Reversing mitochondrial defects in aged hearts: role of mitochondrial calpain activation

Abstract

Aging chronically increases endoplasmic reticulum (ER) stress that contributes to mitochondrial dysfunction. Activation of calpain 1 (CPN1) impairs mitochondrial function during acute ER stress. We proposed that aging-induced ER stress led to mitochondrial dysfunction by activating CPN1. We posit that attenuation of the ER stress or direct inhibition of CPN1 in aged hearts can decrease cardiac injury during ischemia-reperfusion by improving mitochondrial function. Male young (3 mo) and aged mice (24 mo) were used in the present study, and 4-phenylbutyrate (4-PBA) was used to decrease the ER stress in aged mice. Subsarcolemmal (SSM) and interfibrillar mitochondria (IFM) were isolated. Chronic 4-PBA treatment for 2 wk decreased CPN1 activation as shown by the decreased cleavage of spectrin in cytosol and apoptosis inducing factor (AIF) and the α1 subunit of pyruvate dehydrogenase (PDH) in mitochondria. Treatment improved oxidative phosphorylation in 24-mo-old SSM and IFM at baseline compared with vehicle. When 4-PBA-treated 24-mo-old hearts were subjected to ischemia-reperfusion, infarct size was decreased. These results support that attenuation of the ER stress decreased cardiac injury in aged hearts by improving mitochondrial function before ischemia. To challenge the role of CPN1 as an effector of the ER stress, aged mice were treated with MDL-28170 (MDL, an inhibitor of calpain 1). MDL treatment improved mitochondrial function in aged SSM and IFM. MDL-treated 24-mo-old hearts sustained less cardiac injury following ischemia-reperfusion. These results support that age-induced ER stress augments cardiac injury during ischemia-reperfusion by impairing mitochondrial function through activation of CPN1.

INTRODUCTION

Coronary artery disease occurs more frequently in elderly patients (1–3). The severity of ischemia-reperfusion injury is markedly increased in both elderly patients and aged animals (2, 4). The increased cardiac injury in aged hearts is due to mitochondrial dysfunction that occurs in the baseline condition (5). Aging-induced mitochondrial dysfunction leads to decreased oxidative phosphorylation (6–9), increased reactive oxygen species (ROS) production (10), and sensitizes to opening of mitochondrial permeability transition pore (MPTP) (11, 12). These factors contribute to increased cardiac injury in aged hearts during ischemia-reperfusion (6, 13–15).

There are two populations of cardiac mitochondria: subsarcolemmal mitochondria (SSM) are located underneath the plasma membrane and interfibrillar mitochondria (IFM) exist between the myofibrils (6, 16). Aging impairs the mitochondrial electron transport chain (ETC), especially in IFM in aged hearts (6, 17). In aged mice (24 mo), the rate of oxidative phosphorylation is decreased in both SSM and IFM (11, 18). Mechanisms of mitochondrial defects with aging include the oxidative stress (8, 19), mutations and deletion of mitochondrial DNA (20), and decreased mitophagy (21, 22). We and others found that chronically increased endoplasmic reticulum (ER) stress also contributes to mitochondrial dysfunction during aging (11, 18, 23, 24). The ER contributes a pivotal role in the regulation of protein folding, lipid synthesis, and calcium homeostasis (25). Although an initial ER stress response represents an adaptation to restore ER function by decreasing the rate of protein synthesis, prolonged ER stress causes mitochondrial dysfunction and cell injury (26). The increased ER stress occurs earlier than mitochondrial dysfunction in aged hearts, and attenuation of the ER stress improves mitochondrial function in aged hearts (11, 18). These results support that ER stress is a major cause of the mitochondrial dysfunction during aging. However, the mechanisms whereby the increased ER stress damages mitochondria during aging remain unknown.

Calpain 1 (CPN1) and calpain 2 (CPN2) are calcium-sensitive cysteine proteases that exist in both cytosol and mitochondria (27–30). CPN1 and CPN2 (CPN1/2) include a large catalytic subunit and a small regulatory subunit (CPNS1) (31, 32). Genetic removal of CPNS1 eliminates the activities of CPN1 and 2 (31, 32). Thus, CPNS1 knockout mice are used to prevent the activation of CPN1 and 2 (31, 32). Exposure of the purified cardiac mitochondria without cytosolic calpain contamination from C57BL/6 mice to exogenous calcium (25 µM) decreases oxidative phosphorylation. In contrast, calcium treatment does not inhibit oxidative phosphorylation in mitochondria isolated from CPNS1 knockout mice (33). These results provide direct evidence that activation of mitochondrial CPN1 (mCPN1) leads to mitochondrial dysfunction. ER stress increases the intracellular calcium level by disrupting calcium homeostasis (25). The induction of acute ER stress using thapsigargin activates CPN1 and 2 in adult mice (33). ER stress is chronically increased in aged hearts (11). The intracellular calcium level is also increased in aged cardiac myocytes (34) that favors CPN1 and 2 activation. Thus, we propose that chronically increased ER stress during aging contributes to mitochondrial dysfunction by activating the mCPN1.

4-Phenylbutyrate (4-PBA) is a chemical chaperone that is commonly used to decrease ER stress by stabilizing protein conformation within the ER (35–37). Chronic 4-PBA treatment for 2 wk decreases the ER stress and improves mitochondrial function in the aged heart in the baseline condition (11). Thus, 4-PBA was used to study the role of ER stress in mCPN1 activation during aging. Next, the contribution of direct inhibition of mCPN1 to improve mitochondrial function in aged hearts was studied. In the present study, we asked: 1) would attenuation of the ER stress using 4-PBA decrease mCPN1 activation in aged hearts; 2) would attenuation of the ER stress using 4-PBA that improves mitochondrial function in aged hearts in the baseline condition lead to decreased cardiac injury during ischemia-reperfusion; 3) would direct inhibition of CPN1 improve mitochondrial function in aged hearts; 4) would direct inhibition of CPN1 in the aged heart at baseline lead to decreased cardiac injury during subsequent ischemia-reperfusion. The results from the current study support mCPN1 as a key effector of ER stress-mediated mitochondrial dysfunction in the aging heart and provide direction to develop additional strategies to improve mitochondrial function in aged hearts as an intervention to mitigate the age-enhanced cardiac injury that occurs during ischemia and reperfusion (2, 4).

METHODS

Treatment of Aged Mice with 4-Phenylbutyrate or MDL-28170

The experimental procedures conformed to the Guide for the Care and Use of Laboratory Animals and were approved by the Institutional Animal Care and Use Committees of Virginia Commonwealth University (VCU) and the McGuire Department of Veterans Affairs Medical Center. All methods were performed in accordance with the ARRIVE (Animal Research: Reporting of In Vivo Experiments) guidelines. Young (3 mo) and old aged (24 mo) C57BL/6 male mice were provided by National Institutes of Aging colony Charles River Laboratories (Wilmington, MA).

4-PBA (1 g/kg/day) was dissolved in drinking water and fed to 3-mo- and 24-mo-old mice for 2 wk (35) (Fig. 1). Mice were continually supplied with regular drinking water in the vehicle group. Then, mice were used either for mitochondrial isolation or cardiac ischemia-reperfusion study. MDL-28170 (MDL) inhibits both CPN1 and CPN2, but CPN1 is more sensitive to MDL inhibition compared with CPN2 (38, 39). Thus, MDL was used in the current study. MDL (50 mg/kg) was first dissolved in DMSO (50 µL) and then diluted with saline. MDL was administered to 24-mo-old mice through intraperitoneal injection once a day for 2 days. DMSO (50 µL) was used as vehicle treatment and administered to 24-mo-old mice in the same way as MDL. Then, the mice were used either for mitochondrial isolation or ischemia-reperfusion studies (Fig. 1).

Figure 1.

Depiction of experimental protocol. A: the 4-phenylbutyrate (4-PBA) treatment protocol. The 4-PBA (1 g/kg/day) was dissolved in drinking water administered to 3- or 24-mo-old mice for 2 wk. Then, mice were used for direct mitochondrial isolation or ischemia-reperfusion studies. There was no additional 4-PBA treatment during ischemia-reperfusion. B: the MDL-28170 (MDL) treatment protocol. MDL (50 mg/kg) or DMSO (vehicle) was administered to 24-mo-old mice through intraperitoneal injection once a day for 2 days. Then, mice were used for direct mitochondrial isolation or the study of ischemia-reperfusion. There was no additional MDL treatment during ischemia-reperfusion.

Isolation of Cardiac Mitochondria from Mouse Hearts

Mice were anesthetized with pentobarbital (100 mg/kg ip) and the heart was excised for mitochondrial isolation under deep anesthesia. SSM and IFM were isolated using the previously published protocol (11, 40). Briefly, the heart was washed with isolation buffer A [100 mM KCl, 50 mM 3-(N-morpholino) propanesulfonic acid (MOPS), 1 mM EGTA, 5 mM MgSO₄ 7H₂O, 1 mM ATP, and 0.2% bovine serum albumin, pH 7.4] at 4°C. Then, heart tissue was minced and re-suspended in buffer B (buffer A + 0.2% bovine serum albumin) and homogenized with a polytron tissue processor (Brinkman Instruments, Westbury, NY) for 2.5 s at the setting of 10,000 rpm. The polytron homogenate was first centrifuged at 500 g for 10 min to separate supernatant and pellet. The supernatant was collected and centrifuged at 3,000 g for 10 min to sediment SSM. The pellet from the polytron homogenate was used to isolate IFM. The pellet was resuspended in mitochondrial isolation buffer A and homogenized again and incubated with trypsin (5 mg/g, wet wt) for 10 min at 4°C with constant stirring. The homogenate was centrifuged at 500 g for 10 min, and the supernatant was collected and further centrifuged at 3,000 g for 10 min to sediment IFM. SSM and IFM were washed and suspended in KME (100 mM KCl, 50 mM MOPS, and 0.5 mM EGTA) for functional study (41).

Measurement of Oxidative Phosphorylation

The rate of oxidative phosphorylation was measured using a Clark-type oxygen electrode at 30°C as previously described (41). Mitochondria were incubated in oxidative phosphorylation buffer (80 mM KCl, 50 mM MOPS, 1 mM EGTA, 5 mM KH₂PO₄, and 1 mg defatted, dialyzed bovine serum albumin/mL at pH 7.4). Glutamate (20 mM, complex I substrate) and malate (5 mM, complex I substrate), succinate (20 mM, complex II substrate), and TMPD (N,N,N′,N′ tetramethyl p-phenylenediamine, 1 mM)-ascorbate (10 mM, complex IV substrate) were used. Rotenone (7.5 µM) was used to block potential reverse electron flow when succinate and TMPD were used as substrates (41). ADP was used to stimulate oxidative phosphorylation in the isolated mitochondria (41). Dinitrophenol (DNP, 0.3 mM) was used as an uncoupler to assess respiration independent of complex V in the isolated mitochondria (41).

Measurement of Calcium Retention Capacity in Isolated Mitochondria

The calcium retention capacity (CRC) is used to reflect the sensitivity of calcium-induced MPTP opening in isolated mitochondria (42). Mitochondria (250 μg/mL) were incubated in buffer containing 150 mM sucrose, 50 mM KCl, 2 mM KH₂PO₄, 5 mM succinate in 20 mM Tris/HCl, pH 7.4. Calcium (5 nmol/pulse) was sequentially added into the medium to induce MPTP opening. Succinate was used as the substrate for CRC measurement such that the CRC value is higher in mitochondria oxidizing a complex II substrate compared with a complex I substrate (43, 44). Extra mitochondrial Ca²⁺ concentration was recorded with 0.5 µM Calcium Green-5N and fluorescence monitored with excitation and emission wavelengths set at 500 and 530 nm, respectively (42).

Isolated Heart Model of Ischemia-Reperfusion

Mice were anesthetized with pentobarbital sodium (100 mg/kg ip) and anticoagulated with heparin (1,000 IU/kg ip). Hearts were harvested and perfused retrograde via the aorta in the Langendorff mode with modified Krebs–Henseleit (K-H) buffer oxygenated with 95% O₂-5% CO₂. Cardiac function was monitored with a balloon inserted into the left ventricle and recorded and analyzed with ADInstruments (Colorado Springs, CO) (45). The heart was perfused for 15 min with K-H buffer followed by 25 min of global ischemia at 37°C and 30 min of reperfusion. Hearts were paced at 420 beats/min during the 15-min equilibration period and after 10 min of reperfusion (27). Coronary effluent was collected during the entire reperfusion phase, and LDH activity in the coronary effluent was measured (45). Myocardial infarct size was determined using staining with triphenyl tetrazolium chloride (45). In both 4-PBA- and MDL-treated hearts, 4-PBA or MDL was not included in the perfusion buffer to exclude the direct inhibition by 4-PBA or MDL in isolated hearts.

Immunoblotting

Mitochondrial proteins were separated using 7.5%, 12%, or 4%–15% Tris-glycine gels (Bio-Rad, Hercules, CA) based on the molecular weight of the targeted proteins. The separated proteins were transferred to a PVDF membrane (Fisher Scientific, Hampton, NH) using semi-dry transfer (Bio-Rad, Hercules, CA). The PVDF membrane was cut to be used for blotting targeted protein and loading control, respectively. The blots were incubated for 1 h at room temperature in 5% (wt/vol) nonfat dry milk in Tris-buffered saline-Tween 20 (TBST) buffer (10 mM Tris pH 7.5, 150 mM NaCl, 0.1% Tween 20) followed by an overnight incubation at 4°C with primary antibodies (Table 1). The membrane was washed using TBST buffer for three times to remove unattached primary antibodies (27, 33, 38, 39, 46–51). Then, the membrane was incubated with the secondary antibody (27, 33, 38, 39, 46). After 1-h incubation at room temperature with a 1:10,000 dilution of HRP-conjugated anti-mouse or anti-rabbit IgG F(ab)₂ (GE Healthcare Life Sciences, Piscataway, NJ), blots were developed using ECL Plus Western Blotting Detection Reagents (GE Healthcare Life Sciences, Piscataway, NJ). Membranes were digitally analyzed using the Chemidoc Touch Imaging System (Bio-Rad, Hercules, CA).

Table 1.

Antibodies used in the current manuscript

Primary Antibodies				Secondary Antibodies
Antibody Name	Company	Catalog Number	Concentration	Antibody Name	Company	Catalog Number	Concentration
AIF	Cell signaling	4642	1:1,000	anti-Rabbit IgG	Fisher Scientific	45000683	1:5,000
GAPDH	Cell signaling	5174	1:1,000	anti-Rabbit IgG	Fisher Scientific	45000683	1:5,000
PDH α1	Cell signaling	2784	1:1,000	anti-Rabbit IgG	Fisher Scientific	45000683	1:5,000
Spectrin	Santa Cruz	sc-46696	1:100	anti-Mouse IgG	Fisher Scientific	45000679	1:5,000
VDAC	Abcam	ab14715	1:2,500	anti-Rabbit IgG	Fisher Scientific	45000683	1:5,000

AIF, apoptosis inducing factor; PDH, pyruvate dehydrogenase.

Statistical Analysis

Data are expressed as means ± standard error (52). Differences between groups (≥3 groups) were compared by one-way ANOVA after passing a normality test. When a significant F value was obtained, means were compared using the Student–Newman–Keuls test of multiple comparisons. Differences between two groups were compared by unpaired Student t test (SigmaStat 3.5, Systat, Richmond, CA). Statistical significance was defined as a value of P < 0.05.

RESULTS

Aging Activated Cytosolic and Mitochondrial CPN1/2

Spectrin is a substrate of cytosolic CPN1 and 2 (53, 54). A decrease in spectrin content or an increase in cleaved spectrin content is used as the indication of cytosolic CPN activation (38, 53, 54). The content of spectrin was decreased in 24-mo-old hearts compared with 3-mo-old hearts (Fig. 2A), indicating that the cytosolic CPN1 and/or 2 was activated during aging.

Figure 2.

Aging activates cytosolic calpain (CPN)1 and 2 and mitochondrial (m)CPN1. Cytosol and mitochondria were isolated to detect cytosolic and mitochondrial calpain activation in the baseline condition in 3- or 24-mo-old mice. Aging led to decreased spectrin content in cytosol compared with 3-mo-old mice (A), indicating that cytosolic CPN1 and 2 were activated during aging. The content of mature apoptosis inducing factor (mAIF, 62 kDa) was decreased in 24-mo-old IFM (B) compared with 3 mo old mice. The truncated AIF (tAIF, 57 kDa) was also found in 24-mo-old IFM (B). Cytosolic tAIF content was increased in 24-mo-old mice compared with 3-mo-old mice (C). Compared with 3 mo old, the content of the pyruvate dehydrogenase (PDH) α1 subunit was also decreased in 24-mo-old SSM (D) and IFM (E). These results indicate that aging increased the activation of cytosolic CPN1 and 2 and mCPN1. Male mice were used in the current study. Means ± SE. Unpaired Student t test was used for statistical analysis. *P < 0.05 vs. 3 mo old.

Apoptosis inducing factor (AIF) is a substrate of mCPN1 (28). AIF is a nuclear-encoded chaperone protein that is imported into the mitochondrial matrix as a 67 kDa precursor. Mature AIF (mAIF, 62 kDa) is formed after the removal of the mitochondrial leader sequence. The mAIF is anchored on inner mitochondrial membrane (55). Soluble AIF (truncated AIF, tAIF, 57 kDa) is formed after proteolysis of the membrane-bound mAIF by activated mCPN1. The tAIF is located in the mitochondrial intermembrane space (56), and is released from mitochondria into cytosol to trigger caspase-independent apoptosis when the permeability of the mitochondrial outer membrane is increased (28, 39). Compared with 3 mo, the mAIF content was decreased in 24-mo-old IFM (Fig. 2B). A decrease in mAIF contents should lead to increased tAIF contents. Therefore, activation of mCPN1 in 24 mo should lead to decreased mAIF content and increased tAIF content. However, the t-AIF may be further cleaved in 24 mo and its contents were not increased in 24 mo (Fig. 2B). This suggests that tAIF may be released into cytosol. The tAIF content was increased in cytosol in 24-mo-old hearts (Fig. 2C). Activation of the mCPN1 also cleaves the α1 subunit of pyruvate dehydrogenase (PDH) (39). Compared with 3 mo, the content of the α1 subunit of PDH was decreased in 24-mo-old SSM and IFM (Fig. 2, D and E). These results support that aging activates both cytosolic CPN1 and 2 and mCPN1.

4-PBA Treatment Decreased CPN1 Activation in Aged Hearts

The 4-PBA treatment did not alter spectrin content in 3-mo-old mice compared with vehicle treatment (Fig. 3A). However, 4-PBA treatment preserved spectrin content in 24-mo-old mice compared with vehicle (Fig. 3A). The 4-PBA treatment leads to decreased UPR in hearts following ischemia-reperfusion (57). The 4-PBA treatment decreases tunicamycin-induced ER stress accompanied by improved mitochondrial function in rat hearts (58). Our previous study showed that the 4-PBA treatment leads to decreased ER stress in aged hearts (11). These results support that attenuation of the ER stress using 4-PBA decreased cytosolic CPN1 and 2 activation in aged hearts. GAPDH was used as the protein loading control (Fig. 3A).

Figure 3.

The 4-phenylbutyrate (4-PBA) treatment inhibited activation of cytosolic calpain (Ccpn)1 and 2 and mitochondrial (m)CPN1. Male mice (3 or 24 mo old) received 4-PBA or vehicle treatment for 2 wk. Cytosol and mitochondria were isolated to detect cytosolic and mitochondrial calpain activation in the baseline condition. The 4-PBA treatment did not alter spectrin or mature apoptosis inducing factor (mAIF) content in 3-mo-old mice. The 4-PBA treatment increased the content of spectrin in aged hearts (A) compared with vehicle, supporting that 4-PBA treatment to decrease endoplasmic reticulum (ER) stress attenuated the activation of cytosolic CPN1 and 2. The 4-PBA treatment increased the content of mAIF in aged subsarcolemmal mitochondria (SSM) compared with vehicle (B). The 4-PBA treatment also increased the mAIF content in aged interfibrillar mitochondria (IFM) (C). These results indicate that 4-PBA treatment decreased the activation of mCPN1. Means ± SE. One-way ANOVA was used for statistical analysis. *P < 0.05 vs. 3 mo old. †P < 0.05 vs. 24 mo old vehicle.

The 4-PBA treatment did not alter the mAIF content in 3-mo-old SSM (Fig. 3B). However, 4-PBA treatment increased mAIF content in 24-mo-SSM compared with vehicle treatment (Fig. 3C). The mAIF content was decreased in 24-mo-old IFM compared with 3 mo. (Fig. 3C). The 4-PBA treatment increased the mAIF content in 24-mo-old IFM compared with vehicle treatment (Fig. 3C). VDAC was used as the protein loading control (Fig. 3, B and C). In addition, 4-PBA treatment also increased the content of the α1 subunit of PDH in 24-mo-old SSM (Fig. 4A) and IFM (Fig. 4B) compared with vehicle. These results indicate that attenuation of the ER stress with 4-PBA treatment leads to decreased activation of the mCPN1.

Figure 4.

The 4-phenylbutyrate (4-PBA) treatment decreased the degradation of pyruvate dehydrogenase (PDH) α1 subunit. The PDH α1 subunit is a substrate of mitochondrial calpain 1 (mCPN1). Male mice (3 or 24 mo old) received 4-PBA or vehicle treatment for 2 wk. The 4-PBA treatment did not alter PDH α1 subunit content in 3-mo-old mice. The 4-PBA treatment increased the content of PDH α1 subunit content in aged subsarcolemmal mitochondria (SSM; A) compared with vehicle. The 4-PBA treatment also increased the contents of PDH α1 subunit content in aged interfibrillar mitochondria (IFM) compared with vehicle (B). These results provide further evidence that attenuation of the endoplasmic reticulum (ER) stress leads to decreased mCPN1 activation during aging. Means ± SE. One-way ANOVA was used for statistical analysis. *P < 0.05 vs. 3 mo old. †P < 0.05 vs. 24 mo old vehicle. n = 4 in each group.

4-PBA Treatment Decreased the Sensitivity of MPTP Opening in Aged SSM and IFM

The CRC is a reliable indicator of sensitization to MPTP opening in isolated heart mitochondria (59). The CRC was decreased in both 24-mo-old SSM and IFM compared with 3 mo (Fig. 5, A and B), indicating that aging sensitizes to MPTP opening (11, 12). The 4-PBA treatment increased the CRC in 3-mo-old SSM compared with vehicle treatment (Fig. 5A), whereas the CRC in 3-mo-old. IFM was not affected by the 4-PBA treatment (Fig. 5B). The 4-PBA treatment increased the CRC in both SSM and IFM from 24-mo-old mice compared with vehicle treatment (Fig. 5, A and B), suggesting that attenuation of the ER stress with 4-PBA decreased the susceptibility to MPTP opening in aged hearts.

Figure 5.

The 4-phenylbutyrate (4-PBA) treatment decreases the susceptibility to mitochondrial permeability transition pore (MPTP) opening in aged subsarcolemmal mitochondria (SSM) and interfibrillar mitochondria (IFM). Calcium retention capacity (CRC) was used to reflect MPTP opening. A decrease in the CRC indicated the increased sensitivity to MPTP opening. The CRC was decreased in 24-mo-old SSM (A) and IFM (B) compared with 3-mo-old SSM, suggesting that aging sensitizes to MPTP opening. Treatment with 4-PBA (4-phenylbutyric acid) improved the CRC in 3-mo-old SSM (A) but not in IFM (B) compared with untreated 3-mo-old mice. The 4-PBA treatment also improved the CRC in 24-mo-old SSM (A) and IFM (B) compared with untreated 24-mo-old mice. These results support that attenuation of the ER stress decreases the susceptibility to MPTP opening during aging. Male mice were used in the current study. Means ± SE. One-way ANOVA was used for statistical analysis. *P < 0.05 vs. 3-mo-old vehicle, †P < 0.05 vs. 24-mo-old vehicle. n = 10 in 3-mo-old vehicle, 24-mo-old vehicle, and 24 mo old + 4-PBA groups. n = 8 in 3 mo old + 4-PBA group.

4-PBA Treatment Decreased Cardiac Injury in Aged Hearts during Ischemia-Reperfusion

There were no differences in left ventricular developed pressure (LVDP mmHg) (Fig. 6A) or end diastolic pressure (LVEDP mmHg) (Fig. 6B) between vehicle and 4-PBA-treated 24-mo-old hearts before ischemia. Ischemia-reperfusion led to decreased LVDP in both vehicle and 4-PBA-treated hearts compared with the pre-ischemic value (Fig. 6A). Ischemia led to increased LVEDP in both vehicle and 4-PBA-treated hearts compared with the pre-ischemic value (Fig. 6B). The LVEDP remained elevated during reperfusion in both vehicle and 4-PBA-treated hearts compared with before ischemia (Fig. 6B). However, the extent of the increase in LVEDP in the 4-PBA-treated hearts during reperfusion was less than that in vehicle-treated hearts (Fig. 6B). The 4-PBA treatment decreased the infarct size in 24-mo-old hearts compared with vehicle (Fig. 6C). Compared with vehicle treatment, 4-PBA treatment in the baseline condition led to decreased LDH release (mU/mg) into coronary effluent during the 60-min reperfusion period following 25 min of in vitro global ischemia in 24-mo-old hearts [means ± SE, 183 ± 28 (vehicle, n = 8) vs. 101 ± 15* (4-PBA, n = 10), *P < 0.05 vs. vehicle]. These results showed that 4-PBA treatment to decrease age-induced ER stress in the baseline condition before ischemia led to decreased cardiac injury in aged hearts following in vitro ischemia-reperfusion. Treatment of the aging defect in mitochondria present at baseline by treatment to decrease ER stress led to a reduction in cardiac injury from subsequent ischemia-reperfusion injury in the aged heart.

Figure 6.

The 4-phenylbutyrate (4-PBA) treatment in the baseline condition decreased cardiac injury in aged mouse hearts following ischemia-reperfusion. There were no differences in left ventricular developed pressure (LVDP, mmHg) between vehicle and 4-PBA-treated 24-mo-old hearts before ischemia (A). Ischemia-reperfusion markedly decreased the left ventricular developed pressure (LVDP, mmHg) in both vehicle and 4-PBA-treated hearts compared with before ischemia. The 4-PBA treatment did not improve the LVDP during reperfusion compared with vehicle treatment (A). There were no differences in left ventricular end-diastolic pressure (LVEDP, mmHg) between vehicle and 4-PBA-treated hearts before ischemia (B). Ischemia-reperfusion increased the LVEDP in both vehicle and 4-PBA-treated hearts compared with the pre-ischemic value. The 4-PBA treatment attenuated the increase in LVEDP during reperfusion compared with vehicle (B), indicating that 4-PBA treatment to decrease endoplasmic reticulum (ER) stress before ischemia improved diastolic relaxation in aged hearts during reperfusion. The 4-PBA treatment administered only in vivo decreased infarct size in aged hearts during reperfusion, supporting that the attenuation of ER stress to improve mitochondrial function before the onset of ischemia decreased cardiac injury in aged hearts during the subsequent in vitro ischemia and reperfusion. Male mice were used in the current study. Means ± SE. One-way ANOVA was used for statistical analysis for A and B. Unpaired Student t test was used for statistical analysis for C. *P < 0.05 vs. pre-ischemia. †P < 0.05 vs. 24 mo old vehicle.

MDL Treatment Improved Mitochondrial Function in Aged SSM and IFM

MDL was used to inhibit CPN1 and 2 in aged hearts in the baseline condition. The effect of MDL treatment on oxidative function in 24-mo-old SSM and IFM was assessed in the baseline state before ischemia-reperfusion. Compared with vehicle treatment, MDL improved the rate of ADP-stimulated oxidative phosphorylation in SSM and IFM when glutamate + malate was used as complex I substrate (Table 2). MDL treatment also improved the dinitrophenol (DNP)-uncoupled respiration in SSM and IFM using complex I substrates (Table 2). However, in vivo MDL did not improve the rate of respiration in SSM and IFM when succinate + rotenone or TMPD-ascorbate was used as complex II and complex IV substrates, respectively (Table 2). There was no difference in the CRC between vehicle and MDL-treated SSM and IFM (Table 2), suggesting that MDL treatment did not alter the sensitivity to MPTP opening in aged SSM and IFM. The rate of oxidative phosphorylation in IFM was higher compared with corresponding SSM (Table 2), consistent with previous findings in rat (16) and rabbit heart mitochondria (41).

Table 2.

MDL treatment improves oxidative phosphorylation in aged SSM and IFM without ischemia-reperfusion

	24-mo-old SSM		24-mo-old IFM
	Vehicle	MDL	Vehicle	MDL
n	11	10	11	10
Complex I substrates (Glutamate + Malate)
State 3, nAO/min/mg	151 ± 7	177 ± 7*	213 ± 6†	240 ± 5*†
State 4, nAO/min/mg	48 ± 6	54 ± 5	60 ± 4	67 ± 5
RCR	3.3 ± 0.3	3.3 ± 0.2	3.6 ± 0.2	3.7 ± 0.3
ADP/O	2.79 ± 0.16	2.88 ± 0.16	2.97 ± 0.14	3.16 ± 0.20
2 mM ADP, nAO/min/mg	148 ± 10	184 ± 11*	261 ± 13†	300 ± 12*†
0.3 mM DNP, nAO/min/mg	146 ± 9	183 ± 10*	258 ± 13†	296 ± 10*†
Complex II substrates (Succinate + Rotenone)
State 3, nAO/min/mg	442 ± 21	459 ± 12	679 ± 12†	687 ± 18†
State 4, nAO/min/mg	125 ± 10	147 ± 4	184 ± 7†	200 ± 5†
RCR	3.7 ± 0.3	3.1 ± 0.1	3.7 ± 0.1	3.4 ± 0.1
ADP/O	1.63 ± 0.10	1.85 ± 0.12	1.73 ± 0.13	1.83 ± 0.13
2 mM ADP, AO/min/mg	397 ± 48	450 ± 28	638 ± 15†	654 ± 23†
0.3 mM DNP, nAO/min/mg	346 ± 18	404 ± 25	565 ± 14†	575 ± 26†
Complex IV substrates (TMPD-ascorbate + Rotenone)
2 mM ADP, nAO/min/mg	1,365 ± 58	1,378 ± 94	1,928 ± 88†	2,052 ± 68†
24-mo-old SSM: CRC to reflect MPTP opening
CRC, nmol Ca2+/mg	451 ± 29	509 ± 29	789 ± 34†	869 ± 51†

Means ± SE. CRC, calcium retention capacity (nmol calcium/mg protein); DNP, dinitrophenol; IFM, interfibrillar mitochondria; MDL, MDL-28170; MPTP, mitochondrial permeability transition pore; RCR, respiratory control ratio (state3/state 4); SSM, subsarcolemmal mitochondria; TMPD, N,N,N′,N′-tetramethyl-p-phenylenediamine.

^*P < 0.05 vs. corresponding vehicle treatment; †P < 0.05 vs. corresponding SSM.

MDL Treatment Inhibited mCPN1 in Aged SSM and IFM in the Baseline Condition

SSM and IFM were isolated from 24-mo-old mice with or without MDL treatment. The contents of mAIF were increased in SSM from MDL-treated mice compared with vehicle treatment (Fig. 7A). MDL treatment also increased the contents of mAIF in IFM from 24-mo-old mice (Fig. 7B). MDL treatment also increased the content of the PDH α1 subunit in 24-mo-old SSM compared with vehicle (Fig. 7, C and D). VDAC was used as the protein loading control in both SSM and IFM (Fig. 7, A–D). These results indicate that the in vivo MDL treatment regimen used leads to decreased mCPN1 activation.

Figure 7.

MDL-28170 (MDL) treatment decreased mitochondrial calpain 1 (mCPN1) activation in aged heart mitochondria. MDL or vehicle was administered to 24-mo-old male mice for 2 days. Subsarcolemmal mitochondria (SSM) and interfibrillar mitochondria (IFM) were isolated from mouse hearts in the baseline state. Compared with vehicle, the contents of mature apoptosis inducing factor (mAIF) were increased in 24-mo-old SSM with MDL treatment (A). MDL treatment also increased the contents of mAIF in aged IFM compared with vehicle (B). MDL treatment also increased the pyruvate dehydrogenase (PDH) α1 content in aged SSM (C). MDL treatment also tended to increase the PDH α1 content in aged IFM (D). These results show that MDL treatment decreased the activation of mCPN1 in aged mice in the baseline condition. Means ± SE. Unpaired Student t test was used for statistical analysis. †P < 0.05 vs. 24-mo-old vehicle. AIF study, n = 5 in each group. PDH study, n = 4 in each group.

MDL Treatment Decreased Cardiac Injury in Aged Hearts following Ischemia-Reperfusion

Compared with vehicle treatment, MDL administered in vivo only in the baseline condition did not improve cardiac function during the 30-min reperfusion period following 25 min of in vitro global ischemia in 24-mo-old hearts (Table 3). Compared with vehicle treatment, MDL administered in vivo only in the baseline condition led to decreased LDH release (mU/mg) into coronary effluent during the 30-min reperfusion period following 25 min of in vitro global ischemia in 24-mo-old hearts [means ± SE, 286 ± 6 (vehicle, n = 11) vs. 146 ± 6* (MDL, n = 10), *P < 0.05 vs. vehicle]. This result indicates that MDL treatment decreased cardiac injury in 24-mo-old hearts during ischemia-reperfusion, likely by improving mitochondrial function in the baseline condition before ischemia.

Table 3.

Hemodynamic data in 24-mo-old hearts with or without MDL treatment

	n	Pre-Ischemia		End of Ischemia	End of Reperfusion
		LVDP, mmHg	LVEDP, mmHg	LVEDP, mmHg	LVDP, mmHg	LVEDP, mmHg
Vehicle	11	70 ± 6	9 ± 2	25 ± 3	33 ± 6	25 ± 4
MDL	10	60 ± 8	7 ± 2	26 ± 6	33 ± 4	16 ± 4

Means ± SE. P = not significant vs. vehicle-treated hearts. LVDP, left ventricular developed pressure; LVEDP, left ventricular end-diastolic pressure; MDL, MDL-28170.

MDL Treatment Improved Mitochondrial Function in the Aged Heart following Ischemia-Reperfusion

SSM and IFM were isolated from 24-mo-old hearts following ischemia-reperfusion. MDL was only administered to 24-mo-old mice in vivo, and not during the ex vivo ischemia-reperfusion. There was no direct MDL treatment during ischemia-reperfusion. Compared with vehicle, MDL treatment did not improve the respiration stimulated by ADP or DNP following-ischemia and reperfusion in 24-mo-old SSM and IFM using glutamate + malate or succinate with rotenone as complex I and complex II substrates, respectively (Table 4). However, MDL did improve respiration when TMPD-ascorbate was used as complex IV substrate in SSM and IFM (Table 4). These results indicate that inhibition of CPN1/2 using MDL in the baseline condition attenuates the damage in the distal part of the electron transport chain during ischemia and reperfusion in aged SSM and IFM. MDL treatment increased the CRC in both SSM and IFM (Table 4) compared with vehicle treatment, indicating that MDL treatment decreased the sensitivity to MPTP opening in aged SSM and IFM following ischemia-reperfusion. These results suggest that inhibition of CPN1/2 using MDL decreases cardiac injury in aged hearts during ischemia-reperfusion in part by inhibiting MPTP opening. The rate of oxidative phosphorylation in IFM was also higher compared with corresponding SSM following ischemia-reperfusion (Table 4). The different biochemical properties between SSM and IFM may lead to different susceptibility to ischemia-reperfusion injury (6).

Table 4.

MDL treatment improves oxidative phosphorylation in aged SSM and IFM following ischemia-reperfusion

	24 Mo Aged SSM		24 Mo Aged IFM
	Vehicle	MDL	Vehicle	MDL
n	11	10	11	10
Complex I substrates (Glutamate + Malate)
State 3, nAO/min/mg	116 ± 14	116 ± 11	187 ± 15†	206 ± 20†
State 4, nAO/min/mg	49 ± 2	54 ± 2	76 ± 4†	80 ± 6†
RCR	2.3 ± 0.2	2.1 ± 0.1	2.5 ± 0.1	2.6 ± 0.1
ADP/O	2.55 ± 0.07	2.46 ± 0.05	2.64 ± 0.07	2.64 ± 0.03
2 mM ADP, nAO/min/mg	119 ± 17	123 ± 14	198 ± 22†	227 ± 26†
0.3 mM DNP, nAO/min/mg	120 ± 16*	123 ± 13	203 ± 22†	224 ± 26†
Complex II substrates (Succinate + Rotenone)
State 3, nAO/min/mg	311 ± 12	320 ± 13	415 ± 26†	472 ± 24*†
State 4, nAO/min/mg	133 ± 5	141 ± 9	166 ± 8†	189 ± 11†
RCR	2.3 ± 0.1	2.3 ± 0.1	2.5 ± 0.1	2.5 ± 0.1
ADP/O	1.41 ± 0.03	1.50 ± 0.05	1.54 ± 0.04	1.59 ± 0.06
2 mM ADP, nAO/min/mg	319 ± 14	338 ± 21	454 ± 26†	541 ± 31†
0.3 mM DNP, nAO/min/mg	288 ± 12	299 ± 21	406 ± 25†	478 ± 26†
Complex IV substrates (TMPD-ascorbate + Rotenone)
2 mM ADP, nAO/min/mg	1097 ± 49	1311 ± 64*	1493 ± 66†	1859 ± 73*†
24-mo-old SSM: CRC to reflect MPTP opening
CRC, nmol Ca2+/mg	229 ± 11	312 ± 16*	411 ± 26†	500 ± 28*†

Means ± SE. CRC, calcium retention capacity (nmol calcium/mg protein); DNP, dinitrophenol; IFM, interfibrillar mitochondria; MDL, MDL-28170; MPTP, mitochondrial permeability transition pore; RCR, respiratory control ratio (state3/state 4); SSM, subsarcolemmal mitochondria; TMPD, N,N,N′,N′-tetramethyl-p-phenylenediamine.

^*P < 0.05 vs. vehicle-treated ischemia-reperfusion hearts; †P < 0.05 vs. corresponding SSM.

DISCUSSION

In our previous study, we showed that attenuation of the chronically increased ER stress in aged hearts led to improved mitochondrial function (11, 12). In the present study, we found that attenuation of the ER stress in aged hearts decreased the activation of the cytosolic CPN1 and 2 and mitochondria-localized CPN1. In addition, the direct inhibition of CPN1 and 2 using MDL also reversed mitochondrial defects present in aged heart mitochondria in the baseline state. These results support that the ER stress contributes to mitochondrial defects at least in part by activating CPN1 and 2 during aging. The improvement of mitochondrial function in 24-mo-old mice in the baseline condition with 4-PBA (11) or MDL treatment led to decreased cardiac injury during subsequent ischemia and reperfusion, supporting the notion that aging-induced mitochondrial dysfunction in the baseline condition augments cardiac injury during ischemia reperfusion. Thus, our study identified the critical role of CPN activation in the genesis of the mitochondrial dysfunction that results from age-induced ER stress (Fig. 8). Prevention of CPN1 and 2 activation through attenuation of the ER stress or direct administration of CPN inhibitors is a promising strategy to eliminate mitochondrial defects in aged hearts in the baseline condition (12).

Figure 8.

Schematic depiction of a strategy to improve mitochondrial function in aged hearts. Aging increases the endoplasmic reticulum (ER) stress that activates cytosolic and mitochondrial calpain1 and 2 (CPN1/2). Activation of CPN1/2 impairs the electron transport chain (ETC) and sensitizes the mitochondrial permeability transition pore (MPTP) to opening. Activation of cCPN1/2 may enhance the ER stress by cleaving the SERCA. The damaged ETC (45) and increased susceptibility to MPTP opening (59) augments cardiac injury during ischemia-reperfusion. The 4-phenylbutyrate (4-PBA) treatment improves mitochondrial function by decreasing the activation of CPN1/2 through attenuation of the ER stress. Direct inhibition of CPN1/2 using MDL-28170 (MDL) also improves mitochondrial function in aged hearts by reducing damage in the ETC, demonstrating that activation of mitochondrial and cytosolic calpains is a key mediator of ER-stress induced mitochondrial dysfunction with aging in the heart.

Cytosolic Calpain Activation during Aging

The induction of acute ER stress using thapsigargin leads to CPN1 and 2 activation in adult hearts (33). ER stress is progressively and chronically increased during aging (11). The chronically increased ER stress contributes to mitochondrial dysfunction in aged hearts (11). In the present study, we found that spectrin content was decreased in aged hearts compared with younger hearts, supporting that aging activates cytosolic CPN1 and 2. Our previous study showed that treatment of aged mice with 4-PBA decreased the ER stress (11). Here, we found that the 4-PBA treatment preserved spectrin content, supporting that attenuation of the ER stress decreases cytosolic CPN activation (60). Activation of cytosolic CPN1 and 2 increases cardiac injury during ischemia-reperfusion by cleaving functional proteins including Na⁺, K⁺-ATPase (61), Ca²⁺-ATPase (62), and troponin T (63). Cytosolic CPN1 and 2 activation also leads to cleavage of junctophilin-2 (JPH2) that is a key structural protein integral to excitation and contraction coupling in cardiac myocytes (64). A decrease in JPH2 content impairs contractile function in failing hearts (64). Aging leads to decreased cardiac function, especially diastolic function (65). Thus, prevention of cytosolic CPN1 and 2 activation may improve cardiac function in aged hearts by protecting CPN1- and 2-targeted functional and structural proteins.

Mitochondrial CPN1 Activation in Aged Hearts

The mCPN1 exists in the mitochondrial intermembrane space and matrix compartments (30). The activity of mCPN1 is increased in hearts in pathological conditions including ischemia-reperfusion and endotoxemia (31). AIF is a substrate of the mCPN1 (28). Activation of the mCPN1 cleaves mAIF to tAIF that is released from mitochondria to cytosol and nucleus to induce caspase-independent apoptosis (66). Thus, a decrease in mAIF or an increase in tAIF content is used as a marker of mCPN1 activation (27, 28). In the current study, we found that aging leads to decreased mAIF content compared with young hearts, especially in IFM. An activation of mCPN1 should lead to increased tAIF formation within mitochondria. Although the tAIF was also found in 24-mo-old IFM (55), but its content was not increased in mitochondria. The tAIF is not anchored on mitochondrial inner membrane and can be released into cytosol. We find that cytosolic tAIF is increased in 24-mo-old mice, suggesting that aging increases the formation of tAIF. These results indicate that aging activates mCPN1. In addition, the content of the PDH α1 subunit is also decreased in 24-mo-old SSM and IFM. The PDH α1 subunit is another substrate of mCPN1 (12, 39). These results further support that aging leads to the activation of mCPN1.

CPN1/2 Activation and Mitochondrial Dysfunction

Aging damages the ETC including the presence of decreased activities of complexes III and IV (8, 17, 67). In aged mice, the rate of oxidative phosphorylation is decreased in both SSM and IFM with complex I substrates (11, 68). The 4-PBA treatment improved the respiration in SSM and IFM isolated from aged hearts, supporting the notion that the attenuation of the ER stress reduces the damage to the ETC during aging (11). Recent study shows that chronic metformin treatment also improves mitochondrial function in the aged mouse heart mitochondria by decreasing ER stress (18). Our current study showed that inhibition of the mCPN1 also improved mitochondrial function in aged SSM and IFM. The results clearly support that activation of mCPN1 participates in the genesis of mitochondrial dysfunction during aging.

Activation of the mCPN1 during ischemia-reperfusion impairs mitochondrial function by degrading subunits of complex I and pyruvate dehydrogenase (12, 38, 39). The 4-PBA treatment decreases the mCPN1 activation in aged heart mitochondria (Fig. 3). Thus, attenuation of the ER stress using 4-PBA may improve mitochondrial function in aged hearts by protecting complex I and metabolic enzymes through inhibition of the mCPN1. Although AIF is not a subunit of complex I, AIF contributes a critical role in maintaining complex I activity through its chaperone function to assist complex I subunit assembly (55). Therefore, a decrease of mCPN1 activity using 4-PBA to decrease ER stress or MDL to directly inhibit mCPN1 can improve mitochondrial function in aged hearts by protecting AIF.

A recent study shows that aging leads to decreased mitophagy that is a key process to remove dysfunctional mitochondria (65). Activation of cytosolic CPN1/2 leads to decreased mitophagy in hearts following ischemia and reperfusion (38, 69). The 4-PBA treatment leads to decreased activation of cytosolic CPN1 and 2. Thus, downregulation of calpain activity via treatment with 4-PBA or MDL could improve mitochondrial function in aged hearts by improving mitophagy. Taken together, calpain inhibition may improve mitochondrial function in aged hearts by protecting metabolic enzyme activity, preserving AIF content, and potentially upregulating the mitophagy process to remove age-induced dysfunctional mitochondria.

CPN1/2 Activation May Increase the ER Stress

In the current study, 4-PBA treatment leads to decreased activation of cytosolic CPN1 and 2 and mCPN1 in aged hearts, indicating that calpains are key downstream targets of the ER stress during aging. Interestingly, activation of CPN1 also increases the ER stress in cardiac myocytes (70). Inhibition of SERCA using thapsigargin is commonly used to induce acute ER stress by disrupting calcium homeostasis (26). The content of SERCA is decreased in aged hearts at baseline (34) and in adult hearts following ischemia-reperfusion (62). Inhibition of CPN1 protects SERCA in adult hearts following ischemia-reperfusion (62), indicating that SERCA is a CPN1 substrate. Therefore, an increase in CPN1 activity in aged hearts may in turn enhance the ER stress through degradation of SERCA. Taken together, ER stress and calpain activation may form a positive feedback loop leading to mitochondrial dysfunction during aging (Fig. 8).

Inhibition of CPN1/2 in Aged Hearts in the Baseline Condition Improves Mitochondrial Function during Ischemia-Reperfusion

Mitochondrial dysfunction occurs in aged hearts (6, 11, 12). Ischemia-reperfusion leads to superimposed mitochondrial damage in aged hearts (6). Blockade of electron transport using amobarbital before ischemia leads to improved mitochondrial function from 24-mo-old hearts during reperfusion (71), indicating that mitochondrial damage can still be decreased even in the aged hearts. Since the occurrence of heart attack is hard to predict in the clinical setting, it is a challenge to apply treatments before ischemia. Therefore, a practical approach is to target the age-induced mitochondrial dysfunction present at baseline to decrease subsequent ischemia-reperfusion injury. In the present study, we found that administration of MDL in aged mice in the baseline condition not only led to improved mitochondrial function before ischemia, but also improved respiration in hearts following ischemia-reperfusion. MDL treatment improved the oxidation of TMPD-ascorbate in both SSM and IFM following ischemia-reperfusion. TMPD-ascorbate is a complex IV substrate that donates electrons to cytochrome c → complex IV → oxygen (41). The increased TMPD-ascorbate oxidation indicates that MDL treatment decreases damage at the distal portion of the electron transport chain. MDL treatment led to decreased MPTP opening in SSM and IFM following ischemia-reperfusion, consistent with the notion that activation of calpain 1 increases MPTP opening (38, 72). MPTP opening is considered as a final step to induce cell death during ischemia-reperfusion (73, 74). MDL treatment may decrease cardiac injury during ischemia-reperfusion by decreasing MPTP opening.

Reversal of the Mitochondrial Defects to Decrease Cardiac Injury in Aged Hearts

The impaired mitochondrial function is a key contributor to increased cardiac injury in aged hearts during ischemia-reperfusion (5, 75, 76). Thus, the reversal of the age-related mitochondrial defects has the potential to attenuate cardiac injury from a superimposed cardiac insult. Treatment of aged Fisher 344 rats in vivo 3 h before ischemia with acetylcarnitine improved mitochondrial function and decreased cardiac injury in aged hearts following in vitro ischemia-reperfusion (5). A recent study showed that restoration of mitochondrial function with SS-31 treatment improved cardiac function in aged mice in the baseline condition (10). In the current study, we showed that preservation of mitochondrial function in aged mouse hearts with 4-PBA treatment only in the baseline condition led to decreased cardiac injury during subsequent ischemia-reperfusion. In addition, the direct inhibition of calpain with MDL in the baseline condition also improved mitochondrial function and decreased cardiac injury during ischemia-reperfusion. A decrease in ER stress in the baseline state before ischemia may lead to decreased cell injury during ischemia-reperfusion (36). Although the 4-PBA or MDL was not included in the perfusion buffer, some residual 4-PBA or MDL likely still remains in the heart tissue during ischemia-reperfusion. However, the effect of residual 4-PBA or MDL should not have a major protective effect due to a lower concentration present. These results provide further solid evidence that the mitochondrial dysfunction present in aged hearts at baseline predisposes to cardiac injury during subsequent ischemia-reperfusion.

Limitations

The activities of CPN1 and 2 can be measured using fluorescence indicator approaches. In this method, exogenous calcium is used to stimulate CPN1/2 activation (30). Since the CPN1 and 2 were already activated in aged hearts in the baseline state, additional exogenous calcium stimulation may be not necessary. Therefore, we used the alternative approach to measure the contents of spectrin and AIF as indicators of the activation of CPN1 and 2 in the current study (27–29, 53). We believe that the current results provide sufficient evidence to show that aging leads to the activation of CPN1 and 2. CPN2 has also been found in isolated crude mitochondria (29, 77). Compared with CPN1, CPN2 does not have a mitochondrial leader sequence (78). The location of CPN2 within mitochondria needs to be further studied. Thus, we did not pursue the role of mCPN2 activation in mitochondrial dysfunction in the current study. Since we have limited 24-mo-old mice available, we did not measure the infarct size in MDL-treated hearts. A decrease in LDH release into the coronary effluent supports the concept that inhibition of CPN1/2 in the baseline condition leads to decreased cardiac injury following subsequent ischemia-reperfusion.

Although 4-PBA is commonly used to inhibit ER stress, the 4-PBA also has potential off-target effects including urea secretion and histone deacetylase activity (79). The 4-PBA treatment decreases cardiac injury in Adriamycin-treated hearts. Thus, the 4-PBA may decrease cardiac injury by inhibiting histone deacetylase (79). Recent studies show that circadian (sleep/wake) cycles also affect mitochondrial function by altering calcium dynamics and ROS generation (80–82). However, circadian variation will have only a limited effect in our study in that the light cycle is strictly controlled in our animal facility. All experiments involving animals were performed at a similar time in the morning.

Conclusions

The present study shows that an increase in the ER stress during aging leads to activation of mitochondrial calpains that in turn impairs the mitochondrial electron transport chain. Activation of cytosolic calpains may also enhance the ER stress through degradation of SERCA (34). Restoration of mitochondrial function by decreasing the ER stress or via direct inhibition of CPN1 and 2 decreases cardiac injury during a subsequent episode of ischemia-reperfusion, supporting the concept that mitochondrial defects present in aging hearts in the baseline condition augment cardiac injury during ischemia-reperfusion (5) (Fig. 8). Since the occurrence of a myocardial infarction is difficult to predict in patients, pre-emptive treatment applied in the baseline condition to restore the age-induced defect in mitochondrial function can be a translationally feasible approach to prepare aged patients to mitigate the cardiac injury during an unpredictable occurrence of the ischemia and reperfusion associated with acute myocardial infarction and its treatment.

GRANTS

This work was supported by R21 AG049461 (to Q. Chen) from the National Institute on Aging, the Department of Veterans Affairs Office of Research and Development, Medical Research Service Merit Review Award 2IO1BX001355-01A2 and 2IO1BX001355-09A2 (to Q. Chen and E. J. Lesnefsky), and a Pauley Heart Center Pilot Project from Virginia Commonwealth University (to Q. Chen).

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the authors.

AUTHOR CONTRIBUTIONS

Q.C. and E.J.L. conceived and designed research; Q.C., J.T., and Y.H. performed experiments; Q.C., J.T., and Y.H. analyzed data; Q.C. interpreted results of experiments; Q.C. prepared figures; Q.C. drafted manuscript; Q.C. and E.J.L. edited and revised manuscript; Q.C., J.T., Y.H., and E.J.L. approved final version of manuscript.