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The Journal of Physiology logoLink to The Journal of Physiology
. 2016 Nov 29;594(24):7419–7433. doi: 10.1113/JP272885

Role of dynamin‐related protein 1‐mediated mitochondrial fission in resistance of mouse C2C12 myoblasts to heat injury

Tianzheng Yu 1, Patricia Deuster 1, Yifan Chen 1,✉
PMCID: PMC5157065  PMID: 27730652

Abstract

Key points

  • Understanding how skeletal muscles respond to high temperatures may help develop strategies for improving exercise tolerance and preventing heat injury.

  • Mitochondria regulate cell survival by constantly changing their morphology through fusion and fission in response to environmental stimuli. Little is known about the involvement of mitochondrial dynamics in tolerance of skeletal muscle against heat stress.

  • Mild heat acclimation and moderate heat shock appear to have different effects on the mitochondrial morphology and fission protein Drp1 in skeletal muscle cells. Mitochondrial integrity plays a key role in cell survival under heat stress.

Abstract

The regulation of mitochondrial morphology is closely coupled to cell survival during stress. We examined changes in the mitochondrial morphology of mouse C2C12 skeletal muscle cells in response to heat acclimation and heat shock exposure. Acclimated cells showed a greater survival rate during heat shock exposure than non‐acclimated cells, and were characterized by long interconnected mitochondria and reduced expression of dynamin‐related protein 1 (Drp1) for their mitochondrial fractions. Exposure of C2C12 muscle cells to heat shock led to apoptotic death featuring activation of caspase 3/7, release of cytochrome c and loss of cell membrane integrity. Heat shock also caused excessive mitochondrial fragmentation, loss of mitochondrial membrane potential and production of reactive oxygen species in C2C12 cells. Western blot and immunofluorescence image analysis revealed translocation of Drp1 to mitochondria from the cytosol in C2C12 cells exposed to heat shock. Mitochondrial division inhibitor 1 or Drp1 gene silencer reduced mitochondrial fragmentation and increased cell viability during exposure to heat shock. These results suggest that Drp1‐dependent mitochondrial fission may regulate susceptibility to heat‐induced apoptosis in muscle cells and that Drp1 may serve as a target for the prevention of heat‐related injury.

Keywords: apoptosis, heat acclimation, heat stress, mitochondrial division inhibitor‐1, mitochondrial dynamics, reactive oxygen species, skeletal muscle

Key points

  • Understanding how skeletal muscles respond to high temperatures may help develop strategies for improving exercise tolerance and preventing heat injury.

  • Mitochondria regulate cell survival by constantly changing their morphology through fusion and fission in response to environmental stimuli. Little is known about the involvement of mitochondrial dynamics in tolerance of skeletal muscle against heat stress.

  • Mild heat acclimation and moderate heat shock appear to have different effects on the mitochondrial morphology and fission protein Drp1 in skeletal muscle cells. Mitochondrial integrity plays a key role in cell survival under heat stress.

Abbreviations

AR

aspect ratio

Drp1

dynamin‐related protein 1

DHE

dihydroethidium

FF

Form factor

GFP

green fluorescence protein

HA

heat acclimation

HSF1

heat shock transcription factor 1

HSP

heat shock protein

IMF

intermyofibrillar

Mfn1

mitofusin 1

Mfn2

mitofusin 2

Mdivi‐1

mitochondrial division inhibitor 1

OPA1

optic atrophy 1

ROS

reactive oxygen species

SS

subsarcolemmal

TEM

transmission electron microscopy

TMRE

tetramethylrhodamine ethyl ester

Introduction

Skeletal muscle, which makes up ∼40–50% of the body mass in mammals, generates significant amounts of heat during contraction (Block, 1994). Muscle temperature can exceed 40°C during exercise (Taylor et al. 1998; Drust et al. 2005). Understanding the regulation of muscle function under high temperatures may help develop strategies for improving performance and preventing injury. Exposure to a temperature above the physiological range can produce both detrimental and beneficial effects on muscle health. In general, acute exposure to very high temperatures can cause apoptotic damage in muscle cells (Islam et al. 2013) and muscle injury in animals (Abdelnasir et al. 2014), whereas frequent exposures to moderately high temperatures or heat acclimation (HA) may protect muscle cells against a subsequent severe heat insult (Monastyrskaya et al. 2003; Liu & Brooks, 2012). In fact, HA has also been tested for cross‐tolerance or protection against other pathological conditions (Horowitz et al. 2015). Interestingly, HA has been shown to protect against obesity‐induced insulin resistance by increasing heat shock protein 72 in skeletal muscles of mice (Chung et al. 2008; Gupte et al. 2009). How muscle cells paradoxically respond to mild versus severe heat stress remains poorly understood. The majority of studies of the adaptation and resistance of muscle cells to heat have focused on mechanisms involving heat shock transcription factor 1 (HSF1) and heat shock proteins (HSPs) (Tetievsky et al. 2008; Abdelnasir et al. 2014). Emerging evidence suggests that cellular organelles, including mitochondria, endoplasmic reticulum, lysosomes and the Golgi apparatus, serve key roles in stress/damage‐sensing and apoptosis signalling. How they respond to the disruption of cellular homeostasis by high temperatures may ultimately determine the fate of stressed cells (Ferri & Kroemer, 2001).

Mitochondria regulate cell survival and coordinate cell‐wide stress responses by constantly changing their morphology through fusion and fission in response to energy demands and environmental stimuli (Hoppins & Nunnari, 2012; Youle & van der Bliek, 2012). Disruption in these processes causes mitochondrial dysfunction, and leads to the pathogenesis of various acute and chronic diseases (Archer, 2013). High temperatures indeed affect mitochondrial function, and impair mitochondrial electron transport; this condition also induces the production of reactive oxygen species (ROS) under in vitro (Wang et al. 2013) and in vivo (Qian et al. 2004) conditions. Altered mitochondrial morphology has also been observed in heat shock‐exposed cultured mouse embryonic fibroblasts (Sanjuan Szklarz & Scorrano, 2012). How changes in mitochondrial morphology might affect resistance of cultured muscle cells, in particular C2C12 myoblasts, to heat is largely unexplored.

In mammals, mitochondrial morphology is coordinately regulated by several GTPases, dynamin‐related protein 1 (Drp1) for fission and mitofusin isoforms (Mfn1 and 2), and optic atrophy1 (OPA1) for fusion, respectively (Ishihara et al. 2006). The overall morphology and shape of mitochondria are determined by the balance between mitochondrial fission and fusion activities (Hoppins & Nunnari, 2012; Youle & van der Bliek, 2012; Archer, 2013). The role of mitochondrial dynamics proteins in responses to HA and heat stress has yet to be determined.

In this study, we investigated changes in mitochondrial morphology and dynamics of C2C12 mouse skeletal muscle cells exposed to HA or lethal heat shock. We discovered that HA‐induced resistance of these cells against heat injury is associated with mitochondrial elongation mediated by down‐regulation of the fission protein, Drp1. We demonstrate that exposing C2C12 cells to heat shock causes cell damage characterized by excessive mitochondrial fragmentation with increased recruitment of Drp1 to mitochondria. We further show that inhibition of mitochondrial fission by mitochondrial division inhibitor (Drp1 translocation blocker) Mdivi‐1 or by Drp1 gene silencer protects mitochondrial morphology and prevents cell injury during exposure to heat shock. Together, our work reveals a novel mechanism regulating resistance of C2C12 cells to heat stress, which may have relevance to heat injury prevention.

Methods

Cell culture, myogenic differentiation, heat acclimation and heat shock

The mouse skeletal myoblast cell line C2C12 was recently purchased from ATCC (ATCC® CRL‐1772™) and was maintained at 37°C in DMEM containing 10% fetal bovine serum, 100 U ml–1 penicillin and 100 μg ml–1 streptomycin. Myogenic differentiation was initiated upon reaching ∼80% confluence by switching the cells to DMEM containing 2% horse serum supplemented with 1 μM insulin. For heat acclimation, cells were placed in an incubator preset at 39.5°C for 3 h per day for 3 days. Control cells and HA cells (when not being incubated at 39.5°C) were maintained at 37°C for 3 days. After the 3 day incubation, cells were tested for cell viability during exposure to heat shock. For heat shock, cells were maintained in an incubator preset at 43°C for indicated times. Immediately after heat shock, cells were harvested for subsequent assays.

Mouse heat acclimation protocol

Mice (6–8 weeks old, male C57BL/6J) were purchased from Jackson Laboratories (Bar Harbor, ME, USA) and maintained in a temperature‐controlled (21°C) animal facility at the Uniformed Services University (USU), with 12 h light/dark cycle and free access to food and water. After arrival, all the mice were given 1 week to recover. Heat acclimation was initiated by exposing the mice to 33°C in an environmental chamber from 0800 to 0011 h (3 h per day) for ten consecutive days. Control mice were maintained at room temperature (21°C). All procedures were approved by the USU Institutional Animal Care and Use Committee.

TEM analysis of mitochondria in mouse skeletal muscle

Immediately after heat or sham exposure, mice were perfusion fixed under isoflurane anaesthesia. The gastrocnemius muscle was removed and incubated in fixative (5% formaldehyde, 2% glutaraldehyde in 0.1 M PBS, pH 7.4) overnight. The samples were then sent to the Biomedical Instrument Center at USU for a standard transmission electron microscopy (TEM) preparation procedure. The gastrocnemius muscle comprises a mixture of type I, type IIa, type IIb and type IIx (IId) muscle fibres that range in their mitochondrial content (Shortreed et al. 2009; Gouspillou et al. 2014; Mishra et al. 2015). For comparison, only mitochondria (oxidative)‐enriched fibres (IIa, IIx/d and/or I) were selected and areas presenting all the classical structures of sarcomere were imaged. For each animal, six to eight muscle fibres were analysed, and a total of 200 subsarcolemmal (SS) and 200 intermyofibrillar (IMF) mitochondria were individually traced by using NIH‐developed ImageJ software to quantify the following morphological and shape descriptors (Picard et al. 2013): surface area, perimeter, circularity [4π·(surface area/perimeter2)] and Feret's diameter (longest distance between any two points within a given mitochondrion). Form factor (FF), a measure sensitive to the complexity and branching aspect of mitochondria, was calculated as perimeter/(4π·surface area) and aspect ratio (AR), a measure of the ‘length to width ratio’, was calculated as major axis/minor axis.

Cell viability and cell death assays

Cell viability was determined by the trypan blue exclusion test with a Bio‐Rad TC20 automated cell counter per the manufacturer's instruction. Caspase activity was measured by CellEvent™ Caspase‐3/7 Green detection reagent (Molecular Probes, Carlsbad, CA, USA), and dead cells were detected by using an Annexin V Alexa Fluor® 488 apoptosis kit (Molecular Probes).

Mitochondrial morphology analysis and membrane potential and ROS measurement

Mitochondria were labelled with CellLight Mitochondria‐GFP (Molecular Probes) or visualized by MitoTracker Red/Green sCMXRos (Invitrogen) staining. Quantitative analyses of mitochondrial morphology and Drp1 subcellular distribution were performed using ImageJ software (NIH Image) (Yu et al. 2014). Mitochondrial membrane potential was evaluated with tetramethylrhodamine ethyl ester (TMRE, Molecular Probes) and ROS levels were detected by using dihydroethidium, a fluorescent probe (DHE; Invitrogen), as described previously (Yu et al. 2014).

Fluorescence microscopy

Fluorescence images were viewed and acquired with a Leica AF6000 epifluorescence microscope, which was equipped with a digital microscope camera. Excitation/emission wavelengths were 358/461 nm for blue fluorescence (4′,6‐diamidino‐2‐phenylindole, DAPI), 480/535 nm for green fluorescence (GFP, annexin V, Alexa 488, MitoTracker Green and caspase‐3/7 Green), and 555/613 nm for red fluorescence (MitoTracker Red, TMRE, ethidium and Alexa 594).

Mitochondria isolation

Mitochondria were isolated as described previously (Yu et al. 2014). Briefly, the cells were suspended in cold isolation buffer (10 mM Hepes, pH 7.2, 1 mM EDTA, 320 mM sucrose) containing protease inhibitor and homogenized in a Dounce homogenizer. The homogenate was centrifuged at 700 g for 8 min. The first supernatant was saved, and the pellet was homogenized and centrifuged again. The two supernatants were pooled and centrifuged together at 17,000 g for 15 min to obtain the mitochondrial pellet.

Western blotting and immunofluorescence

Western blotting was performed with 1:1000 of the following primary antibodies (Yu et al. 2014): mouse anti‐Drp1 (BD Biosciences, Franklin Lakes, NJ, USA), rabbit anti‐ phospho‐Drp1‐Ser616 (Cell Signaling Technology, Danvers, MA, USA), rabbit anti‐Mfn1 (Santa Cruz Biotech, Santa Cruz, CA, USA), rabbit anti‐Mfn2 (Cell Signaling Technology), mouse anti‐OPA1 (BD Biosciences), rabbit anti‐VDAC (Cell Signaling Technology), mouse anti‐cytochrome c (Santa Cruz Biotech) and mouse anti‐actin (Santa Cruz Biotech). Horseradish peroxidase‐conjugated anti‐rabbit and anti‐mouse antibodies were used as secondary antibodies.

Indirect immunofluorescence was performed as described previously (Yu et al. 2014). Briefly, cells cultured on coverslips were fixed in 4% paraformaldehyde and permeabilized with 0.1% Triton X‐100. Mouse anti‐Drp1 (BD Biosciences) antibody was used for the primary antibody and Alexa 594‐conjugated antibodies (Invitrogen) were used for the secondary antibody. Fluorescence images were acquired and adjusted by using ImageJ software (NIH Image).

Statistical analysis

Data are expressed as mean ± standard deviations. Statistical significance was established by a t test, or one‐ or two‐way ANOVA followed by post hoc test for comparisons. Comparisons of mitochondrial morphological parameters were carried out using the non‐parametric Mann–Whitney test.

Results

Heat acclimation improved cell viability during heat shock exposure and modified mitochondrial morphology in C2C12 myoblasts

Following treatment with HA, C2C12 myoblasts showed significantly higher survival rates during exposure to heat shock compared to control cells (Fig. 1 A). Morphological examination revealed that HA‐treated myoblasts contained overly elongated mitochondrial networks, whereas control cells maintained predominantly tubular mitochondria (Fig. 1 B). Furthermore, expression of mitochondrial fission protein Drp1 was lower in the mitochondrial fractions from HA‐treated cells compared to control cells (Fig. 1 C). We also performed Western blotting of VDAC and actin in whole cell lysate and found no differences in the ratio of VDAC to actin between control and HA cells (1.13 ± 0.05 vs 1.16 ± 0.04, P > 0.05). No changes in mitochondrial fusion proteins OPA1, and Mfn 1 and 2 were found in HA‐treated cells (Fig. 1 D). Mitochondrial morphology and dynamics were further examined in the gastrocnemius muscles of control and HA‐exposed mice. TEM images revealed larger SS and IMF mitochondria in the muscles of HA‐exposed mice, compared to control mice (Fig. 2 A). Further quantitative analysis of major mitochondrial shape parameters indicated an increase in size of SS and IMF mitochondria in the muscles of HA‐exposed mice (Table 1 and Fig. 2 B). Finally, expression of Drp1 was lower in the mitochondrial fractions of the gastrocnemius muscles from HA‐exposed mice compared to control mice (Fig. 2 C).

Figure 1. Heat acclimation (HA) improves cell viability during heat shock exposure and affects mitochondrial morphology and Drp1 in C2C12 myoblasts.

Figure 1

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A, viability of control and HA‐treated C2C12 cells during incubation at 43°C. Control cells were maintained at 37°C for 3 days, whereas HA‐treated cells were incubated at 39.5°C for 3 h per day (at 37°C for all other times) for 3 days. Data were obtained from three experiments; * P < 0.01 versus control by two‐way ANOVA/Bonferroni post hoc test. B, representative immunofluorescence images of mitochondria (green) and Drp1 distribution (red), and percentages of cells with elongated mitochondria in control and HA‐treated C2C12 cells following a 4 h incubation at 43°C. Data were obtained from three independent experiments and ∼300 cells were counted. DAPI: blue fluorescent DNA dye; scale bar = 5 μm. C, Western blot analysis of Drp1 in mitochondrial fractions from C2C12 cells following 3 days of heat acclimation. Experiments were repeated at least three times. * P < 0.001 versus control. D, representative Western blot of OPA1, Mfn1 and Mfn2 in control and HA‐treated C2C12 cells. [Colour figure can be viewed at wileyonlinelibrary.com]

Figure 2. Heat acclimation (HA) causes morphological changes in mitochondria and reduces Drp1 in the mitochondrial fraction of mouse gastrocnemius muscles.

Figure 2

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A, representative transmission electron micrographs of subsarcolemmal (SS) and intermyofibrillar (IMF) mitochondria in the gastrocnemius muscles of control and HA mice. Scale bar = 5 μm. B, bar charts of morphological parameters for SS and IMF mitochondria in the gastrocnemius muscles of control and HA mice; n = 200 SS and 200 IMF mitochondria. C, Western blot analysis of Drp1 in mitochondrial fractions from the gastrocnemius muscles of control and HA mice. HA mice were exposed to 33°C in an environmental chamber for 3 h per day (at 21°C all other times) for ten consecutive days; control mice were maintained at 21°C for ten consecutive days. * P < 0.001 versus control, n = 3 per group.

Table 1.

Morphological parameters for SS and IMF mitochondria in the gastrocnemius muscles of control and HA mice

Median Mean (SD) Min. Max. Skewness
SS
Surface area (μm2) Control 0.18 0.24 (0.21) 0.04 1.19 1.61
HA 0.39 * 0.44 (0.26) 0.10 1.54 1.76
Perimeter (μm) Control 1.89 2.32 (1.33) 0.92 9.32 2.00
HA 3.08 * 3.25 (1.28) 1.27 9.32 1.77
Circularity (0–1) Control 0.56 0.56 (0.14) 0.20 1 0.20
HA 0.54 0.55 (0.15) 0.21 1 0.22
Feret's diameter (μm) Control 0.70 0.92 (0.52) 0.33 3.30 1.49
HA 1.17 * 1.25 (0.48) 0.44 3.30 1.41
Form factor Control 1.78 1.91 (0.55) 1.00 4.98 1.45
HA 1.85 1.98 (0.62) 1.00 4.76 1.18
Aspect ratio Control 2.66 2.86 (1.10) 1.02 6.20 0.87
HA 2.45 * 2.67 (1.17) 1.02 7.78 1.52
IMF
Surface area (μm2) Control 0.06 0.07 (0.05) 0.02 0.41 2.99
HA 0.18 * 0.25 (0.22) 0.04 1.23 1.77
Perimeter (μm) Control 1.14 1.21 (0.36) 0.58 3.39 2.10
HA 1.89 * 2.32 (1.34) 0.92 9.32 2.00
Circularity (0–1) Control 0.61 0.60 (0.12) 0.28 1 0.20
HA 0.56 * 0.56 (0.14) 0.20 1 0.20
Feret's diameter (μm) Control 0.44 0.47 (0.15) 0.22 1.29 1.91
HA 0.70 * 0.92 (0.53) 0.33 3.30 1.49
Form factor Control 1.65 1.73 (0.36) 1.00 3.61 1.11
HA 1.78 * 1.91 (0.55) 1.00 4.98 1.45
Aspect ratio Control 2.40 2.54 (0.81) 1.02 5.80 0.75
HA 2.57 2.80 (1.11) 1.02 6.20 0.96
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*

P < 0.01 versus control by non‐parametric Mann–Whitney test. n = 200 SS and 200 IMF mitochondria for each of control and HA groups.

Exposure to heat shock caused apoptotic cell death

The survival rates of both C2C12 myoblasts and myotubes decreased with duration of heat shock exposure in a similar manner: < 70% survived after exposure to heat shock for 4 h (Fig. 3 A). We examined mitochondrial events, known to occur during programed cell death (apoptosis), in C2C12 myoblasts following exposure to heat shock. Immunofluorescence staining and Western blotting showed that cytochrome c was located inside mitochondria under normal incubation, but was released from mitochondria into the cytosol of C2C12 myoblasts during heat shock exposure (Fig. 3 B). Cell injury was further assessed by using Caspase 3/7 Green and Annexin V Apoptosis Detection kits. Heat shock‐exposed C2C12 myoblasts became Caspase 3/7‐ (Fig. 3 C) and Annexin V‐positive (Fig. 3 D).

Figure 3. Heat shock exposure induces apoptotic cell death.

Figure 3

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A, viability of C2C12 myoblasts and myotubes during incubation at 43°C. Cell viability was measured by trypan blue exclusion assay. Data were obtained from three experiments; * P < 0.01 versus 0 h by one‐way ANOVA with Dunnett's post hoc test. B, representative immunofluorescence images of cytochrome c (red) and cytosolic expression of cytochrome c in C2C12 myoblasts following a 4 h incubation at 37 and 43°C. DAPI: blue fluorescent DNA dye; scale bar = 5 μm. C, representative fluorescence images and quantitative analysis of caspase‐3/7 activities in C2C12 myoblasts following a 4 h incubation at 37 and 43°C. Experiments were repeated at least three times; * P < 0.01 versus 37°C. Scale bar = 2 μm. D, representative fluorescence images of Annexin V in C2C12 myoblasts and quantitative analysis of Annexin V‐positive C2C12 myoblasts and myotubes following a 4 h incubation at 37 and 43°C. Experiments were repeated at least three times and data were obtained from ten observation fields for each group; * P < 0.001 versus 37°C. Scale bar = 5 μm. [Colour figure can be viewed at wileyonlinelibrary.com]

Exposure to heat shock caused mitochondrial fragmentation in C2C12 myoblasts

We examined mitochondrial morphology in C2C12 cells after exposure to heat shock. Mitochondria in C2C12 myotubes were small and densely packed, and very difficult to discern. C2C12 myoblasts contained more conventional tubular mitochondria under our experimental conditions and thus were used for mitochondrial morphology analysis. Mitochondria form filamentous and often interconnected networks under normal incubation at 37°C, but the mitochondrial networks became mostly small and punctate units in C2C12 myoblasts after exposure to heat shock (Fig. 4 A). We conducted time course experiments and found that mitochondria rapidly became fragmented when exposed to heat shock (Fig. 4 B): the number of cells containing fragmented mitochondria increased from ∼10% at 0 min to ∼47% at 15 min and reached a plateau of ∼91% at 30 min and thereafter.

Figure 4. Heat shock exposure causes mitochondrial fragmentation in C2C12 myoblasts.

Figure 4

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A, representative fluorescence images showing mitochondria morphology (red) in C2C12 myoblasts following a 30 min incubation at 37 and 43°C. Insets, higher magnification of indicated area. DAPI: blue fluorescent DNA dye; scale bar = 5 μm. B, percentage of C2C12 myoblasts with tubular and fragmented mitochondria following a 15–120 min incubation at 43°C. Experiments were repeated at least three times and ∼300 cells were counted per experiment. * P < 0.001 versus Tubular; # P < 0.001 versus 0 min by two‐way ANOVA with Bonferroni post hoc test. [Colour figure can be viewed at wileyonlinelibrary.com]

Exposure to heat shock caused activation of mitochondrial fission in C2C12 myoblasts

Mitochondrial structural dynamics are regulated by the fusion and fission of the organelles with an increase in fission activity, which results in mitochondrial fragmentation. Therefore, we assessed mitochondrial fission protein Drp1. Whole cell lysate immunoblotting showed no significant changes in total Drp1 after exposure to heat shock (Fig. 5 A). We further examined cytosolic and mitochondrial Drp1 levels in C2C12 cells after incubation at 43°C for 0, 15, 30 and 60 min (Fig. 5 A). We found a time‐dependent increase in the mitochondrial fractions of Drp1 and concurrent decrease in its cytosolic fractions. To verify heat‐induced mitochondrial translocation of Drp1, we performed immunofluorescence image and intensity analysis of Drp1 and mitochondria in C2C12 cells (Fig. 5 B). Under normal incubation, Drp1 predominantly resided throughout the cytosol, with limited association with the tubular mitochondria. After exposure to heat shock, cytosol Drp1 decreased, whereas the majority of Drp1 punctate units were co‐localized with the fragmented mitochondria. Western blot analysis also showed significantly increased phosphorylation of Drp1 at serine‐616 in heat shock‐exposed C1C12 myoblasts (Fig. 5 C).

Figure 5. Heat shock exposure induces mitochondrial translocation and phosphorylation of Drp1 in C2C12 myoblasts.

Figure 5

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A, Western blot analysis of Drp1 in whole cell lysates (total) and in mitochondrial (mito) and cytosol (cyto) fractions from C2C12 myoblasts during a 60 min incubation at 43°C. * P < 0.01 versus 37°C by one‐way ANOVA with Tukey's post hoc test. B, immunofluorescence images (left) and intensity profiles (right) of Drp1 (red) and mitochondria (green) in C2C12 myoblasts following a 30 min incubation at 37 and 43°C. Immunofluorescence intensities along the arrows were calculated and plotted (Drp1 in red and mitochondria in green) against the corresponding distances (pixels) of the arrows (right). N: nucleus. Scale bar = 2 μm. C, Western blot analysis of Drp1 phosphorylation at Ser616 in C2C12 myoblasts during a 60 min incubation at 43°C. * P < 0.001 versus 37°C, # P < 0.01 versus 15 or 30 min by one‐way ANOVA with Tukey's post hoc test. [Colour figure can be viewed at wileyonlinelibrary.com]

Inhibition of mitochondrial fission protected cell viability and mitochondrial structural integrity against heat shock

To determine whether resistance of cells to heat injury is mediated by mitochondrial dynamics, we tested the effects of inhibiting mitochondrial fission on cell viability by Mdivi‐1 and Drp1 shRNA, which inhibits Drp1 assembly and GTPase activity (Cassidy‐Stone et al. 2008) and suppresses the transcription of the Drp1 gene in infected cells, respectively. Pretreatment with Mdivi‐1 or Drp1 shRNA significantly improved the survival rates of C2C12 myoblasts during exposure to heat shock and significantly reduced the number of Annexin V‐positive apoptotic cells (Fig. 6 A). Drp1 shRNA also prevented cytochrome c release and caspase activation in C2C12 myoblasts during exposure to heat shock (Fig. 6 B).

Figure 6. Inhibition of mitochondrial fission protects cell viability and mitochondrial morphology during heat shock exposure.

Figure 6

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A, cell viability of C2C12 myoblasts during incubation at 43°C and quantitative analysis of Annexin V‐positive cells following a 4 h incubation at 43°C. Cells were labelled with an Annexin V apoptosis detection kit and were pretreated with vehicle or Mdivi‐1 or infected with Drp1 shRNA. Data were obtained from ten observation fields for each group; * P < 0.01, shRNA versus vehicle; # P < 0.01, Mdivi‐1 versus vehicle by two‐way ANOVA with Bonferroni post hoc test for cell viability. * P < 0.001 versus vehicle for Annexin V by one‐way ANOVA with Dunnett's post hoc test. B, effects of Drp1 shRNA on cytosolic cytochrome c (cyt c) and caspase 3/7 activity in C2C12 myoblasts following a 4 h incubation at 43°C. C2C12 myoblasts were infected with vehicle (veh) or Drp1 shRNA before being exposed to 43°C. C, representative images of mitochondrial morphology in C2C12 myoblasts and quantitative analysis of C2C12 myoblasts with fragmented mitochondria following 30 min of incubation at 43°C. Cells were pretreated with vehicle or Mdivi‐1 or infected with Drp1 shRNA. Data were obtained from three experiments and ∼300 cells were counted; * P < 0.001 versus vehicle by one‐way ANOVA with Dunnett's post hoc test. Scale bar = 5 μm. D, representative Western blot of Drp1 in C2C12 myoblasts pretreated with vehicle or GFP or infected with Drp1 shRNA.

Mitochondrial morphology analysis revealed a significant reduction in the number of fragmented mitochondria in C2C12 myoblasts pretreated with Mdivi‐1 or infected with Drp1 shRNA following heat shock exposure (Fig. 6 C). Only ∼5.3% of Mdivi‐1‐treated cells contained fragmented mitochondria compared to ∼82.5% of cells pretreated with vehicle. A similar reduction in mitochondrial fragmentation (> 93% cells maintained tubular mitochondrial networks) was found in cells infected with Drp1 shRNA. Western blot analysis showed that expression of Drp1 protein was reduced by ∼80% in Drp1 shRNA‐treated cells, compared to cells infected with vehicle control, i.e. Lentiviral Particle carrying copGFP (Santa Cruz) (Fig. 6 D).

Inhibition of mitochondrial fission reduced loss of mitochondrial membrane potential and production of ROS in C2C12 myoblasts caused by heat shock

We measured mitochondrial membrane potential magnitude in C2C12 myoblasts labelled with MitoTracker Green and TMRE. Exposure to heat shock caused a significant decline of mitochondrial TMRE, but not MitoTracker Green fluorescence in C2C12 myoblasts (Fig. 7 A). Time‐lapse experiments further revealed that the decrease in mitochondrial TMRE fluorescence occurred as early as ∼20–25 min (Fig. 7 B). Pretreatment with Mdivi‐1 or Drp1 shRNA prevented heat shock‐induced loss of mitochondrial membrane potential in C2C12 cells (Fig. 7 C).

Figure 7. Inhibition of mitochondrial fission reduces loss of mitochondrial membrane potential and production of ROS in C2C12 myoblasts during heat shock exposure.

Figure 7

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A, representative images and quantitative analysis of MitoTracker Green (mito) and TMRE fluorescence in C2C12 myoblasts following a 30 min incubation at 37 and 43°C. Cells were stained with MitoTracker Green FM and TMRE, which accumulate in mitochondria based on mass and proton electrochemical gradient, respectively. MitoTracker and TMRE fluorescence signals were acquired simultaneously. B, real time measurements of TMRM fluorescence intensity in C2C12 cells during incubation at 37 and 43°C. n = 9. The validity of TMRM as an indicator of mitochondrial membrane potential was verified by using a mitochondria uncoupler, carbonyl cyanide 4‐trifluoro‐methoxyphenylhydrazone (FCCP, 2 μM). C, quantitative analysis of TMRE fluorescence as a function of incubation time at 43°C. Cells were pretreated with vehicle or Mdivi‐1 or infected with Drp1 shRNA. n = 30; * P < 0.01 versus 37°C by one‐way ANOVA with Dunnett's post hoc test. D, representative DHE fluorescence images of C2C12 myoblasts following incubation at 43°C for 4 h and quantitative analysis of DHE fluorescence as a function of incubation time at 43°C. Cells were pretreated with vehicle or Mdivi‐1 or infected with Drp1 shRNA. n = 30; * P < 0.01, shRNA versus vehicle; # P < 0.01, Mdivi‐1 versus vehicle by two‐way ANOVA with Bonferroni post hoc test. [Colour figure can be viewed at wileyonlinelibrary.com]

Exposing C2C12 myoblasts to heat shock caused a time‐dependent increase in DHE fluorescence, an indicator of ROS (Fig. 7 D). The fluorescence signal increased from baseline to ∼2‐fold at 1 h and ∼5‐fold at 4 h of exposure to heat shock. Pretreatment with Mdivi‐1 or Drp1 shRNA prevented an increase in ROS in C2C12 cells induced by heat shock.

Exposure to heat shock caused OPA1 cleavage and loss of mitochondrial membrane potential in C2C12 myoblasts in a time‐dependent manner

We examined mitochondrial fusion proteins in C2C12 myoblasts exposed heat shock. No significant changes in Mfn1 and Mfn2 levels were found in C2C12 cells following up to 60 min of heat shock exposure (Fig. 8 A). Western blot analysis of whole cell lysates with an OPA1 antibody (BD Biosciences) revealed at least one long and one short form of OPA1 in C2C12 cells before exposure to heat shock (Fig. 8 B). No changes in OPA1 levels were detected in cells 15 min into heat exposure. The long form of OPA1 significantly decreased and a new short form band appeared, which indicates cleavage of high molecular weight forms of OPA1 at 30 min into heat exposure.

Figure 8. Heat shock exposure causes OPA1 cleavage and concurrent loss of mitochondrial membrane potential in C2C12 myoblasts.

Figure 8

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A, Western blot analysis of mitochondrial outer membrane fusion proteins, Mfn1 and Mfn2, in C2C12 cells following incubation at 43°C for 0–60 min. B, Western blot analysis of short (S) and long (L) forms of mitochondrial inner membrane fusion protein OPA1 in C2C12 cells following incubation at 43°C for 0–60 min. * P < 0.01 versus 37°C by one‐way ANOVA with Tukey's post hoc test.

Discussion

Fusion–fission dynamics adapt the morphology of the mitochondrial compartment to various metabolic needs of cells and enable them to regain homeostasis and survive under many stress conditions. The present study showed that HA and heat shock affects mitochondrial morphology in C2C12 cells primarily by reducing and increasing recruitment of the fission mediator Drp1 to mitochondria. Our results demonstrate key mitochondrial events involved in the HA and heat shock responses and provide evidence supporting a new mechanism – Drp1‐mediated mitochondrial fission – underlying the susceptibility of muscle cells to heat‐induced injury.

Mitochondria typically form a reticular network radiating from the nucleus to create an interconnected system for ensuring essential energy supplies to the cell; they are able to adapt to environmental challenges by remodelling their morphology. The formation of elongated mitochondrial networks is a conserved mechanism engaged during chronic cellular stress (e.g. nutrient deprivation) as a means to maintain ATP production, avoid mitophagy and improve tolerance of mtDNA mutations by diluting damaged mitochondrial contents across the mitochondrial network (Tondera et al. 2009; Rambold et al. 2011; Mishra et al. 2015; Senyilmaz et al. 2015). On the other hand, fragmented mitochondria are associated with various pathological conditions (Yoon et al. 2011; Galluzzi et al. 2012; Hoppins & Nunnari, 2012; Youle & van der Bliek, 2012; Archer, 2013). In the present study, we expanded previous findings by showing that HA‐exposed mouse skeletal muscle tissue and cells shared a common feature of elongated mitochondria: HA resulted in increased resistance against heat shock‐induced injury in muscle cells. Mitochondrial networks are maintained through the complex coordination of fission and fusion regulated by a number of key mitochondrial morphology proteins. Our results indicate that HA‐induced mitochondrial morphological remodelling is due to an imbalance favouring fusion over fission, which is caused by down‐regulation of Drp1. The mechanisms behind these changes remain unclear. We are not aware of any studies linking HA to cellular energy stress, but HA may probably have an indirect effect on mitochondrial morphology. For instance, HA is known to cause an adaptation response with up‐regulation of HSF1 and HSP70s (Tetievsky et al. 2008). Expression of HSP72 is critical in promoting mitochondrial fusion in skeletal muscle (Drew et al. 2014). A single bout of 1 h of mild heat exposure (40°C) is reportedly sufficient to cause up‐regulation of HSP72 in C2C12 cells (Liu & Brooks, 2012). In the present study, HA‐treated cells probably had upregulated HSP72, which helped maintain mitochondrial homeostasis and morphology during heat shock exposure.

Although fused or elongated mitochondrial networks help cells survive starvation, it is unknown whether this morphological adaptation in mitochondria is associated with resistance of cells against heat insults. The present study suggests that elongation of mitochondria with decreased fission may be advantageous under lethal heat conditions. We showed that heat shock caused mitochondrial fragmentation and Drp1 translocation in control but not in HA‐treated muscle cells. The homeostatic alterations in mitochondrial fission and morphology in HA‐treated cells may limit recruitment of Drp1 and resist mitochondrial breakdown under heat stress. In addition, as discussed above, HA is known to cause overexpression of heat stress proteins. HSP70 can inhibit stress protein kinases (Gabai et al. 1997) that activate Drp1 under mitochondrial stress (Kashatus et al. 2015; Park et al. 2015). Thus, through complex mechanisms, HA may alter mitochondrial morphology and dynamics to protect cells against mitochondria‐dependent apoptosis during heat exposure.

Our results provide evidence that cells exposed to lethal heat shock undergo apoptotic death. Limited information is available on how heat stress initiates apoptosis signalling. Apoptotic cell death is initiated and activated by diverse internal or external pro‐apoptotic insults. Depending on the type of stimulus, the apoptotic signalling cascades undergo a mitochondrial‐dependent, ligand‐mediated death receptor, or an endoplasmic reticulum stress‐induced pathway (Adams, 2003). The execution phase of apoptosis usually involves activation of a family of protease proteins called caspases. Heat‐induced apoptosis of C2C12 cells is probably activated primarily through an intrinsic, mitochondria‐dependent pathway. Several events, including mitochondrial oxidative stress, observed in cells exposed to heat shock are linked to activation of caspases, which probably serve as the primary mediators of apoptosis (Primeau et al. 2002; Palmer et al. 2011; Rigoulet et al. 2011). These cellular events ultimately lead to changes in the integrity of the mitochondrial membrane (Westphal et al. 2011). Loss of mitochondrial integrity results in release of pro‐apoptotic proteins, including cytochrome c, which facilitate recruitment of caspases to form the apoptosome. This downstream caspase activation results in subsequent apoptotic cell death under heat conditions (Katschinski et al. 2000; Wang et al. 2013). Thus, several primary pro‐apoptotic factors clearly contributed to initiation and activation of heat‐induced apoptosis of C2C12 cells.

In the present study, we demonstrated that HA improved the viability of C2C12 cells against heat. Reduced mitochondrial Drp1 and increased mitochondrial size were found in both C2C12 cells and mouse gastrocnemius muscles following HA. Whether these changes directly contributed to HA‐induced heat tolerance of cells remains unclear. Mitochondrial apoptotic susceptibility to heat stress is probably dependent upon the ratio of pro‐ and anti‐apoptotic Bcl‐2 family proteins, including pro‐apoptotic Bax and anti‐apoptotic Bcl‐2 (Primeau et al. 2002). Further investigation of HA effects on Bcl‐2 family proteins should provide information about the improved ability of HA‐treated cells to survive subsequent heat stress. Mitochondrial fission is involved in skeletal muscle differentiation and growth. Inhibition of mitochondrial fission has been shown to impair myogenesis (Kim et al. 2013), whereas severe burn injury has been shown to induce skeletal muscle regeneration (Fry et al. 2016). Whether heat shock would stimulate and HA would inhibit muscle regeneration processes has yet to be examined.

Our results revealed proteolytic conversion of larger into smaller isoforms of OPA1 in C2C12 cells during heat exposure. Whether this was caused directly by heat or indirectly by other mitochondrial events remains unclear. It is known that OPA1 plays a critical role in maintaining mitochondrial inner membrane integrity (Lee & Yoon, 2014). Disruption of OPA1 assembly can cause an increase of mitochondrial permeability (Frezza et al. 2006; Yamaguchi et al. 2008) and dissipation of mitochondrial membrane potential (Lee & Yoon, 2014). On the other hand, loss of mitochondrial membrane potential may also lead to the cleavage of OPA1 and fusion inhibition (Duvezin‐Caubet et al. 2006; Ishihara et al. 2006; Liu et al. 2009). The fact that mitochondrial membrane potential (Fig. 7 A) in C2C12 cells declined well before, not concurrently with, proteolysis of OPA1 (Fig. 8 B) during heat exposure seems to negate a direct link between them. Instead, Drp1‐dependent mitochondrial fission (Fig. 4 B) preceded all the mitochondrial events, including release of ROS, loss of membrane potential and cleavage of OPA1 during heat exposure (Figures 7 A, D and 8 B). Overall, our data support Drp1 over OPA1 as the primary mediator involved in the regulation of cell susceptibility to heat‐induced apoptosis. First, translocation and phosphorylation of Drp1 and mitochondrial fragmentation preceded the cleavage of OPA1, and second, HA affected expression of Drp1, but not OPA1 in muscle cells and tissues. However, factors that may act as an upstream Drp1 signal in heat stress have yet to be determined.

In conclusion, the findings of the present study provide novel insights into the role of mitochondria in the regulation of heat‐induced apoptosis of muscle cells. Our results demonstrate that HA and heat shock cause changes in mitochondrial morphology of muscle cells by acting on Drp1, which results in favouring mitochondrial fusion and fission, respectively. Inhibition of Drp1‐dependent mitochondrial division improves mitochondrial morphology and cell viability during heat exposure. Thus, we propose that Drp1 may serve as a biomarker for susceptibility to heat stress and is a candidate for preventing heat injury.

Additional information

Competing interests

The authors declare that there are no competing interests. The information in this paper represents the opinions of the authors and not those of the Uniformed Services University of or the Department of Defense.

Author contributions

T.Y. designed and performed the experiments, analysed the data and helped prepare the manuscript. P.D. provided editorial advice. Y.C. developed the concept and project, analysed the data and wrote the manuscript.

Funding

This work was supported by Congressionally Directed Medical Research Program Award W81XWH‐14‐2‐0133.

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