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. 2020 Jul 21;150(9):2257–2267. doi: 10.1093/jn/nxaa201

Curcumin Ameliorates Heat-Induced Injury through NADPH Oxidase–Dependent Redox Signaling and Mitochondrial Preservation in C2C12 Myoblasts and Mouse Skeletal Muscle

Tianzheng Yu 1,2,✉, Jacob Dohl 3,4, Li Wang 5, Yifan Chen 6, Heath G Gasier 7,8, Patricia A Deuster 9
PMCID: PMC7919340  PMID: 32692359

ABSTRACT

Background

Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase and the mitochondrial electron transport chain are the primary sources of reactive oxygen species (ROS). Previous studies have shown that severe heat exposure damages mitochondria and causes excessive mitochondrial ROS production that contributes to the pathogenesis of heat-related illnesses.

Objectives

We tested whether the antioxidant curcumin could protect against heat-induced mitochondrial dysfunction and skeletal muscle injury, and characterized the possible mechanism.

Methods

Mouse C2C12 myoblasts and rat flexor digitorum brevis (FDB) myofibers were treated with 5 μM curcumin; adult male C57BL/6J mice received daily curcumin (15, 50, or 100 mg/kg body weight) by gavage for 10 consecutive days. We compared ROS levels and mitochondrial morphology and function between treatment and nontreatment groups under unheated or heat conditions, and investigated the upstream mechanism and the downstream effect of curcumin-regulated ROS production.

Results

In C2C12 myoblasts, curcumin prevented heat-induced mitochondrial fragmentation, ROS overproduction, and apoptosis (all P < 0.05). Curcumin treatment for 2 and 4 h at 37°C induced increases in ROS levels by 42% and 59% (dihydroethidium-derived fluorescence), accompanied by increases in NADPH oxidase protein expression by 24% and 32%, respectively (all P < 0.01). In curcumin-treated cells, chemical inhibition and genetic knockdown of NADPH oxidase restored ROS to levels similar to those of controls, indicating NADPH oxidase mediates curcumin-stimulated ROS production. Moreover, curcumin induced ROS-dependent shifting of the mitochondrial fission–fusion balance toward fusion, and increases in mitochondrial mass by 143% and membrane potential by 30% (both P < 0.01). In rat FDB myofibers and mouse gastrocnemius muscles, curcumin preserved mitochondrial morphology and function during heat stress, and prevented heat-induced mitochondrial ROS overproduction and tissue injury (all P < 0.05).

Conclusions

Curcumin regulates ROS hormesis favoring mitochondrial fusion/elongation, biogenesis, and improved function in rodent skeletal muscle. Curcumin may be an effective therapeutic target for heat-related illness and other mitochondrial diseases.

Keywords: antioxidant, apoptosis, heat stress, mitochondrial fission, mitochondrial fusion, myoblast, myofiber, NOX, curcumin

Introduction

Prolonged exposure to heat can lead to heat-related illnesses including cramps (painful muscle spasms), exhaustion, and stroke (1). According to the US CDC, extreme heat often results in the greatest number of annual deaths among all weather-related hazards. Despite the fact that heat-related illnesses are preventable, each year the number of heat-related deaths in the United States is ∼1500 (2). Understanding the pathophysiology of heat-related illnesses will enable the development of new prevention and treatment strategies. Our previous studies have determined that severe heat exposure damages mitochondria and causes overproduction of reactive oxygen species (ROS) generated by the mitochondrial electron transport chain (ETC), which contribute to the pathogenesis of heat-related illnesses (3–5).

ROS are chemically reactive molecules containing oxygen, e.g., singlet oxygen (1O2), superoxide (•O2−), hydroxyl radicals (•OH), and hydrogen peroxide (H2O2). ROS can oxidize lipids, proteins, RNA, and DNA, to result in tissue injury and organ dysfunction (6, 7). Excessive ROS production has been implicated in the pathogenesis of many diseases, such as diabetes, cancer, neurodegeneration, and even heat-related illnesses (4, 8–10). In disease-free and normal conditions, ROS are maintained at low levels by antioxidant defenses and serve as important cellular signaling molecules (8, 11). In mammalian cells, ROS are primarily generated from mitochondrial respiration and nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (located in plasma membranes) during respiratory bursts (12–14). During respiration, a small amount of •O2− is produced and undergoes 1 electron oxidation and dismutation to H2O2 by superoxide dismutase 2 (15). When mitochondrial ROS production exceeds antioxidant defenses, however, ROS can induce membrane permeabilization and cytochrome c release into the cytoplasm, increasing caspase activity and cell death (16).

Curcumin is the major bioactive component of turmeric (Curcuma longa) and widely used as an additive in many Asian foods. Curcumin is reported to possess antioxidant, anti-inflammatory, and antitumor properties, and has been used in >100 clinical trials in the past decade (17, 18). Interestingly, curcumin can also increase ROS levels (19, 20). Curcumin properties can be either antioxidant or pro-oxidant; which property pertains is dependent on the concentration of curcumin and cell sensitivity (19, 21, 22). The mechanism by which curcumin stimulates ROS production, however, remains unknown.

Skeletal muscle is highly susceptible to heat injury (3, 5, 23). In the present study, we cultured mouse C2C12 myoblasts (in vitro) and isolated rat primary flexor digitorum brevis (FDB) myofibers (ex vivo) at normal temperature (37°C) and under heat stress (43°C), and used an in vivo mouse model of heat stress. We aimed to examine curcumin's role in modulating ROS production during heat stress, and test whether curcumin is effective in preventing heat-induced ROS production and cell injury. Moreover, we wanted to elucidate the upstream mechanisms responsible for the downstream effect of curcumin-induced ROS production, with particular emphasis on mitochondrial morphology and function. Our results show that curcumin ameliorates heat-induced skeletal muscle injury through NADPH oxidase–dependent redox signaling and mitochondrial preservation.

Methods

C2C12 myoblast culture, in vitro heat exposure, and curcumin treatment

The mouse myoblast C2C12 cell line (ATCC® CRL-1772™) was cultured in DMEM supplemented with 10% FBS, 100 unit penicillin/mL, and 100 μg streptomycin/mL at 37°C in a 5% CO2 humidified incubator. The C2C12 cell in vitro heat exposure (43°C) has been described previously (5). Curcumin was purchased from Alfa Aesar and added to the cell culture medium (5 μM) 30 min before heat exposure. Cells treated with 0.1% DMSO served as controls.

Animals and housing

All procedures involving animals were approved by the Uniformed Services University of the Health Sciences Institutional Animal Care and Use Committee. Six-week-old male Sprague Dawley rats and C57BL/6J mice were purchased from Charles Rivers and Jackson Laboratories, respectively. All animals were housed in a conventional facility (∼21°C) with a 12-h light/dark cycle and had free access to water. The rats received a standard rodent diet comprising 68%, 19%, and 13% of calories from carbohydrate, protein, and fat (Teklad Custom, Harlan Laboratories; TD.08485), respectively. Mice were fed a regular diet (Envigo) ad libitum comprising 58%, 29%, and 13% of calories from carbohydrates, protein, and fat, respectively.

FDB myofiber isolation and ex vivo heat exposure

Rat FDB single myofibers were isolated by collagenase A (3 mg/mL, Sigma; SKU# 11088793001) and dispase II (1 mg/mL, Sigma; SKU# D4693), as described previously (24). Isolated myofibers were allowed to recover for 1 h, then pretreated with 5 μM curcumin or DMSO (control), and exposed to 43°C for 30 min. Our previous study showed that 43°C heat exposure for 30 min caused hypercontraction in most of the fibers, indicating muscle injury (3).

In vivo mouse model of heat stress

All mice were surgically implanted with the BioTherm13 tag temperature transponder (Biomark Inc.) in the abdominal cavity under isoflurane anesthesia and allowed 1 wk to recover. Mice were then divided into the following groups (n = 6/group): control nonheat, curcumin nonheat, control heat, and curcumin heat. Control mice received olive oil only; curcumin mice received curcumin (15, 50, or 100 mg/kg body weight) in olive oil by gavage once per day for 10 consecutive days before heat exposure.

Mice were ∼10 wk old on the day of heat exposure and tissue collection. Heat exposure was conducted in an environmental chamber (Model 3950, Thermo Forma) with the temperature preset at 39.5°C (25). Heat exposure was sustained up to a maximum tolerable period of 3 h or terminated when core temperature (Tc) reached 42.4°C, whichever occurred first. Immediately after heat exposure, blood and tissues were collected under isoflurane anesthesia for subsequent analysis.

Inhibition of NADPH oxidase

NADPH oxidase activity was inhibited with a chemical inhibitor and small interfering RNA (siRNA). Apocynin, a known inhibitor of NADPH oxidase (26), was purchased from Santa Cruz Biotechnology and added to the cell medium (200 μM) 30 min before treatment with curcumin. NADPH oxidase 2 (NOX2) is highly expressed in C2C12 myoblasts and a main source of skeletal muscle ROS production (27). To genetically knock down NADPH oxidase, we transfected C2C12 cells with NOX2 gp91phox siRNA (Santa Cruz) as per the manufacturer's instructions. A scrambled siRNA was used as control.

Determination of cellular and mitochondrial ROS levels

Cellular ROS levels were determined with 5 μM dihydroethidium (DHE; detects •O2−) and 25 μM 2’,7’-dichlorodihydrofluorescein diacetate (H2DCFDA; nonspecific ROS detection), and mitochondrial ROS levels were determined with 5 μM MitoSOX™ Red (detects •O2−). To detect ROS levels in tissues, fresh mouse gastrocnemius muscle was snap-frozen in a Tissue-Plus OCT compound (Fisher Scientific) and cut into 5-μm sections. Cryostat sections were mounted on SuperFrost Plus™ slides and stained with 5 μM DHE. H2DCFDA, DHE, and MitoSOX™ Red were purchased from Invitrogen.

Assessment of mitochondrial morphology, mass, and membrane potential

In cells, mitochondria were visualized using MitoTracker Red CMXRos (Invitrogen) and mitochondrial membrane potential (ΔΨm) was measured with the cationic fluorescent dye tetramethylrhodamine ethyl ester (TMRE) (Invitrogen). Both fluorescent dyes (100 nM) were added to the cells 15 min before imaging. Mitochondrial mass was determined from the MitoTracker Red fluorescence intensity, and mitochondrial morphology was assessed as previously described (19, 28, 29). Because changes in mitochondrial mass may alter the fluorescence signal in the mitochondria, ΔΨm was calculated as mean TMRE intensity per mitochondrial area. Mitochondrial morphology in gastrocnemius muscle was evaluated by transmission electron microscopy as previously described (5).

Cell lysate, subcellular fractionation, western blot, and immunofluorescence

Cell lysate was obtained by incubating cells with ice-cold radioimmunoprecipitation assay buffer buffer containing protease inhibitors, and subcellular fractionation was performed as described previously (30). Cell lysate or cytosolic (used to determine cytochrome c) protein (∼10 μg) was loaded into polyacrylamide gels (Bio-Rad Laboratories) and separated by SDS-PAGE.

The following primary antibodies were used: mouse anti-dynamin related protein 1 (DRP1) (BD Biosciences, Catalog #611112), mouse anti-optic atrophy protein 1 (OPA1) (BD Biosciences, #612606), rabbit anti-mitofusin (MFN)-1 (#14739) and anti-MFN2 (#9482, Cell Signaling), rabbit anti-NOX2 (Santa Cruz, sc-130543 or Abcam, ab80508), rabbit anti-peroxisome proliferator-activated receptor gamma coactivator 1α (PGC1α) (Abcam, ab54481), rabbit anti-cytochrome c (Cell Signaling, #11940), rabbit anti-voltage-dependent anion-selective channel (VDAC) (Cell Signaling, #4661), and mouse anti-actin (Santa Cruz, sc-47778). Horseradish peroxidase–conjugated anti-rabbit and anti-mouse antibodies were used as secondary antibodies. With the exception of actin (1:5000), primary antibodies were 1:1000 and secondary antibodies 1:10,000. Protein expression was visualized with Western ECL Blotting Substrates (Bio-Rad), and image acquisition was performed using a Bio-Rad ChemiDoc MP Imaging System. Densitometry was performed using ImageJ software (NIH).

For indirect immunofluorescence, cells were cultured on coverslips (∼40% confluency), fixed with 4% paraformaldehyde, and permeabilized using 0.1% Triton X-100. The cells were then incubated with rabbit anti-cytochrome c (1:1000, Cell Signaling) at 37°C for 1 h, washed 3 times in PBS, and incubated with goat anti-rabbit Alexa 594 secondary antibody (Invitrogen) for 1 h at room temperature. Fluorescence images were acquired and adjusted using ImageJ (NIH).

Cell viability, caspase 3/7 activities, and cell death assays

Cell viability was determined using the trypan blue exclusion test with a TC20 automated cell counter (Bio-Rad) per the manufacturer's instructions, and confirmed by cell counting with a light microscope. Caspase 3/7 activity was measured with CellEvent™ Caspase-3/7 Green detection reagent (Invitrogen) following the manufacturer's instructions. Apoptosis was determined by using an Annexin V Alexa Fluor® 488 apoptosis kit (Invitrogen). To detect cell injuries in situ, an Alexa Fluor® 594 Terminal deoxynucleotidyl transferase dUTP Nick End Labeling assay (Molecular Probes) was used to label apoptotic nuclei in fresh-frozen sections of mouse skeletal muscle.

Fluorescence microscopy

Fluorescence images were viewed and acquired with a Nikon Eclipse Ti epifluorescence microscope equipped with a digital camera. Excitation/emission wavelengths were 480/535 nm for green fluorescence (annexin V, caspase-3/7 Green) and 555/613 nm for red fluorescence (MitoTracker red, TMRE, ethidium, MitoSOX, and Alexa 594). The images were analyzed using ImageJ software (NIH). For presentation, some images were adjusted for sharpness/contrast and these adjustments were applied equally across the entire image.

Statistical analysis

All data are presented as mean ± SDs. Statistical analyses were performed by using GraphPad Prism 8 Software. A 2-factor ANOVA was used to compare condition (heat compared with unheated), treatment (curcumin compared with control), and the interaction (condition × treatment) in C2C12 myoblasts, rat FDB myofibers, and mice. A 1-factor ANOVA was used to compare mean group differences for ROS levels, NADPH oxidase, mitochondrial morphology, fission/fusion proteins, mitochondrial biogenesis in C2C12 cells with or without ROS and NOX2 inhibitors at 37°C, and in mice treated with different doses of curcumin. A repeated-measures 2-factor ANOVA was used to determine the main effect of curcumin treatment on C2C12 cell viability with heat exposure from 0 to 6 h. When a significant main effect was observed, multiple comparisons testing was completed using Tukey's, Bonferroni, and Dunnett's tests, and indicated where used. When variances were heterogeneous, Welch's ANOVA test was performed and indicated when used. Values of P < 0.05 were considered statistically significant.

Results

Curcumin preserved mitochondrial integrity and function during heat stress in C2C12 myoblasts

When C2C12 myoblasts were exposed to 43°C for 2 h, mitochondria transitioned from a tubular to a fragmented appearance (Figure 1A, B). The percentage of cells containing short fragmented mitochondria was decreased by ∼64% in myoblasts pretreated with 5 μM curcumin then exposed to 43°C (Figure 1B). ΔΨm was significantly decreased in myoblasts exposed to 43°C (Figure 1C), indicative of mitochondrial dysfunction. Curcumin treatment similarly increased ΔΨm in cells incubated at 37°C and 43°C (P < 0.0001) (Figure 1C).

FIGURE 1.

FIGURE 1

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Curcumin preserves mitochondrial integrity and function during heat stress in C2C12 myoblasts. (A) Representative fluorescent microscopy images of C2C12 myoblasts labeled with MitoTracker Red. The cells were pretreated with DMSO (control) or 5 μM curcumin and then incubated at 37°C or 43°C for 2 h. Scale bar: 10 μm. (B) Percentage of myoblasts with short fragmented mitochondria, and (C) ΔΨm in those cells. Values are means ± SDs; n = 3 independent experiments. Data were analyzed using 2-factor ANOVA with Tukey's multiple comparisons test. Labeled means without a common letter differ, P < 0.05. ΔΨm, mitochondrial membrane potential.

Curcumin prevented heat-induced cellular ROS production and cell injury in C2C12 myoblasts

In cells exposed to 43°C, ROS levels increased by 290%. Curcumin caused a 47% increase in ROS levels at 37°C (P = 0.03) and inhibited ROS production in cells incubated at 43°C (Figure 2A, B). In addition, curcumin inhibited heat-induced mitochondrial release of cytochrome c (Figure 2C), caspase 3/7 activation (Figure 2D), and apoptotic cell death (Figure 2E), and improved cell viability (Figure 2F).

FIGURE 2.

FIGURE 2

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Curcumin prevents heat-induced ROS production and cell injuries in C2C12 myoblasts. (A) Representative fluorescent microscopy images of C2C12 myoblasts labeled with dihydroethidium and (B) ROS quantification. (C) Representative western blot images (top panel) of cyt c in the cytosolic fraction (actin served as a loading control) and immunostaining (bottom panel) of C2C12 cells. (D) Quantification of caspase 3/7 activities, (E) annexin V positive (apoptotic) cells, and (F) cell viability. The cells were incubated with DMSO (control) or 5 μM curcumin and exposed to 37°C or 43°C for 2 h or the indicated times. Scale bars: 10 μm. Values are means ± SDs; n = 3 independent experiments. Data were analyzed using 2-factor ANOVA with Tukey's multiple comparisons test (B, D, E) or repeated-measures 2-factor ANOVA with Bonferroni's multiple comparisons test (F). Labeled means without a common letter differ, P < 0.05. *Different from control at the indicated time points, P < 0.05. au, fluorescence arbitrary units; cyt c, cytochrome c; ROS, reactive oxygen species.

Curcumin induced a time-dependent increase in cellular ROS production in C2C12 myoblasts, and mitochondria were not the source of curcumin-induced ROS production

The curcumin-induced ROS production was time dependent, peaking 2–6 h after treatment (Figure 3A, B). Consistent with previous findings that showed mitochondria are the main source of heat-stimulated ROS production (3–5), cells incubated at 43°C had increased MitoSox Red fluorescence (Figure 3C). In cells incubated at 37°C, MitoSox Red fluorescence was not influenced by curcumin (Figure 3C, D). MitoTEMPO, a mitochondria-targeted superoxide dismutase mimetic, also failed to reduce curcumin-induced ROS production (Figure 3E, F). In contrast, N-acetyl-l-cysteine (1 mM), a glutathione precursor, prevented curcumin-induced ROS production. Taken together, these data suggest that mitochondria are not the source of curcumin-induced ROS production.

FIGURE 3.

FIGURE 3

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Curcumin induces a time-dependent increase in ROS production and mitochondria are not the source of curcumin-induced ROS production in C2C12 myoblasts. Quantification of cellular ROS levels was performed by determining the fluorescence intensity of (A) DHE and (B) H2DCFDA. (C) Representative fluorescent microscopy images of C2C12 myoblasts labeled with MitoSOX™ Red to determine mitochondrial ROS levels. The cells were incubated with DMSO (control), 5 μM curcumin, or exposed to 43°C (heat stress) for 2 h. (D) Quantification of MitoSOX™ Red fluorescence intensity as the mitochondrial (mito) ROS levels. (E) Representative fluorescent microscopy images of C2C12 myoblasts labeled with DHE. The cells were pretreated with DMSO (control), MitoTEMPO (mitochondrial antioxidant), or NAC (cellular antioxidant), and then incubated with DMSO or 5 μM curcumin for 2 h. (F) Quantification of cellular ROS levels. Scale bar: 10 μm. Values are means ± SDs; n = 3 independent experiments. Data were analyzed using 1-factor ANOVA with Tukey's multiple comparisons test (A), Welch's ANOVA with the Games-Howell multiple comparisons test for the unequal variance (B), or 1-factor ANOVA with Dunnett's post hoc test to compare with the control (D, F). Labeled means without a common letter differ, P < 0.05. *Different from control, P < 0.05. DHE, dihydroethidium; ROS, reactive oxygen species; NAC, N-acetyl-L-cysteine.

NADPH oxidase mediated curcumin-induced ROS production in C2C12 myoblasts

NADPH oxidase, an enzyme complex that transports electrons across the plasma membrane and reduces oxygen to superoxide, is another source of cellular ROS production (31). As depicted in Figure 4A and B, 2- and 4-h curcumin incubations increased NOX2 protein expression in a time-dependent manner. The NADPH oxidase inhibitor apocynin abolished curcumin-induced ROS production (Figure 4C, D). To confirm the role of NADPH oxidase activity in generating ROS, we transfected C2C12 cells with NOX2 gp91phox siRNA which suppressed NOX2 protein expression by ∼80% and blocked the curcumin-induced increase in NOX2 (Figure 4E). Consistent with the findings when using apocynin, NOX2 gp91phox siRNA prevented curcumin-induced ROS production (Figure 4F). These results indicate that NADPH oxidase may be involved in curcumin-induced ROS production in C2C12 cells.

FIGURE 4.

FIGURE 4

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NADPH oxidase mediates curcumin-induced ROS production in C2C12 myoblasts. (A) Representative western blot images of NOX2; actin served as a loading control. (B) Densitometry of NOX2 protein expression. Values are NOX2 relative to actin. (C) Representative fluorescent microscopy images of C2C12 myoblasts labeled with DHE and (D) quantified ROS levels. Scale bar: 10 μm. Apocynin is an NADPH oxidase inhibitor. (E) Representative western blot images of NOX2; C2C12 cells were transfected with NOX2 gp91phox siRNA or scramble siRNA (sham). (F) Quantified cellular ROS levels. Cells were incubated with 5 μM curcumin for 2 h or the indicated time. Values are means ± SDs; n = 3 independent experiments. Data were analyzed using 1-factor ANOVA with Tukey's post hoc test. Labeled means without a common letter differ, P < 0.05. DHE, dihydroethidium; NOX2, nicotinamide adenine dinucleotide phosphate oxidase 2; ROS, reactive oxygen species; siRNA, small interfering RNA.

Curcumin induced mitochondrial elongation through elevated levels of cellular ROS in C2C12 myoblasts

Because mitochondria are a target for cellular ROS (3, 14), we examined whether curcumin-induced ROS production influences mitochondrial morphology. Mitochondrial shape is variable among C2C12 myoblasts, consisting of small spheres and tubular filaments. In curcumin-treated cells, mitochondria were mostly in an elongated tubular form with an interconnected reticular network (Figure 5A, B). Curcumin incubation increased the expression of the fusion proteins MFN2 and OPA1 and decreased the expression of the fission protein DRP1 (Figure 5C–E). In addition, the OPA1 long-to-short isoform ratio increased when cells were incubated in curcumin for 4 h (Figure 5F). When cells were treated with N-acetyl-l-cysteine (NAC), however, the aforementioned changes induced by curcumin were prevented (Figure 5G, H). These results indicate curcumin acts on mitochondria by altering the fission–fusion balance toward fusion through elevated levels of ROS.

FIGURE 5.

FIGURE 5

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Curcumin induces ROS-dependent mitochondrial elongation in C2C12 myoblasts. (A) C2C12 cells were labeled with MitoTracker Red. Scale bar: 10 μm. (B) Percentage of cells with elongated mitochondria. (C) Depiction of regulatory control over mitochondrial fission and fusion. (D) Representative western blot images of proteins involved in fission and fusion. Actin served as a loading control for cytosolic DRP1 and VDAC for mitochondrial MFN1, MFN2, and OPA1. (E) Densitometry of DRP1, MFN1, MFN2, and OPA1 protein expression. Values are DRP1 relative to actin, MFN1, and MFN2, and OPA1 relative to VDAC. (F) The percentage of OPA1-L relative to OPA1-S. Inhibition of cellular ROS production with 1 mM NAC prevented the increase in (G) curcumin-induced expression of the mitochondrial fusion proteins MFN2 and OPA1 and (H) curcumin-induced mitochondrial elongation. The cells were incubated with DMSO (control) or 5 μM curcumin for 2 h or the indicated times. Values are means ± SDs; n = 3 independent experiments. Data were analyzed using 1-factor ANOVA with Tukey's post hoc test. Labeled means without a common letter differ, P < 0.05. DRP1, dynamin related protein 1; MFN, mitofusin; OPA1, optic atrophy 1; OPA1-L, OPA1-long form; OPA1-S, OPA1-short form; VDAC, voltage-dependent anion channels; ROS, reactive oxygen species, NAC, N-acetyl-L-cysteine.

Curcumin induced mitochondrial biogenesis and bioenergetics through elevated cellular ROS production in C2C12 myoblasts

We investigated whether curcumin is involved in mitochondrial biogenesis and function. When C2C12 myoblasts were treated with 5 μM curcumin ≤4 h, mitochondrial content, as determined by the protein expression ratio of VDAC-to-actin (Figure 6A) and MitoTracker Red (Figure 6B, C), increased in a time-dependent manner. ΔΨm increased in curcumin-treated cells (Figures 1C, 6D). In addition, curcumin increased the protein expression of PGC1α, the master regulator of mitochondrial biogenesis, and AMP-activated protein kinase-α1 (AMPKα1), a cellular energy sensor that regulates mitochondrial activities. Inhibition of cellular ROS production by NAC prevented curcumin-induced increases in mitochondrial mass (Figure 6C), ΔΨm (Figure 6D), and protein expression of PGC1α and AMPKα1 (Figure 6E, F). These findings suggest that curcumin stimulates ROS-dependent mitochondrial biogenesis and alters mitochondrial function.

FIGURE 6.

FIGURE 6

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Curcumin induces reactive oxygen species–dependent mitochondrial biogenesis and alters bioenergetics in C2C12 myoblasts. (A) Mitochondrial content levels (voltage-dependent anion channels-to-actin protein expression ratio). (B) Representative fluorescent microscopy images of C2C12 myoblasts labelled with MitoTracker Red to determine mitochondrial mass. Scale bar: 10 μm. (C) Mitochondrial mass (D) and ΔΨm. (E) Representative western blot images of PGC1α and AMPKα1; actin served as a loading control. (F) Densitometry of PGC1α and AMPKα1 protein expression. Values are PGC1α and AMPKα1 relative to actin. Cells were incubated with DMSO, 5 μM curcumin, or 5 μM curcumin + 1 mM NAC for 2 h or the indicated times. Values are means ± SDs; n = 3 independent experiments. Data were analyzed using 1-factor ANOVA with Tukey's post hoc test. Labeled means without a common letter differ, P < 0.05. AMPK, AMP-activated protein kinase; ΔΨm, mitochondrial membrane potential; NAC, N-acetyl-L-cysteine; PGC, peroxisome proliferator-activated receptor gamma coactivator.

Curcumin ameliorated heat-induced mitochondrial dysfunction, ROS production, and muscle hypercontraction in rat FDB myofibers

In FDB myofibers incubated at 37°C, curcumin resulted in a trend for an increase (P = 0.08) in ROS production (Figure 7B). ΔΨm was decreased in fibers incubated at 43°C; however, curcumin attenuated the increase in membrane depolarization and ROS production (Figure 7A, B). In addition, curcumin decreased the number of hypercontracted fibers induced by heat: 27% compared with 63% in control (Figure 7C, D). These data suggest curcumin preserves mitochondrial function and protects against heat-induced ROS production and skeletal muscle injury ex vivo.

FIGURE 7.

FIGURE 7

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Curcumin ameliorates heat-induced mitochondrial dysfunction, ROS production, and muscle hypercontraction in rat FDB myofibers. Quantifications of (A) ΔΨm and (B) cellular ROS levels were performed by determining the fluorescence intensity of tetramethylrhodamine ethyl ester and ethidium, respectively. (C) Representative images of normal (arrowhead) and hypercontracted (arrow) myofibers and (D) quantitative analysis. FDB myofibers were pretreated with DMSO (control) or 5 μM curcumin and incubated at 37°C or 43°C for 30 min. Scale bar: 10 μm. Values are means ± SDs; n = 3 independent experiments. Data were analyzed using 2-factor ANOVA with Tukey's post hoc test. Labeled means without a common letter differ, P < 0.05. FDB, flexor digitorum brevis; ROS, reactive oxygen species; ΔΨm, mitochondrial membrane potential.

Curcumin preserved mitochondrial integrity and ameliorated heat-induced skeletal muscle injury in a mouse model of heat stress

We validated the aforementioned effects of curcumin in vivo in a mouse model of heat injury (4, 5). Curcumin treatment had no effect on mouse behavior, food intake, body weight, basal Tc, and heat-induced hypothermia (data not shown). Curcumin increased protein expression of PGC1α in mouse gastrocnemius muscle and PGC1α remained higher during heat stress (Supplemental Figure 1). When myofibers from heat-exposed (39.5°C) mice were examined, mitochondria appeared disorganized with increased irregularity (Figure 8A). Curcumin preserved mitochondrial networks in myofibers from mice exposed to 39.5°C: mitochondria remained well organized and showed distinct rings evenly distributed throughout the fibers, similarly to those in nonheated mice. Images obtained from transmission electron microscopy provided further details of the effects of heat-induced injury on skeletal muscle mitochondria ultrastructure, and provided evidence of reduced mitochondrial density, cristae number, and surface area (Figure 8B). As documented in myofibers, curcumin preserved the fine details of mitochondrial cristae in mice exposed to 39.5°C. In a dose-dependent manner, curcumin also reduced heat-induced ROS production (Figure 8C, D) and caspase activation (Figure 8E) and ameliorated apoptotic cell death (Figure 8F).

FIGURE 8.

FIGURE 8

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Curcumin preserves mitochondrial integrity and ameliorates heat-induced skeletal muscle injury in mice. (A) Representative fluorescent microscopy images of mouse flexor digitorum brevis myofibers labeled with MitoTracker Red. Scale bar: 10 μm. (B) Representative electron micrographs showing ultrastructure of mitochondria (m) in mouse gastrocnemius muscles. (C) Representative fluorescent microscopy images of fresh-frozen section of gastrocnemius muscle labeled with DHE and (D) the quantified ROS levels. Scale bar: 10 μm. (E) Quantification of caspase 3/7 activities and (F) terminal deoxynucleotidyl transferase dUTP Nick End Labeling positive (apoptotic) myofibers. Values are means ± SDs; n = 6 mice/group. Data were analyzed using 1-factor ANOVA (D, E) or 2-factor ANOVA (F) with Tukey's post hoc test. Labeled means without a common letter differ, P < 0.05. au, fluorescence arbitrary units; DHE, dihydroethidium; ROS, reactive oxygen species.

Discussion

Our group and others have previously documented a role for curcumin in regulating the redox state, improving health, and treating illnesses and disease (19, 21, 22). The goal of this research was to explore the source of ROS production induced by curcumin and the consequent effects on mitochondrial morphology and function in myocytes, and whether curcumin could protect against heat-induced injury. From this body of work, we report 3 novel findings. First, curcumin induces increased ROS production via NADPH oxidase and not the mitochondrial ETC. Second, curcumin-induced ROS production acts as a signal to modulate mitochondrial fusion, biogenesis, and bioenergetics. Third, by preserving mitochondrial morphology and function, curcumin reduces the impact of heat-induced cellular stress and injury. These data provide a mechanism whereby curcumin alters mitochondrial structure and function in myocytes and affords protection during periods of heat stress.

The NOX family of ROS-generating NADPH oxidases is located within plasma membranes and transfers electrons from NADPH to reduce oxygen to superoxide (31). In contrast to mitochondria, where ROS are produced as byproducts of oxidative phosphorylation, the main function of NADPH oxidase is to produce ROS (26, 31). The thioredoxin system (comprised of thioredoxin, NADPH, and thioredoxin reductase) is considered to be one of the most important regulators of cellular redox homeostasis (32). In addition to catalyzing the NADPH-dependent reduction of thioredoxin, thioredoxin reductase interacts with, and modulates, other plasma membrane proteins, such as NADPH oxidase (33, 34). Curcumin has been reported to irreversibly inhibit thioredoxin reductase activity in a dose- and time-dependent manner, thereby increasing NADPH oxidase activity (34). In the present study, curcumin increased the expression of NOX2 and ROS production, and this may have occurred by repressing thioredoxin reductase activity.

Curcumin is known to have a low oral bioavailability (35). Both the absorption and distribution of curcumin have been extensively studied (19). Oral administration of curcumin can increase plasma curcumin concentrations ≤2 μM in humans and ≤4 μM in rodents (36, 37). Once absorbed into the blood, curcumin is distributed across tissues including skeletal muscle (38). For in vitro cultured cells, curcumin is a highly lipophilic molecule that accumulates within plasma membranes; uptake is much higher in tumor cells and other fast-proliferating cells than in healthy cells (39, 40). In addition to cell sensitivity, curcumin-induced ROS production is also concentration dependent. At high concentrations (>20 μM), curcumin induces a pronounced increase in ROS levels associated with apoptosis and anticarcinogenic properties (19, 20, 22). At lower concentrations, such as used in the present investigation (5 μM), the magnitude of ROS production is lower. Because ROS production is kept in balance by antioxidant defenses, the differences that occur with high compared with low concentrations may be attributed to the activity of antioxidant enzymes, i.e., superoxide dismutase, catalase, glutathione peroxidase, and heme oxygenase 1 (21, 41–44). A controlled increase in ROS production through a concomitant increase in antioxidant defenses may, therefore, serve as a potent downstream signal.

A minor increase in ROS within a physiological range activates signaling pathways critical for regulating cell proliferation and survival—a process termed redox signaling (8, 11). Mitochondria are a primary target of cellular ROS (14), and redox signaling activates transcription factors involved in mitochondrial biogenesis (45, 46). In skeletal muscle, ROS increases nuclear expression of mitochondrial transcription factor A and nuclear respiratory factor 1, activates AMPK, and enhances the expression of PGC1α, a master regulator of mitochondrial biogenesis (47, 48). Consistent with these documented downstream effects of ROS, curcumin increased protein expression of PGC1α and AMPKα1, and subsequently increased mitochondrial mass and membrane potential. Reducing ROS levels by administering the cellular antioxidant NAC abolished all of the aforementioned mitochondrial responses, which confirms that curcumin induces mitochondrial biogenesis and alters bioenergetics through increased ROS production.

Mitochondria are dynamic organelles that constantly change their morphology through fission and fusion (14, 15, 49). DRP1 regulates mitochondrial fission (15, 50), whereas mitofusins (MFN1 and 2) and OPA1 mediate fusion of the mitochondrial outer and inner membranes, respectively (51, 52). Redox signaling may modify these fission/fusion proteins posttranslationally (49, 53, 54). One proposed hypothesis is that during stress, a mild increase in ROS causes nuclear transcriptional changes and subsequent regulation of mitochondrial fission and fusion (55). Indeed, in the present study curcumin increased the protein expression of MFN2 and OPA1, but decreased DRP1. In addition, curcumin increased the OPA1 long-to-short isoform ratio. The long form of OPA1 promotes mitochondrial fusion and maintains cristae structure, whereas short forms are associated with fission (56, 57). The data presented herein imply curcumin induces a mild increase in ROS and this shifts the mitochondrial fission–fusion balance toward fusion, and thereby increases the number of elongated and tubular mitochondria.

The benefits of elongated mitochondria remain to be fully understood, but this elongation may enhance the efficiency of ATP production as a prosurvival adaptation (15). Mitochondrial fission and dysfunction are commonly documented in pathologic conditions. In severe heat stroke, for instance, DRP1 activation and cleavage of the fusion protein OPA1 contribute to excessive mitochondrial ROS production (4, 5, 58). When cellular ROS levels exceed a threshold, the redox balance is perturbed and oxidative damage can ensue. Accumulation of ROS within the mitochondria causes opening of the mitochondrial permeability transition pore and subsequent release of cytochrome c into the cytosol, caspase activation, and cell death (3–5). As aforementioned, curcumin shifts the mitochondrial fission–fusion balance toward fusion, thus preventing mitochondrial fragmentation and a reduction in membrane potential during heat stress. In addition, curcumin ameliorates the heat-induced mitochondrial ROS production and injury.

The data presented herein suggest curcumin regulates ROS hormesis favoring mitochondrial biogenesis, fusion/elongation, and improved function (Supplemental Figure 2). Uncontrolled stress, such as severe heat exposure, damages mitochondria and can cause overproduction of ROS from the mitochondrial ETC, which then contributes to the pathogenesis of many diseases, including heat-related illnesses. Curcumin induces a mild increase in ROS formation through NADPH oxidase, which results in mitochondrial elongation and improved function. Curcumin can, therefore, preserve mitochondrial morphology and bioenergetics, a potential benefit in pathological conditions where mitochondrial ROS production can become uncontrolled and lethal to cells. Curcumin may be an effective therapeutic target for heat-related illness and other mitochondrial diseases.

Supplementary Material

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Acknowledgments

The authors’ responsibilities were as follows—TY and PAD: designed the research; TY, JD, LW, YC, and HGG: conducted the research; TY: analyzed the data, wrote the paper, and had primary responsibility for the final content; TY, HGG, and PAD: edited the paper; and all authors: read and approved the final manuscript.

Notes

Supported by US Department of Defense (DoD) Defense Health Program Nutritional and Dietary Supplement Working Group grant HU0001-14-1-003 and NIH Office of Dietary Supplements grant F1919759.

Author disclosures: The authors report no conflicts of interest.

The opinions and assertions expressed herein are those of the authors and do not necessarily reflect the official policy or position of the Uniformed Services University or the DoD. The contents of this publication are the sole responsibility of the authors and do not necessarily reflect the views, opinions, or policies of The Henry M Jackson Foundation for the Advancement of Military Medicine, Inc. Mention of trade names, commercial products, or organizations does not imply endorsement by the US Government.

Supplemental Figures 1 and 2 are available from the “Supplementary data” link in the online posting of the article and from the same link in the online table of contents at https://academic.oup.com/jn/.

Abbreviations used: AMPK, AMP-activated protein kinase; DHE, dihydroethidium; DRP, dynamin related protein; ETC, electron transport chain; FDB, flexor digitorum brevis; H2DCFDA, 2’,7’-dichlorodihydrofluorescein diacetate; MFN, mitofusin; NAC, N-acetyl-L-cysteine; NADPH, nicotinamide adenine dinucleotide phosphate; NOX2, nicotinamide adenine dinucleotide phosphate oxidase 2; OPA, optic atrophy; PGC, peroxisome proliferator-activated receptor gamma coactivator; ROS, reactive oxygen species; siRNA, small interfering RNA; Tc, core temperature; TMRE, tetramethylrhodamine ethyl ester; VDAC, voltage-dependent anion channels; ΔΨm, mitochondrial membrane potential.

Contributor Information

Tianzheng Yu, Consortium for Health and Military Performance, Department of Military and Emergency Medicine, F Edward Hébert School of Medicine, Uniformed Services University, Bethesda, MD, USA; Henry M Jackson Foundation for the Advancement of Military Medicine, Bethesda, MD, USA.

Jacob Dohl, Consortium for Health and Military Performance, Department of Military and Emergency Medicine, F Edward Hébert School of Medicine, Uniformed Services University, Bethesda, MD, USA; Henry M Jackson Foundation for the Advancement of Military Medicine, Bethesda, MD, USA.

Li Wang, Department of Biochemistry and Molecular Biology, University of Maryland School of Medicine, Baltimore, MD, USA.

Yifan Chen, Consortium for Health and Military Performance, Department of Military and Emergency Medicine, F Edward Hébert School of Medicine, Uniformed Services University, Bethesda, MD, USA.

Heath G Gasier, Consortium for Health and Military Performance, Department of Military and Emergency Medicine, F Edward Hébert School of Medicine, Uniformed Services University, Bethesda, MD, USA; Department of Anesthesiology, Center for Hyperbaric Medicine & Environmental Physiology, Duke University School of Medicine, Durham, NC, USA.

Patricia A Deuster, Consortium for Health and Military Performance, Department of Military and Emergency Medicine, F Edward Hébert School of Medicine, Uniformed Services University, Bethesda, MD, USA.

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