• Users Online: 199
  • Print this page
  • Email this page

 
Table of Contents
ORIGINAL ARTICLE
Year : 2022  |  Volume : 65  |  Issue : 5  |  Page : 226-232

Calpain inhibitors inhibit mitochondrial calpain activity to ameliorate apoptosis of cocultured myoblast


1 Department of Gastrointestinal Surgery, The First Affiliated Hospital of Fujian Medical University, Fuzhou, Fujian, China
2 Department of Dermatology, The First Affiliated Hospital of Fujian Medical University, Fuzhou, Fujian, China
3 Department of Thyroid and Breast Surgery, The First Affiliated Hospital of Fujian Medical University, Fuzhou, Fujian, China

Date of Submission24-Feb-2021
Date of Decision20-Jun-2022
Date of Acceptance09-Jul-2022
Date of Web Publication27-Oct-2022

Correspondence Address:
Dr. Lingjun Kong
Department of Thyroid and Breast Surgery, The First Affiliated Hospital of Fujian Medical University, 20 Chazhong Road, Fuzhou 350004, Fujian
China
Prof. Sizeng Chen
Department of Gastrointestinal Surgery, The First Affiliated Hospital of Fujian Medical University, 20 Chazhong Road, Fuzhou 350004, Fujian
China
Login to access the Email id

Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0304-4920.359797

Rights and Permissions
  Abstract 


Cancer cachexia is a fatal syndrome associated with muscle regeneration disability. Tumor factors induce the apoptosis of myoblasts to impair the regeneration of skeletal muscle. Cancer cachectic myoblast apoptosis is associated with mitochondria injury. It has been reported that activated mitochondrial calpain caused mitochondria injury in mouse cardiomyocytes and pulmonary smooth muscle. We wondered if mitochondrial calpains exist in skeletal myoblast and their potential role in myoblast apoptosis of cancer cachexia. We used a transwell to build a novel myoblast-carcinoma cell coculture model to simulate the cancer cachexia environment in vitro. Calpain inhibitors, calpastatin (CAST) and calpeptin (CAPT), were used during coculture. We found for the first time that two calpains (calpain-1 and calpain-2) and CAST were present in the mitochondria of myoblast. The activation of mitochondrial calpain decreased mitochondrial complex I activity, promoted mitochondrial permeability transition pore opening, and impaired mitochondrial membrane potential in myoblast during coculture, which induced myoblasts apoptosis. CAST and CAPT protected myoblasts from apoptosis by inhibiting mitochondrial calpain activity, which may attenuate or even reverse cancer cachectic muscle atrophy by improving muscle regeneration ability. Our study provides a new perspective for understanding the mechanism of cancer cachexia, and will further contribute to treat cancer cachexia by focusing on the mitochondrial calpain activity.

Keywords: Apoptosis, calpain, cancer cachexia, mitochondria, myoblast


How to cite this article:
Zeng X, Zhao L, Chen Z, Kong L, Chen S. Calpain inhibitors inhibit mitochondrial calpain activity to ameliorate apoptosis of cocultured myoblast. Chin J Physiol 2022;65:226-32

How to cite this URL:
Zeng X, Zhao L, Chen Z, Kong L, Chen S. Calpain inhibitors inhibit mitochondrial calpain activity to ameliorate apoptosis of cocultured myoblast. Chin J Physiol [serial online] 2022 [cited 2022 Nov 26];65:226-32. Available from: https://www.cjphysiology.org/text.asp?2022/65/5/226/359797




  Introduction Top


Cancer cachexia is a complicated metabolic syndrome. It is mainly characterized by muscle atrophy.[1] Nearly 85% of terminal cancer patients eventually develop cancer cachexia, and nearly 20% of cancer patients die of cancer cachexia.[2] Cancer cachexia cannot be fully reversed by conventional nutritional support, and it has no effective treatment.[3] So far, the pathogenesis of cancer cachexia remains unclear.

Cancer cachectic muscle atrophy (CCMA) is induced by multiple mechanisms, including muscle protein metabolism disorder,[4] myocyte damage,[5] and myocyte regeneration dysfunction.[6] The sarcolemma damage of myocytes is an important feature of CCMA. Myocyte regeneration by muscle satellite cells (MSCs) is the main mechanism of repairing myocyte damage. MSCs are undifferentiated mononuclear myogenic cells.[7] When myocytes damage happens, MSCs become activated and proliferate to produce myoblasts, which are muscle precursor cells.[8] The myoblasts then fuse into the damaged myocytes for repair.[9] In cancer cachectic animals and humans, tumor factors impair not only the myocytes but also myoblasts, causing myoblasts apoptosis, and myocyte regeneration damage.[6],[10]

The calpain family consists of 15 members including two ubiquitously expressed calpain-1 (μ-calpain) and calpain-2 (m-calpain), and one muscle-specific expressed calpain-3.[11],[12] Calpastatin (CAST) is the only known endogenous calpain inhibitor which is ubiquitously expressed. Calpains and CAST are mainly reported in the cytoplasm and participated in a variety of physiological and disease processes.[13],[14] Calpains can induce apoptosis by mediating the cleavage apoptosis-inducing factor (AIF), which leads to the activation of the mitochondrial apoptosis pathway.[15] Recently, calpain-1, calpain-2, and CAST have been found in the mitochondria of cardiomyocyte and pulmonary smooth muscle cell, involving in mitochondria injury and cell apoptosis.[16],[17],[18]

To the best of our knowledge, the role of mitochondrial calpain in myoblast of cancer cachexia has not yet been investigated before. In this study, we used a myoblast-carcinoma cell coculture model to simulate the cancer cachexia environment in vitro. We investigated whether calpains exist in the mitochondria of myoblast, and their potential role in cancer cachectic myoblast apoptosis.


  Materials and Methods Top


Cell culture

Mouse C2C12 myoblasts and CT26 colon carcinoma cells were obtained from ATCC (Manassas, VA, USA). To build the coculture model, myoblasts were seeded (20,000 cells/cm2) in the plate and CT26 cells were seeded (20,000 cells/cm2) in transwells (Corning, #3450) on another plate. After 24 h of independent culture, the transwells were placed into the plates containing myoblasts and fresh medium with or without calpain inhibitor (CAST, Millipore, 208902, 1 μM; calpeptin (CAPT), Sigma-Aldrich, C8999, 50 μM). After 24 h of coculture, the myoblasts were stained with JC-1 or harvested for further analysis.

Our study consists of five groups: NC group, sham myoblasts (empty transwell and no calpain inhibitor); CO group, myoblasts cocultured with CT26 cells (no calpain inhibitor); CAST group, myoblasts cocultured with CT26 cells (with CAST); CAPT group, myoblasts cocultured with CT26 cells (with CAPT), and CC group, myoblasts cocultured with CT26 cells (with CAST and CAPT).

Flow cytometry analysis

An Annexin V/PI Staining Kit (Beyotime, C1062) was used to quantitatively detect myoblast apoptosis as described.[19] A MitoProbe Transition Pore Assay Kit (Thermo Fisher, M34153) was used to measure mitochondrial permeability transition pore (MPTP) opening according to the manufacturer's instructions as described.[20] Accuri C6 flow cytometer (BD Biosciences, USA) was used to detect fluorescence intensity. A minimum of 1 × 104 cells was recorded in each sample and analyzed with FlowJo software (version 7.6.2, FlowJo LLC, USA).

JC-1 staining assay

JC-1 dye (Beyotime; C2006) was used to assess mitochondrial membrane potential (Δψm) in myoblasts. For fluorescence microscopy, myoblasts were incubated with JC-1 according to the manufacturer's instructions as described.[19] Images were acquired with a fluorescence microscope (Nikon; Japan). For fluorescence labeling assay, myoblasts were collected with trypsin (Gibco, 25200072) and incubated with JC-1 according to the manufacturer's instructions as described.[21] SpectraMax M5 microplate reader (Molecular Devices, USA) was used to read the assay plate.

Isolation of myoblast mitochondria

Myoblast cytoplasm and mitochondria were isolated and collected by Mitochondria Isolation Kit for Cultured Cells (Abcam; ab110170) as described[22] for further analysis.

Western blot analysis

Myoblast cytosolic proteins and mitochondrial proteins were separated by electrophoresis and stained with primary antibody and HRP-conjugated secondary antibody (Abcam, ab97051) as previously described.[19] Images of the membranes were recorded with ChemiDoc XRS+ system (Bio-Rad, USA), and analyzed with Quantity One software (version 4.6.6; Bio-Rad, USA). Primary antibodies used: Abcam: caspase-3 (ab184787); calpain-1 (ab108400); calpain-2 (ab126600); CAST (ab28252); NDUFS3 (ab177471); Cyclophilin (ab181983); GAPDH (ab181602); VDAC (ab154856); Santa Cruz Biotechnology: calpain-3 (sc-365277); spectrin (sc-46696); and Cell Signaling Technology: AIF (4642S).

Calpain activity assay

Myoblast cytosolic and mitochondrial calpain activity were detected by the calpain activity assay kit (Millipore; QIA120) as described.[23] SpectraMax M5 was used to read the assay plate.

Complex I enzyme activity assay

Mitochondrial complex I enzyme activity was detected by Complex I Enzyme Activity Microplate Assay Kit (Abcam, ab109721) in samples of myoblast mitochondria as described.[22] SpectraMax M5 was used to read the assay plate.

Statistical analysis

Data are presented as the mean ± standard error of the mean and analyzed with IBM SPSS Statistics for Windows, Version 19.0. Armonk, NY, USA, IBM Corp. When equal variance was assumed, variation among groups was evaluated using one-way analysis of variance followed by the Tukey test. When equal variance was not assumed, Dunnett's T3 test was applied. Statistical significance was assumed for two-sided P < 0.05.


  Results Top


Calpain inhibitors attenuated coculture-induced apoptosis in myoblasts

As shown in [Figure 1]a and [Figure 1]b, coculture significantly increased the apoptosis ratio of myoblasts, which was attenuated by CAST/CAPT/CAST+CATP treatment. Moreover, as shown in [Figure 1]c and [Figure 1]d, coculture dramatically increased the cleavage of caspase-3 in myoblasts. Compared with CO group, CAST/CAPT/CAST+CATP treatment markedly attenuated those changes. These results indicated that the activation of calpain during coculture activated caspase-3 and induced myoblast apoptosis.
Figure 1: Calpain inhibitors decreased myoblast apoptosis. (a) Myoblast apoptosis was assessed by flow cytometry (n = 6). Q2 and Q3 quadrants represent the apoptotic myoblasts. (b) Statistical analysis of myoblast apoptosis. Significant differences were detected between CO and NC groups (P < 0.001), between CO and CAST/CAPT/CC groups (P = 0.001, P = 0.002, P < 0.001, respectively). (c) Representative blots of cleaved caspase-3, pro-caspase-3, and GAPDH in myoblasts (n = 3). (d) Quantification of cleaved caspase-3/pro-caspase-3 ratio. Statistical differences were detected between CO and NC groups (P < 0.001), between CO and CAST/CAPT/CC groups (P < 0.001). Data are represented as mean ± SEM. (*P < 0.05, **P < 0.01, ***P < 0.001). CAPT: Calpeptin, CAST: Calpastatin.

Click here to view


Calpain inhibitors inhibited coculture-induced calpain activation

Coculture markedly increased cytosolic and mitochondrial calpain-1 content compared with the NC group [Figure 2]a,[Figure 2]b,[Figure 2]c and [Figure 2]j. Coculture also increased cytosolic and mitochondrial calpain enzyme activity [Figure 2]m and [Figure 2]n. Moreover, the cytosolic cleaved spectrin (a marker of cytosolic calpain activation) was increased by coculture [Figure 2]a and [Figure 2]g,[Figure 2]h,[Figure 2]i, supporting that cytosolic calpain was activated during coculture. Compared with CO group, CAST/CAPT/CAST+CATP treatment markedly attenuated those changes. These data suggested that calpain inhibitors attenuated cytosolic and mitochondrial calpain activation. We also found calpain-2 and CAST in myoblast mitochondria, but their protein expressions were not significantly altered by coculture or calpain inhibitors [Figure 2]b, [Figure 2]k and [Figure 2]l.
Figure 2: Calpain inhibitors prevented calpain activation during coculture. (a and b) Representative blots of cytosolic (a) and mitochondrial (b) protein contents (n = 3). (c) Densitometric analysis of cytosolic calpain-1. Statistical differences were detected between CO and NC groups (P = 0.005), between CO and CAST/CAPT/CC groups (P = 0.014, P = 0.019, P = 0.011, respectively). (d) Densitometric analysis of cytosolic calpain-2. Statistical differences were detected between CO and CC groups (P = 0.018). (e and f) Densitometric analysis of cytosolic calpain-3 and cytosolic CAST. No statistical differences were detected between five groups. (g) Densitometric analysis of cytosolic cleaved spectrin (C-spectrin). Statistical difference was detected between CO and NC groups (P = 0.003). (h) Densitometric analysis of cytosolic full-length spectrin (F-spectrin). Statistical difference was detected between CO and NC groups (P < 0.001). (i) Statistical analysis of C-spectrin/F-spectrin ratio. Statistical differences were detected between CO and NC groups (P < 0.019), between CO and CAST/CAPT/CC groups (P < 0.011, P = 0.027, P < 0.030, respectively). (j) Densitometric analysis of mitochondrial calpain-1. Statistical differences were detected between CO and NC groups (P < 0.001), between CO and CAST/CAPT/CC groups (P = 0.003, P = 0.002, P = 0.001, respectively). (k and l) Densitometric analysis of mitochondrial calpain-2 and mitochondrial CAST. No statistical difference was detected between five groups. (m) Statistical analysis of cytoplasmic calpain activity. Statistical differences were detected between CO and NC groups (P < 0.001), between CO and CAST/CAPT/CC groups (P = 0.002, P = 0.004, P = 0.001, respectively). (n) Statistical analysis of mitochondrial calpain activity. Statistical differences were detected between CO and NC groups (P < 0.001), between CO and CAST/CAPT/CC groups (P = 0.01, P = 0.019, P = 0.009, respectively). Data are represented as mean ± SEM. (*P < 0.05, **P < 0.01, ***P < 0.001). CAPT: Calpeptin, CAST: Calpastatin, SEM: standard error of the mean.

Click here to view


Calpain inhibitors protected the Δψm of myoblast during coculture

As shown in [Figure 3]a and [Figure 3]b, the JC-1 red/green ratio in CO group was dramatically decreased compared with NC group, which was markedly attenuated by calpain inhibitors, indicating that activated calpain during coculture impaired Δψm in myoblast.
Figure 3: Calpain inhibitors prevented mitochondria injury during coculture. (a) Myoblast JC-1 staining (n = 9). Scale bar represents 200 μm. (b) Statistical analysis of JC-1 red/JC-1 green ratio. Statistical differences were detected between CO and NC groups (P = 0.001), between CO and CAST/CAPT/CC groups (P = 0.021, P = 0.001, P = 0.002, respectively). (c and d) MPTP opening of myoblasts was detected by flow cytometry (n = 3). Statistical differences were detected between CO and NC groups (P = 0.005), between CO and CAST/CAPT/CC groups (P = 0.046, P = 0.036, P = 0.033, respectively). (e) Representative blots of protein contents (n = 3). (f) Densitometric analysis of mitochondrial CYCD. Statistical difference was detected between CO and NC groups (P = 0.013). (g) Densitometric analysis of mitochondrial NDUFS3. No statistical difference was detected between five groups. (h) Statistical analysis of mitochondrial complex I activity (n = 9). Statistical differences were detected between CO and NC groups (P < 0.001), between CO and CAPT groups (P = 0.033). (i) Densitometric analysis of mitochondrial AIF. Statistical differences were detected between CO and NC groups (P < 0.001), between CO and CAST/CAPT/CC groups (P = 0.039, P = 0.047, P = 0.043, respectively). Data are represented as mean ± SEM. (*P < 0.05, **P < 0.01, ***P < 0.001). CAPT: Calpeptin, CAST: Calpastatin, SEM: Standard error of the mean.

Click here to view


Calpain inhibitors attenuated the mitochondrial permeability transition pore opening during coculture

MPTP opening causes mitochondrial membrane permeation that induces Δψm damage. As shown in [Figure 3]c and [Figure 3]d, coculture significantly decreased the fluorescence intensity in myoblast, which was ameliorated by CAST/CAPT/CAST+CATP treatment. These data indicated that activated calpain can induce MPTP opening.

Cyclophilin D (CYCD) is an important regulator of MPTP opening. As shown in [Figure 3]e and [Figure 3]f, the mitochondrial CYCD content was dramatically decreased by coculture. However, calpain inhibitors did not significantly alter mitochondrial CYCD content. These results indicated that activated calpain decreased CYCD content, which might lead to MPTP opening in mitochondria.

Calpain inhibitors increased mitochondrial complex I activity during coculture

As shown in [Figure 3]h, coculture dramatically decreased mitochondrial complex I activity compared with NC group. Calpain inhibitors improved complex I activity compared with CO group, but only CAPT treatment reach statistically difference. NDUFS3 is a core subunit of complex I and is essential to maintain complex I activity. There were no differences in NDUFS3 content between all groups [Figure 3]e and [Figure 3]g, suggesting that the decreased complex I activity in mitochondria is not due to altered NDUFS3 content.

Calpain inhibitors preserved apoptosis-inducing factor content in myoblast mitochondria

In addition to inducing MPTP opening, calpain plays a more direct role in the cleaving of AIF and induces its release from mitochondria to cytoplasm.[24],[25] Similar to these findings, the mitochondrial AIF content was significantly decreased by coculture [Figure 3]e and [Figure 3]i, which was dramatically attenuated by calpain inhibitors. However, we failed to detect the cleaved AIF in both cytoplasm and mitochondria.


  Discussion Top


Cancer cachexia is a fatal syndrome associated with muscle regeneration disability. Tumor-bearing-induced myoblast apoptosis impairs muscle regeneration. In the present study, we first found that calpain-1, calpain-2, and CAST were present in the mitochondria of C2C12 myoblast. Next, we found that coculture increased mitochondrial calpain activity and MPTP opening, caused Δψm damage, mitochondria injury, and myoblast apoptosis. Calpain inhibitors attenuated mitochondrial calpain activation, inhibited MPTP opening, and decreased Δψm damage during coculture, which protected the myoblasts from apoptosis. In our study, we found that CAST reduced the protein level of calpain-1. It is now clear that CAST can bind calpains to inhibit their activities, and the inhibitory activity of CAST is specific for calpains.[11],[14],[26] However, it remains to be unknown that whether CAST affects the expression or degradation of calpain-1, which needs further investigation.

The MPTP is located on the membrane of mitochondria.[27] The opening of MPTP increases mitochondrial membrane permeability and Δψm loss, which induced mitochondria injury.[28] In mouse hearts, ischemia-reperfusion-induced Δψm damage could be attenuated by calpain inhibitors.[23] Moreover, the Δψm damage induced by microcystin-LR was also attenuated by calpain inhibitors in cultured hepatocytes,[29] which suggested that activated calpain could induce MPTP opening. Consistent with these findings, we found that coculture induced the activation of calpain in myoblast cytoplasm and mitochondria, caused the MPTP opening and Δψm damage. The administration of calpain inhibitors ameliorated these changes. CYCD is a key regulator of the MPTP opening. Coculture also markedly reduced mitochondrial CYCD content, which was ameliorated by calpain inhibitors, suggesting that activated mitochondrial calpain may induce CYCD cleavage to increase MPTP opening. However, we did not detect any cleaved band of CYCD in myoblast.

Complex I is crucial in mitochondrial electron transport.[30] Activated mitochondrial calpain damages complex I activity, which leads to MPTP opening and cardiac injury during reperfusion.[16],[31] In our study, we found that coculture impaired mitochondrial complex I activity, which was ameliorated by calpain inhibitors, suggesting that activated mitochondrial calpain might impair complex I activity to induce MPTP opening.

The subunit damage and posttranslational modification can both induce complex I dysfunction.[32] NDUFS3 is a complex I subunit which is crucial for NADH oxidation and subsequent electron transfer.[16] We found that NDUFS3 content is not changed by coculture or calpain inhibitors, suggesting that it is not responsible for complex I disability. Conformational change (induced by a sulfhydryl oxidation of cysteine in complex I) can also induce complex I disability,[33] and the impaired complex I activity in our study might also be due to this posttranslational modification, which needed further investigation.

A mitochondrial-center control pathway is the most common mechanism of apoptosis.[34] In this scenario, apoptotic signals converge at mitochondrial membranes and cause MPTP opening and mitochondrial proteins (such as AIF and cytochrome C) release.[35] The released cytochrome C triggers the caspase proteolytic cascade in the cytoplasm, which in turn, activates the downstream pathway to induce cell apoptosis.[36],[37] In this study, we found that activation of calpain during coculture induced MPTP opening and activated caspase-3 in myoblast, which led to myoblast apoptosis.

AIF is a mitochondrial flavoprotein that acts as an antioxidant in the intermembrane space of mitochondria.[13],[38] When mitochondria are damaged, AIF can be cleaved and released from mitochondria, translocates to the nuclear, and induces DNA degradation and cell apoptosis.[39] Calpains can promote apoptosis by mediating AIF cleavage. In mouse hearts, activated mitochondrial calpain cleaves AIF to truncated AIF (t-AIF). Then, t-AIF was released from mitochondria to cytoplasm, which triggers caspase-independent apoptotic cell death.[16] In our study, coculture caused MPTP opening and decreased mitochondrial AIF content, while the administration of calpain inhibitors ameliorated these changes, suggesting that the activation of mitochondrial calpain might induce the cleavage of mitochondrial AIF and the release of t-AIF from mitochondria. However, we failed to detect t-AIF in the myoblast, whether myoblast apoptosis is induced by t-AIF needs further investigation.


  Conclusion Top


To the best of our knowledge, we demonstrated for the first time that calpain-1, calpain-2, and CAST are present in mouse myoblast mitochondria. Calpain inhibitors prevent myoblast apoptosis during coculture by inhibiting mitochondrial calpain activity. Based on our results, we speculate that calpain inhibitors can maintain the regeneration capability of muscle tissue by inhibiting myoblast apoptosis and thereby attenuate CCMA. Although CAST and CAPT are classic calpain inhibitors, they still have off-target effects. A genetic approach will be needed to further investigate the role of mitochondrial calpain in CCMA myoblast apoptosis, which will provide new insights to understand the mechanism of CCMA. Calpain inhibitors may attenuate or even reverse CCMA by improving muscle regeneration ability. Our results will further contribute to develop focused approaches to attenuate CCMA by manipulating the mitochondrial calpain activity.

Acknowledgments

We are grateful to our laboratory staff for their help with this study.

Financial support and sponsorship

The work was supported by Joint Funds for the innovation of science and Technology, Fujian province (Grant number: 2018Y9083).

Conflicts of interest

There are no conflicts of interest.



 
  References Top

1.
Fearon K, Strasser F, Anker SD, Bosaeus I, Bruera E, Fainsinger RL, et al. Definition and classification of cancer cachexia: An international consensus. Lancet Oncol 2011;12:489-95.  Back to cited text no. 1
    
2.
Donohoe CL, Ryan AM, Reynolds JV. Cancer cachexia: Mechanisms and clinical implications. Gastroenterol Res Pract 2011;2011:601434.  Back to cited text no. 2
    
3.
Yoshida T, Semprun-Prieto L, Sukhanov S, Delafontaine P. IGF-1 prevents ANG II-induced skeletal muscle atrophy via Akt- and Foxo-dependent inhibition of the ubiquitin ligase atrogin-1 expression. Am J Physiol Heart Circ Physiol 2010;298:H1565-70.  Back to cited text no. 3
    
4.
Schiaffino S, Dyar KA, Ciciliot S, Blaauw B, Sandri M. Mechanisms regulating skeletal muscle growth and atrophy. FEBS J 2013;280:4294-314.  Back to cited text no. 4
    
5.
Busquets S, Figueras MT, Fuster G, Almendro V, Moore-Carrasco R, Ametller E, et al. Anticachectic effects of formoterol: A drug for potential treatment of muscle wasting. Cancer Res 2004;64:6725-31.  Back to cited text no. 5
    
6.
He WA, Berardi E, Cardillo VM, Acharyya S, Aulino P, Thomas-Ahner J, et al. NF-κB-mediated Pax7 dysregulation in the muscle microenvironment promotes cancer cachexia. J Clin Invest 2013;123:4821-35.  Back to cited text no. 6
    
7.
Bossola M, Marzetti E, Rosa F, Pacelli F. Skeletal muscle regeneration in cancer cachexia. Clin Exp Pharmacol Physiol 2016;43:522-7.  Back to cited text no. 7
    
8.
Chargé SB, Rudnicki MA. Cellular and molecular regulation of muscle regeneration. Physiol Rev 2004;84:209-38.  Back to cited text no. 8
    
9.
Talbert EE, Guttridge DC. Impaired regeneration: A role for the muscle microenvironment in cancer cachexia. Semin Cell Dev Biol 2016;54:82-91.  Back to cited text no. 9
    
10.
He WA, Calore F, Londhe P, Canella A, Guttridge DC, Croce CM. Microvesicles containing miRNAs promote muscle cell death in cancer cachexia via TLR7. Proc Natl Acad Sci U S A 2014;111:4525-9.  Back to cited text no. 10
    
11.
Dókus LE, Yousef M, Bánóczi Z. Modulators of calpain activity: Inhibitors and activators as potential drugs. Expert Opin Drug Discov 2020;15:471-86.  Back to cited text no. 11
    
12.
Huang J, Zhu X. The molecular mechanisms of calpains action on skeletal muscle atrophy. Physiol Res 2016;65:547-60.  Back to cited text no. 12
    
13.
Ozaki T, Tomita H, Tamai M, Ishiguro S. Characteristics of mitochondrial calpains. J Biochem 2007;142:365-76.  Back to cited text no. 13
    
14.
Goll DE, Thompson VF, Li H, Wei W, Cong J. The calpain system. Physiol Rev 2003;83:731-801.  Back to cited text no. 14
    
15.
Ozaki T, Yamashita T, Ishiguro S. ERp57-associated mitochondrial μ-calpain truncates apoptosis-inducing factor. Biochim Biophys Acta 2008;1783:1955-63.  Back to cited text no. 15
    
16.
Chen Q, Thompson J, Hu Y, Dean J, Lesnefsky EJ. Inhibition of the ubiquitous calpains protects complex I activity and enables improved mitophagy in the heart following ischemia-reperfusion. Am J Physiol Cell Physiol 2019;317:C910-21.  Back to cited text no. 16
    
17.
Kar P, Chakraborti T, Roy S, Choudhury R, Chakraborti S. Identification of calpastatin and mu-calpain and studies of their association in pulmonary smooth muscle mitochondria. Arch Biochem Biophys 2007;466:290-9.  Back to cited text no. 17
    
18.
Kar P, Samanta K, Shaikh S, Chowdhury A, Chakraborti T, Chakraborti S. Mitochondrial calpain system: An overview. Arch Biochem Biophys 2010;495:1-7.  Back to cited text no. 18
    
19.
Zeng X, Chen S, Lin Y, Ke Z. Acylated and unacylated ghrelin inhibit apoptosis in myoblasts cocultured with colon carcinoma cells. Oncol Rep 2018;39:1387-95.  Back to cited text no. 19
    
20.
Chen X, Li X, Zhang W, He J, Xu B, Lei B, et al. Activation of AMPK inhibits inflammatory response during hypoxia and reoxygenation through modulating JNK-mediated NF-κB pathway. Metabolism 2018;83:256-70.  Back to cited text no. 20
    
21.
Hendriks KD, Joschko CP, Hoogstra-Berends F, Heegsma J, Faber KN, Henning RH. Hibernator-derived cells show superior protection and survival in hypothermia compared to non-hibernator cells. Int J Mol Sci 2020;21:1864.  Back to cited text no. 21
    
22.
Zhang XL, Wang ZZ, Shao QH, Zhang Z, Li L, Guo ZY, et al. RNAi-mediated knockdown of DJ-1 leads to mitochondrial dysfunction via Akt/GSK-3ß and JNK signaling pathways in dopaminergic neuron-like cells. Brain Res Bull 2019;146:228-36.  Back to cited text no. 22
    
23.
Thompson J, Hu Y, Lesnefsky EJ, Chen Q. Activation of mitochondrial calpain and increased cardiac injury: Beyond AIF release. Am J Physiol Heart Circ Physiol 2016;310:H376-84.  Back to cited text no. 23
    
24.
Cao G, Xing J, Xiao X, Liou AK, Gao Y, Yin XM, et al. Critical role of calpain I in mitochondrial release of apoptosis-inducing factor in ischemic neuronal injury. J Neurosci 2007;27:9278-93.  Back to cited text no. 24
    
25.
Polster BM, Basañez G, Etxebarria A, Hardwick JM, Nicholls DG. Calpain I induces cleavage and release of apoptosis-inducing factor from isolated mitochondria. J Biol Chem 2005;280:6447-54.  Back to cited text no. 25
    
26.
Ono Y, Saido TC, Sorimachi H. Calpain research for drug discovery: Challenges and potential. Nat Rev Drug Discov 2016;15:854-76.  Back to cited text no. 26
    
27.
Halestrap AP. What is the mitochondrial permeability transition pore? J Mol Cell Cardiol 2009;46:821-31.  Back to cited text no. 27
    
28.
Argilés JM, Busquets S, Stemmler B, López-Soriano FJ. Cancer cachexia: Understanding the molecular basis. Nat Rev Cancer 2014;14:754-62.  Back to cited text no. 28
    
29.
Ding WX, Shen HM, Ong CN. Calpain activation after mitochondrial permeability transition in microcystin-induced cell death in rat hepatocytes. Biochem Biophys Res Commun 2002;291:321-31.  Back to cited text no. 29
    
30.
Laughlin TG, Bayne AN, Trempe JF, Savage DF, Davies KM. Structure of the complex I-like molecule NDH of oxygenic photosynthesis. Nature 2019;566:411-4.  Back to cited text no. 30
    
31.
Karamanlidis G, Lee CF, Garcia-Menendez L, Kolwicz SC Jr., Suthammarak W, Gong G, et al. Mitochondrial complex I deficiency increases protein acetylation and accelerates heart failure. Cell Metab 2013;18:239-50.  Back to cited text no. 31
    
32.
Hollander JM, Thapa D, Shepherd DL. Physiological and structural differences in spatially distinct subpopulations of cardiac mitochondria: Influence of cardiac pathologies. Am J Physiol Heart Circ Physiol 2014;307:H1-14.  Back to cited text no. 32
    
33.
Galkin A, Abramov AY, Frakich N, Duchen MR, Moncada S. Lack of oxygen deactivates mitochondrial complex I: Implications for ischemic injury? J Biol Chem 2009;284:36055-61.  Back to cited text no. 33
    
34.
Hotchkiss RS, Strasser A, McDunn JE, Swanson PE. Cell death. N Engl J Med 2009;361:1570-83.  Back to cited text no. 34
    
35.
Kroemer G, Galluzzi L, Brenner C. Mitochondrial membrane permeabilization in cell death. Physiol Rev 2007;87:99-163.  Back to cited text no. 35
    
36.
Li P, Nijhawan D, Budihardjo I, Srinivasula SM, Ahmad M, Alnemri ES, et al. Cytochrome c and dATP-dependent formation of Apaf-1/caspase-9 complex initiates an apoptotic protease cascade. Cell 1997;91:479-89.  Back to cited text no. 36
    
37.
Zou H, Henzel WJ, Liu X, Lutschg A, Wang X. Apaf-1, a human protein homologous to C. elegans CED-4, participates in cytochrome c-dependent activation of caspase-3. Cell 1997;90:405-13.  Back to cited text no. 37
    
38.
Yu SW, Wang H, Poitras MF, Coombs C, Bowers WJ, Federoff HJ, et al. Mediation of poly(ADP-ribose) polymerase-1-dependent cell death by apoptosis-inducing factor. Science 2002;297:259-63.  Back to cited text no. 38
    
39.
Ye H, Cande C, Stephanou NC, Jiang S, Gurbuxani S, Larochette N, et al. DNA binding is required for the apoptogenic action of apoptosis inducing factor. Nat Struct Biol 2002;9:680-4.  Back to cited text no. 39
    


    Figures

  [Figure 1], [Figure 2], [Figure 3]



 

Top
 
  Search
 
    Similar in PUBMED
   Search Pubmed for
   Search in Google Scholar for
 Related articles
    Access Statistics
    Email Alert *
    Add to My List *
* Registration required (free)  

 
  In this article
Abstract
Introduction
Materials and Me...
Results
Discussion
Conclusion
References
Article Figures

 Article Access Statistics
    Viewed844    
    Printed6    
    Emailed0    
    PDF Downloaded139    
    Comments [Add]    

Recommend this journal