EFAVIRENZ, ATAZANAVIR, AND RITONAVIR DISRUPT SARCOPLASMIC RETICULUM Ca²⁺ HOMEOSTASIS IN SKELETAL MUSCLES

Abstract

Muscle fatigue, pain and weakness are co-morbidities in people living with HIV-1 infection (PLWH), yet an underlying cause remains poorly understood. Herein we evaluated whether concentrations of efavirenz (EFV), atazanavir (ATV), and ritonavir (RTV) that reflect what is present in blood of PLWH could be contributing to the reported skeletal muscle co-morbidites by perturbing sarcoplasmic reticulum (SR) Ca²⁺ cycling. In live-cell imaging, EFV (6.0 μM), ATV (6.0 μM), and RTV (3.0 μM) elicited Ca²⁺ transients and blebbing of the plasma membranes of C2C12 skeletal muscle myotubes. Pretreating C2C12 skeletal muscle myotubes with the SR Ca²⁺ release channel blocker ryanodine (50 μM), slowed the rate and amplitude of Ca²⁺ release from and reuptake of Ca²⁺ into the SR. EFV, ATV and RTV (1 nM - 20 μM) potentiated and then displaced [³H] ryanodine binding to rabbit skeletal muscle ryanodine receptor Ca²⁺ release channel (RyR1). The drugs at concentrations 0.25 to 31.2 μM also increased and or decreased the open probability of RyR1 by altering its gating and conductance. ATV (≤ 5 μM) potentiated also inhibited the ability of sarco(endo)plasmic reticulum Ca²⁺-ATPase (SERCA1) to hydrolyze ATP and transport Ca²⁺. RTV (2.5 – 31.5 μM) dose-dependently inhibited SERCA1-mediated, ATP-dependent Ca²⁺ transport. EFV (0.25 – 31.5 μM) had no measurable effect on SERCA1’s ability to hydrolyze ATP and transport Ca²⁺. These data support the notion that EFV, ATV and RTV could be contributing to skeletal muscle co-morbidities in PLWH by modulating SR Ca²⁺ homeostasis.

INTRODUCTION

Anti-retroviral therapy (ART) has increased life expectancy for people living with HIV-1 infection (PLWH) [1]. However, chronic usage of some antiretroviral drugs (ARVs) is linked to skeletal muscle fatigue, weakness and pain that are independent of neurologic diseases [3–11]. Kietrys et al., reported that fifty one percent of middle aged PLWH have muscle pain and weakness [12]. In poorly managed patients, this dynapenia can progress to muscle loss (sarcopenia) and early onset frailty despite ARV-induced viral suppression [13]. The Multicenter AIDS Cohort Study also found HIV-1 infected individuals over the age of fifty-five years were twice as likely to be frail compared to uninfected controls [14]. Not only do these muscle deficits result in multiple non-specific health complaints, falls, and post-operative complications requiring more frequent and longer hospital stays, but they also decrease the overall day-to-day quality of life for PLWH [9].

Three ARVs that are associated with muscle fatigue, weakness and pain, are the non-nucleoside reverse transcriptase inhibitor, efavirenz (EFV) and the protease inhibitors, atazanavir (ATV), and ritonavir (RTV) but specific mechanisms by which they do remain undefined [14–22]. The World Health Organization (WHO) and the National Institutes of Health (NIH) recommend using EFV with two nucleoside reverse transcriptase inhibitors (NRTIs) to treat adults and adolescents with HIV-1 RNA >500,000 copies/mL or with HBV coinfection [23–24]. This recommendation is based on data from large randomized controlled trials and cohort studies that showed EFV plus two NRTIs efficiently suppressed plasma viral replication and were superior or non-inferior to comparable regimens in ART-naive patients [24]. EFV is given at a daily dose of up to 600 mg/day to attain plasma C_max concentrations of ~2.5 μg/ml (or 8 μM). In patients of child-bearing age, and those with metabolic diseases such as atherosclerosis, metabolic syndrome and diabetes that have HIV RNA levels <100,000 copies/mL, the WHO and NIH also recommend including ATV as a component of ART therapy [23–24]. This recommendation is also based on cohort studies showing that ATV decreased the risk of cardiovascular events and slowed the progression of atherosclerosis, as measured by carotid artery intima medial thickness [16]. Daily dosing of 300 mg/day ATV achieves a plasma C_max of ~ 2.2 μg/ml (3.0 μM). RTV (100 mg/day) is usually co-administered with ATV (and other protease inhibitors) to boost ATV’s plasma C_max to ~ 6.0 μM. Boosted ATV, also has relatively fewer metabolic adverse effects [24].

Efficient skeletal muscle contraction/relaxation depends critically on the timely and adequate release of Ca²⁺ from and reuptake of Ca²⁺ into the sarcoplasmic reticulum (SR) [25]. Ca²⁺ release from the SR is initiated by depolarization-induced mechanical interactions between L-type Ca²⁺ channels on the plasma membrane and type 1 ryanodine receptor release channels (RyR1) on the SR. Contraction is terminated when Ca²⁺ released from the SR is returned inside the SR via sarco(endo)plasmic reticulum ATPase (SERCA1). Defects in and/or aberrant activation of these processes are established causes of skeletal muscle weakness and associated diseases [26–30].

Thus, the objective of the current study is to use live-cell Ca²⁺ imaging, displacement [³H]ryanodine binding assay, lipid bilayer single channel assay, ATP-hydrolysis and ATP-dependent Ca²⁺ transport assays to determine if the muscle fatigue, weakness and pain associated with EFV, ATV and RTV is due, in part, to the drug’s abilities to perturb SR Ca²⁺ homeostasis.

METHODS and EXPERIMENTAL PROCEDURES

Antibodies, reagents, and drugs

[³H]Ryanodine and ⁴⁵Ca²⁺ were purchased from GE-Healthcare (Chicago, IL). Phosphatidylserine, phosphatidylcholine and phosphatidylethanolamine were obtained from Avanti Polar Lipids Inc. (Alabaster, AL). Free-base efavirenz (EFV) was obtained from Shengda Pharmaceutical Co (Zhejiang, China), Atazanavir (ATV) sulfate was purchased from Gyma Laboratories of America, Inc (Westbury, NY). Dialysis membranes were obtained from Spectrum Laboratories Inc (Rancho Dominguez, CA). Mouse monoclonal RyR1 antibodies (34C), mouse monoclonal SERCA1 antibodies (VE121G9) and FKBP12 Monoclonal Antibody (OTI3B3) were obtained from Fisher Scientific (Waltham, Massachusetts). All other reagents and solvents used were of the highest grade commercially available.

Preparation of stock solutions of EFV, ATV and RTV

EFV (7.85 mg, M_w = 315.67 Da), ATV (17.65 mg, M_w = 704.86 Da) and RTV (14.4 mg, Mw 720.95) were dissolved in 2.5 ml of ethanol. Water (2.5 ml) was then added and the suspensions were vortexed for 5 min at room temperature (22˚C) and centrifuged at 20,000 × g for 30 min. The supernatants were removed and diluted 10 X with methanol and high-performance liquid chromatography (HPLC) was used to determine the concentrations of these drugs in solutions. Samples (20 μl) were analyzed using a YMC Octyl C8 column (Waters Inc., Milford, MA) with a mobile phase consisting of 52% 25mM KH₂PO₄, pH 4.15/48% acetonitrile at a flow rate of 0.4 ml/min and UV/Vis detection at 212 nm.

C2C12 cell cultures

Mouse skeletal C2C12 myoblasts were obtained from ATCC^® (CRL-1772, Manassas, VA) and grown in Dulbecco’s modified Eagle’s medium containing 1.8 mM CaCl₂ (DMEM; Invitrogen, Carlsbad, CA), supplemented with 10% fetal bovine serum (Sigma-Aldrich, St. Louis, MO) and antibiotics (100 units/ml penicillin, 100 μg/ml streptomycin, 100 μg/ml gentamicin, pH 7.3) with 5% CO₂/95% air at 37°C. At 70–80% confluency, cells were sub-cultured onto laminin-coated glass-bottom chambers for 24 hrs. The media were changed to DMEM with 2% fetal bovine serum to differentiate myoblast into myotubes (96 hrs) as described earlier [31].

Preparation SR vesicles

Sarcoplasmic reticulum (SR) membrane vesicles were prepared from male New Zealand white rabbits (3 – 4.5 kg, Charles River Laboratories, Wilmington, MA) as described earlier [31, 32, 33, 34], with approval from the Institutional Animal Care and Use Committee, University of Nebraska Medical Center and in accordance with the Guide for the Care and Use of Laboratory Animals.

(a). Crude SR vesicles:

New Zealand White rabbits were anesthetized with Inactin (sodium thiobutabarbital, 200mg/kg, via an ear vein) and then sacrificed by exsanguination [31, 32, 33, 34]. White muscle from the back and hind legs were then removed and placed in isolation buffer (0.3 M sucrose, 5 mM imidazoleMC1 (pH 7.4 at 4 “C) containing the protease inhibitors, phenylmethylsulfonyl fluoride (PMSF, 230 μM) and leupeptin (1.1 μM). The muscle (~300 g) was then cut into small pieces, placed into six Beckman type JA- 10 centrifuge tubes (~ 50 g each) with 250 ml of isolation buffer and homogenized 3 × 30 sec each time, using a Kinematica PT-600 Polytron at a setting of 4.5. The homogenates were centrifuged at 7500 × g_av for 20 mm and the supernatants were discarded. The pellets were then rehomogenized (3 × 30 sec each time) in 250 ml of isolation buffer, using the Polytron at a setting of 5.5. The homogenates were then centrifuged at 11,000 × g_av for 20 min. After filtering of the supernatants through four layers of cheesecloth, crude microsomal vesicles were obtained by sedimentation at 85,000 × g_av for 30 min in a Beckman type 45Ti rotor. The pellets were resuspended (using a 20-ml syringe and a blunt-tipped stainless steel needle) in 0.3 M sucrose, 10 mM imidazole/HC1, pH 7.4, containing freshly diluted PMSF and leupeptin. Crude microsomal vesicles were divided into 100 ml aliquots, flash frozen in dry ice/acetone, and stored at −80°.

(b). Junctional and longitudinal SR vesicles:

Crude microsomal vesicles (5 ml) were layered onto discontinuous sucrose gradients consisting of (from bottom to top) 5 ml of 1.5 M, 7 ml of 1.2 M, 7 ml of 1.0 M, and 7 ml of 0.8 M sucrose in isolation buffer containing freshly dissolved PMSF and leupeptin [31–34]. The gradients were then centrifuged in a Beckman SW-28 swinging bucket-type rotor at 110,000 × g_av for 2 hr. The membrane fractions sedimenting at the 1.2 M/1.5 M and 0.8M/1.0M sucrose interfaces were collected by aspiration. They were then diluted 3 X with 10 mM imidazole/HC1 buffer, the vesicles were recovered by sedimentation at 100,000 × g_av for 30 min in a Beckman type 45Ti rotor. The pellets were resuspended at a protein concentration of 4–8 mg/ml in buffer containing 0.3 M sucrose, 10 mM imidazole/HC1, pH 7.4, with PMSF and leupeptin, flash frozen and stored at −70°C. A [³H]ryanodine binding assay was conducted to determine the amount of [³H]ryanodine bound per mg junctional SR protein (1.5 M/1.2 M sucrose interface) compared to crude membrane vesicles. ATP-dependent Ca²⁺ uptake assays (spiked with ⁴⁵Ca²⁺) were also performed to determine the amount ⁴⁵Ca²⁺ transported from the solution into the light SR vesicles (0.8 M/1.2 M sucrose) per mg protein compared to crude membrane vesicles.

Proteoliposomes containing RyR1

Junctional SR vesicles (19 ml of 4 mg/ml) were solubilized in buffer containing 1.0 M NaCl, 0.05 mM EGTA, 0.35 mM Ca²⁺, 5 mM AMP, 20 mM Na/PIPES, pH 7.4, 0.3 mM Pefabloc, 0.03 mM leupeptin, 0.9 mM dithiothreitol (DTT), 1.5% CHAPS, and 5 mg/ml phosphatidylcholine as described earlier [31]. Solubilized samples were centrifuged at 26,000 × g_av for 30 min, and the supernatant was layered onto a 7–15% linear sucrose gradient (3 ml of solubilized protein onto 34 ml per gradient, 6 tubes) and centrifuged at 89,500 × g_av (Beckman Ultra Centrifuge, SW28 rotor) for 17 h at 4°C. [³H]ryanodine (3.3 nM) was placed into one of the tubes to for locating RyR1. After centrifugation, 2 ml aliquots from the [³H]ryanodine-labeled tube were collected, starting from the bottom of the tube assayed for [³H]ryanodine (scintillation counter) to determine location of RyR1. Aliquots (15 μl) were also mixed with gel dissociation medium, electrophorese using 4–15% SDS-polyacrylamide gels and silver stained to verify purified RyR1 in fractions containing high [³H]ryanodine binding. Fractions containing purified RyR1 were pooled and dialyzed at 4°C for 44 h in buffer containing 0.5 M NaCl, 0.1 mM EGTA, 0.2 mM CaCl₂, 10 mM Na/PIPES, pH 7.4, 0.1 mM DTT, and 0.1 mM PMSF with three buffer changes at 2, 6, and 15 h. Proteoliposomes containing purified RyR1 were then centrifuged at 163,500 × g_av for 2 hrs, and the pellet was then resuspended in buffer containing 10 mM Na/PIPES, pH 7.4, 0.1 mM DTT and 0.3 M sucrose and 100 μl aliquots were quick-frozen and stored in the vapor phase of liquid nitrogen until use [31].

Western blot

Western blot assays were conducted to determine the presence of RyR1, the immunophilin protein FKBP12.0 (a protein associated with RyR1) and SERCA1 in SR membrane preparations and in proteoliposome fractions using methods described earlier [35, 36, 37]. Primary antibody concentrations were 1:1000 for 16 hrs. at 4°C and secondary antibody concentrations were 1:2500 for 2 hrs. at room temperature. β-actin served as the internal reference.

Effects of EFV, ATV, and RTV on intracellular Ca²⁺ homeostasis in C2C12 cells

Differentiated C2C12 myotubes in DMEM were loaded with Fluo 3-AM (5 μM) for 30 min at 37°C, washed, and placed on the stage of a laser confocal microscope (Zeiss Confocal LSM 510 confocal microscope equipped with an Argon-Krypton Laser, 25 mW argon laser, 2% intensity, Thornwood, NJ, excitation wavelength 488 nm, and emission wavelengths of 515 nm) [31]. EFV (6 μM, 12 μM), ATV (6 μM, 12 μM), and RTV (3.0 μM, 6.0 μM) were manually added to a corner of the glass chamber and images were recorded every 2 sec for 3 min after addition. Experiments were repeated with a second set of cells that were pre-treated with ryanodine (50 μM) for 20 min prior to the addition of the anti-retroviral agents. Data were analyzed using LSM Meta 5.0, Microsoft Excel (Microsoft, Seattle, WA) and Prism (GraphPad, Version 7, La Jolla, CA).

Effects of EFV, ATV, and RTV on activity of RyR1

(a). [³H]ryanodine binding assays:

SR membrane vesicles (0.1 mg/ml) were incubated in binding buffer (500 mM KCl, 20 mM Tris·HCl, 0.03 mM Ca²⁺, 2 mM reduced glutathione, and 100 μM EGTA, 6.7 nM [³H]ryanodine, pH 7.4) with varying concentrations of EFV (1 – 20000 nM), ATV (1 −20000 nM), RTV (1 – 20000 nM), or ryanodine (0 to 300 nM) for 2 hr at 37°C. Non-specific [³H]ryanodine binding was determined by incubating a separate set of vesicles with 1 μM unlabeled ryanodine [31, 32, 33, 34]. After the two-hour incubation, membranes were rapidly filtered, washed and [³H]ryanodine on filter paper were counted using a liquid scintillation counter. Nonlinear regression analyses were then used to fit the data to a one- and a two-site competition models using GraphPad Prism (Version 7).

(b). Single channel recordings:

For this, phosphatidylethanolamine, phosphatidylserine and phosphatidylcholine in a ratio of 5:3:2 (35 mg/mL of lipid) in n-decane were painted across a 250 μm diameter hole of the bilayer cup as described earlier [31,36,37]. Proteoliposomes containing purified RyR1 was then added to the cis side (equivalent to the cytoplasm) to allow a single RyR1 to fuse into the bilayer. The other side of the bilayer cup was designated the trans side (equivalent to the lumen of the SR). All recordings were performed in symmetric KCl buffer solutions (0.25 mM KCl, 20 mM K/HEPES, pH 7.4) with the cis chamber containing 3.0 – 3.3 μM Ca²⁺. EFV (0 – 31.2 μM), ATV (0 – 30 μM) and RTV (0 – 30 μM) were added sequentially as boluses to the cis chamber at 100 times higher than the final concentrations and the chamber were vigorously stirred for 15 s prior to obtaining a 2 min recording at ± 30 mV. All experiments were conducted at room temperature (23–25°C) and electrical signals were filtered at 2 kHz and digitized at 10 kHz. Data were acquired using commercially available equipment and software (Axopatch 1D, Digidata 1322A and pClamp 10.2, Axon Instruments, Burlingame, CA, USA). Data were analyzed using pClamp 10.2 (Axon Instruments, Burlingame, CA, USA), Sigma Plot 10.0 (Stystat Software Inc, Chicago, IL, USA) and GraphPad Prism (Ver 7).

Effects of EFV, ATV and RTV on SERCA1 Activity

(a). ATPase activity:

Longitudinal SR vesicles (3.0 ml of 15 μg/ml) were incubated in buffer (10 mmol/L HEPES, pH 7.3; 0.1 mol/L KCl; 5 mmol/L Mg²⁺; 100 μmol/L Ca²⁺; 100 μmol/L EGTA; and 2.5 mmol/L Na₂-ATP) with varying concentrations of EFV, ATV and RTV (0 – 20 μM) for 20 min at 37°C in the absence or presence of the Ca²⁺ ionophore, A23187 (3 μg/mL) [37, 38]. After 20 mins, the reaction was stopped by the addition of 0.05 ml of 5M perchloric acid. Inorganic phosphate (Pi) generated from the hydrolysis of ATP was assessed using the malachite green colorimetric assay described by Lanzetta et al., [39] Data are expressed as micromoles of inorganic phosphate (P_i) liberated per 100 μg protein per 20 min. Longitudinal vesicles incubated with the SERCA1 inhibitor thapsigargin was also used to determine non-SERCA1 ATPase activity.

(b). ATP-dependent Ca²⁺ accumulation (steps 1–6 of the POST-ELBERS cycle [E₁→E₂]):

Longitudinal SR vesicles (3.0 ml of 15 μg/ml) enriched in SERCA1 were incubated in buffer containing 100 mM KCl, 50 mM histidine, 6.5 mM MgCl₂, 3.0 mM Tris oxalate, 0.5 mM EGTA, 50 μM ryanodine, 0.5 M Ca²⁺ (spiked with 0.5 to 1.0 μCi of ⁴⁵Ca²⁺ per ml of incubation buffer) [35, 36] with varying amounts of EFV (0 – 31.2 μM), ATV (0 – 31.2 μM) and RTV (0 – 31.2 μM). Na₂ATP (3 mM) was then added to initiate Ca2+ uptake and after 20 min at 37°C, samples were diluted in ice-cold wash buffer (100 mM KCl, 50 mM histidine, 6.5 mM MgCl₂, 3.0 mM Tris oxalate, 0.5 mM EGTA, 50 μM ryanodine and 5 mM ruthenium red (wash buffer), rapidly filtered and washed 3X with 2 ml of wash buffer. The amount of ⁴⁵Ca²⁺ remaining on the filter paper subtracted from the amount remaining in the presence of the inhibitor cocktail was used as a measure of ATP-dependent Ca²⁺ transport by SERCA1. A separate set of longitudinal vesicles were also incubated with the SERCA1 inhibitor thapsigargin to determine non-SERCA1 Ca²⁺ uptake [40].

Statistical Analysis

Paired Student T-tests were used to compare data with and without drug treatment using Microsoft Excel (Microsoft Corporation, Seattle WA). One-way analysis of variance (ANOVA) followed by the Bonferroni’s post-hoc test was also used for some studies employing GraphPad Prism 7.0 (La Jolla, CA). Data are presented in text and graphs as the mean ± S.E.M. Significance was determined at the 95% confidence interval.

RESULTS

EFV, ATV and RTV stock levels

Following HPLC analyses, 6.3% of EFV, 12.9% of ATV and 1.5% of RTV dissolved in ethanol/water (1:1), affording stock concentrations of 1.8 mM EFV, 3.2 mM ATV, and 0.39 mM RTV, respectively.

EFV, ATV and RTV effects Ca²⁺ homeostasis

EFV (6 μM) added to antiretroviral-naïve C2C12 skeletal muscle myotubes induced a rapid elevation in cytoplasmic Ca²⁺ that originated perinuclear and dispersed to the ends of myotubes (Fig. 1A, red line, see also Supplemental video #1A). The mean peak amplitude of the Ca²⁺ transient was 3.9 ± 0.3 fluorescence units (f.u.) change with a rate of Ca²⁺ rise of 0.14 ± 0.01 f.u./sec. and a rate of Ca²⁺ transient decay of 0.04 ± 0.00 f.u./sec, Fig.1B, red lines). The duration of the global Ca²⁺ transient elicited by EFV also lasted for about 90 sec (Fig. 1A). EFV-induced elevation in cytoplasmic Ca²⁺ was also blebbing of the skeletal muscle cell membrane (Supplemental video, #1A). Addition of a second dose of EFV (6 μM) to EFV-exposed C2C12 cells did not elicit a second Ca²⁺ transient. Addition of caffeine (20 mM) to the EFV-treated cells also failed to elevate intracellular Ca²⁺, suggesting that EFV may be depleting Ca²⁺ levels inside the SR (data not shown). Addition of 6 μM EFV to antiretroviral-naïve C2C12 cells that was pretreated with 50 μM ryanodine for 20 min generated an attenuated Ca²⁺ transient, i.e., the rate of rise was slower and the peak Ca²⁺ transient amplitude were reduced (Figs. 1A and 1B, blue lines). Pre-treating C2C12 cells with ryanodine also attenuated EFV-induced blebbing of the plasma membrane, indicating that the blebbing of the plasma membrane arises from elevation in cytoplasmic Ca²⁺ (compare Supplemental videos #1A and #1B). Exposing antiretroviral-naive C2C12 cells to an initial 10 μM EFV, afforded a Ca²⁺ transients with similar kinetics to that elicited by 6 μM (data not shown). (ii) Exposing antiretroviral-naïve C2C12 cells to 6 μM ATV also elicited Ca²⁺ transients. However, the rate of Ca²⁺ transient rise was 7.7 × faster than that of EFV and the rate of decay of the Ca²⁺ transient was 3x faster than that of EFV (Fig. 1C and 1D, red lines, Supplemental video #1C). The amplitude of ATV Ca²⁺ transient was similar to EFV (4.4 ± 0.1 f.u.). The rise in cytoplasmic Ca²⁺ by ATV was also associated blebbing of the plasma membrane of C2C12 cells. However, the diameter of the blebs was ~1.5 × larger than that of EFV (Supplemental fig.1C). Addition of a second dose of ATV (6 μM) to C2C12 cells that had previously been exposed to ATV, did not elicit a second Ca²⁺ transient. Addition of caffeine (20 mM) to ATV-treated to C2C12 cells also failed to trigger intracellular Ca²⁺ transients, suggesting that EFV is also depleting SR Ca²⁺ stores (data not shown). Exposing antiretroviral-naive C2C12 cells to an initial 10 μM ATV, afforded a Ca²⁺ transients with similar kinetics to that elicited by 6 μM ATV (data not shown).

Figure 1: ARV-induced elevation in cytoplasmic Ca²⁺ in C2C12 skeletal muscle myotubes.

Panel A shows the mean Ca²⁺ transient (twenty C2C12 cells from six separate chambers, done on three different days) elicited by 6 μM efavirenz (EFV) in antiretroviral-naïve C2C12 skeletal muscle myotubes (red), and the mean Ca²⁺ transients elicited in C2C12 cells that have been pre-treated with 50 μM ryanodine prior to addition of 6 μM EFV (blue). Black arrow point to Panel B shows the mean rates of cytoplasmic Ca²⁺ rise and cytoplasmic Ca²⁺ decay triggered by EFV in antiretroviral-naïve C2C12 skeletal muscle myotubes (red) and in C2C12 cells that have been pre-treated with 50 μM ryanodine prior to addition of EFV (blue lines). Each time point from Ca²⁺ transients and kinetic measurements were mean ± SEM. Panel C shows the mean Ca²⁺ transient (eighteen C2C12 cells from six separate chambers, done on three different days) elicited by 6 μM ATV in antiretroviral-naïve C2C12 skeletal muscle myotubes (red), and the mean Ca²⁺ transients elicited in C2C12 cells that have been pre-treated with 50 μM ryanodine prior to addition of 6 μM ATV (blue). Panel D shows the mean rates of cytoplasmic Ca²⁺ rise and cytoplasmic Ca²⁺ decay triggered by ATV in antiretroviral-naïve C2C12 skeletal muscle myotubes (red) and in C2C12 cells that have been pre-treated with 50 μM ryanodine prior to addition of ATV (blue lines). Each time point from Ca²⁺ transients and kinetic measurements were mean ± SEM. Panel E shows the mean Ca²⁺ transients (eighteen C2C12 cells from six separate chambers, done on three different days) elicited by 3 μM ritonavir (RTV) in antiretroviral-naïve C2C12 skeletal muscle myotubes (red), and the mean Ca²⁺ transient elicited in C2C12 cells that have been pre-treated with 50 μM ryanodine prior to addition of 3 μM RTV (blue). Panel F shows the mean rates of cytoplasmic Ca²⁺ rise and cytoplasmic Ca²⁺ decay triggered by RTV in antiretroviral-naïve C2C12 skeletal muscle myotubes (red) and in C2C12 cells that have been pre-treated with 50 μM ryanodine prior to addition of RTV (blue lines). Each time point from Ca²⁺ transients and kinetic measurements were mean ± SEM.

Pre-treatment of C2C12 cells with 50 μM ryanodine for 20 min attenuated the amplitude of Ca²⁺ transient elicited by ATV (6 μM), indicating that ATV is also mobilizing Ca²⁺ from the SR via RyR1 to elevate cytoplasmic Ca²⁺ (Figs. 1C and 1D, blue lines, Supplemental video #1D). Inhibiting RyR1 also attenuated ATV-induced blebbing of the plasma membrane of C2C12 cells, emphasizing an association between the elevation in cytoplasmic Ca²⁺ and plasma membrane blebbing (compare Supplemental videos #1C and #1D). Addition of RTV (3 μM) to antiretroviral-naïve C2C12 cells also elicited Ca²⁺ transients with mean rate of Ca²⁺ rise of 0.54 ± 0.06 f.u./sec peak amplitude 4.3 ± 0.2 f.u that lasted for about 20 sec (Fig. 1E, red line, Supplemental video #1E). The rate of decay of RTV’s Ca²⁺ transient was similar to ATV’s, but 3x faster than that of EFV (Fig. 1E, red line). The rise in cytoplasmic Ca²⁺ induced by RTV was also associated with blebbing of the plasma membranes of C2C12 cells. Unlike EFV and RTV, addition of a second dose of RTV (3 μM) to C2C12 cells, triggered a second Ca²⁺ transient, with peak amplitude greater than the first and with faster decay kinetics (Fig. 1E, red, black arrow), suggesting enhanced Ca²⁺ uptake into the SR and/or increased effusion of Ca²⁺ from the cytoplasm. Pre-treating C2C12 cells with 50 μM ryanodine for 20 min also attenuated the amplitude of the Ca²⁺ transient elicited by RTV (6 μM), indicating that RTV is elevating cytoplasmic Ca²⁺ by mobilizing it from the SR via RyR1 (Figs. 1E and 1F, blue lines, Supplemental video #1F).

EFV, ATV and RTV effects on RyR1

Having, established that EFV, ATV and RTV can trigger Ca²⁺ release from the SR of skeletal muscle cells, displacement binding and single channel approaches were then used to delineate mechanisms of actions of these drugs on RyR1.

(a) [³H]ryanodine displacement assays offer a rapid and highly sensitive assay to screen and to assess the interactions of drugs with RyR1. Since the high affinity ryanodine binding site lies within the pore-forming region of RyR1 and the degree of “openness” of the channel can be modulated in vitro by the [Ca²⁺] in the binding buffer, we selected a [Ca²⁺] in the binding buffer to maintain RyR1 in a partially opened state [31, 32, 34]. If a ligand binds to an open RyR1, then amount of [³H]ryanodine bound per unit time will be enhanced over baseline. Conversely, if a ligand deactivates or closes RyR1, the amount of [³H]ryanodine bound per unit time will be lowered.

All three drugs enhanced then reduced the amount of [³H]ryanodine bound to RyR1 after two hours of incubation. EFV at concentrations between 1–10 nM, ATV at concentrations between 1–20 nM, and RTV at concentrations between 1–60 nM enhanced [³H]ryanodine binding to RyR1, with EC_50s’ of 5.2 ± 1.4 nM, 14.9 ± 1.4 nM and 18.1 ± 2.2 nM, and maximum percent enhancement of 20.5 ± 2.2 %, 40.5 ± 5.2 % and 62.3 ± 6.2 % respectively, over control (Fig. 2A). Higher concentrations of EFV (20 nM - 20 μM), displaced [³H]ryanodine from RyR1 with the displacement curve spanning < two log units (Fig. 2A, open triangles). The displacement curve for EFV fitted to a two-site model (r² = 0.96) with an IC₅₀ value for site 1 of 730.2 ± 40.0 μM and IC₅₀ value for site 2 of 4200.1 ± 400 nM. Using the Cheng-Prussoff equation defined by K_i = IC₅₀/(1 + (L/K_L)), where L is the concentration of [³H]ryanodine used (6.7 nM), and K_L is the equilibrium dissociation constant of [³H]ryanodine = 2.4 nM for RyR1, the K_i of EFV for the higher affinity site responsible for displacement of [³H]ryanodine was 52.8 ± 2.4 nM and 1100 ± 200 nM for the lower affinity site [31, 34, 41, 42]. The Hill slope was 1.6, consistent with positive cooperativity. At higher concentrations (> 100 nM), ATV and RTV also displaced [³H]ryanodine from RyR1, but their displacement curves fitted to a single site with IC_50s’ of 6200.8 ± 800 nM for ATV and 8800.1 ± 1200.5 nM for RTV. K_is’ (displacement) for ATV and RTV calculated using the Cheng-Prussoff equation were 1600.5 ± 200 nM and 2300.4 ± 300.1 nM. For comparison, the prototype ligand ryanodine (0.2 – 300 nM) also displaced [³H]ryanodine from RyR1 with the displacement curve spanning over two log units (Fig. 2A, open circles). The displacement curve fitted to a one-site model (r² = 0.98) with an IC₅₀ value of 5.3 ± 0.4 μM. Using the Cheng-Prussoff equation, the K_i of ryanodine for RyR1 was 1.4 ± 0.2 nM.

Figure 2: Effects of EFV, ATV and RTV on the binding of [³H]ryanodine to RyR1 and purification of RyR1 from rabbit skeletal muscles.

Panel A shows curves for the effects of increasing concentrations of EFV (Δ), ATV (■), RTV (□) and the prototype ligand ryanodine (ο) on equilibrium [³H]ryanodine binding to RyR1 in a buffer containing 0.03 mM CaCl₂. Each point for each compound is from n=6 experiments done in duplicate using three separate SR membrane preparations. Curves were fitted using the binding analysis programs of Prism 7.0 (GraphPad Inc., San Diego, CA) for a one or a two-site fit for potentiation and displacement parts of the curves. Panels B and C are to demonstrate our ability to generate proteoliposomes containing RyR1 and that the immunophilin FKBP12.0 remain bound to RyR1 after solubilization and linear sucrose gradient centrifugation. Panel B shows a representative silver-stained gel for the protein profile for 20 μl volumes from solubilized junctional SR proteins and proteoliposome aliquots collected after linear sucrose gradient centrifugation. Aliquots were mixed with gel dissociation medium also electrophoresed on 4 –15% linear gradient Tris-glycine polyacrylamide gel at 150 V for 180 min. Arrows show purified and partly degraded RyR1. Panel C shows a representative autoradiogram for the immunophilin FKBP12.0 in solubilized junctional SR proteins and proteoliposome aliquots collected after linear sucrose gradient centrifugation.

(b) Single channel studies were then conducted using proteoliposomes containing RyR1 reconstituted into lipid bilayers to gain insights into the effects of EFV, ATV and RTV on the gating, open probability (P_o) and conductance of RyR1. Fig. 2B shows that after solubilization and linear sucrose gradient centrifugation of junctional SR membranes, fractions 2 and 7 contained purified RyR1 on high-sensitivity silver-stained polyacrylamide gel. Fig. 2C show these fractions also contain the FKBP12.0, an immunophilin that helps to stabilize the closed state of the channel and reduces sub-conductance (sub-state) opening of RyR1 [43, 44, 45, 46].

(i). EFV and single RyR1:

Prior to addition of EFV, mean P_o of RyR1 in 3.0 μM cis Ca²⁺ at room temperature and at + 30 mV was 0.01 ± 0.00 (Fig. 3 top, also see Supplemental, Table 1). The current amplitude was also 21.8 ± 1.8 p A, equivalent to a conductance = 726 ± 60 pS, consistent with earlier studies [31, 35]. Addition of 240 nM EFV to the cis chamber, increased the P_o of RyR1 eight-fold (13/15 separate RyR1 channels) within 15 sec after addition (Table 1 and Supplemental Figs. 1A–1K). The other two channels did not respond to EFV. The increase in P_o arose at ± 30 mV holding potentials from increases in the frequency of transitions from closed and full open states, with no significant changes in current amplitude/channel conductance (Fig. 3). The solvent used (1 % ethanol) and lower concentrations of EFV had no effect on the P_o of RyR1 up to 5 minutes after cis addition. Increasing the concentration of cis EFV to 2.5 μM, increased the P_o of RyR1 further by decreasing the dwell time in the closed state (Fig. 3 and Table 1, also see Supplemental Fig. 1A–K for gating parameters). At 2.5 μM cis, EFV also induced a reversible state of reduced conductance (R1, 33% of maximum, 233 ± 15 pS, Fig. 5). R1 at −30 mV lasted for ~125 msec, and three times that (~375 msec) at +30 mV. Transitions from R1 to the fully opened state were not observed. Increasing cis EFV to 5.0 μM increased the P_o of RyR1 further by decreasing the dwell time in the closed state (Fig. 3, Table 1 and Supplemental Figs 1A–K). At 5.0 μM, EFV also induced a second reversible state of reduced conductance with a current amplitude of 66 % of maximum (R2, 479 ± 24 pS, Fig. 3, also see Supplemental Fig 1A–K). Transitions from R2 to full opened state and from the full opened to R1 were observed at +30 mV. Increasing cis EFV concentration to 12.5 μM, increased the P_o of RyR1 further by increasing the dwell time in the open state (Fig, 5). At 12.5 μM cis EFV, both R1 and R2 were also observed, although R2 was the dominant sub-state. Increasing EFV in the cis chamber to 20.0 μM cis, did not increase the P_o of RyR1 (Fig. 3), but the channel was more likely to transition from the fully opened to the R2 state. Increasing EFV in the cis chamber further to 31.2 nM decreased the P_o of RyR1 by decreasing the dwell time in the open state and by increasing the dwell time in the closed state. R1 but not R2 were also observed at ± 30 mV. After 90 sec, the channel went into an irreversible shut state (Fig. 3A, bottom panels).

Figure 3: Effects of increasing concentrations of EFV on gating and conductance of rabbit skeletal muscle RyR1.

Single-channel currents were recorded at +30 mV (left, upward deflections) and −30 mV (right, downward deflections) in symmetric KCl buffer solution (0.25 mM KCl, 20 mM K-HEPES, pH 7.4) with 3.0 μM cis Ca²⁺ as described in the text. The top right and top left panels show representative recordings of a RyR1 channel in the absence of EFV over 0.5 seconds and after additive concentrations of EFV added to the cis chamber. O, open; C, closed; R1 are states of reduced conductance induced by EFV. The gating parameters for n = 15 separate channels are shown in Table 1.

Table 1:

Effects of antiretroviral drugs on RyR1 function

Efavirenz (μM)	Po (+30 mV)	Dwell time open state	Dwell time closed state	Closed- maximally opened transitions (Hz)
0.00	0.01 ± 0.00	0.62 ± 0.05	6.34 ± 0.28	532 ± 47
0.24	0.08 ± 0.01*	0.61 ± 0.07	4.91 ± 0.43*	1125 ±24*
2.50	0.22 ± 0.01*	0.74 ± 0.06	1.38 ± 0.08*	1122 ±22*
5.00	0.59 ± 0.03*	1.01 ± 0.01*	0.56 ± 0.07*	1046 ±30*
12.50	0.69 ± 0.04*	1.03 ± 0.05*	0.53 ± 0.46*	1005 ±16*
20.00	0.67 ± 0.04*	0.99 ± 0.04*	0.57 ± 0.06*	1060 ±25*
31.20	0.38 ± 0.01*	0.73 ± 0.06*	1.53 ± 0.02*	1064 ±25*
Atazanavir (μM)	Po (+30 mV)	Dwell time open state (ms)	Dwell time closed state (ms)	Closed- maximally opened transitions (Hz)
0.00	0.09 ± 0.00	0.33 ± 0.01	4.63 ± 0.30	623 ± 40
1.56	0.14 ± 0.01*	0.66 ± 0.02*	1.97 ± 0.03*	789 ± 25*
3.12	0.56 ± 0.03*	2.83 ± 0.08*	0.62 ± 0.03*	1102 ±22*
6.78	0.92 ± 0.10*	25.12 ± 1.52*	0.55 ± 0.07*	206 ± 30*
13.20	0.82 ± 0.04*	5.70 ± 0.20*	0.43 ± 0.05*	225 ±16*
20.00	0.60 ± 0.04*	1.44 ± 0.05*	2.22 ± 0.15*	750 ± 55
30.00	0.11 ± 0.01	0.33 ± 0.01	5.65 ± 0.06	142 ±20*
Ritonavir (μM)	Po (+30 mV)	Dwell time open state (ms)	Dwell time closed state (ms)	Close - maximally opened transitions (Hz)
0.00	0.02 ± 0.00	0.37 ± 0.03	5.26 ± 0.68	567 ± 53
0.24	0.07 ± 0.01*	0.35 ± 0.02	4.25 ± 0.11	773 ± 47*
2.50	0.29 ± 0.03*	0.68 ± 0.06*	1.01 ± 0.22*	998 ± 89*
5.00	0.07 ± 0.04	1.22 ± 0.15*	13.2 ± 1.81*	762 ± 45*
12.50	0.11 ± 0.03*	1.64 ± 0.03*	07.67 ± 0,74	828 ± 40*
20.00	0.11 ± 0.02*	1.01 ± 0.06*	10.57 ± 1.24*	848 ± 37*
31.20	0.92 ± 0.01*	1.13 ± 0.09*	0.72 ± 0.09*	880 ± 30*

Data shown are mean ± SEM for n=15 channels for EFV, n= 13 channels for ATV and n= 11 channel for RTV.

^*denotes significantly different (p<0.05) from no drug.

Figure 5: Effects of increasing concentrations of RTV on gating and conductance of rabbit skeletal muscle RyR1.

Single-channel currents were recorded at +30 mV (left, upward deflections) and −30 mV (right, downward deflections) in symmetric KCl buffer solution (0.25 mM KCl, 20 mM K-HEPES, pH 7.4) with 3.0 μM cis Ca²⁺ as described in the text. The top right and top left panels show representative recordings of a RyR1 channel in the absence of RTV over 0.5 seconds and after additive concentrations of RTV added to the cis chamber. O, open; C, closed; R1 are states of reduced conductance induced by RTV. The gating parameters for n = 11 separate channels are shown in Table 1.

(ii). ATV and single RyR1:

In 3.3 μM cis Ca²⁺ (slightly higher than EFV), the mean P_o of thirteen RyR1 channels at +30 mV was 0.09 ± 0.01 (Fig. 4 top, also see Table 1 and Supplemental Figs 2A–K). The current amplitude was 21.6 ± 2.0 pA, equivalent to a conductance of 720 ± 67 pS. Addition of 1.6 μM ATV to the cis chamber, increased the P_o of RyR1 by 40% in 12/13 RyR1 channels within 15 sec. The other channel did not respond to cis ATV. The increase in P_o arose from increases in the frequency of transitions from closed and full open states (gating frequency) and the dwell time in the opened state, and from a decrease in the dwell time in the closed state (Fig. 4, also see Supplemental Figure 2A–K). These changes were seen equally at both ± 30 mV holding potentials, with no significant change in current amplitude/channel conductance. Lower concentrations of ATV did not elicit changes in the P_o of RyR1 up to five minutes after addition. Increasing the concentration of ATV in cis chamber to 3.2 μM, increased the P_o of RyR1 further by increasing the number of transitions from the closed to opened state, decreasing the dwell time in the closed state and increasing the dwell time in opened state (Fig. 4, also see Table 1 and Supplemental Figure 2A–K). At this concentration, ATV did not alter the conductance of RyR1. Increasing cis ATV further to 6.8 μM, increased the P_o of RyR1 to near full open (0.92 ± 0.10) by decreasing the dwell time in the closed state and increasing the dwell time in the opened state (> 25 msec at ± 30 mV holding potential). At this concentration, ATV also decreased the current amplitude of RyR1 to 74% of maximum (i.e., reduced conductance state, R’1, 532 ± 34 pS, Fig. 4). Increasing cis ATV to 13.2 μM, attenuated the P_o of RyR1 by increasing the number of transitions from the opened to closed state (Fig. 4, also see Table 1). The channel also opened only to R’1 reduced conductance state. Increasing cis ATV further to 20.0 μM and 30.0 μM cis, decreased the P_o of RyR1 by decreasing the number of transitions from the closed to the opened state and increasing the dwell time in the closed state (Fig. 4). The current amplitude of RyR1 in the presence of 20.0 μM and 30.0 μM cis ATV remained at 74% of maximum (R’1).

Figure 4: Effects of increasing concentrations of ATV on gating and conductance of rabbit skeletal muscle RyR1.

Single-channel currents were recorded at +30 mV (left, upward deflections) and −30 mV (right, downward deflections) in symmetric KCl buffer solution (0.25 mM KCl, 20 mM K-HEPES, pH 7.4) with 3.3 μM cis Ca²⁺ as described in the text. The top right and top left panels show representative recordings of an RyR1 channel in the absence of ATV over 0.5 seconds and after additive concentrations of ATV added to the cis chamber. O, open; C, closed; R1 are states of reduced conductance induced by ATV. The gating parameters for n = 13 separate channels are shown in Table 1.

(iii). RTV and single RyR1:

Mean P_o of RyR1 in 3.0 μM cis Ca²⁺ before the addition of RTV was 0.02 ± 0.00 (Fig. 5, Table 1 and Supplementary Figure 3A–K). The current amplitude was also 21.5 ± 1.4 pA, equivalent to a conductance = 717 ± 45 pS. Addition of 720 nM RTV to the cis chamber, increased the P_o of RyR1 three-fold in all eleven RyR1 channels assayed. The increase in P_o arose from an increase in the frequency of transitions from closed and fully open state and the associated decrease in the dwell time in the closed state at ± 30 mV holding potentials (Fig. 5, Table 1 and Supplementary Figure 3A–K). Lower concentrations of RTV did not elicit changes in the P_o of RyR1 up to five minutes after addition. Increasing the concentration of RTV in cis chamber to 1.6 μM, increased the P_o of RyR1 further by increasing the number of transitions from the closed to opened state (Fig. 5). Increasing cis RTV to 3.2 μM, did not changed the P_o of RyR1 (0.15 ± 0.02). However, the gating of RyR1 changed significantly to fewer but longer lasting transitions from the closed to opened state. The current amplitude of RyR1 was also reduced by 16% to 84 % of maximum (R’2, 599 ± 24 pS, Fig. 6 and Supplementary Figure 3A–K). There were fewer but longer lasting transitions from the closed to opened state and the reversible state of reduced conductance (R’2,) persisted with 7.8 μM and 15.6 μM cis RTV (Fig. 5). Increasing the concentration of RTV in the cis chamber further to 31.2 μM, increased the P_o of RyR1 to near fully open (0.92 ± 0.01, +30 mV) by increasing the dwell time in the opened state, by decreasing the dwell time in the closed state and decreasing the number of transitions from the closed to the opened state (Fig. 6). The current amplitude remained at 84 % of maximum, R’2.

Figure. 6. The effects of antiretroviral drugs on the ability of SERCA1 to hydrolyze ATP and transport Ca²⁺.

Panels A, B and C show the effects of efavirenz, atazanavir and ritonavir on their ability of SERCA1 to hydrolyze ATP (steps 2–3 of the Post-Elber’s cycle), respectively. Graphs are means ± SEM from n=6 experiments from three separate longitudinal SR membrane preparations with (■) and without (□) the ionophore A28137. * denotes significantly different (p < 0.05) from no drug. Panels D, E and F show the effect of EFV, ATV and RTV on the ability of SERCA2a to transport Ca2+ (E1→E2 of the Post-Elbers cycle). Graphs are means ± SEM from n = 6 experiments from three separate longitudinal SR membrane preparations. * denotes significantly different (p<0.05) from no drug.

EFV, ATV and RTV effect SERCA1 activity

Having, observed attenuations in the rate of Ca²⁺ transient decay by EFV, ATV and RTV in ryanodine-treated C2C12 cell, experiments were conducted to assess the effects of EFV, ATV and RTV on the ability of SERCA1 to hydrolyze ATP and transport Ca²⁺. The light fraction at the interface of 0.8 M and 1.0 M from the discontinuous sucrose gradient centrifugation was used as it is enriched in SERCA1 protein [35].

(b). ATPase activity:

Treating longitudinal vesicles with EFV (0 – 20 μM) did not show any significant effect the ATPase activity of SERCA1 as assessed from the hydrolysis of ATP (Fig. 6A). As expected, total ATPase activity was higher in the presence of Ca²⁺ ionophore, A23187 since during isolation vesicles can be folding outside and resulting in SERCA1 on the inside the vesicles. The effect of ATV on the ATPase activity of SERCA1 was also biphasic with concentrations of 0.25 and 5.0 μM potentiated the ATPase activity of SERCA1, while higher concentrations decreased the ATPase activity of SERCA1 in a dose dependent manner (Fig. 6B). The biphasic effect was seen both in the presence and in absence of the Ca²⁺ ionophore, A23187. RTV at concentrations ranging from 0.25 to 20 μM, dose-dependently decreased the ATPase activity of SERCA1, in the presence or absence of the Ca²⁺ ionophore, A23187 (Fig. 6C). Incubating longitudinal membrane vesicles with EFV (0 – 31.2 μM) had no effect on ATP-dependent Ca²⁺ transport by SERCA1 (Fig. 6D). Interestingly, incubating longitudinal membrane vesicles with ATV (0 – 31.2 μM) resulted in a biphasic ATP-dependent Ca²⁺ transport by SERCA1 with up to 5.0 μM potentiating Ca²⁺ accumulation and > 5 μM inhibiting Ca²⁺ accumulation via SERCA1 (Fig. 6E). RTV (0 – 31.2 μM) dose-dependently inhibited the ability of SERCA1 to transport Ca²⁺ (Fig. 6F).

DISCUSSION

The principal finding of the present study is that the muscle fatigue, pain and weakness reported by PLWH taking the anti-retroviral drugs, EFV, ATV and RTV, may be due, in part, to disruption of skeletal muscle SR Ca²⁺ homeostasis. This conclusion is based on several observations. Using single cell confocal imaging we showed that the ARVs tested can induce global Ca²⁺ transients in antiretroviral naïve skeletal muscle cells. The amplitude of the Ca²⁺ transients were >5-fold rise over basal and lasted for >25 seconds. The elevation in cytoplasmic Ca²⁺ induced by ATV, EFV and RTV were attenuated by pretreating C2C12 skeletal muscle cells with the SR Ca²⁺ release channel blocker ryanodine, providing direct evidence that these drugs are mobilizing Ca²⁺ from the SR via activation of RyR1. The smaller amplitude Ca²⁺ transients seen in antiretroviral naïve, ryanodine-treated C2C12 cells may be due in part to EFV, ATV and RTV activating plasma membrane, store-operated Ca²⁺ and TRP channels [47, 48, 49, 50]. Exposing skeletal muscles C2C12 cells to a second dose of ATV and EFV did not elicit additional Ca²⁺ transients suggesting that these drugs may be closing RyR1. In the future, a temperature- and CO₂-regulated head stage will be attached to our microscope to prolong cell viability and determine if lower concentrations of ATV, EFV and RTV also induce subcellular Ca²⁺ changes.

In this study we also showed for the first time that the global elevation in cytoplasmic Ca²⁺ elicited by ATV, EFV and RTV are resulting in blebbing of their plasma-membrane of C2C12 skeletal muscle cells. The size of “blebs” distortion were different amongst the drugs, with ATV exposure resulting in the largest blebs followed and EFV and then RTV. Prior studies have shown that blebbing of the plasma membranes of skeletal muscle cells usually occurs above areas of hyper-contraction and arises primarily from an increase in oxidative stress [51, 52]. Additional work is needed to determine mechanisms by which ATV, EFV and RTV trigger oxidative stress in skeletal muscle cells.

Having identified RyR1 as a target for EFV, ATV and RTV, additional studies were conducted to gain insights into how these drugs are activating/deactivating RyR1. Using [³H]ryanodine displacement assays, we discovered that EFV binds to three distinct sites on RyR1; a high affinity site (nM) that activates or opens to increase [³H]ryanodine binding, and two lower affinity sites (high nM and low μM) that deactivate or close RyR1 and reduce [³H]ryanodine binding. [³H]ryanodine displacement assays also revealed two binding sites each for ATV and RTV; a high affinity (nM) that activates RyR1 and increase [³H]ryanodine binding and a μM affinity site that closes RyR1 and decreases [³H]ryanodine binding. To gain further insights into the mechanisms by which ATV, EFV and RTV activate and deactivate RyR1, we then used lipid bilayer single channel assays. In these assays we found that addition of nM concentrations of ATV, EFV and RTV to the cis chamber (equivalent to the cytoplasmic side of the channel), increased the frequency of transitions of RyR1 from closed to full opened state. These drugs also induced reversible states of sub-maximal conductance with long open times (>100 msec). EFV induced two sub-optimal conductance states (R1, 33%, and R2, 66% of maximum), while ATV and RTV induced one sub-optimal conductance state each of 74% of maximum and 84% of maximum, respectively. To date, the mechanisms by which EFV, ATV and RTV induced sub-maximal conductance states remain poorly understood. One simplistic possibility is that these drugs exists in multiple energetically favored conformations and binding of the different conformers alter the pore size of RyR1 [42]. Another possibility is that these drugs are competing/displacing the immunophilin FKBP12.0 bound to RyR1, akin to that seen with the immuno-suppressant agents tacrolimus (FK506) and sirolimus (rapamycin) that lead to states of sub-maximal conductance [44, 45]. In future studies we will use FK506/rapamycin to dissociate FKBP12.0 from RyR1 prior to evaluating the effects EFV, ATV and RTV on gating and conductance of RyR1. We may also create photo-activatable derivatives of EFV, ATV and RTV and use them to identify the locations of their binding sites on RyR1 [32, 40]. While mechanical interactions with L-type Ca²⁺ channnels servesas the physiological trigger for RyR1 activation, RyR1 is also activated by Ca²⁺ [53]. As such, activation of RyR1 by ATV, EFV and RTV and the subsequent release of Ca²⁺ from the SR could result in aberrant skeletal muscle contraction, occlusion of blood vessels and pain [54]. Others have shown that “leaky” RyR1 arising point mutations, oxidation stress and mechanical ventillation is also a common cause of muscle weakness in central core disease, muscular dystrophy and aging [55–61].

In addition to nM concentrations activating/opening RyR1, low micromolar amounts of ATV and EFV also dose-dependently reduced the open probability (P_o) of RyR1 by decreasing the frequency of transitions from the closed to the fully opened state, although transitions from the R1 state to the R2 and fully opened states were seen. Low micromolar EFV also irreversibly shut RyR1 (Fig.3 bottom left and right panels). Deactivation of RyR1 could a reason for the lack of Ca²⁺ transients in C2C12 cells by repeated doses of EFV and ATV. Inactivation and/or closure of RyR1 will attenuate the rate and amplitude of Ca²⁺ release from the SR which in turn will impair muscle contraction [62]. Although low micromolar concentrations of RTV did not close RyR1, it altered its gating pattern to one in which openings and closings events were infrequent but long lasting, akin to “intermittent bursting”. These intermittent bursts could also lead to aberrant skeletal muscle contraction.

In addition to their effects on RyR1, in this study we also investigated the effects of EFV, ATV and RTV on the ability of SERCA1 to hydrolyze ATP and transport Ca²⁺ to better understand their functional impact on SR Ca²⁺ cycling and skeletal muscle contraction/weakness. SERCA1 belongs to a family of highly conserved ATP-dependent Ca²⁺ pumps that transport Ca²⁺ against a concentration gradient via the Post-Elbers, multiple-step mechanisms [63, 64, 65]. By lowering cytosolic Ca²⁺, SERCA1 initiates muscle relaxation and at the same time replenishes the SR with Ca²⁺ for the next muscle contraction. In this study, we also showed for the first time that RTV dose-dependently inhibited the ability of SERCA1 to hydrolyze ATP and transport Ca²⁺, while ATV potentiated and then attenuated the ATPase activity of SERCA1 and its ability to transport Ca²⁺. EFV did not have any significant effect on the ability of SERCA1 to hydrolyze ATP and transport Ca²⁺. While it is tempting to speculate that ATV and RTV are modulating step 2 (E1(Ca²⁺) E1(ATP)) and/or step 3 (E1(ATP) E1(ADP) of the Post-Elbers cycle, since longitudinal SR vesicles were used, more studies are needed to determine if these drugs triggering the dissociation of the endogenous inhibitors from SERCA1 including phospholamban, sarcolipin and ankyrin 1 [66, 67]. In future studies, recombinant SERCA1 will be expressed in sf9 insect cells, and the effects of ATV and RTV on SERCA1 devoid of its inhibitor proteins will be investigated.

In conclusion, the present study show for the first time that the muscle fatigue, weakness and pain reported in PLWH that are ATV, EFV and RTV is due in part to these drugs disrupting SR Ca²⁺ homeostasis. By activating RyR1, nM concentrations of these drugs are depleting the Ca²⁺ content inside the SR and by closing RyR1, low micromolar concentrations of these drugs are preventing Ca²⁺ release from the SR. By inhibiting SERCA1, low micromolar concentrations of these drugs are preventing the reuptake of Ca²⁺ release from the SR in preparation for the next contraction. These drugs are also inducing blebbing of the plasma membrane of skeletal muscle cells, which is arising from an increase in oxidative stress. More work is needed to determine which of the other antiretroviral drugs are perturbing/not perturbing SR Ca²⁺ cycling at therapeutic doses so as to minimize ART-induced skeletal muscle fatigue, weakness and pain in PLWH.

Muscle fatigue and weakness are side effects of the anti-HIV drugs efavirenz (EFV), atazanavir (ATV) and ritonavir (RTV)

Here we show EFV, ATV and RTV are causing these muscle defects by disrupting sarcoplasmic reticulum (SR) Ca²⁺ homeostasis

More work is needed to determine if other anti-HIV drugs are also perturbing skeletal muscle SR Ca²⁺ homeostasis