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. 2011 Jan 4;589(Pt 4):855–861. doi: 10.1113/jphysiol.2010.202036

Cyclophilin-D is dispensable for atrophy and mitochondrial apoptotic signalling in denervated muscle

Frederic N Daussin 1, Richard Godin 1, Alexis Ascah 1, Sonia Deschênes 1, Yan Burelle 1
PMCID: PMC3060365  PMID: 21224232

Abstract

In the present study, we specifically determined whether the regulatory protein cyclophilin-D (CypD), and by extension opening of the permeability transition pore (PTP), is involved in the activation of mitochondria-derived apoptotic signalling previously described in skeletal muscle following loss of innervation. For this purpose, CypD-defficient (CypD-KO) mice and their littermate controls were submitted to unilateral sciatic nerve transection, and mitochondrial resistance to Ca2+-induced opening of the PTP, and muscle apoptotic signalling were investigated 14 days post-surgery. Denervation caused atrophy, facilitated Ca2+-induced opening of the PTP in vitro in permeabilized muscle fibres, and activation of the apoptotic proteolytic cascade in the whole muscle of both mouse strains. In CypD-KO mice, mitochondrial resistance to Ca2+-induced PTP opening was greater than in WT mice, in both the normal and the denervated state, indicating that lack of CypD desensitized to PTP opening. However, denervation in CypD-KO mice still resulted in a facilitation of PTP opening compared to normally innervated contralateral muscle, indicating that in vitro additional factors could poise mitochondria from denervated muscle toward PTP opening. At the whole muscle level, lack of CypD, despite conferring greater resistance to PTP opening, did not protect against atrophy, release of mitochondrial pro-apoptotic factors and activation of caspases following denervation. Altogether, these results provide direct evidence that CypD-dependent PTP opening is dispensable for atrophy and apoptotic signalling in skeletal muscle following denervation.

Non-technical summary

Activation of apoptotic cell death signalling by mitochondria is involved in mediating skeletal muscle atrophy following denervation. However, the underlying molecular mechanisms remain unclear, in particular the role of the permeability transition pore (PTP), a high conductance non specific channel of the mitochondrial inner membrane involved in cell death. Using Ppif−/− mice, which are devoid of the PTP sensitizing protein cyclophilin-D and thus more resistant to pore opening, we directly tested the hypothesis that this pore is involved in atrophy and activation of apoptotic proteolytic signalling following denervation. Our results demonstrate that cyclophilin-D, and by extension opening of the PTP, is dispensable for atrophy and activation of apoptotic proteolytic signalling induced by denervation.

Introduction

Activation of apoptotic signalling by mitochondria is believed to be involved in muscle remodelling in several progressive neuromuscular diseases that are associated with atrophy, including denervation disorders, muscular dystrophy, mitochondrial myopathies, inactivity and ageing-induced sarcopaenia (Siu & Alway, 2005; Aure et al. 2006; Angelin et al. 2007; Marzetti et al. 2010). A key event in this process is the induction of permeation pathways across the inner mitochondrial membrane (IMM) and/or the outer mitochondrial membrane (OMM), which can occur independently or in a coordinated fashion. Several studies performed in denervation models have led to the suggestion that permeabilization of the OMM by pro-apoptotic members of the Bcl-2 protein family plays a role in this process by allowing the release of pro-apoptotic factors and subsequent activation of caspases 9 and 3 (Siu & Alway, 2005, 2006; Adhihetty et al. 2007). Direct support for this hypothesis was in fact provided by experiments which showed that genetic ablation of the pro-apoptotic Bcl-2 protein Bax attenuated denervation-induced apoptosis and atrophy (Siu & Alway, 2006).

Increased susceptibility to opening of the IMM permeability transition pore (PTP), another important event in the mitochondrial cell death cascade, as well as increased intracellular Ca2+ and mitochondrial oxidative stress, which are the primary factors promoting PTP opening, was also reported in muscle following denervation (Csukly et al. 2006; Adhihetty et al. 2007). We have also shown (Csukly et al. 2006) that in isolated mitochondria from denervated muscle, the expression of cyclophilin-D (CypD), a mitochondrial chaperone-like protein acting as an important endogenous sensitizer of the PTP under conditions of Ca2+ overload and oxidative stress (Basso et al. 2005), is increased. In this regard, several studies have shown that inhibition of CypD provides efficient protection against cell death in several cell types (Crompton, 1999; Baines et al. 2005). Moreover, inhibition of CypD was recently shown to ameliorate muscle pathology in several muscular dystrophies associated with Ca2+ dysregulation and oxidative stress, including Duchenne muscular dystrophy (Millay et al. 2008), Bethlem muscular dystrophy and Ulrich muscular dystrophy (Angelin et al. 2007; Palma et al. 2009). However, the role of CypD, and more generally of the PTP, in denervation-induced muscle remodelling has not been established.

In the present study, Ppif−/− mice, which are devoid of CypD, were therefore used to clarify the contribution of this protein, and by extension PTP opening, to atrophy and apoptotic signalling in denervated muscle. More specifically, we hypothesized that ablation of CypD, by increasing resistance to PTP opening, would attenuate denervation-induced atrophy and activation of apoptotic signalling events located immediately downstream of mitochondrial membrane permeation.

Methods

Ethics approval and animal care

All procedures were approved by the animal ethics committee of the Université de Montréal and were in accordance with the guidelines of the Canadian Council of Animal Care. The authors have also complied with the policies and regulations outlined by The Journal of Physiology (Drummond, 2009). Ppif+/− mice (provided by Dr M. Forte, University of Oregon) were bred to generate Ppif−/− (CypD-KO) and Ppif+/+ (WT) mice. At 8 weeks old, animals were submitted to unilateral transection of the sciatic nerve as per Csukly et al. (2006), while sham surgery was performed on the contralateral leg. Fourteen days after the surgery, gastrocnemius muscles were extracted, weighed and used for the assays described below.

Mitochondrial functional assays in permeabilized muscle fibres

Dissection and permeabilization of fibre bundles with saponin were performed using the white part of gastrocnemius as described previously (Picard et al. 2008). Ghost fibres were prepared as per Picard et al. (2008) by incubating saponin permeabilized bundles in a high KCl medium, which allows one to extract myosin. Permeabilized myofibres and ghost fibre bundles were then kept on ice until use. All mitochondrial parameters described below were determined at least in duplicate.

Calcium retention capacity

Ghost fibres were incubated at 23°C in q quartz microcuvette with continuous stirring in 600 μl of CRC buffer (in mm: 250 sucrose, 10 Mops, 0.005 EGTA, 10 Pi-Tris, pH 7.3) supplemented with glutamate–malate (5:2.5 mm) and 0.5 nm oligomycin. After addition of fibre bundles and respiratory substrates, mitochondria were exposed to a single pulse of 20 nm Ca2+. Changes in extra-mitochondrial calcium concentration were monitored fluorimetrically using Calcium green 5N. PTP susceptibility was assessed by measuring time required for PTP opening and calcium retention capacity (CRC). CRC was taken as the total amount of Ca2+ accumulated by mitochondria before its release by PTP opening. CRC values were expressed in nanomoles of Ca2+ per milligram of dry weight. A standard curve relating [Ca2+] to the fluorescence of Calcium green was performed to calculate the [Ca2+] in the microcuvette.

Caspase activity assay

Caspase activity in whole muscle homogenates was measured fluorimetrically on a plate reader using specific substrates for caspase 3 (Ac-DEVD-AFC) and caspase 9 (Ac-LEHD-AFC). Activity was expressed as Δfluorescence per second per milligram protein and normalized as a percentage of WT control.

Immunoblotting

Proteins in whole lysates and cytosolic fractions were resolved on 12.5% polyacrylamide mini-gels, and transferred onto PVDF membranes as per (Siu & Alway, 2005). Membranes were then exposed to the following antibodies: Bax (sc-526, Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), Bcl-2 (sc-492, Santa Cruz), Smac/Diablo (ab-9709, Abcam Inc., Cambridge, MA, USA), cytochrome c (556433, BD Biosciences, Franklin Lakes, NJ, USA), and lactate dehyrodgenase (LDH; sc-33781, Santa Cruz), which was used as a loading control.

Statistical analyses

Results are expressed as means ±s.e.m. Statistical differences were analysed using two-way ANOVA (Statistica package; StatSoft Inc., Tulsa, OK, USA). Tukey's post hoc test was used to identify the location of significant differences when the ANOVA yielded a significant F ratio. P values less than 0.05 were considered statistically significant.

Results

Morphometric data

In WT mice, 14 days of denervation caused an atrophy of the gastrocnemius muscle as evidenced by a 54% reduction in absolute muscle mass (112.6 ± 2.7 vs. 61.1 ± 1.3 mg, control vs. denervated respectively, P < 0.01). However, loss of muscle mass was of similar magnitude in CypD-KO mice (53%: 110.7 ± 3.6 vs. 58.6 ± 2.1 mg, control vs. denervated respectively, P < 0.01).

Mitochondrial sensitivity to PTP opening in permeabilized muscle fibres

In WT mice, denervation led to an expected reduction of time to PTP opening and CRC in response to the in vitro Ca2+ challenge (Fig. 1), indicating an increased sensitivity to permeability transition compared to mitochondria from normally innervated muscle fibres. In mitochondria from CypD-KO mice, time to PTP opening and CRC were 60–95% greater than in mitochondria from WT mice, in both the normal and the denervated state, indicating that lack of CypD generally desensitized the mitochondria to PTP opening. Despite this relative desensitization, denervation in CypD-KO mice still resulted in a reduction of time to PTP opening (−33% in CypD-KO vs.−31% in control) and CRC (−55% in CypD-KO vs.−56% in control) compared to the normally innervated contralateral muscle fibres.

Figure 1. Effect of denervation in Ca2+-induced PTP opening in vitro.

Figure 1

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A, typical mitochondrial Ca2+ uptake and release kinetics following a single pulse of 20 nmol Ca2+ in ghost fibres. For the sake of clarity only the tracings from CypD-KO mice are shown. B, time required to trigger PTP opening following addition of a single pulse of exogenous 20 nmol Ca2+. C, mitochondrial Ca2+ retention capacity expressed in nmol (mg dry weight)−1. Values are means ±s.e.m. for n= 6–9 in each group. Different from non-denervated within the same group: *P < 0.05, **P < 0.01; different from WT within the same treatment group (i.e. control or denervated): #P < 0.05, ##P < 0.01.

Cell death signalling in whole muscle

At the level of the whole muscle, the activities of caspase 9 and caspase 3, two downstream consequences of mitochondrial apoptotic signalling, were consistently increased following denervation in WT mice (Fig. 2A). This was associated with an increased abundance in the cytosolic fractions of some (Smac/Diablo), but not all (cytochrome c) mitochondrial pro-apoptotic factors involved in the activation of caspases (Fig. 2B), which is consistent with previously published data (Siu & Alway, 2005, 2006; Adhihetty et al. 2007). However, the magnitude of changes in caspase activities, and abundance of mitochondrial pro-apoptotic factors observed following denervation was similar in WT and CypD-KO mice, despite the fact that sensitivity to PTP opening was lower in the latter. Denervation also induced a significant increase in Bax content and a downregulation of Bcl-2 expression, which was unaffected by lack of CypD (Fig. 2C).

Figure 2. Effect of denervation on mitochondrial apoptotic signalling.

Figure 2

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A and B, respectively, the activity of caspase 3 and caspase 9 measured in the cytosolic fraction, and the amount of cytochrome c and Smac/Diablo in the cytosolic fraction from normal and denervated muscle in WT and CypD deficient mice. C, the expression of Bax and Bcl-2 in whole muscle homogenate. Expression of proteins is normalized against that of LDH, which was used as a loading control. Caspase activities are expressed in fluorescence units min−1 (mg protein)−1. All values represent means ±s.e.m. and are expressed as a percentage of WT control values. *Different from non-denervated within the same group: *P < 0.05, **P < 0.01.

Discussion

Previous studies have led to the suggestion that CypD, and by extension opening of the PTP (Csukly et al. 2006; Adhihetty et al. 2007), could be involved in the activation of mitochondria-derived apoptotic signalling previously described in skeletal muscle following loss of innervation (Tews, 2002; Siu & Alway, 2005, 2006; Adhihetty et al. 2007). However, this hypothesis was never tested directly. In the present study we provide novel evidence that genetic ablation of CypD, despite enhancing resistance to PTP, confers no protection against denervation-induced muscle atrophy, and activation of apoptotic proteolytic cascades. We also show that although CypD deficiency increases the Ca2+ threshold for PTP opening in permeabilized fibres, sensitization still occurs in response to denervation, suggesting that in the absence of CypD, additional factors poise mitochondria toward PTP opening in vitro.

Atrophy and mitochondrial apoptotic signalling in denervated muscle

Membrane permeabilization, and subsequent activation of the mitochondrial apoptotic pathway, is a complex mechanism involving distinct permeation events in the OMM (mediated by the Bcl-2 protein family) and IMM (mediated by PTP opening) (Crow et al. 2004; Tomasello et al. 2009). Although both events can occur independently, several lines of experimental evidence indicate that depending of the cellular insult, they can also occur in a coordinated fashion, with PTP opening sometime acting upstream, and sometime downstream of Bax recruitment (Tomasello et al. 2009), thus making it difficult to determine the respective contribution of OMM and IMM permeabilization in a situation such as denervation, in which numerous factors promoting apoptotic signalling are present.

Studies in humans and animals models, including the present study (Fig. 2B), have reported that Bax expression is strongly upregulated in denervated muscle, while Bcl-2 expression is reduced (Tews, 2002; Siu & Alway, 2005, 2006; Adhihetty et al. 2007). Furthermore, Siu & Alway (2006) showed that genetic inactivation of Bax attenuated muscle atrophy, mitochondrial release of pro-apoptotic factors, and activation of caspase 3 and 9 measured 14 days after denervation. These previous observations contrast with our current results, which indicate that ablation of CypD, despite causing an upward shift (by 80–95%) in the threshold for PTP opening, does not have these beneficial effects on mitochondrial apoptotic signalling and muscle atrophy. Taken together these results would thus suggest that permeabilization of the OMM by Bax is the main factor triggering mitochondrial apoptotic signalling in denervated muscle, and that opening of the PTP does not play a major role, despite the existence in denervated muscle fibres of the two primary conditions known to directly promote PTP opening, namely increased intracellular [Ca2+] (Joffe et al. 1981; Csukly et al. 2006) and oxidative stress (Abruzzo et al.; Adhihetty et al. 2007; Bhattacharya et al. 2009). Although we cannot completely rule out the possibility that the absence of CypD was insufficient to prevent PTP opening following denervation due to overwhelming conditions within muscle in vivo, we consider this unlikely considering that genetic ablation of CypD was previously shown to attenuate muscle pathology in other disease models associated with severe Ca2+ overload and oxidative stress including ischaemia–reperfusion (Baines et al. 2005; Nakagawa et al. 2005), various muscular dystrophies (Angelin et al. 2007; Millay et al. 2008; Palma et al. 2009), and overexpression of sarcolemmal L-type Ca2+ channels (Nakayama et al. 2007).

In the present experiments, we observed robust and systematic increases in caspases 9 and 3 in denervated muscle, which are hallmarks of mitochondrial apoptotic signalling. However, changes in the cytosolic content of mitochondrial pro-apoptotic factors involved in the activation of these caspases were less consistent, as Smac/Diablo content was increased by denervation, while cytochrome c was paradoxically reduced. This latter phenomenon appears to be due to the fact that denervation causes a reduction of whole muscle cytochrome c content, which may limit the amount available for release in the cytosol. Indeed, in additional experiments we observed a reduction in whole muscle cytochrome c content following denervation in the two mouse strains (−40 ± 3% compared to normally innervated muscle, pooled data from WT and CypD-KO mice, P= 0.01, n= 7 in each group), but no change in Smac/Diablo content. In a previous study, a similar reduction in muscle cytochrome c content was also reported following denervation, along with an increase in apoptosis inducing factor (AIF) content (Adhihetty et al. 2007). In addition, the rise in cytosolic cytochrome c content observed following denervation in other studies (Siu & Alway, 2005) was modest and substantially lower than for Smac/Diablo and AIF. Overall, these data therefore indicate that the measure of cytosolic cytochrome c content yields variable results in denervated muscle and may not be the best indicator of mitochondrial membrane permeabilization in this condition.

Susceptibility to PTP opening in vitro

Previous studies conducted in CypD-KO mice have shown that CypD acts as an endogenous ‘sensitizer’ to Ca2+-induced PTP opening (Baines et al. 2005; Basso et al. 2005; Nakagawa et al. 2005; Schinzel et al. 2005). Our data showing that time to PTP opening and CRC were systematically higher in muscle mitochondria from CypD-KO vs. control mice both in the normal and the denervated state are in line with these previous reports. Despite this upward shift in resistance to PTP opening in CypD-KO mice, denervation was still able to reduce time to PTP opening and CRC, which indicates that additional factors can poise mitochondria toward PTP opening. However, importantly, this relative sensitization to PTP opening was observed in vitro under conditions where mitochondria are submitted to excessive conditions of Ca2+ overload which are not present in vivo.

Although investigating the factors underlying denervation-induced sensitization in detail was not the objective of this study, previously published data from our laboratory and others have shown that basal Ca2+ levels are higher in isolated mitochondria from denervated muscle compared to levels observed in mitochondria from normally innervated muscle (Joffe et al. 1981; Csukly et al. 2006) bringing them closer to the Ca2+ threshold for PTP opening. Furthermore, a recent study (Bhattacharya et al. 2009) reported that mitochondria isolated from denervated muscle display enhanced activity of calcium-dependent cytosolic phospholipase A2 (cPLA2), which was previously shown to promote Ca2+-induced PTP opening through the release of fatty acids (Broekemeier & Pfeiffer, 1995; Zoratti & Szabo, 1995). These factors could contribute to explaining why in CypD-defficient mice, denervation still leads to a relative reduction in the amount of Ca2+ required to trigger PTP opening in mitochondria in vitro.

Conclusion

Prevention of PTP opening through inhibition of CypD was recently shown to reduce cell death signalling and muscle damage in several disease models associated with Ca2+ dysregulation and oxidative stress (Baines et al. 2005; Nakagawa et al. 2005; Angelin et al. 2007; Nakayama et al. 2007; Millay et al. 2008; Palma et al. 2009). However, results from the present study indicate that genetic ablation of CypD and the ensuing increase in resistance to PTP opening does not beneficially affect muscle atrophy, and activation of mitochondrial apoptotic signalling following the loss of muscle innervation. These novel data therefore provide direct evidence that CypD-dependent PTP opening is dispensable for apoptotic signalling in skeletal muscle following denervation.

Acknowledgments

This work was funded by a grant from the Natural Sciences and Engineering Council of Canada (NSERC) to Y.B. Y.B. is a Junior II Investigator of the Fonds de Recherche en Santé du Québec (FRSQ). The authors are grateful to Dr Micheal Forte for providing Ppif mice.

Glossary

Abbreviations

CRC

calcium retention capacity

cPLA2

cytosolic phospholipase A2

CypD

cyclophilin-D

IMM

inner mitochondrial membrane

OMM

outer mitochondrial membrane

PTP

permeability transition pore

Author contributions

All authors contributed to conception and design, or analysis and interpretation of data, and drafting the article or revising it critically for important intellectual content, and gave final approval of the version to be published.

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