Mitochondrial m-calpain opens the mitochondrial permeability transition pore in ischemia–reperfusion

International Journal of Cardiology 197 (2015) 26–32

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Mitochondrial m-calpain opens the mitochondrial permeability transition pore in ischemia–reperfusion Kaori Shintani-Ishida ⁎,1, Ken-ichi Yoshida 1,2 Department of Forensic Medicine, Graduate School of Medicine, the University of Tokyo, Japan

a r t i c l e

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Article history: Received 28 January 2015 Received in revised form 14 May 2015 Accepted 12 June 2015 Available online 15 June 2015 Keywords: Calpain Mitochondria Mitochondrial permeability transition pore Calcium Ischemia–reperfusion injury

a b s t r a c t Background/objectives: : Opening of the mitochondrial permeability transition pore (mPTP) is involved in ischemia–reperfusion injury. Isoforms of Ca2+-activated cysteine proteases, calpains, are implicated in the development of myocardial infarction in ischemia–reperfusion. Growing evidence has revealed the presence of calpains in the mitochondria. We aimed to characterize mitochondrial calpains in the rat heart and to investigate the roles of calpains in mPTP opening after ischemia–reperfusion. Methods and results: : Western blotting analysis showed the expression of μ-calpain, m-calpain and calpain 10 in mitochondria isolated from male Sprague-Dawley rats, but casein zymography detected only m-calpain activity. Subcellular fractionation of mitochondria demonstrated the distribution of m-calpain to the matrix fraction. Addition of N500 μM of Ca2+ to isolated mitochondria induced mitochondrial swelling, reflecting mPTP opening, and calpain activation. Ca2+-induced mitochondrial swelling was inhibited partially by the calpain inhibitor calpeptin. These results support a partial contribution of calpain in the opening of the mPTP. The addition of Ca2+ to the mitochondria induced inactivation of complex I of the electron transport chain, and cleavage of the ND6 complex I subunit, which were inhibited by calpeptin. Mitochondria isolated from rat hearts that underwent 30 min of coronary occlusion followed by 30 min of reperfusion showed activation of mitochondrial calpains, ND6 cleavage, complex I inactivation, and mPTP opening, which were inhibited by pretreatment with calpain inhibitor 1. Conclusions: : We demonstrated for the first time the presence of mitochondrial matrix m-calpain, and its contribution to complex I inactivation and mPTP opening after postischemic reperfusion in the rat heart. © 2015 Elsevier Ireland Ltd. All rights reserved.

1. Introduction The mitochondrial permeability transition pore (mPTP) is a nonspecific pore in the inner mitochondrial membrane that is permeable to small molecules. Opening of the mPTP during ischemia–reperfusion (IR) induces apoptosis through the mitochondrial release of proapoptotic proteins and necrosis through ATP depletion (for a review, see [1]). Inhibitors of mPTP such as cyclosporine A (CsA) [2], sanglifehrin-A [3], and NIM811 [4], limit the size of myocardial infarction (MI) in an ex vivo IR Abbreviations: AIF, apoptosis-inducing factor; AK2, adenylate kinase 2; BSA, bovine serum albumin; CsA, cyclosporine A; DMF, N,N-dimethylformamide; IM, inner membrane; IMS, intermembrane space; IR, ischemia–reperfusion; LAD, left anterior descending coronary artery; MI, myocardial infarction; mPTP, mitochondrial permeability transition pore; NCX,Na+/Ca2+ exchanger;OM, outermembrane;PDH, pyruvatedehydrogenase; SR, sarcoplasmic reticulum; TBS-T, Tris-buffered saline with Tween-20; VDAC, voltage-dependent anion channel. ⁎ Corresponding author at: Department of Forensic Medicine, Graduate School of Medical Science, Kyoto Prefectural University of Medicine, 602-8566 Kyoto, Japan. E-mail address: [email protected] (K. Shintani-Ishida). 1 These authors take responsibility for all aspects of the reliability and freedom from bias of the data presented and their discussed interpretation. 2 Present address: Department of Forensic Medicine, Tokyo Medical University, 1608402 Tokyo, Japan.

http://dx.doi.org/10.1016/j.ijcard.2015.06.010 0167-5273/© 2015 Elsevier Ireland Ltd. All rights reserved.

injury model. mPTP opening is induced physiologically by Ca2+ and is enhanced by mitochondrial Ca2+ overload in IR. The uniporter blocker Ru360, attenuates mitochondrial Ca2+ uptake and prevents IR injury through inhibition of mPTP opening in vitro [5] and ex vivo [6]. Calpains, Ca2+-activated cysteine proteases, are thought to contribute to the development of MI in IR [7]. Ca2+ overload after reperfusion induces the activation of calpains, which renders the membrane fragile through proteolysis of the cytoskeletal protein fodrin [8]. Several calpain inhibitors reduce MI size in IR injury models [9–12]. Among the 15 calpain isoforms, 10 isoforms, including conventional calpain 1 (μ-calpain) and calpain 2 (m-calpain), are expressed in the heart [13]. Although calpains are generally thought to reside in the cytosol, in a few years, growing evidence has supported a role for mitochondrial calpains in mitochondrial dysfunction. In bovine pulmonary artery smooth muscle, the Ca2+ ionophore A23187 induces the activation of μ-calpain in the mitochondrial intermembrane space (IMS), thereby cleaving the Na+/Ca2 + exchanger (NCX) in the mitochondrial inner membrane (IM) [14]. In rat liver mitochondria, μ-calpain induces the truncation and subsequent cytosolic release of apoptosis-inducing factor (AIF) in the mitochondrial IMS, whereas m-calpain cleaves voltage-dependent anion channels (VDACs) in the mitochondrial outer membrane (OM) [15]. In the rat renal mitochondrial matrix,

K. Shintani-Ishida, K. Yoshida / International Journal of Cardiology 197 (2015) 26–32

calpain 10 induces Ca2+-induced mitochondrial swelling (mPTP opening) and inhibition of complex I activity on the electron transport chain (ETC.) through cleavage of complex I subunits (ND6 and NDUFC2) [16]. In heart mitochondria, only one report showed the presence of μ-calpain in the mitochondrial IMS [17]. However, the role of mitochondrial calpains in mPTP opening, mitochondrial dysfunction, and MI development has not been studied in the heart after IR. Recently, we found that enhanced Ca2+ loading in the sarcoplasmic reticulum (SR) blocks cytosolic Ca2 + overload and fodrin proteolysis after IR [18]. However, the SR-loaded Ca2+ was transferred to the mitochondria, resulting in mitochondrial Ca2 + overload, acceleration of mPTP opening, and MI development [18]. These findings led us to hypothesize that mitochondrial calpains are activated through SRmitochondria Ca2+ transfer, and are implicated in mPTP opening and MI development after IR. To test this hypothesis, we characterized mitochondrial calpains and investigated their targets and roles in mPTP opening in rat hearts after IR. 2. Materials and methods This study was performed according to the Guide for the Care and Use of Laboratory Animals (NIH publication 85-23, revised 1996) and was approved by the Institutional Animal Care and Use Committee of the University of Tokyo. The animal experiment protocols are summarized in Fig. S1 (Supplementary Data).

2.1. Isolation and subfractionation of mitochondria All of the procedures were carried out at 4 °C. Cardiac mitochondria were isolated from 8-week-old male Sprague-Dawley rats using a Mitochondria Isolation Kit for Tissue and Cultured Cells (#K288-50; BioVision, Milpitas, CA), and the isolated mitochondria underwent subfractionation according to Ozaki et al. [19]. Briefly, the isolated mitochondria were suspended in 2 volumes of 20 mM of potassium phosphate buffer containing 0.2 mg/ml of bovine serum albumin (BSA; Sigma-Aldrich, St. Louis, MO) at pH 7.4 and allowed to stand at 4 °C for 1 h. The suspension was centrifuged at 3000 ×g for 10 min, and the supernatant was centrifuged at 105,000 ×g for 30 min. The pellet and supernatant were defined as the OM and IMS fractions, respectively. The 3000 ×g pellet was resuspended in potassium phosphate buffer, sonicated (15 s, 4 times), and centrifuged at 77,000 ×g for 60 min. The pellet and supernatant were defined as the IM and matrix fractions, respectively. The mitochondria and their subfractions were used immediately for assays for both mitochondrial swelling and Complex I activity. The residual samples were stored at −80 °C until use for other assays. Protein concentrations of these fractions were determined with a Coomassie Protein Assay Kit (Thermo Fisher Scientific, Waltham, MA) after solubilization with 1 M NaOH. Human mitochondria were prepared from post-mortem heart tissue collected in two forensic autopsies performed at Kyoto Prefectural University: a 21-year-old woman at 29 h after death (case 1) and a 48-yearold woman at 40 h after death (case 2). The use of autopsy materials was approved by the Ethical Review Board of Kyoto Prefectural University. Mitochondrial isolation was performed immediately after heart tissue collection. 2.2. Percoll/OptiPrep density gradients The isolated mitochondria were layered over a combination gradient of 6% (v/v) Percoll® (Santa Cruz Biotechnology, Santa Cruz, CA) and 17% and 35% (v/v) OptiPrepTM (Axis-Shield Poc AS, Oslo, Norway) according to Ogbi et al. [20]. After centrifugation at 50,000 ×g for 30 min, the gradient was fractionated into 12 tubes.

27

2.3. Western blot analysis The mitochondria and their subfractions were solubilized in Laemmli sample buffer and subjected to SDS-PAGE on either 12.5% gels for the detection of voltage-dependent ion channel 1 (VDAC1), glyceraldehyde-3phosphate dehydrogenase (GAPDH), and pyruvate dehydrogenase (PDH), 7.5% gels for the detection of AIF, m-calpain, μ-calpain, and calpastatin, or 15% gels for the detection of VDAC1, calpain 10, adenylate kinase 2 (AK2), and ND6. Blots were probed with antibodies specific to AK2, calpastatin (Santa Cruz Biotechnology), VDAC1, calpain 10 (abcam, Tokyo, Japan), PDH, ND6, NDUFV2 (Sigma-Aldrich, St. Louis, MO), AIF (Merck Millipore, Billerica, MA), GAPDH (Applied Biological Materials, Richmond, BC), DAP13 (Aviva System Biology, San Diego, CA), NDUFS7 (GeneTex, Hsinchu City, Taiwan), or m-calpain and μ-calpain (kindly provided by K. Inomata, Tokyo Metropolitan Institute of Gerontology). These primary antibodies were diluted 1000-fold with 1% BSA in Tris-buffered saline with Tween-20 (BSA/TBS-T). Immune complexes were detected with horse radish peroxidase-conjugated goat antibodies to mouse or rabbit IgG (1:5000 dilution with 1% BSA/TBS-T; Promega, Madison, WI) and chemiluminescence reagents (Western Lightning-ECL; PerkinElmer, Waltham, MA). Band intensities were measured using ImageQuant™ LAS 4000 mini (GE Healthcare, Little Chalfont, UK). 2.4. Zymography Casein zymography was performed according to the protocol described by Arthur and Mykles [21]. Briefly, FITC-casein (0.45 mg/ml; AnaSpec, Fremont, CA) was copolymerized with the separating gel containing 10% (w/v) acrylamide (acrylamide:bisacrylamide ratio = 74:1) and 225 mM of Tris–HCl (pH 8.8), and then a stacking gel containing 4.8% acrylamide and 125 mM of Tris–HCl (pH 6.8) was poured. After pre-running of the gel at 125 V for 30 min, mitochondrial lysate (10 μg) was loaded and separated at 125 V for 2 h. The gel was incubated with either 5 mM of CaCl2 or 5 mM of EDTA (for control) in 50 mM Tris– HCl (pH 7.3) containing 10 mM of 2-mercaptoethanol for 30 min. The caseinolytic activity was viewed on a UV transilluminator. The proteins on the Ca2+(+)-zymogram were transferred to a nitrocellulose membrane for Western blot analysis of m-calpain. 2.5. Evaluation of mPTP opening Opening of the mPTP was evaluated by swelling of isolated cardiac mitochondria [18]. The isolated mitochondria (approximately 0.2 mg protein) were preincubated at 30 °C for 10 min in medium containing 110 mM of KCl, 20 mM of MOPS, 10 mM of Tris–HCl, 0.5 μM of rotenone, and 0.5 μM of antimycin A (pH 7.4). To determine Ca2+-induced mitochondrial swelling, CaCl2 was added and absorbance at 520 nm (A520) was monitored for 15 min using a microplate reader (Infinite® 200 PRO; Tecan, Männedorf, Switzerland). 2.6. Calpain activity assay The mitochondria (0.2 mg protein) were incubated with 10 μM of tBOC-LM-CMAC (cell-permeable fluorogenic calpain substrate; Life Technologies Corporation, Carlsbad, CA) at room temperature for 30 min, and then were centrifuged at 10,000 ×g for 15 min to remove the substrate. The mitochondrial pellet was resuspended, and the fluorescence of hydrolyzed CMAC was measured using a microplate reader (Infinite® 200 PRO; Tecan) with excitation at 360 nm and emission at 460 nm. 2.7. Exogenous m-calpain treatment The mitochondria (30 μg protein) were sonicated 3 times for 30 s with an interval of 30 s on ice and incubated with either 1, 5, or 10 μg

28

K. Shintani-Ishida, K. Yoshida / International Journal of Cardiology 197 (2015) 26–32

of rat recombinant m-calpain (Calbiochem®, Merck Millipore, Billerica, MA) and 100 mM of Ca2+ for 30 min at 30 °C.

A

2.8. Assay of complex I enzyme activity Complex I enzyme activity was measured as described previously [22]. Briefly, the mitochondria (0.2 mg protein) were incubated with assay medium containing 25 mM of potassium phosphate (pH 7.2), 5 mM of MgCl2, 2 mM of KCN, 2.5 mg/ml of BSA (fraction V; SigmaAldrich), 130 μM of NADH, 65 μM of ubiquinone and 2 μg/ml of antimycin A for 3 min, and the NADH oxidation was measured at 340 nm with a microplate reader (Infinite® 200 PRO; Tecan). At 3 min after the addition of 2 μg/ml of rotenone, the change in absorbance was measured, and rotenone-sensitive NADH:ubiquinone activity was determined. 2.9. IR model The left anterior descending coronary artery (LAD) of 8-week-old male Sprague-Dawley rats was occluded, and the heart was harvested after demarcation of the risk area by injection of Evans blue dye, as described previously [23]. In separate experiments, a selective calpain inhibitor, calpain inhibitor-1 (total of 0.5 mg ml−1 kg−1 in 10% N,Ndimethylformamide [DMF]) was injected intravenously for 1 min at a constant rate at 10 min before LAD occlusion [9]. The vehicle rats were injected with 10% DMF before LAD occlusion.

kDa

VDAC (mitochondria OM)

32

AIF (mitochondria IM)

67

GAPDH (cytosol)

37

µ-calpain

80

m-calpain

80

calpain-10

B

Zymograph + Ca2+ Ca2+ free

3.1. m-Calpain activity is present in the heart mitochondrial matrix Western blot analysis showed the presence of μ- and m-calpains in the mitochondrial and cytosolic fractions (Fig. 1A). Calpain 10 was detected mainly in the mitochondrial fraction, while its faint band was also observed in the cytosolic fraction (Fig. 1A), consistent with a previous report [16]. The mitochondrial fraction did not contain the cytosolic marker GAPDH, confirming no contamination with the cytosol. FITCcasein zymography showed two bands (μ- and m-calpain) in the cytosolic fraction after incubation with Ca2+, which were not visible in the absence of Ca2+ incubation (Fig. 1B, left vs. middle panel). In the mitochondrial fraction, only the faster migrating band was detected on the zymogram (Fig. 1B, left panel), which was identified subsequently as m-calpain by Western blot analysis (Fig. 1B, right panel). μ-Calpain and calpain 10 were detected in the mitochondrial fraction by Western blot analysis, but the bands reflecting the activity of these calpains were not detected by zymography either with FITC-casein (Fig. 1B) or with a known in vitro mitochondrial calpain substrate SLLVY-AMC [16] (data not shown). The endogenous calpain inhibitor calpastatin was not detected in the mitochondrial fraction (Fig. 1C). In the Percoll/OptiPrep gradient of the isolated mitochondria, the distribution of m-calpain was parallel to that of the mitochondrial marker VDAC, but not that of the SR marker SERCA2a, that of the plasma membrane marker Ncadherin, nor that of the Golgi marker syntaxin 6, as demonstrated by Western blot analysis (Fig. 2). After analysis of the mitochondrial subfractions, m-calpain was found in the matrix fraction, as demonstrated by Western blot analysis (Fig. 3A). We also found an unknown m-calpain-immunoreactive band in the IMS fraction. However, the zymogram showed caseinolytic activity only in the matrix fraction (Fig. 3B), suggesting that this band might be caused by non-specific

S

Western blot m-calpain

m-calpain -

S

C

Mt

calpastatin

S

Mt

S

Mt

kDa

107 Cont

3. Results

Mt

µ-calpain -

2.10. Statistical analysis Quantitative data are presented as means ± SE and were analyzed by ANOVA followed by either the Dunnett or Tukey–Kramer test. P b0.05 was considered statistically significant.

78 W

W

Mt

Fig. 1. Identification of mitochondrial calpains in the rat heart. Panel A shows representative Western blots of VDAC1 (OM marker), AIF (IM marker), GAPDH (cytosol marker), μ- and m-calpains, and calpain-10 in the whole homogenate (W) and in both the mitochondrial (Mt) and cytosolic (S) fractions. Five micrograms of total protein from each fraction was applied to SDS-PAGE. Panel B shows representative casein zymographs with or without Ca2+. The proteins on the Ca2+(+)-zymographs were transferred to a membrane for Western blot analysis of m-calpain. Panel C shows a representative Western blot of calpastatin in the W and Mt fractions. Control (Cont), mouse calpastatin transfected 293T cell lysate (#sc-118967; Santa Cruz Biotechnology). Three independent experiments were performed with similar findings.

immunoreactive binding. These results indicate that the mitochondria contain active m-calpain in the matrix fraction in the rat heart. 3.2. Ca2+-induced mPTP opening is dependent partly on calpain activation Ca2+-induced mPTP opening was monitored by a reduction in the absorbance at 520 nm, which was significant with N500 μM of Ca2+ (Fig. 4). The addition of 500 μM of Ca2+ also induced calpain activation in the isolated mitochondria incubated with the cell-permeable fluorogenic calpain substrate t-BOC-LM-CMAC (Fig. 5A). The Ca2+-induced calpain activation was diminished by the calpain inhibitor calpeptin (1 μM), but not by the mPTP blocker CsA (Fig. 5A), indicating calpain activation independent of mPTP opening. Additionally, calpeptin and the more specific calpain inhibitor, calpastatin, significantly, but only partly, inhibited the Ca2+-induced mitochondrial swelling, in contrast with the greater inhibition by CsA (Fig. 5B). These results indicate that mitochondrial calpains are activated by Ca2 + overload and contributes partially to Ca2+-induced mPTP opening. 3.3. Ca2+-induced calpain activation decreases mitochondrial complex I activity by cleaving a complex I subunit In cardiac mitochondria, the addition of 500 μM of Ca2 + reduced complex I activity to 60% of the control level (Fig. 6A). Complex I inactivation was prevented completely by calpeptin, but was not affected by

K. Shintani-Ishida, K. Yoshida / International Journal of Cardiology 197 (2015) 26–32

Density

Low

1

29

2

3

4

5

6

High

7

8

9

10 11 12

kDa

VDAC (mitochondria)

32

SERCA2a (SR)

97

Syntaxin 6 (Golgi)

32

N-cadherin (plasma membrane)

130

m-calpain

80

Fig. 2. Representative Western blots of VDAC, SERCA2a (SR marker), syntaxin 6 (Golgi marker), N-cadherin (plasma membrane [PM] marker), and m-calpain in the Percoll/OptiPrep gradient fractions. Two independent experiments were performed with similar findings.

3.4. IR induces mitochondrial calpain activation and calpain-dependent mPTP opening in vivo Mitochondria were isolated from rat hearts that underwent ischemia for 30 min and reperfusion for 30 min. IR increased mitochondrial calpain activity 8-fold over that of sham-operated rats (Fig. 7A). IR also induced mitochondrial swelling (1.5-fold over that of sham-operated rats) (Fig. 7B) [18], ND truncation (Fig. 7C), and complex I inactivation (Fig. 7D). Calpain inhibitor I is the only commercially available calpain inhibitor, whose in vivo effects were confirmed by a substantial reduction in MI size after IR [9]. IR-induced mPTP opening, ND truncation, and complex I inactivation were inhibited by pretreatment with calpain inhibitor I. These results demonstrate a role for calpain in mPTP opening and in complex I inactivation in IR. Western blot analysis of the mitochondria isolated from post-mortem heart tissues

A

kDa

VDAC1 (OM)

32

AK2 (IMS)

26

AIF (IM)

67

PDH (Matrix)

54

m-calpain

80 rix

at M

IM S IM M O t M

B

OM

IMS

IM

Matrix Mt

showed human heart mitochondria similarly express m-calpain (Fig. 8). 4. Discussion Growing evidence supports the mitochondrial localization of μcalpain [14,15,17], m-calpain [15], and calpain 10 [16]. Furthermore, mitochondrial μ-calpain isolated from the rat cerebral cortex and from SHSY5Y neuroblastoma cells has a mitochondrial targeting sequence in its N-terminal region [24]. Additionally, relatively high calpain activity is detected in both the mitochondrial IMS and matrix, as well as in the cytosol, in swine liver [19]. In bovine pulmonary smooth muscle mitochondria, μ-calpain in the IMS cleaves NCX on the IM after Ca2 + ionophore treatment [14]. In Ca2+-loaded rat liver mitochondria [15] and in mitochondria isolated from the mouse heart submitted to IR in Langendorff system [17], μ-calpain in the IMS truncates and releases AIF on the IM. In contrast, we demonstrated that m-calpain in the matrix is activated in Ca2+-loaded mitochondria and in mitochondria isolated from the heart after IR. Western blot analysis showed the presence of μ-calpain, m-calpain, and calpain 10 in the mitochondria; however, casein zymography showed only m-calpain activity. Consistent with the lack of μ-calpain activity, we did not find truncation of the known μ-calpain substrates NCX and AIF in rat heart mitochondria after treatment with 500 μM of Ca2+ (data not shown). Mitochondrial μ-calpain activity is likely to differ by species and organs. Reports on the presence of mitochondrial m-calpain are limited. Previously, we reported that IR induces the activation of m-calpain in the rat heart membrane fraction, which can contain mitochondria [25]. In rat hepatic mitochondria, m-calpain is localized mainly in the OM and IMS, while it appears slightly in the matrix [15]. We found m-calpain in the matrix but not in the OM in the rat heart. Additionally, the addition of Ca2 + to isolated hepatic mitochondria induced truncation of the OM protein VDAC [15,26], while its addition to heart mitochondria truncated the IM protein ND6 (Fig. 5) but did not affect the OM protein VDAC (data not shown). m-Calpain localization might differ by cell type. 12

Decrease in A520 (%)

CsA. Consistent with a previous report on the renal mitochondria, the complex I subunit ND6 was truncated by the addition of Ca2 +, and calpeptin inhibited this truncation (Fig. 6B). However, the other subunits identified as potential calpain substrates, namely NDUFV2, DAP13, and NDUFS7 [16], were not truncated by Ca2 + incubation (Fig. 6C). Consistent with the m-calpain activity in the cardiac mitochondria (Fig. 1B), ND6 was truncated by exogenous m-calpain in a concentration-dependent manner (Fig. 6D), which was blocked by calpeptin (Fig. 6E).

** **

10 8 6 4 2 0 -2

0

20

50

100

200

500

1000

2+

Fig. 3. Localization of m-calpain in mitochondrial subfractions. Panel A shows representative Western blots of VDAC1 (OM marker), AK2 (IMS marker), AIF (IM marker), PDH (matrix marker), and m-calpain in the whole mitochondrial (Mt), OM, IMS, IM and matrix fractions. Panel B shows a representative casein-zymograph of the submitochondrial fractions. Three independent experiments were performed with similar findings.

Ca (µM) Fig. 4. Ca2+-induced mitochondrial swelling. A decrease in the A520 at 15 min after the addition of Ca2+ was determined. Data are means ± SE (n = 4 experiments). **P b 0.01 vs. the value for 0 μM Ca2+ (Dunnett's test).

K. Shintani-Ishida, K. Yoshida / International Journal of Cardiology 197 (2015) 26–32

A

A

Calpain activity (ΔFU)

60

*

*

50 40 30 20

††

†

10 0

Complex I activity (% of control)

30

140

††

120

††

100 80

40 20 0

Control -10

Control

Vehicle

+ CP

Cleaved ND6 (% of total)

*

*

60 40

+ CS

+ CsA

*

intact (24 kDa) cleaved

40

*

30 20 10 0

20

+ CS

Control Vehicle + CP 500 µM Ca2+

0

1

1

1

sA

C

S

C

P

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P

C

µM

µM

µM

1

e

cl

hi

µM

0.

Ve

Mt swelling (% of vehicle in each experiment)

100 80

+ CP

500 µM Ca2+

B

B *

Vehicle

+ CsA

500 µM Ca2+

**

**

60

Fig. 5. Effects of calpain inhibitors on Ca2+-induced mitochondrial calpain activation and swelling. Panel A shows calpain activity at 15 min after the addition of 500 μM of Ca2+. Either a calpain inhibitor, calpeptin (+CP, 1 μM), or an mPTP blocker, cyclosporine A (+CsA, 1 μM), was added to the mitochondrial preparation at 10 min before the addition of Ca2+. Data are means ± SE (n = 7 experiments). *P b 0.05 vs. the value for control, †P b 0.05; †† P b 0.01 vs. the value for vehicle (Tukey–Kramer test). Panel B shows the effects of calpain inhibitors (CP and calpastatin [CS]) and of an mPTP blocker (CsA) on mitochondrial swelling at 15 min after the addition of 500 μM of Ca2+. Data are means ± SE (n = 6 experiments). *P b 0.05 vs. the value for vehicle (Tukey–Kramer test).

C

D

NDUFV2 (24 kDa)

kDa 25

DAP13 (15 kDa)

20

intact cleaved

15

NDUFS7 (20 kDa) –

+

–

+

0

500 µM Ca2+

1

Cleaved ND6 (% of total)

E Complex I is an L-shaped multi-subunit protein consisting of a membrane arm and a matrix arm [27]. ND6 can be a substrate of the matrix m-calpain, assuming that it is exposed to the matrix. Although mcalpain is abundant in the cytosolic fraction [28], the lack of the cytosolic marker GAPDH in this fraction excluded contamination of the mitochondrial preparation with cytosolic proteins in our studies. The mitochondrial m-calpains are not likely to be derived from the SR, Golgi [29] or plasma membrane [30], according to Percoll/OptiPrep gradient analysis of isolated mitochondria. In the rat and rabbit kidney, calpain 10 is implicated in complex I inactivation through truncation of the complex I substrates ND6 and NDUFV2 [16]. Mitochondrial calpain 10 is required for normal renal function and cell viability; however its expression decreases with age in the kidney, but not in rodent and human liver [31]. In rat cardiac mitochondria, calpain 10 was detected by Western blotting, but its activity was not detected by zymography using either FITC-casein or SLLVYAMC (an in vitro known mitochondrial calpain substrate) [16]. Several lines of evidence have supported the causality of complex I inactivation to mPTP opening in cardiac mitochondria. Mitochondrial complex I dysfunction is observed in several heart diseases such as heart failure [32], cardiomyopathy [33], and MI [34]. In the mouse, cardiac-specific knockout of the complex I subunit Ndufs4 leads to sensitization of mPTP and accelerates heart failure after either pressure overload or repeated pregnancies [35]. Additionally, the complex I inhibitor rotenone induces mPTP opening through mitochondrial overproduction of reactive oxygen species [36]. We found that calpain inhibitors prevented mPTP opening, reduction in complex I activity, and truncation of the complex I subunit ND6 in the Ca2 +-loaded

5

10

m-calpain (µg)

intact cleaved 40

*

30 20 10 0

– (control)

+

–

+

m-calpain (10 µg)

+ inhibitor

Fig. 6. Ca2+-induced complex I inactivation and ND6 truncation in the mitochondria. Panel A shows Complex I activity at 15 min after the addition of 500 μM of Ca2+. Either a calpain inhibitor, calpeptin (+CP, 1 μM) or an mPTP blocker, cyclosporine A (+CsA, 1 μM), was added to the mitochondrial preparation at 10 min before the addition of Ca2+. Data are means ± SE (n = 7 experiments). *P b 0.05 vs. the value for control, † P b 0.05; ††P b 0.01 vs. the value for vehicle (Tukey–Kramer test). Panel B shows a representative Western blot of ND6 and the quantitative data. The intensities of the bands for cleaved ND6 were determined as percentages of the total (intact + cleaved) ND6. Data are means ± SE (n = 5 experiments). *P b 0.05 vs. the value for control (Tukey–Kramer test). Panel C shows representative Western blots of NDUFV2, DAP13, and NDUFS7 in the mitochondria at 15 min after the addition of 500 μM of Ca2+. Panel D shows a representative Western blot of ND6 after exogenous m-calpain addition. Panel E shows the effects of a calpain inhibitor (1 μM calpeptin) on ND6 truncation by exogenous mcalpain. Data are means ± SE (n = 4 experiments). *P b 0.05 vs. the value for control (Tukey–Kramer test).

mitochondria (Figs. 5, 6A, B) and in mitochondria isolated from the heart after IR (Fig. 7). These findings suggest that calpain-mediated complex I inactivation contributes to mPTP opening.

K. Shintani-Ishida, K. Yoshida / International Journal of Cardiology 197 (2015) 26–32

B **

1200

Mt swelling (% of total)

Calpain activity (% of sham)

A 1000 800 600 400 200

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intact cleaved 50

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120

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Complex I activity (% of sham)

Cleaved ND6 (% of total)

31

40 30 20 10

*

100 80 60 40 20 0

0

Sham Vehicle CI IR

Sham Vehicle CI IR

Fig. 7. Mitochondrial calpain activation and mitochondrial dysfunction after IR in vivo. Panel A shows calpain activity in isolated mitochondria from hearts after ischemia for 30 min followed by 30 min of reperfusion. Data are means ± SE (n = 8 preparations from 4 rats). **P b 0.01 vs. the value for sham-operated rats (Dunnett's test). Panels B, C and D show mitochondrial swelling, cleaved ND6, and complex I activity, respectively, in mitochondria isolated from hearts at 30 min after ischemia for 30 min followed by 30 min of reperfusion. Calpain inhibitor-1 (CI) was injected at 10 min before ischemia. Data are means ± SE (n = 8 preparations from 4 rats). *P b 0.05, **P b 0.01 vs. the value for sham-operated rats (Tukey–Kramer test).

In IR, calpain is activated by cytosolic Ca2+ overload and disrupts the membrane skeleton and the intercalated disk through the proteolysis of fodrin [8]. Calpain inhibitors reduce MI size [9–12]. On the other hand, mPTP plays a pivotal role in the development of MI after IR. Numerous studies have shown that mPTP blockers suppress MI development [2–4]. Mitochondrial Ca2 + uniporter blockers also reduce MI by preventing mPTP opening [6]. The results of the present study suggest that not only sarcolemmal fragility/disruption by cytosolic calpains, but also mPTP opening by mitochondrial calpains are implicated in MI development during IR. Consistent with this hypothesis, we demonstrated previously that the introduction of an anti-phospholamban antibody into the heart not only promotes Ca2 + uptake into the SR and prevents fodrin proteolysis after IR, but also induces mitochondrial Ca2 + overload through SR-mitochondria microdomains, resulting in the acceleration of mPTP opening and MI development [18]. Ding et al. reported that hepatotoxin (microcystin-LR)-induced mPTP opening induces the release of mitochondrial Ca2+ into the cytosol, leading to the activation of cytosolic calpain and the promotion of cell death in rat hepatocytes [37]. However, we found previously that either CsA or Ru360 fails to inhibit fodrin proteolysis, but decelerates mPTP opening and

limits MI size in the rat heart after IR [18]. These results suggest the independent progression of calpain activation in the mitochondria and cytosol. In conclusion, m-calpain in the mitochondrial matrix induces complex I inactivation through ND6 truncation and mPTP opening in rat cardiac mitochondria after either Ca2 + loading or IR. Mitochondrial mcalpain is also expressed in the human heart; therefore, further study on the role of mitochondrial m-calpain in the human heart is required. Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.ijcard.2015.06.010. Sources of funding This study was supported by JSPS KAKENHI Grant Numbers 24659334 and 25293162. Conflicts of interest None. References

kDa

m-calpain

80

VDAC (mitochondria)

32

GAPDH (cytosol)

37 W Mt case 1

W Mt case 2

Fig. 8. Western blot analysis of m-calpain, VDAC and GAPDH in the whole homogenates (W) and the mitochondrial (Mt) fractions from human myocardia. Case 1, a 21-year-old woman at 29 h after death; case 2, a 48-year-old woman at 40 h after death.

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