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Published in final edited form as: Nanomedicine. 2021 Jul 10;37:102439. doi: 10.1016/j.nano.2021.102439

Coenzyme Q nanodisks counteract the effect of statins on C2C12 myotubes

Anthony Moschetti 1, Ruben K Dagda 2, Robert O Ryan 2,*
PMCID: PMC8464493  NIHMSID: NIHMS1723388  PMID: 34256063

Abstract

Depletion of coenzyme Q (CoQ) is associated with disease, ranging from myopathy to heart failure. To induce a CoQ deficit, C2C12 myotubes were incubated with high dose simvastatin. This resulted in a concentration-dependent inhibition of cell viability. Simvastatin-induced effects were prevented by co-incubation with mevalonic acid. When myotubes were incubated with 60 μM simvastatin, mitochondrial CoQ content decreased while co-incubation with CoQ nanodisks (ND) increased mitochondrial CoQ levels and improved cell viability. Incubation of myotubes with simvastatin also led to a reduction in oxygen consumption rate (OCR). When myotubes were co-incubated with simvastatin and CoQ ND, the decline in OCR was ameliorated. The data indicate that CoQ ND represent a water soluble vehicle capable of delivering CoQ to cultured myotubes. Thus, these biocompatible nanoparticles have the potential to bypass poor CoQ oral bioavailability as a treatment option for individuals with severe CoQ deficiency syndromes and/or aging-related CoQ depletion.

Keywords: coenzyme Q, simvastatin, nanodisk, C2C12 myotubes, mitochondria

Graphical Abstract narrative

Simvastatin was used to inhibit coenzyme Q biosynthesis in cultured C2C12 myotubes, resulting in decreased cell viability and reduced oxygen consumption. Coenzyme Q formulated into nanodisk particles conferred aqueous solubility to this hydrophobic lipid. Co-incubation of cultured myotubes with simvastatin and coenzyme Q nanodisks restored coenzyme Q levels, ameliorated deleterious simvastatin-induced effects on cell viability and increased cellular oxygen consumption rate. Coenzyme Q nanodisks represent an injectable form of coenzyme Q that has the potential to circumvent the poor oral bioavailability of coenzyme Q.

graphic file with name nihms-1723388-f0007.jpg

Introduction

Statins are one of the most widely prescribed classes of drug in the United States [1]. They are highly effective at reducing plasma cholesterol levels and have been shown to lower mortality from coronary heart disease by as much as 30% [2]. Statins function by inhibiting the rate-limiting enzyme in the mevalonate pathway, 3-hydroxy, 3-methylglutaryl (HMG) CoA reductase, thereby inhibiting the biosynthesis of cholesterol and other metabolites, including coenzyme Q (CoQ). Clinically, statin treatment (20 mg/day) consistently results in a decline in plasma and skeletal muscle CoQ levels [3,4]. Statin-dependent lowering of CoQ content has led to the hypothesis that a correlation exits between reduced CoQ and statin-induced myopathy [4,5]. The rationale for this postulate is based on the essential role CoQ plays in the electron transport chain where it ferries electrons between Complex I and Complex III, and Complex II and Complex III [6]. When CoQ levels decline, aerobic respiration is adversely affected, decreasing ATP production efficiency. It is considered that myopathy arises from the inability of mitochondria to meet the energy demands of myocytes, especially during exercise.

In support of this hypothesis, studies indicate patients receiving CoQ as a dietary supplement (100-600 mg/day) along with a statin, experience a 40% reduction in myopathy [7,8]. At the same time, other studies have produced inconsistent results with respect to the ability of CoQ supplements to counteract statin-induced myopathy [4,7]. Because of its intrinsic hydrophobicity, CoQ is poorly absorbed from the human digestive track [9]. Thus, it is plausible that CoQ’s lack of aqueous solubility and poor oral bioavailability contribute to disparate results observed with respect to the effect of CoQ supplements on statin-induced myopathy [10]. To counteract this, various attempts to enhance CoQ bioavailability have been pursued. Fore example, CoQ supplements are currently available as tablets, powder-filled capsules, and oil suspensions in soft gel capsules. To improve absorption, Chopra et al. [11] added an emulsifier to CoQ10 supplements. Langsjoen and Langsjoen [12] showed that ubiquinol, the reduced form of CoQ, displays superior bioavailability compared to its oxidized form, ubiquinone, although both have been used in clinical studies.

An alternative strategy to increase CoQ bioavailability involves solubilization of CoQ in a delivery vehicle suitable for intramuscular or intravenous administration. A candidate vehicle for this purpose is reconstituted high density lipoprotein (rHDL). These nanoparticles are organized as a disk-shaped bilayer that is stabilized by a scaffold protein. To distinguish nanoparticles harboring exogenous bioactive agents from nascent discoidal HDL, the term nanodisk (ND) has been adopted. ND have been shown to confer aqueous solubility to several hydrophobic bioactive agents, including amphotericin B [13] all trans retinoic acid [14] curcumin [15] and others [16]. ND self-assembly occurs upon incubation of specific glycerophospholipids with members of the class of exchangeable apolipoprotein or amphipathic peptides [17]. Recently, Moschetti et al [18] reported the formulation and characterization of CoQ10 ND. In the present study, C2C12 myotubes were treated with a high-dose simvastatin and its effect on mitochondrial CoQ content determined. When C2C12 myotubes were co-incubated with simvastatin and CoQ ND, the deleterious effects of high dose simvastatin treatment on mitochondrial function were ameliorated.

Methods

Materials.

CoQ10, CoQ9, mevalonic acid lithium salt, stigmasterol, simvastatin and egg yolk phosphatidylcholine (PC) were purchased from Sigma Chemical Co. (St Louis, MO, USA) CoQ8 was from Avanti Polar Lipids Inc. (Alabaster AL, USA)

CoQ ND Formulation.

CoQ ND were formulated as described by Moschetti et al [18]. Briefly, five mg PC was dispersed in 0.5 mL 20 mM sodium phosphate, 150 mM NaCl, pH 7.4; PBS), followed by the addition of 1.0 mg CoQ9 or CoQ10 (from a 10 mg/mL stock solution in dimethylformamide) and 2 mg recombinant human apolipoprotein (apo) A-I in 0.5 mL PBS [19–21]. Following this, the mixture was bath sonicated (between 43°C and 47°C) until the sample transitioned from opaque to clear, indicating ND formation. Empty ND, lacking CoQ, were prepared using the same protocol. Once formed, ND were dialyzed and sterile filtered prior to use in experiments.

C2C12 Cell Culture.

Murine C2C12 myoblasts were purchased from UC Berkeley Biosciences Divisional Services. Cells were plated at 25,000 cells/well in 96-well plates and cultured in Dulbecco’s minimal essential medium (DMEM) supplemented with 10% fetal bovine serum, 50 U/mL penicillin, and 50 μg/mL streptomycin. After 48 h culture at 37°C, DMEM was replaced with DMEM containing 2.0 mM GlutaMAX, 50 U/mL penicillin, 50 μg/mL streptomycin, and 2% horse serum to promote myoblast differentiation into multinucleated myotubes as described [22]. After 3–5 days culture, differentiation was complete. Cell protein was determined using the BCA assay (ThermoFisher, Sparks NV, USA).

Cell viability assays.

C2C12 myotube viability was assessed using the CellQuanti-Blue assay (BioAssay Systems, Hayward CA, USA). Simvastatin was introduced to the cell culture medium from a 10 mg/ml stock solution in DMSO. Cells were cultured in 96-well plates and treated with DMSO only, simvastatin or simvastatin plus indicated amounts of CoQ ND. Following 72 h incubation, assays were conducted as per the manufacturer’s instructions and sample fluorescence emission at 590 nm (excitation 530 nm) was measured on a Spectramax M5 plate reader. In other experiments, myotubes were incubated with simvastatin (60 μM) in the absence or presence of mevalonic acid, followed by cell viability assay.

CoQ extraction from isolated mitochondria.

Mitochondria were isolated from C2C12 myotubes as described [18]. Isolated mitochondria were centrifuged at 12,000 x g for 5 min, the supernatant was removed and the pellet dried under vacuum. Following addition of an internal standard (CoQ8; 300 ng), 1-propanol was added. Samples were homogenized, transferred to 1.5 mL microfuge tubes and centrifuged at 3000 x g for 5 min. The 1-propanol supernatants were recovered and filtered through a 0.22 μm Nylon membrane prior to HPLC analysis.

CoQ analysis by HPLC.

1-propanol extracts were chromatographed on a Shimadzu Prominence HPLC fitted with a Kinetex 5 μm EVO C18 100 Å, 150 × 4.6 mm column and SecurityGuard ULTRA guard column. Samples were separated using an isocratic mobile phase of 2:1 methanol:2-propanol (vol/vol) eluted at 0.7 mL/min (30°C column temperature). Absorbance was monitored at 275 nm utilizing an SPD-M20A photodiode array detector.

C2C12 myotube cholesterol analysis.

C2C12 myotubes were washed twice with PBS, centrifuged at 700 x g for 5 min and the supernatant removed. An internal standard (15 μg stigmasterol) was added and the pellet extracted [23]. An aliquot (10 μl) of the extract was subjected to gas chromatography (GC) on a Shimadzu Model GC-2010 Plus system-fitted with an Agilent HP-5 fused silica capillary column (30 m x 0.32 mm id, 7-inch format, 0.25 μm film thickness). Oven temperature was programmed from 250° to 275°C at 2°C/min and held for 12 min. The injection port temperature was 300°C and the flame ionization detector temperature was 300°C.

C2C12 oxygen consumption rate (OCR).

C2C12 Myoblasts were cultured in 24-well XF24 microplates at a density of 25,000 cells / well. Following differentiation, myotubes were incubated under specified conditions for 72 h. Myotubes were then analyzed on a XF24 Seahorse Extracellular Flux Analyzer (Agilent Technologies) as described [18]. At the end of each assay, cells were fixed in 4% paraformaldehyde for 30 min, washed with PBS and stained with 1.25 μg/mL 4’,6-diamidino-2-phenylindole (DAPI). DAPI-stained nuclei were visualized and imaged using an EVOS-FL Cell Imaging System equipped with EVOS Light Cubes specific for UV (Ex 357 nm / Em 447 nm), at 20x magnification (0.45 numerical aperture), as described [24]. Up to 10 different regions per well were analyzed (blinded to the observer) by counting the number of nuclei per field using NIH Image J software (version 1.44). Raw OCR values were normalized to the average number of nuclei per field.

Statistical methods.

Statistical analyses were performed by two-way ANOVA to compare treatment samples versus control samples. Statistical tests were performed using GraphPad Prism version 9.0.0 for Windows (GraphPad Software, San Diego, CA, USA).

Results

Objective and Rationale.

The present investigation was designed to assess the ability of CoQ ND to prevent simvastatin-induced CoQ depletion and mitochondrial dysfunction. Because C2C12 cells are of murine origin, they possess predominantly CoQ9, with much lower amount of CoQ10 [25]. CoQ9 differs from CoQ10 only in the number of 5 carbon isoprene units present in its hydrophobic tail (9 units versus 10). By contrast, human cells possess a single major CoQ species, CoQ10. We used both CoQ9 and CoQ10 in the present studies and, for each experiment, the specific CoQ species is indicated. When the term CoQ is used, it refers to either CoQ9 or CoQ10.

Effect of simvastatin on C2C12 myotube cell viability.

C2C12 myotubes were incubated in the absence or presence of media supplemented with simvastatin for 72 h. Simvastatin concentration-dependent effects on cell viability were then measured (Figure 1). The results obtained indicate that the vehicle used for simvastatin administration (i.e. DMSO) had no effect on cell viability. At the same time, the supra-pharmacological doses of simvastatin used led to a concentration-dependent decline in cell viability. At simvastatin doses of 20, 40, 60, and 80 μM, cell viability decreased by 41, 45, 51 and 79 %, respectively. Simvastatin doses above 60 μM reduced viability to a near-lethal threshold. Based on these results, 60 μM simvastatin was determined to approximate the LD50 and this dose was used in all subsequent experiments.

Figure 1. Effect of simvastatin dose on C2C12 cell viability.

Figure 1.

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C2C12 myotubes were incubated in media containing indicated amounts of simvastatin (Statin) for 72 h as described in Materials and Methods. Following incubation, cell viability was assayed using the CellQuanti-Blue assay. Sample fluorescence emission was monitored at 590 nm (excitation 530 nm). Values reported are the mean ± standard error (n = 6) **** p < 0.0001 versus 20, 40, 60, and 80 μM simvastatin.

Effect of mevalonic acid on simvastatin-induced loss of C2C12 myotube cell viability.

C2C12 myotubes were incubated with simvastatin for 72 h in the absence or presence of increasing amounts of mevalonic acid. Following incubation, cell viability was measured (Figure 2). Control incubations with DMSO or mevalonic acid, but no simvastatin, had no effect on cell viability. C2C12 myotubes incubated with simvastatin manifested the expected decline in cell viability while myotubes incubated with simvastatin plus mevalonic acid showed no decline in cell viability at all mevalonic acid concentrations tested. Thus, it is concluded that mevalonic acid prevents manifestation of the deleterious effects of high-dose simvastatin treatment on C2C12 myotubes.

Figure 2. Effect of mevalonic acid on simvastatin-induced loss of C2C12 myotube cell viability.

Figure 2.

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C2C12 myotubes were incubated for 72 h in media containing either 0.3% DMSO, simvastatin, mevalonic acid (Mev), or simvastatin plus mevalonic acid. Following incubation, cell viability was assayed. Values reported are the mean ± standard error (n = 12) ns, not significant; ****, p < 0.0001 vs 60 μM simvastatin.

Impact of simvastatin on the CoQ content of C2C12 myotube mitochondria.

A question arising from the results described above relates to the extent to which mitochondrial CoQ content is affected by simvastatin treatment? To examine this, following incubation of C2C12 myotubes with simvastatin, mitochondria were isolated, extracted and analyzed by reversed phase HPLC (Figure 3). The data show that, compared to control myotubes incubated with DMSO, mitochondria from simvastatin-treated myotubes manifest a sharp decline in CoQ9 content. When myotubes were incubated with simvastatin plus CoQ10 ND (20 μM as CoQ10), mitochondrial CoQ10 levels increased. As expected, CoQ9 levels were largely unaffected by incubation of myotubes with CoQ10 ND.

Figure 3. Impact of simvastatin on the CoQ content of C2C12 myotube mitochondria.

Figure 3.

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C2C12 myotubes were incubated for 72 h in media containing either 0.3% DMSO carrier, simvastatin, or simvastatin plus CoQ10 ND. Following incubation, myotube mitochondria were isolated and, after addition of CoQ8 internal standard (IS), extracted with 1-propanol. Panel a) Representative HPLC traces of myotube mitochondrial extracts including 0.3% DMSO (blue line), 60 μM simvastatin (dotted line), and 60 μM simvastatin plus 20 μM CoQ10 ND (orange line). Panel b) Histogram depicting ng CoQ10 per μg mitochondrial protein following myotube incubation under specified conditions. Panel c) same as Panel b except ng CoQ9 per μg mitochondrial protein is reported. Values are the mean ± standard error (n = 3) *, p < 0.05 vs 60 μM simvastatin.

Effect of simvastatin on C2C12 myotube free cholesterol content.

To determine the effect of simvastatin on myotube cholesterol content, GC analysis of myotube lipid extracts was performed. C2C12 myotubes were incubated in the presence and absence of simvastatin for 72 h followed by introduction of an internal standard (i.e. stigmasterol) and extraction. GC analysis (Figure 4) revealed, under the conditions of this experiment, simvastatin treatment did not affect the free cholesterol content of these cells.

Figure 4. Effect of simvastatin on C2C12 myotube free cholesterol content.

Figure 4.

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C2C12 myotubes were incubated for 72 h in media containing either 0.3% DMSO carrier or simvastatin in DMSO. Following incubation, the cells were washed, internal standard (stigmasterol) added and extracted. An aliquot of each extract was then analyzed by GC. Values reported are the mean ± standard error (n = 3); ns, not significant.

Effect of CoQ ND on simvastatin-induced loss of C2C12 myotube cell viability.

As seen in Figure 3, incubation of simvastatin-treated C2C12 myotubes with CoQ10 ND led to an increase in mitochondrial CoQ10 content. To assess whether the enhanced CoQ10 content of these mitochondria influences simvastatin-induced effects on cell viability, C2C12 myotubes were incubated with simvastatin in the absence or presence of CoQ10 ND, followed by assay of cell viability (Figure 5). The results obtained reveal that, compared to incubation with simvastatin only, myotubes incubated with simvastatin plus CoQ10 ND (20 μM as CoQ10) manifested a significant improvement in cell viability. A virtually identical result was obtained when myotubes were incubated with simvastatin plus CoQ9 ND. The observation that there was no difference between CoQ9 ND and CoQ10 ND in terms of their relative ability to protect C2C12 myotubes from simvastatin-induced loss of cell viability indicates these two CoQ species are largely interchangeable in murine cells. Parallel incubations with lower concentrations of CoQ ND (10 μM as C0Q10 or CoQ9) did not yield a statistically significant change in cell viability versus myotubes incubated with simvastatin alone. Moreover, the observed enhancement in cell viability observed for myotubes incubated with 20 μM CoQ ND remained significantly below that of control myotubes (0 μM simvastatin).

Figure 5. Effect of CoQ ND on simvastatin-induced loss of C2C12 myotube cell viability.

Figure 5.

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C2C12 myotubes were incubated for 72 h in media containing either 0.3% DMSO carrier, simvastatin, simvastatin plus CoQ110 ND or simvastatin plus CoQ9 ND. Following incubation, cell viability was measured and sample fluorescence emission at 590 nm determined (excitation 530 nm). Values reported represent the mean ± standard error (n = 6); ****, p < 0.0001 versus 0 μM simvastatin; ***, p < 0.0004 versus 0 μM CoQ ND; ns, not significant.

Effect of CoQ10 ND on C2C12 myotube OCR.

Given the ability of CoQ10 ND to replenish the CoQ10 content of simvastatin-treated C2C12 myotube mitochondria, a stress test was performed to assess the effect of exogenous CoQ10 on OCR. C2C12 myotubes were incubated for 72 h with DMSO, simvastatin, simvastatin plus CoQ10 ND or simvastatin plus “empty” ND. Following incubation, OCR was measured in an XF24 Seahorse Analyzer. Control cells incubated with DMSO only had robust basal- and FCCP-induced maximal OCRs, respectively (Figure 6). By contrast, basal and maximal OCRs in simvastatin-treated cells were much lower, indicating simvastatin elicited an adverse effect on mitochondrial respiration in these myotubes. In myotubes treated with simvastatin plus CoQ10 ND, basal and maximal OCRs were intermediate between control cells and simvastatin-treated cells. These data indicate that ND-mediated replenishment of mitochondrial CoQ content attenuated the damaging effects of simvastatin on cellular respiration. To verify that the effects observed were due to CoQ enrichment, a control incubation of simvastatin plus empty ND showed no improvement in OCR over simvastatin alone.

Figure 6. Effect of simvastatin and CoQ10 ND on C2C12 myotube oxygen consumption rate (OCR).

Figure 6.

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C2C12 myotubes were incubated for 72 h in media supplemented with 0.3% DMSO (blue), 60 μM simvastatin (red), simvastatin plus empty ND (magenta) or simvastatin plus CoQ10 ND (green). Following incubation, cells were subjected to a mitochondrial stress test assay on a Seahorse FX24 analyzer with intervals of drug additions (Oligomycin, FCCP, and Rotenone/Antimycin A). The OCR was determined for each incubation condition and normalized to nucleus count, as visualized by DAPI stain. Panel a) Representative OCR traces for each incubation condition; Panel b) histogram depicting the mean basal OCR for each treatment condition and Panel c) histogram depicting the maximal OCR for each treatment condition. Values reported are the mean ± standard error (n = 3) * p < 0.05 vs empty ND.

Discussion

In a recent study, Moschetti et al [18] reported the successful formulation of CoQ10 into reconstituted HDL-like nanoparticles (i.e. ND). These particles are organized as a disk-shaped phospholipid bilayer circumscribed by an apolipoprotein scaffold [20]. The CoQ component is considered to integrate into the hydrophobic milieu of the bilayer, effectively conferring CoQ with aqueous solubility. While the product particles are structurally similar to nascent HDL, given their compositional differences, the term nanodisk (ND) is used [20]. The ability to solubilize the highly hydrophobic CoQ in ND has prompted an examination of its potential effectiveness as a delivery vehicle. Initially, we sought to develop a cell culture system in which CoQ can be depleted, resulting in some measurable cellular defect. The model selected was cultured C2C12 myotubes treated with a supra-pharmacological dose of simvastatin. Subsequently, the ability of CoQ ND administration to prevent, or rescue, any deleterious effects could be evaluated. Using cell viability as a readout, C2C12 myotubes were incubated with increasing doses of simvastatin for 72 h. The data obtained revealed a dose-dependent decline in cell viability. Sixty μM simvastatin was selected as a dose anticipated to inhibit CoQ biosynthesis. Because the dose of simvastatin used in these studies is well above the normal dose administered to patients seeking to lower their plasma cholesterol levels, it was important to evaluate potential off target effects on cell viability. To test this, myotubes were incubated with simvastatin plus mevalonic acid. The finding that mevalonate prevented simvastatin-induced loss of cell viability indicates the effects of simvastatin are due to inhibition of HMG CoA reductase rather than an off target, statin-related phenomenon.

The next question addressed was whether this high-dose simvastatin treatment affects mitochondrial CoQ content. When mitochondria were isolated, extracted and analyzed by reversed phase HPLC, it was revealed that simvastatin induced a marked decline in CoQ content. In this case, both CoQ9 and CoQ10 levels decreased. When C2C12 myotubes were co-incubated with CoQ10-containing ND and simvastatin, CoQ10 levels increased while, as expected, CoQ9 levels remained low. This result supports the view that exogenous CoQ, administered as aqueous soluble ND, prevents statin-induced loss of CoQ in this cell culture model. Given the effects of high dose simvastatin on C2C12 myotube CoQ content, we sought to evaluate whether free cholesterol levels were affected. When statin-treated C2C12 cells were extracted and analyzed, the data revealed that free cholesterol levels were the same as control cells. Thus, C2C12 myotubes likely compensated for simvastatin-induced inhibition of the mevalonate pathway by either hydrolyzing available cholesteryl ester stores or taking up cholesterol from the culture medium. In terms of the latter process, a well known response of cells deprived of cholesterol is to up-regulate the low density lipoprotein receptor, thereby promoting increased uptake of exogenous cholesterol [26]. In any case, the observed statin-induced effects on cell viability cannot be attributed to a loss of free cholesterol content.

Having established that CoQ ND prevent simvastatin-dependent depletion of CoQ, we sought to determine the extent to which replenishment of mitochondrial CoQ prevents statin-induced effects on cell viability. The results showed a distinct, albeit partial, prevention of the decline in cell viability induced by incubation with simvastatin alone. Of note, when simvastatin was co-incubated with either CoQ9 or CoQ10 ND, their relative abilities to prevent simvastatin-induced loss of cell viability were equivalent.

Based on the ability of high dose simvastatin treatment to deplete mitochondrial CoQ levels, it was hypothesized that this will lead to a measurable decline in aerobic respiration. Compared to control cells, myotubes treated with simvastatin showed a marked decrease in basal and maximal OCR. When myotubes were incubated with simvastatin plus CoQ10 ND, but not empty ND, enhanced basal and maximal OCRs were detected, indicating that exogenous, ND-derived CoQ10, is functional, and counteracts the deleterious effects of simvastatin on cellular respiration.

In humans, primary CoQ10 deficiency is caused by mutations in genes that encode enzymes involved in CoQ10 biosynthesis [27]. Moreover, almost every patient suffering from the deleterious, potentially fatal, effects of primary CoQ10 deficiency have been reported to show clinical improvement in response to high-dose oral CoQ10 supplementation [28,29]. Depending on the severity of the mutational variant, patients with primary CoQ deficiency are administered a loading dose of CoQ10 followed by a daily supplement (100-600 mg/day). Despite this intervention, some mutational variants prove fatal within a year [29,30]. Given its poor oral bioavailability [31], it is conceivable that intravenous / intramuscular CoQ10 ND therapy could provide an alternate treatment strategy for patients suffering from primary CoQ10 deficiency.

Aside from inherited deficiencies in CoQ10 biosynthetic enzymes, suboptimal CoQ10 levels have been proposed to contribute to heart failure [32], a problem anticipated to be exacerbated by statin therapy. Indeed, beneficial effects of CoQ supplementation were manifest only when statins were discontinued [33]. Thus, it is conceivable that, if the bottleneck of poor CoQ bioavailability can be surmounted, statin therapy may not have to be halted, thereby preserving its beneficial effects. This goal may be achievable in the form of an injectable CoQ formulation. The availability of CoQ ND provides an opportunity to pursue in vivo studies of CoQ bioavailability and, potentially, expand treatment options for genetic, statin-induced or age-related CoQ insufficiency.

The likely reason that very high oral CoQ10 supplement concentrations are required is poor oral bioavailability [9]. It is well known that the 50-carbon isoprene tail of CoQ10 confers significant hydrophobicity, rendering the molecule completely insoluble in aqueous media. This is in keeping with the fact that the bulk of CoQ present in mitochondria arises from de novo biosynthesis. Despite poor oral bioavailability, CoQ10 supplementation has proven vital for the survival of patients suffering from the destructive, and potentially fatal, consequences of a primary CoQ10 deficiency [30,32].

The effects of statin-mediated inhibition of the mevalonate pathway appear to be more complicated than merely a deficiency in CoQ10. The mevalonate pathway is utilized for production of several other biomolecules that contain isoprene units, including dolichol [34], farnesyl [35] and geranylgeranyl [36] moieties as well as isopentenyladenosine tRNAs [37]. Since inhibition of the mevalonate pathway by statins is expected to result in inhibition of all downstream isoprenoid products, CoQ supplementation alone is unlikely to protect against all statin-related effects.

Although promising, the experiments reported herein require further investigation. As a logical progression, CoQ ND administration to a statin-treated murine model would permit key questions related to the efficacy of CoQ ND in vivo to be addressed. This could lead to intravenous or intramuscular administration CoQ administration methods for treatment of primary CoQ deficiency syndromes or age-related CoQ depletion-related disorders [38].

Acknowledgements

Supported by a grant from the National Institutes of Health (R37 HL64159). We thank Dr. Irina Romenskaia for assistance with cell culture and Dr. Marina MacLean with operation of the GC.

Abbreviations:

ND

nanodisks

OCR

oxygen consumption rate

CoQ

coenzyme Q

HMG

3-hydroxy, 3-methylglutaryl

PC

phosphatidylcholine

PBS

phosphate buffered saline

GC

gas chromatography

DAPI

4’,6-diamidino-2-phenylindole

Footnotes

The authors have no potential, perceived or real conflict of interest disclosures to declare

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References

  • 1.Ioannidis JP. More than a billion people taking statins?: Potential implications of the new cardiovascular guidelines. JAMA 2014;311(5):463–464. [DOI] [PubMed] [Google Scholar]
  • 2.Scandinavian Simvastatin Survival Study Group. Randomized trial of cholesterol lowering in 4444 patients with coronary heart disease: The Scandinavian Simvastatin Survival Study (4S). Lancet 1994;344(8934):1383–1389. [PubMed] [Google Scholar]
  • 3.Ghirlanda G, Oradei A, Manto A, Lippa S, Uccioli L, Caputo S, et al. Evidence of plasma CoQ10-lowering effect by HMG-CoA reductase inhibitors: a double-blind, placebo-controlled study. J Clin Pharmacol 1993;33(3):226–229. [DOI] [PubMed] [Google Scholar]
  • 4.Marcoff L, Thompson PD. The role of coenzyme Q10 in statin-associated myopathy: a systematic review. J Am Coll Cardiol, 2007;49(23):2231–2237. [DOI] [PubMed] [Google Scholar]
  • 5.Sirvent P, Mercier J, Lacampagne A. New insights into mechanisms of statin-associated myotoxicity. Curr Opin Pharmacol 2008;8(3):333–338. [DOI] [PubMed] [Google Scholar]
  • 6.Curry L, Almukhtar H, Alahmed J, Roberts R, Smith PA. Simvastatin Inhibits L-Type Ca2+− Channel Activity Through Impairment of Mitochondrial Function. Toxicol Sci 2019;169:543–552. [DOI] [PubMed] [Google Scholar]
  • 7.Caso G, Kelly P, McNurlan MA, Lawson WE. Effect of coenzyme q10 on myopathic symptoms in patients treated with statins. Am J Cardiol 2007:99(10):1409–1412. [DOI] [PubMed] [Google Scholar]
  • 8.Joy TR, Hegele RA. Narrative review: statin-related myopathy. Ann Intern Med 2009; 150(12):858–868. [DOI] [PubMed] [Google Scholar]
  • 9.Kommuru TR, Gurley B, Khan MA, Reddy IK. Self-emulsifying drug delivery systems (SEDDS) of coenzyme Q10: formulation development and bioavailability assessment. Int J Pharm 2001;212(2), 233–246. [DOI] [PubMed] [Google Scholar]
  • 10.Greenberg S, Frishman WH. Coenzyme Q10: a new drug for cardiovascular disease. J Clin Pharmacol 1990; 30(7): 596–608. [DOI] [PubMed] [Google Scholar]
  • 11.Chopra RK, Goldman R, Sinatra ST, Bhagavan HN. Relative bioavailability of coenzyme Q10 formulations in human subjects. Int J Vitam Nutr Res 1998;68(2): 109–113. [PubMed] [Google Scholar]
  • 12.Langsjoen PH, Langsjoen AM. Comparison study of plasma coenzyme Q10 levels in healthy subjects supplemented with ubiquinol versus ubiquinone. Clin Pharmacol Drug Dev 2014;3.1:13–17. [DOI] [PubMed] [Google Scholar]
  • 13.Oda MN, Hargreaves P, Beckstead JA, Redmond KA, van Antwerpen R, Ryan RO. Reconstituted high-density lipoprotein enriched with the polyene antibiotic, amphotericin B. J Lipid Res 2006;47:260–267. [DOI] [PubMed] [Google Scholar]
  • 14.Redmond KA, Nguyen TS, Ryan RO. All-trans retinoic acid nanodisks. Int J Pharm 2007;339:246–250. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 15.Ghosh M, Singh ATK, Xu W, Sulchek T, Gordon LI, Ryan RO. Curcumin nanodisks: formulation and characterization. Nanomedicine 2011;7:162–167. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 16.Ikon N, Hsu FF, Shearer J, Forte TM, Ryan RO. Evaluation of cardiolipin nanodisks as lipid replacement therapy for Barth syndrome. J Biomed Res. 2018;32:107–112. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 17.Tufteland M, Pesavento JB, Bermingham RL, Hoeprich PD Jr., Ryan RO. Peptide stabilized amphotericin B nanodisks. Peptides 2007;28:741–746. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 18.Moschetti A, Vine LN, Lethcoe K, Dagda RK, Ellison P, Ryan RO. Assembly and characterization of biocompatible coenzyme Q10-enriched lipid nanoparticles. Lipids 2020;55:141–149. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 19.Ryan RO, Forte TM, Oda MN. Optimized bacterial expression of human apolipoprotein A-I. Protein Expr Purif 2003;27:98–103. [DOI] [PubMed] [Google Scholar]
  • 20.Ryan RO. Nanodisks: Hydrophobic drug delivery vehicles. Expert Opin Drug Deliv 2008;5:343–351. [DOI] [PubMed] [Google Scholar]
  • 21.Ryan RO. Nanobiotechnology applications of reconstituted high density lipoprotein. J Nanobiotechnology 2010;8:28. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 22.Mullen PJ, Lüscher B, Scharnagl H, Krähenbühl S, Brecht K. Effect of simvastatin on cholesterol metabolism in C2C12 myotubes and HepG2 cells, and consequences for statin-induced myopathy. Biochem Pharmacol 2010;79(8):1200–1209. [DOI] [PubMed] [Google Scholar]
  • 23.Bligh EG, Dyer WJ. A rapid method of total lipid extraction and purification. Can J Biochem Physiol 1959;37:911–917. [DOI] [PubMed] [Google Scholar]
  • 24.Lujan B, Kushmerick C, Banerjee TD, Dagda RK, Renden R. Glycolysis selectively shapes the presynaptic action potential waveform. J Neurophysiology 2016;116:2523–2540. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 25.Lass A, Sohal RS. Effect of coenzyme Q10 and α-tocopherol content of mitochondria on the production of superoxide anion radicals. FASEB J 2000;14:87–94. [DOI] [PubMed] [Google Scholar]
  • 26.Goldstein JL, Brown MS.Regulation of the mevalonate pathway. Nature 1990;343:425–430 [DOI] [PubMed] [Google Scholar]
  • 27.Wang Y, Hekimi S. The Complexity of Making Ubiquinone. Trends Endocrinol Metab 2019;30:929–943. [DOI] [PubMed] [Google Scholar]
  • 28.Quinzii CM, DiMauro S, Hirano M. Human coenzyme Q 10 deficiency. Neurochem Res 2007;32(4-5):723–727. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 29.Quinzii CM, Naini A, Salviati L, Trevisson E, Navas P, DiMauro S, et al. A mutation in parahydroxybenzoate-polyprenyl transferase (COQ2) causes primary coenzyme Q10 deficiency. Am J Hum Genet 2006;78(2):345–349. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 30.López LC, Schuelke M, Quinzii CM, Kanki T, Rodenburg RJ, Naini A, et al. Leigh syndrome with nephropathy and CoQ10 deficiency due to decaprenyl diphosphate synthase subunit 2 (PDSS2) mutations. Am J Hum Genet 2006;79(6):1125–1129. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 31.Pastor-Maldonado CJ, Suárez-Rivero JM, Povea-Cabello S, Álvarez-Córdóba M, Villalón-García I, Munuera-Cabeza M, et al. Coenzyme Q (10): novel formulations and medical trends. Int J Mol Sci 2020;21:E8432. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 32.Sharma A, Fonarow GC, Butler J, Ezekowitz JA, Felker GM. Coenzyme Q10 and heart failure: a state-of-the-art review. Circ Heart Fail 2016;9:e002639. [DOI] [PubMed] [Google Scholar]
  • 33.Langsjoen PH, Langsjoen JO, Langsjoen AM, Lucas LA. Treatment of statin adverse effects with supplemental Coenzyme Q10 and statin drug discontinuation. Biofactors 2005;25(1-4):147–152. [DOI] [PubMed] [Google Scholar]
  • 34.Glomset JA, Gelb MH, Farnsworth CC. Prenyl proteins in eukaryotic cells: a new type of membrane anchor. Trends Biochem Sci 1990;15(4):139–142. [DOI] [PubMed] [Google Scholar]
  • 35.Schafer WR, Kim R, Sterne R, Thorner J, Kim SH, Rine J. Genetic and pharmacological suppression of oncogenic mutations in ras genes of yeast and humans. Science 1989; 245(4916):379–385. [DOI] [PubMed] [Google Scholar]
  • 36.Whyte DB, Kirschmeier P, Hockenberry TN, Nunez-Oliva I, James L, Catino JJ, et al. K-and N-Ras are geranylgeranylated in cells treated with farnesyl protein transferase inhibitors. J Biol Chem 1997;272(22):14459–14464. [DOI] [PubMed] [Google Scholar]
  • 37.Schweizer U, Bohleber S, Fradejas-Villar N. The modified base isopentenyladenosine and its derivatives in tRNA. RNA Biol 2017;14:1197–1208. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 38.Gutierrez-Mariscal FM, Yubero-Serrano EM, Villalba JM, Lopez-Miranda J. Coenzyme Q10: From bench to clinic in aging diseases, a translational review. Crit Rev Food Sci Nutr 2019;59:2240–2257. [DOI] [PubMed] [Google Scholar]

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