, in a manner reminiscent of catalase. To better elucidate the mechanism behind KN93-, catalase-, and tunicamycin-induced cardiomyocyte responses, the fluorescence dye Fura-2 was employed to examine intracellular Ca²⁺ homeostasis in cardiomyocytes from FVB and catalase transgenic mice with or without tunicamycin or KN93 treatment in vitro. Our data revealed that ER stress triggered overtly elevated resting intracellular Ca²⁺ levels, increased the rise in intracellular Ca²⁺ in response to electrical stimuli (ΔFFI), and prolonged intracellular Ca²⁺ clearance, the effects of which were significantly attenuated or mitigated by catalase enzyme. Interestingly, KN93 significantly attenuated prolongation of intracellular Ca²⁺ decay without affecting elevated basal FFI and ΔFFI levels under ER stress. Last but not least, neither KN93 nor catalase altered intracellular Ca²⁺ homeostasis (Fig. 7). These data suggest a possible partial role of intracellular Ca²⁺ handling in catalase- and CaMKII inhibition-induced beneficial mechanical responses under ER stress.

Fig. 6.

Effect of CaMKII inhibition on endoplasmic reticulum (ER) stress-induced cardiomyocyte contractile dysfunction. Freshly isolated cardiomyocytes from FVB control mice were incubated with tunicamycin (3 μg/ml) for 3 h in the presence or absence of the CaMKII inhibitor KN93 (0.5 μM) prior to assessment of cardiomyocyte mechanical function. A: representative cell shortening traces. B: peak shortening. C: +dL/dt. D: −dL/dt. E: TPS. F: TR₉₀. Values are means ± SE; n = 116–118 cells per group. *P < 0.05 vs. control. #P < 0.05 vs. tunicamycin group.

Fig. 7.

Effect of catalase overexpression or CaMKII inhibition on ER stress-induced intracellular Ca²⁺ homeostasis. FVB and catalase mice were challenged with tunicamycin (3 mg/kg ip) prior to assessment of intracellular Ca²⁺ handling. A cohort of cardiomyocytes from FVB mice with or without tunicamycin treatment were incubated with the CaMKII inhibitor KN93 (0.5 μM, 30-min pretreatment) prior to intracellular Ca²⁺ fluorescence assessment. A: representative traces from FVB cardiomyocytes with or without tunicamycin treatment. B: resting fura-2 fluorescence intensity (FFI). C: electronically stimulated rise in FFI (ΔFFI). D: intracellular Ca²⁺ decay rate. Values are means ± SE; n = 55–61 cells per group. *P < 0.05 vs. FVB. #P < 0.05 vs. tunicamycin group.

CaMKII is responsible for ER stress-induced apoptosis and but not mitochondrial function.

Our results further suggested that tunicamycin challenge suppressed cell viability, which was prevented by KN93 or catalase. Neither KN93 nor catalase affected cell survival. Since ER stress was shown to be associated with mitochondrial dysfunction (28), mitochondrial membrane potential was monitored in cardiomyocytes from FVB and catalase transgenic mice treated with or without tunicamycin or KN93. To our surprise, neither tunicamycin nor KN93 or catalase overexpression affected mitochondrial membrane potential (Fig. 8).

Fig. 8.

Effect of catalase overexpression or CaMKII inhibition on ER stress-induced apoptosis and change in mitochondrial membrane potential. FVB and catalase mice were challenged with tunicamycin (3 mg/kg ip) prior to biochemical assessment. A cohort of cardiomyocytes from FVB mice with or without tunicamycin treatment were incubated with the CaMKII inhibitor KN93 (0.5 μM, 30-min pretreatment) prior to biochemical assessment. A: cell viability (n = 8 isolations). B: mitochondrial membrane potential using JC-1 fluorescence (n = 15 fields). C: schematic diagram depicting proposed mechanism responsible for ER stress-induced cardiac dysfunction and apoptosis. ER stress triggers reactive oxygen species (ROS) production leading to oxidation of CaMKII, en route to cardiac dysfunction and apoptosis. Catalase prevents oxidation of CaMKII whereas KN93 inhibits CaMKII activation to alleviate cardiac dysfunction and apoptosis under ER stress. Values are means ± SE. *P < 0.05 vs. FVB. #P < 0.05 vs. tunicamycin.

DISCUSSION

The salient finding from our study showed that ER stress triggers generation of O₂⁻ and upregulated p47phox NADPH oxidase, leading to abnormal echocardiographic, cardiomyocyte contractile function, and intracellular Ca²⁺ homeostasis. In addition, prolonged ER stress triggered cardiomyocyte apoptosis and oxidation of CaMKII. These abnormalities resulted from ER stress were rescued by the cardiac-specific overexpression of antioxidant catalase or CaMKII inhibition, indicating a pivotal role of CaMKII and oxidative stress in ER stress-induced cardiac anomalies.

ER stress is initially protective, aiming to restore ER homeostasis. However, prolonged periods of ER stress can be deleterious and damaging, especially to the heart. ER stress has been noted in a wide array of pathological conditions, including cardiac hypertrophy, alcoholic cardiomyopathy, and ischemia-reperfusion (12, 25, 29). Preventing ER stress with the ER chaperone tauroursodeoxycholic acid is capable of preventing obesity-induced cardiac dysfunction, implicating the therapeutic potential of ER stress perpetuation in heart diseases (7). In this study, tunicamycin triggered ER stress as evidenced by BiP and CHOP. In our hands, induction of ER stress triggered cardiac dysfunction as manifested by significantly reduced fractional shortening and ejection fraction, and reduced peak shortening and velocities of shortening and relengthening in cardiomyocytes. Further analysis of intracellular Ca²⁺ homeostasis revealed increased basal and electrically stimulated rise in intracellular Ca²⁺ levels along with prolonged Ca²⁺ clearance, suggesting a role of intracellular Ca²⁺ mishandling in ER stress-elicited cardiac mechanical abnormalities. Given the close link between ER stress and apoptosis, apoptotic protein markers and TUNEL staining were examined in myocardium from tunicamycin-treated mice. Western blot analysis revealed that ER stress induction significantly increased the levels of Bax and decreased Bcl-2-to-Bax ratio with unchanged levels of Fas and Bcl-2. Bax is a protein that permeabilizes the outer mitochondrial membrane to allow release of cytochrome c and other proapoptotic proteins into cytosol, initiating apoptosis (3). The elevated Bax levels and decreased Bcl-2/Bax ratio suggest ER stress-induced apoptosis may be initiated by intrinsic mitochondrial machinery as opposed to the extrinsic machinery (associated with Fas protein). The elevated Bax levels also depicted that ER stress may trigger mitochondrial dysfunction although this is not supported by our analysis of mitochondrial membrane potential. The role of apoptosis in ER-stressed hearts was further confirmed using TUNEL labeling, corroborating the Western blotting results.

Data from our study indicated that the accumulation of ROS accompanied ER stress and may be primarily responsible for the observed myocardial apoptosis and contractile dysfunction. Oxidative stress compromises cardiac performance and is usually present concurrently with ER stress (33, 44, 45). With ER stress, it has been suggested that ER oxidoreductases, mitochondria, and NADPH oxidase may serve as the sources of ROS production although exact mechanisms are still unclear (33). Tunicamycin induced upregulation of NADPH oxidase (p47phox), which may serve as the primary source of O₂⁻ and other ROS (1). Oxidative stress and ER stress are often considered mutual to each other with increased production of O₂⁻ destabilizing the luminal environment of the ER, leading to ER stress. On the other hand, ER stress may promote ROS production and subsequently cell damage or death (7, 33, 42). Findings from our present study revealed that the antioxidant catalase ablated or attenuated ER stress-induced cardiac abnormalities triggered by tunicamycin, an inhibitor of N-linked glycosylation, favoring a role of ROS production downstream of ER stress. One interesting observation is that overexpression of catalase accelerated maximal relaxation velocity under tunicamycin treatment compared with that of the FVB group. Although the precise nature behind such phenomenon still remains to be elucidated, it is possible that the interaction between tunicamycin and catalase may affect the sarcoplasmic reticulum (SR) Ca²⁺ pump to speed up SR Ca²⁺ uptake. Our data also suggest that overexpression of catalase reduces O₂⁻ and its generating enzyme NADPH oxidase. This somewhat unexpected finding may be related to the effect of catalase on H₂O₂ reduction. Although catalase does not directly neutralize O₂⁻, the antioxidant may alleviate O₂⁻ levels (and possibly p47phox levels) indirectly via reduction of H₂O₂ (20). It has been suggested that catalase serves as the rate-limiting step in the reaction from O₂⁻ to H₂O, and its overexpression has been shown to retard O₂⁻-induced deleterious effects reminiscent of superoxide dismutase (16, 26, 39).

Although oxidative stress can impose cytotoxicity directly, ROS may disturb the redox status to alter specific enzymes through posttranslational modification (15). CaMKII is a redox-sensitive enzyme where activation through oxidation leads to cardiac dysfunction and apoptosis in response to ANG II (14). It was also shown recently that oxidation of CaMKII may be the primary mechanism for myocardial rupture following myocardial infarction (22). Therefore, oxidation of CaMKII may be a unique facilitator of oxidative injury in the heart whose importance is only beginning to be explored. In our hands, levels of oxidized CaMKII were elevated in response to ER stress, the effect of which was mitigated by catalase, suggesting a pivotal role of oxidative stress in ER stress-induced changes in CaMKII activity. These data are in line with previous data where ER-stressed macrophages exhibit increased CaMKII oxidation and ER stress-induced apoptosis is CaMKII dependent (34). A role of CaMKII in the heart has been identified in various disease conditions (2, 8). Phosphorylation and/or oxidation of CaMKII changes the CaMKII activity in a Ca²⁺/calmodulin-independent manner, leading to phosphorylation of Ca²⁺ regulatory proteins such as ryanodine receptor and ion channel proteins, resulting in excitation-contraction coupling defect (5, 13). Activated CaMKII has also been linked to increased Bax expression, leading to apoptosis, correlating with our observations (37).

Perhaps the most intriguing finding from our present study was that overexpression of catalase mitigated or attenuated ER stress-induced changes in cardiac contraction, intracellular Ca²⁺ handling, and cell survival without alleviating tunicamycin-induced ER stress, favoring a key role of ROS accumulation in ER stress-induced primary mechanism for cardiac dysfunction. Catalase overexpression is known to be beneficial in antagonizing ROS accumulation in several disease models (17, 31, 35). In conjunction with the reduced ROS, and improved echocardiographic and cardiomyocyte mechanics in tunicamycin-treated catalase mice, overexpression of catalase also reversed the elevated levels of apoptotic proteins Bax (and Bax-to-Bcl-2 ratio) along with TUNEL-positive cells, indicating a permissive role of ROS in ER stress-induced apoptosis. Given the rise of ROS production, p47phox levels, and CaMKII oxidation following tunicamycin treatment, it is plausible to speculate that oxidation of CaMKII is responsible for ER stress-induced cardiac mechanical dysfunction and apoptosis. This is in line with the findings that catalase overexpression ablated oxidation of CaMKII and thus activation of CaMKII. Our in vitro observation that the specific CaMKII inhibitor KN93 obliterated tunicamycin-induced cardiomyocyte contractile dysfunction further consolidated an essential role of CaMKII in ER stress-elicited cardiac mechanical anomalies. It is noteworthy that KN93 failed to reconcile tunicamycin-induced changes in baseline and electrically stimulated rise in intracellular Ca²⁺, distinct from the response elicited by catalase overexpression. Although the precise mechanism responsible for such a discrepancy is still unclear, it is possible that catalase may inhibit other cell signaling machinery in addition to oxidation of CaMKII. Additionally KN93 has been shown previously to have off-target effects on voltage-gated ion channels (13). However, due to our finding that KN93 alone had no significant effect on myocyte shortening or calcium levels, it appears these effects are minimal.

In summary, our finding demonstrated an essential role of CaMKII as an intermediate in ER stress-induced cardiac dysfunction and its oxidative activation is necessary for the ER stress-elicited cardiac anomalies. Our findings of the beneficial effect of catalase against ER stress-induced cardiac dysfunction, apoptosis, and intracellular Ca²⁺ mishandling should shed some light toward a better understanding the role of CaMKII as a potential target in cardiac diseases afflicted with ER stress.

GRANTS

This work was supported by National Institutes of Health Grants 5P20-RR-016474 and 8P20-GM-103432.

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the author(s).

AUTHOR CONTRIBUTIONS

Author contributions: N.D.R. and J.R. conception and design of research; N.D.R. performed experiments; N.D.R. and J.R. analyzed data; N.D.R. and J.R. interpreted results of experiments; N.D.R. prepared figures; N.D.R. and J.R. drafted manuscript; N.D.R. and J.R. edited and revised manuscript; N.D.R. and J.R. approved final version of manuscript.