Cell Calcium 38 (2005) 35–44

Rapamycin and FK506 reduce skeletal muscle voltagesensor expression and function

Guillermo Avilaa, Robert T. Dirksenb,∗a Department of Biochemistry, Cinvestav-IPN, AP 14-740. Mexico City, DF 07000, Mexicob Department of Pharmacology and Physiology, University of Rochester Medical Center,

601 Elmwood Avenue, Rochester, NY 14642, USA

Received 7 March 2005; received in revised form 4 May 2005; accepted 9 May 2005Available online 13 June 2005

Summary

FK506 and rapamycin are immunosuppressant drugs that disrupt the interaction of FK506-binding proteins (FKBPs) with ryanodine recep-tors (RyR1), which form homotetrameric Ca2+ release channels in the sarcoplasmic reticulum (SR) of skeletal muscle. Here, we characterizedthe effects of short-term treatment (2 h) of skeletal myotubes with either 20�M FK506 or 20�M rapamycin on excitation–contraction (EC)c ina a(cc fecto rs and thei©

K

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Ceo(fcrmt

rs

r

Ca,

chin-

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oupling, sarcolemmal dihydropyridine receptor (DHPR) function, resting intracellular Ca2+, and levels of SR Ca2+ content. Both rapamycnd FK506 produced remarkably similar effects. Specifically, both drugs reduced the maximal amplitude of voltage-gated SR C2+ release(�F/F)max) by 70–75% in parallel with a 50% reduction in both maximal immobilization resistant charge movement (Qmax) and L-type Ca2+

hannel conductance (Gmax). Neither immunosupressant significantly altered steady-state levels of either resting myoplasmic Ca2+ or SR Ca2+

ontent. Thus, store depletion does not account for the observed reduction in Ca2+ release during EC coupling. Instead, the inhibitory efn voltage-gated SR Ca2+ release is explained by significant reductions in both the number of functional sarcolemmal voltage senso

ntrinsic gain of voltage-gated Ca2+ release (i.e. the maximal rate of Ca2+ release per unit gating charge).2005 Elsevier Ltd. All rights reserved.

eywords:FKBP12; Immunophilins; Dihydropyridine receptor; Excitation–contraction coupling

. Introduction

A unique physical interaction between two types ofa2+ channels is a central feature of skeletal musclexcitation–contraction (EC) coupling. In triadic junctionsf skeletal muscle, a set of four dihydropyridine receptorDHPR) particles, termed a tetrad, are located directly acrossrom a homotetramere of ryanodine receptors (RyR1) thatomprise the SR Ca2+ release channel of the sarcoplasmiceticulum (SR). The principal role of the DHPR in skeletaluscle is to act as “voltage sensors”, which directly trigger

he opening of opposing RyR1 release channels in response

Abbreviations:FKBP, FK506-binding protein; DHPR, dihydropyridineeceptor; L-currents, L-type Ca2+ currents; RyRs, ryanodine receptors; SR,arcoplasmic reticulum; EC coupling, excitation–contraction coupling∗ Corresponding author. Tel.: +1 585 275 4824; fax: +1 585 273 2652.E-mail addresses:gavila@cinvestav.mx (G. Avila),

obertdirksen@urmc.rochester.edu (R.T. Dirksen).

to membrane depolarization. Once activated, the SR2+

release channel transports Ca2+ from the SR to the myoplasmwhere these ions activate proteins of the contractile maery (reviewed in[1–3]).

RyRs are composed of four identical protomers and fmacromolecular complexes with a wide variety of pteins (including FK506-binding proteins, calmodulin, hommAKAP, spinophilin) that regulate release channel acity. For example, each RyR1 protomer is associated wsingle 12-Da FK506-binding protein (FKBP12, also knoas calstabin1; for recent reviews, see[4,5]), which modi-fies the activity of RyR1 channels reconstituted intonar lipid bilayers[6–9]. Interestingly, in three-dimensionreconstitutions of the release channel, FKBP12 bindsregion of RyR1 that is thought to be close to the interacsite of RyR1 with the DHPR[10]. This suggests that thFKBP–RyR1 interaction might influence the efficacyDHPR triggered SR Ca2+ release. Accordingly, we foun

143-4160/$ – see front matter © 2005 Elsevier Ltd. All rights reserved.oi:10.1016/j.ceca.2005.05.001

36 G. Avila, R.T. Dirksen / Cell Calcium 38 (2005) 35–44

that the FKBP12–RyR1 interaction profoundly influences thegain of voltage-gated SR Ca2+ release. Specifically, com-pared to wild-type RyR1, the magnitude of voltage-gatedSR Ca2+ release is reduced∼50% following expression inRyR1 “knock-out” (or dyspedic) myotubes of RyR1 proteinscontaining point mutations that abolish FKBP12 binding[11]. More recently, the conclusion that FKBP12 stronglyinfluences the gain of EC coupling in skeletal muscle wascorroborated using myotubes derived from muscle-specificFKBP12-deficient mice[12].

FKBP12 is a member of a highly conserved familyof proteins termed immunophilins, which arecis–transpeptidyl–prolyl isomerases that were identified on the basisof their ability to bind immunosuppressant drugs, such asFK506 and rapamycin. Indeed, incubation of SR vesicleswith rapamycin or FK506 removes FKBP12 from the RyR1[13]. Although these drugs are largely known for theirimmunosuppressive effects, limited information is availablewith regard to their effects on skeletal muscle EC cou-pling, in spite of the pivotal role that the FKBP–RyR1interaction plays in this process[11,12]. To our knowledge,the work of Lamb and Stephenson[14] is the only pre-vious report in which effects of these agents on skeletalmuscle EC coupling were investigated. In that study, both20�M rapamycin and 20�M FK506 significantly reduceddepolarization-induced contractile responses in mechani-c tudyd le fort trac-t ofr onale d them Car

ar argem y-s rm ow-i( ease( c-t red ss atedC tion( ages ar main-i drugsap tb de ov en-s ge-g

2. Methods

2.1. Primary cultures of myotubes

Skeletal muscle from the extremities of newborn normalC57Bl6 mice was minced and digested in a Ca–Mg freeRinger’s solution, containing 0.3% trypsin and 0.01% DNAse(at 37◦C, 45 min). The digested tissue was then dispersed inplating medium consisting of Dulbecco’s Modified Eagle’sMedium (DMEM) supplemented with HS (10%), FBS(10%), penicillin (100 U/ml) streptomycin (100�g/ml) andl-glutamine (4 mM). The suspension was subsequently pre-plated (60 min at 37◦C) in order to reduce fibroblast content.The resulting myoblast-enriched supernatant was then platedon 35 mm Petri dishes at 10,000 cells/cm2 and transferred todifferentiation medium (identical to plating medium exceptwith total serum content set to 2% HS) 1 day later. Experi-ments were carried out on myotubes that were grown for 5–10days in differentiation medium. Treatments with immunosup-pressants consisted of 2-h incubations (at 37◦C) with either20�M rapamycin or 20�M FK506. Control experimentswith 2-h incubation in vehicle alone (0.2% DMSO) failedto significantly modify DHPR function (data not showed).

2.2. Measurements of resting Ca2+ and caffeine-inducedCa2+ release

re-v noI ope( d at3 ech-n d ofi mina ction2bf andF rd-i wasm ,c tem,aflc ribedpm on-c

2

s-t Cat thep

ally skinned rat skeletal muscle fibers. However, this sid not assess the precise mechanism(s) responsib

he observed reduction in depolarization-induced conile activity. Thus, here we characterized the effectsapamycin and FK506 on skeletal muscle DHPR functixpression (e.g. charge movement and L-currents) anagnitude/voltage dependence of voltage-gated SR2+

elease.We compared DHPR function (i.e. voltage-gated SR C2+

elease, immobilization-resistant intramembrane chovements and L-type Ca2+ channel activity) and stead

tate myoplasmic and SR Ca2+ levels in myotubes eitheaintained under standard conditions (control) or foll

ng 2 h exposure with either rapamycin (20�M) or FK50620�M). The results show that both drugs markedly decr70–75%) maximal voltage-gated Ca2+ release. The reduion in voltage-gated Ca2+ release did not result from stoepletion since maximal caffeine-induced Ca2+ release waimilar under all conditions. The decrease in voltage-ga2+ release could only partially be explained by a reduc

∼50%) in the number of functional sarcolemmal voltensors. In addition, the reduction in voltage-gated C2+

elease also involved a decrease in the efficacy of the reng voltage sensors to activate release, since bothlso reduced (∼50%) the maximal rate of SR Ca2+ releaseer unit gating charge ((�F/�t)/Qmax). We conclude thaoth immunosuppressant drugs decrease the magnituoltage-gated SR Ca2+ release by reducing both voltage sor functional expression and the intrinsic gain of voltaated SR Ca2+ release.

f

Resting intracellular Ca2+ levels were measured as piously described[11,16]. Briefly, intact myotubes grown glass coverslips were loaded with the Ca2+-sensitive dye

ndo-1 AM, mounted on an inverted Olympus microscIX70), bathed in Ringer’s solution (see below) and exite50 nm using a DeltaRam Illumination System (Photon Tology Inc., Princeton, NJ). The loading protocol consiste

ncubating control and pre-treated myotubes for 30–45t room temperature in rodent Ringer’s solution (see Se.4 below) supplemented with 6�M Indo-1 AM followedy an additional incubation for 20–30 min at 37◦C to allow

or complete de-esterification of the dye. RapamycinK-506 were not included in the indo-1 loading or reco

ng solutions. Fluorescence emission at 405 and 485 nmonitored using a 40× (1.35 NA) oil immersion objective

ollected at 100 Hz using a photomultiplier detection sysnd presented as the ratio (R) of F405/F485. Resting indo-1uorescence ratios (F405/F485) were converted to free Ca2+

oncentrations using an in situ calibration approach descreviously[15]. Relative changes in SR Ca2+ content wereonitored following application of a maximal activating c

entration of caffeine (10 mM).

.3. Voltage-clamp experiments

Voltage-gated L-type Ca2+ currents, immobilization resiant intramembrane charge movement and intracellular2+

ransients were elicited using the whole-cell variant ofatch clamp technique as previously described[16,17].

G. Avila, R.T. Dirksen / Cell Calcium 38 (2005) 35–44 37

Briefly, L-currents were acquired in response to 200 mstest pulses of variable amplitude from a holding potential of−80 mV. A 1s prepulse to−20 mV preceded each test pulsein order to inactivate endogenous T-type Ca2+ channels. L-currents were normalized by total cell membrane capacitance(Cm), plotted as a function of the test potential and fitted tothe following equation:

I = Gmax(Vm − Vrev)

1 + exp[(VG(1/2) − Vm)/kG](1)

whereVrev is the extrapolated L-current reversal potential,Vm the test potential,Gmax the maximal L-channel conduc-tance,VG(1/2) the voltage for half-activation ofGmax andkGis a slope factor. The average values ofCm were 270± 20 pF(n= 41), 327± 38 pF (n= 13) and 267± 27 pF (n= 13) forcontrol, rapamycin- and FK506-treated myotubes, respec-tively (p> 0.2).

For simultaneous measurements of gating currents andintracellular Ca2+ transients, the ionic composition of theexternal recording solution was altered by replacing 10 mMCaCl2 with 8 mM NiCl2 plus 2 mM CaCl2 (8Ni/2Ca) to elim-inate inward ionic currents. Under these conditions, it waspossible to simultaneously measure both gating charge move-ment and voltage-dependent SR Ca2+ release in the absenceof extracellular Ca2+ influx through the DHPR. Charge move-ments and Ca2+ transients were elicited by 30 ms test pulses.T entw thatmsT argeme

Q

w ithr e-ma ole-c elyd gh-s po-r dye( age-c ed toe ses.F long-p tereda atingi sureda es aree es-c st mpli-t ction

of membrane potential and fitted according to the followingequation:

�F

F= (�F/F )max

1 + exp[(VF (1/2) − Vm)/kF ](3)

where (�F/F)max, VF(1/2) and kF have their usual mean-ings with regard to Ca2+ transients. For calculations of thegain of voltage-gated Ca2+ release at saturating voltages(40–70 mV), the maximal rate of SR Ca2+ release approxi-mated from the peak of the first derivative of the myoplasmicCa2+ transient (�F/�t) was normalized to the maximal amountof charge movement recorded for that cell (Qmax). The result-ing quotient ([�F/�t]/Qmax) was then used as a quantitativeindex of the maximal gain of voltage-gated Ca2+ release (i.e.maximal rate of Ca2+ release per unit of gating charge) incontrol, rapamycin- and FK506-treated myotubes.

2.4. Recording solutions

L-currents were recorded using a pipette solution con-taining (mM): 140 Cs-aspartate, 5 MgCl2, 10 Cs2EGTA, 10HEPES and pH 7.4. The external solution contained (mM):145 TEA-Cl, 10 CaCl2, 0.003 TTX, 10 HEPES and pH 7.4.For simultaneous measurements of intracellular Ca2+ tran-sients and gating currents, a minimal Ca2+ buffering pipettesolution was used that contained (mM): 145 Cs-aspartate, 10CH peri-mT aa entR , 2C ataa atp

3

yR1i R1e alm e ift edf nga thee dis-r aged tedC on-dS elyb ns,v dea nce ofi -

he amount of immobilization-resistant charge movemas determined by integrating the transient of chargeoved outward after the onset of the test pulse (QON) and was

ubsequently normalized to total cell capacitance (nC/�F).he voltage dependence of immobilization-resistant chovement was estimated by fittingQON data to the followingquation:

ON = Qmax

1 + exp[(VQ(1/2) − Vm)/kQ](2)

hereQmax, VQ(1/2) andkQ have their usual meanings wegard to charge movement. Ca2+ transients and charge movents were recorded following a waiting period of∼5 minfter rupture of the cell membrane and entry into the whell mode in order to allow the dye (fluo-3) to completiffuse into the cell interior. A 75 Watt xenon bulb and hipeed DeltaRAM illuminator (Photon Technology Incorated, Monmouth Junction, NJ) were used to excite the480 nm) present in a small rectangular region of the voltlamped myotube. A computer controlled shutter was usliminate illumination during intervals between test pulluorescence emission was measured using a dichroicass mirror centered at 505 nm, an emission filter cent 535 nm, and a photomultiplier detection system oper

n analogue mode. Background fluorescence was meand canceled by analog subtraction. Fluorescence tracxpressed as�F/F, whereF represents the baseline fluorence immediately prior to depolarization and�F representhe fluorescence change from baseline. Fluorescence audes at the end of each test pulse were plotted as a fun

sCl, 0.1 Cs2EGTA, 1.2 MgCl2, 5 MgATP, 0.2 K5Fluo-3, 10EPES and pH 7.4. The external solution for these exents contained (mM): 145 TEA-Cl, 8 NiCl2, 2 CaCl2, 0.003TX, 10 HEPES and pH 7.4. Measurements of resting C2+

nd SR Ca2+ content were carried out in a normal rodinger’s solution consisting of (mM): 145 NaCl, 5 KClaCl2, 1 MgCl2, 10 HEPES and pH 7.4. Experimental dre presented as mean± S.E. with significance accepted< 0.05 (unpaired Student’st-test).

. Results

Using a molecular approach to disrupt the FKBP–Rnteraction, we previously found that FKBP binding to Rynhances the gain of voltage-gated Ca2+ release in skeletyotubes[11]. To probe this further, we set out to determin

he efficiency of voltage-gated Ca2+ release was also reducollowing disruption of the FKBP–RyR1 interaction usipharmacologic approach. Accordingly, we determinedffects of rapamycin and FK506, two drugs known toupt FKBP binding to RyR1, on the magnitude and voltependence of SR Ca2+ release. We measured voltage-gaa2+ release in whole-cell voltage clamp experiments cucted in the presence of 8 mM Ni2+ and 2 mM Ca2+ (seeection2) in the extracellular solution in order to completlock inward ionic Ca2+ currents. Under these conditiooltage-gated Ca2+ transients exhibit a similar magnitund voltage dependence as that observed in the prese

onic Ca2+ currents recorded in 10 mM Ca2+ (compare con

38 G. Avila, R.T. Dirksen / Cell Calcium 38 (2005) 35–44

trol (�F/F)–V data inTable 1with ref. [11]). Compared tothat observed for control myotubes (Fig. 1 A), the magni-tude of voltage-gated SR Ca2+ release was reduced 70–75%at all potentials following short-term treatment with eitherrapamycin (Fig. 1B) or FK506 (Fig. 1C). Rapamycin andFK506 significantly reduced the maximal values of voltage-gated SR Ca2+ release (�F/Fmax) in the absence of significantalterations in the voltage dependent parameters of releaseactivation (see ((�F/F)–V) data inTable 1). These resultscould arise either from drug-induced reductions in RyR1-releasable SR Ca2+ content and/or the ability of the voltagesensor to trigger Ca2+ release during depolarization.

We next determined if the drug-induced reduction involtage-gated Ca2+ release arises from alterations in steady-state myoplasmic and/or SR Ca2+ levels. As can be seenin Fig. 2, short-term exposure of myotubes to rapamycin(20�M) or FK506 (20�M) did not significantly alter restingCa2+ (Fig. 2B) or the level of RyR1-releasable SR Ca2+ con-tent (as determined from peak increase in myoplasmic Ca2+

in response to 10 mM caffeine;Fig. 2C).

Table 1Parameters of fitted�F/F–V,Q–V andI–V curves

Control Rapamycin FK506

(�F/F)–V data(�F/F)max 2.8 ± 0.4 * 0.7 ± 0.3 * 1.0 ± 0.2VF(1/2) (mV) 9.7 ± 2.5 14.5± 2.1 6.8± 1.6kF (mV) 6.4 ± 0.6 7.8± 0.6 5.3± 0.6n (8) (8) (9)

Q–V dataQmax (nC/�F) 11.5± 1.5 4.8± 0.7* 6.3 ± 0.9*

VQ(1/2) (mV) 10.3± 2.7 −4.5 ± 3.0* −0.2 ± 1.5*

kQ (mV) 16.2± 1.2 14.5± 1.1 13.9± 0.6n (8) (8) (13)

I–V dataGmax (nS/nF) 289± 22 154± 24* 199 ± 36*

VG(1/2) (mV) 15.9± 1.2 18.9± 1.6 21.5± 1.2*

kG (mV) 5.4 ± 0.2 7.1± 0.9* 6.8 ± 0.4*

Vrev (mV) 81 ± 0.8 79± 2.2 78± 2.1n (22) (11) (13)

∗ Compared with controlp< 0.05.

Fe(crav

ig. 1. Rapamycin and FK506 significantly decrease the amplitude of voltage-gated SR Ca+ release. (A–C) Representative voltage-gated Ca2+ transientslicited by 30 ms test depolarizations to the indicated membrane potentials (shown in A) in a control myotube (A) and myotubes treated with either rapamycinB) or FK506 (C). Voltage-gated Ca2+transients were obtained concurrently with gating current measurements following complete blockade of ionic Ca2+

urrents using an 8Ni2+/2Ca2+ extracellular solution (see alsoFig. 3). (D) Average (±S.E.) voltage-dependence of Ca2+ transients in control (white circles),apamycin-treated (black circles), and FK506-treated (gray circles) myotubes. The average values for the parameters obtained by fitting each myotube withingroup separately to Eq.(3) are given inTable 1((�F/F)–V data). The solid lines through each data set were generated using Eq.(3) and the corresponding

alues given inTable 1.

G. Avila, R.T. Dirksen / Cell Calcium 38 (2005) 35–44 39

Fig. 2. Rapamycin and FK506 do not alter resting intracellular Ca2+ or SR Ca2+ content. (A) Representative Indo-1 fluorescence ratio (F405/F485) traces fromintact normal myotubes under control conditions (top) or following treatment (2 h, 20�M) with either rapamycin (middle) or FK506 (bottom). Fluorescenceratios were recorded in the absence (∼first 40 s) and presence of 10 mM caffeine (grey bars). (B and C) Average (±S.E.) values of intracellular resting Ca2+

(B) and peak caffeine-induced Ca2+ release (i.e. caffeine response, C) from control, rapamycin-, and FK506-treated myotubes. Neither rapamycin nor FK506significantly (p> 0.2) altered intracellular resting Ca2+ (B), peak caffeine-induced Ca2+ release (C), or the following properties of the caffeine response: timeto peak, plateau ratio and recovery to baseline following caffeine removal (data not shown).

Since ionic L-type Ca2+ currents were blocked inthe presence of the 8Ni2+/2Ca2+ extracellular solution,measurements of intramembrane charge movements couldbe simultaneously recorded in the experiments shown inFig. 1. Short-term treatment of myotube cultures with eitherrapamycin or FK506 caused a significant reduction (∼50%)in the maximum magnitude of immobilization-resistantcharge movement orQmax (Fig. 3andQ–V data inTable 1).This reduction in voltage sensor charge movement at least

partially accounts for the observed∼70–75% reduction involtage-gated SR Ca2+ release following drug treatment(Fig. 1). However, in contrast to the absence of drug effectson the voltage dependence of SR Ca2+ release, rapamycinand FK506 also produced a 10–15 mV hyperpolarizing shiftin the voltage dependence for one-half activation of chargemovement (seeQ–V data inTable 1).

We next determined whether the observed reduction inmaximal immobilization-resistant charge movement was also

40 G. Avila, R.T. Dirksen / Cell Calcium 38 (2005) 35–44

Fig. 3. Rapamycin and FK506 significantly decrease the magnitude of immobilization-resistant intramembrane charge movement. (A–C) Representative non-linear capacitative currents (or gating currents) recorded in response to 30 ms membrane depolarizations to the indicated test potentials (shown inA) in a controlmyotube (A) and myotubes treated with either rapamycin (B) or FK506 (C). Data were obtained concurrently with Ca2+ transient measurements followingblockade of ionic Ca2+ currents using an 8Ni2+/2Ca2+ extracellular solution (see alsoFig. 1). (D) Average (±S.E.) voltage-dependence of immobilization-resistant charge movements (QON) recorded from control (white circles), rapamycin-treated (black circles) and FK506-treated myotubes (gray circles).QON

represents the integral of the currents at the onset of depolarization. The average values for the parameters obtained by fitting each myotube within agroupseparately to Eq.(2) are given inTable 1(Q–V data). The solid lines through each data set were generated using Eq.(2) and the corresponding values given inTable 1.

associated with significant alterations in the magnitude of L-type Ca2+ channel current density (Fig. 4). Treatment witheither rapamycin or FK506 reduced L-type Ca2+ current den-sity to a very similar extent as that observed for chargemovement (∼50%). Effects on L-current density occurred inthe absence of changes in kinetics of channel activation (com-pare traces inFig. 4A–C) and the few differences observed inthe voltage dependence of L-channel activation (VG(1/2) andkG) were modest in magnitude and did not reflect any orderedor systematic dependence (Fig. 4D; Table 1). Fitting theI–Vdata shown inFig. 4D to a modified Boltzmann equation (Eq.(1)) revealed that both drugs caused a similar∼50% reductionin the magnitude of maximal L-channel conductance (Gmax)without causing consistent changes in the other parameterof the fit (VG(1/2), kG, or Vrev) (seeI–V data inTable 1).To test for possible direct effects of immunosuppressants onDHPR L-channel activity in the absence of its interaction withRyR1, we also assessed effects of rapamycin treatment on L-channel activity in dyspedic myotubes (which lack RyR1 pro-teins). Rapamycin treatment failed to alter L-channel activityin dyspedic myotubes. Specifically, peak L-current ampli-tude (at +40 mV) in dyspedic myotubes was similar in bothcontrol (−0.92± 0.29 pA/pF,n= 5) and rapamycin-treated(−0.89± 0.26 pA/pF,n= 4, respectively) myotubes.

Together, results presented inFigs. 1–4indicate that thedrug-induced reductions in maximal L-current conductance(Gmax) arise largely from a reduction in the number of func-tional sarcolemmal DHPRs (Qmax). However, this reductionin the number of functional voltage sensors can only par-tially account for the larger 70–75% reduction in maximalvoltage-gated SR Ca2+ release observed following treatmentwith either rapamycin or FK506. An approach for assessingthe gain of Ca2+-induced–Ca2+-release in cardiac myocytesis to calculate the ratio of Ca2+ release and the integral ofthe triggering L-type Ca2+ current[18]. Therefore, we deter-mined the efficacy of voltage sensor activation of SR Ca2+

release (i.e. gain of voltage-gated Ca2+ release) by calcu-lating the ratio of the maximal rate of voltage-gated SRCa2+ release and the magnitude of voltage sensor chargemovement (i.e. (�F/�t)/Qmax) in control and drug-treatedmyotubes (Fig. 5). For this analysis, maximal voltage-gatedSR Ca2+ release flux (i.e.�F/�t) at saturating voltages (i.e.40–70 mV) was determined from the peak value of the firstderivative of the Ca2+ transient, which represents a reason-able approximation of the maximum rate of SR Ca2+ release(Fig. 5A). In control myotubes, maximal values of the rate ofSR Ca2+ release (�F/�t) and charge movement (Qmax) corre-lated well (Fig. 5B; white circles,R2 = 0.94), confirming that

G. Avila, R.T. Dirksen / Cell Calcium 38 (2005) 35–44 41

Fig. 4. Rapamycin and FK506 significantly decrease L-type Ca2+ current density. (A–C) Representative L-type Ca2+ currents elicited by 200 ms test depo-larizations to the indicated membrane potentials (shown in A) in a control myotube (A) and myotubes treated with either rapamycin (B) or FK506 (C). (D)Average (±S.E.) voltage-dependence of peak L-type Ca2+ current density in control (white circles), rapamycin-treated (black circles) and FK506-treated (graycircles) myotubes. The average values for the parameters obtained by fitting each myotube within a group separately to Eq.(1) are given inTable 1(I–V data).The solid lines through each data set were generated using Eq.(1) and the corresponding values given inTable 1.

voltage-gated Ca2+ release is linearly related to the numberof functional voltage sensors present in the membrane. Themaximal gain of voltage-gated Ca2+ release was then calcu-lated for each experimental condition. This analysis revealedthat the maximal gain of voltage-gated Ca2+ release wassignificantly (p< 0.05) reduced following short-term treat-ment with either rapamycin or FK506 ([�F/�t]/Qmax was35.8± 3.4, 19.5± 6.7 and 18.8± 7.9 for control, rapamycinand FK506, respectively;Fig. 5C).

4. Discussion

We show here that depolarization-induced SR Ca2+ releaseis severely compromised following short-term incubation(2 h) with either 20�M of rapamycin or FK506 (Fig. 1). Thisreduction in voltage-gated Ca2+ release does not result fromincreased SR Ca2+ leak and subsequent store depletion sincecaffeine-induced Ca2+ release is unaltered under these con-ditions (Fig. 2). Rather, our results indicate that rapamycinand FK506 treatment both reduce the number of functionalsarcolemmal voltage sensors (manifested as a parallel∼50%reduction in bothQmax andGmax (Figs. 3 and 4), respec-tively) and the intrinsic ability of the remaining sensors toefficiently activate SR Ca2+ release (manifested as an addi-tional ∼50% reduction in the maximal rate of Ca2+ releasep l-

edge, this is the first characterization of the mechanisms bywhich immunosuppressant drugs alter DHPR function, asboth Ca2+ permeable L-channels and voltage sensors for ECcoupling. We conclude that the observed drug-induced reduc-tions in voltage-gated Ca2+ transients (Fig. 1) result from botha decrease in the number of functional sarcolemmal volt-age sensors (Fig. 3) and a reduction in the intrinsic gain ofvoltage-gated Ca2+ release or rate of Ca2+ release per unit ofgating charge (Fig. 5). The following discussion provides acomparison of our results with those of previous studies, aswell as a consideration of the possible underlying molecularmechanisms involved.

Lamb and Stephenson[14] investigated the effects ofrapamycin and FK506 on depolarization-induced forceresponses in mechanically skinned muscle fibers of the rat.Interestingly, this study found that at low concentrations (i.e.1–2�M), both drugs induced a dual effect on muscle contrac-tion during stimulation with caffeine or depolarization: an ini-tial, fast and reversible potentiation of contraction (∼30–60 s)followed by a slower, irreversible reduction in contractil-ity (∼5–10 min) during continuous stimulation. However, athigher concentrations (20�M), the drugs only produced theslower reduction in contractility (∼80% reduction in forceresponse), even in the absence of continuous stimulation. Thefast potentiation was attributed to a possible direct effect ofrapamycin and FK506 on RyR1 that results in activation andi by

er unit of gating charge (Fig. 5)). To the best of our know ncreased SR Ca2+ release. This conclusion is supported

42 G. Avila, R.T. Dirksen / Cell Calcium 38 (2005) 35–44

Fig. 5. Rapamycin and FK506 significantly decrease the gain of voltage-gated Ca2+ release. (A) Approximations of the maximal rate of voltage-gated Ca2+

release were obtained by determining the first derivative of the Ca2+ transients (�F/�t [(�F/F)/s]) elicited at saturating test potentials (i.e. 40–70 mV). (B)Correlation analysis for the maximum values for the rate of SR Ca2+ release (�F/�t) and the magnitude of immobilization-resistant charge movement (Qmax).Maximum rate of release is plotted as a function of the correspondingQmax value. Correlation analysis revealed coefficients (R2) of 0.94 and 0.30 for control(white circles) and treated (gray triangles) myotubes, respectively. (C) The maximum gain of voltage-gated Ca2+ release was determined from the ratio of thepeak rate of Ca2+ release per gating charge ([�F/�t]/Qmax) at potentials where both parameters reached saturation (i.e. 40–70 mV) in control myotubes (n= 37)and myotubes treated with either rapamycin (n= 13) or FK506 (n= 9). Results are presented from the experiments shown inFigs. 1 and 3plus additionalexperiments in which other simultaneous determinations of peak Ca2+ release and charge movement were obtained (*p< 0.05).

prior studies in which rapamycin increases the activity ofFKBP-stripped RyR1 release channels incorporated into pla-nar lipid bilayers[19].

Interestingly, the slower∼80% reduction in contractileforce response following treatment of mechanically skinnedrat muscle fibers[14] is equivalent to our observation that 2 hincubations of mouse myotubes with either 20�M rapamycinor FK506 results in a 70–75% decrease in maximal voltage-gated SR Ca2+ release (Fig. 1andTable 1). In both cases, thedecrease in voltage-gated contractility and SR Ca2+ releasecannot be explained by SR Ca2+ store depletion ([14] andFig. 2). Rather, our results indicate that the observed reduc-tion in depolarization-induced Ca2+ release arises from dualeffects of rapamycin and FK506: (1) a marked reductionin the number of functional sarcolemmal voltage sensorsand (2) an additional reduction in the intrinsic ability ofthe remaining voltage sensors to efficiently activate release.Thus, the drugs act by both reducing the number of voltagesensors and the intrinsic ability of each sensor to trigger Ca2+

release.To the best of our knowledge, compromised skeletal

muscle function is not a serious clinical complication forpatients given immunosuppressants during organ transplan-tation. This is most likely due to the fact that clinicallyeffective doses of rapamycin and FK-506 required to achieveoptimal immunosuppression (5–10 nM in blood) are more

than three orders of magnitude lower than the concentra-tions used here to disrupt the FKBP12/RyR interaction inmyotubes (20�M). The low dose required during organtransplantation is most likely due to the requirement ofonly a small fraction of cellular FKBPs to be occupiedfor clinical immunosuppression[20]. In fact, FK506 andrapamycin are administered in humans and animals at dosesthat do not attain stoichiometric concentrations in muscle.Thus, the immunosuppressive effects result from a “gain offunction” rather than stoichiometric actions[21]. In addition,pharmacokinetic and pharmacodynamic studies have shownthat blood concentrations of rapamycin between 5 and65 nM support maximal prolongation of implants withoutcomplication in rabbits, dogs, pigs and rats[22]. Hence,rapamycin is effective in preventing graft rejection with awide separation between efficacious and toxic doses, basedon a projected therapeutic-dose range of 5–10 nM[22].

What are the possible molecular mechanisms that couldaccount for the rapamycin- and FK506-induced reductionsin DHPR function? FK506, but not rapamycin, is known toinhibit the protein phosphatase, calcineurin[23]. However,an inhibition of calcineurin is unlikely to explain our resultssince identical effects on voltage-gated SR Ca2+ release(Fig. 1), DHPR charge movement (Fig. 3), L-channel activity(Fig. 4) and the gain of voltage-gated Ca2+ release (Fig. 5)are observed for both rapamycin and FK506.

G. Avila, R.T. Dirksen / Cell Calcium 38 (2005) 35–44 43

A second possibility for the strong inhibition of SR Ca2+

release by rapamycin and FK506 is that this effect resultsfrom a drug-induced disruption of a critical FKBP12–RyR1interaction. Removal of FKBP12 from RyR1 profoundlyalters the activity of RyR1 channels incorporated into planarlipid bilayers. Specifically, FKBP12-stripped RyR1 channelsexhibit longer mean open times, enhanced open probability,increased gating frequency, reduced coupled gating andan increased incidence of subconductance activity[6–9].Extension of these observations to intact cells predicts thatdisruption of the FKBP–RyR1 interaction might result inincreased basal channel activity sufficient to lead to SRCa2+ leak/depletion (for recent reviews see[4,5]). However,our results (Fig. 2) and those of previous studies[11,12,14]indicate that disruption of the FKBP–RyR1 interaction inintact skeletal muscle cells does not lead to a significantreduction in RyR1-releasable store content. Apparently,effects of FKBP depletion on junctional RyR1 activity inintact muscle cells either differs significantly from thatobserved upon isolation and incorporation of these channelsinto planar lipid bilayers or intact cells are able to adequatelycompensate for these changes in basal release channelactivity. However, we did not find evidence of compen-satory alterations in Ca2+ reuptake following short-termdrug exposure since all of the parameters describing thecaffeine-induced release and reuptake (i.e. peak amplitude,t feiner bes.T R1i d SRd orto to besa ucedr ultf les

2–R tionb -n gaino ingd ingF edicmm P12k icd nc-t udiest rac-t dwp yR1i effi-c alm

An interesting, but yet unresolved issue, is whether theobserved parallel reductions in sarcolemmal DHPR chargemovement and the gain of voltage-gated Ca2+ release arisefrom different but parallel signaling pathways or representtwo sequentially linked events (i.e. one being the conse-quence of the other). Interestingly, we previously foundthat sarcolemmal DHPR expression is positively correlatedwith Ca2+ release through RyR1[17]. Thus, it is possi-ble that the primary effect of rapamycin and FK506 is toreduce the gain of voltage-gated Ca2+ release by disrupt-ing the FKBP12–RyR1 interaction. The resulting decreasein Ca2+ release through RyR1 might then alter a heretoforeuncharacterized Ca2+-dependent signaling pathway that con-trols steady-state sarcolemmal density of DHPRs. However,given the relatively brief (2 h) periods of drug exposure usedhere, the pronounced effects of rapamycin and FK-506 onfunctional DHPR expression more likely result from alter-ations in channel insertion/retrieval or degradation, since itis unlikely that the rate of DHPR transcription/translationwould be sufficiently altered during such a brief exposureperiod. Experiments designed to characterize the time-courseand mechanisms that govern sarcolemmal DHPR turnoverand degradation will be required in order to rigorously testthe validity of this possibility.

Alternatively, our data cannot exclude possible directeffects of rapamycin and FK506 on the DHPR that are inde-p ect,d thel inw singd dt sity∼ enta thatr ity,o ctionw andr res-s sar-c

A

nalI anH rant(

R

him.

ep-0.

ime to peak, plateau amplitude and recovery upon cafemoval) were similar in control and drug-treated myotuhus, predictions that disruption of the FKBP12–Ry

nteraction lead to increased basal channel activity anepletion in intact skeletal muscle is unlikely. In suppf this assertion, caffeine responses were also foundimilar in normal and FKBP12-deficient myotubes[12]. Inny event, our results indicate that the observed drug-indeduction in voltage-gated SR Ca2+ release does not resrom a decrease in the Ca2+ content of RyR1-releasabtores.

Alternatively, drug-induced disruption of the FKBP1yR1 interaction might alter the efficiency of communicaetween the voltage sensor and the SR Ca2+ release chanel. Consistent with this idea, a decrease in the intrinsicf skeletal muscle EC coupling has been reported followisruption of FKBP12 binding to RyR1 either by expressKBP binding-deficient RyR1 release channels in dyspyotubes[11] or by comparing voltage-gated Ca2+ release inyotubes derived from normal and muscle-specific FKB

nockout mice[12]. The inhibitory effects of pharmacologisruption of the FKBP12–RyR1 interaction on DHPR fu

ion described here are consistent with these previous sthat employed molecular approaches to disrupt this inteion. Taken together, our results ([11], this study) combineith those of Lamb and Stephenson[14] and Tang et al.[12],rovide strong support for the assertion that the FKBP–R

nteraction imparts a strong modulatory influence on theacy and gain of voltage-gated SR Ca2+ release in skeletuscle.

endent of FKBP12 removal from RyR1. Indeed, a dirrug-mediated effect on DHPR function would explain

ack of a reduction in L-current density in prior studieshich the FKBP12–RyR1 interaction was disrupted urug-free, molecular approaches[11,12]. However, we foun

hat while rapamycin treatment reduced L-current den50% in normal myotubes, it was without effect on L-currmplitude in dyspedic myotubes. These results indicateapamycin does not directly alter DHPR L-channel activr at least in a manner that does not depend on its interaith RyR1. Together, our results indicate that FK-506

apamycin either specifically reduce the functional expion of RyR1-coupled DHPRs or they alter steady-stateolemmal DHPR insertion/retrieval or degradation.

cknowledgments

This work was supported by grants from the Nationstitutes of Health (AR44657 to R.T.D.), an Americeart Association Grant-in-Aid (R.T.D.) and a Conacyt g

39512 to G.A.).

eferences

[1] W. Melzer, A. Herrmann-Frank, H.C. Luttgau, The role of Ca2+ ionsin excitation–contraction coupling of skeletal muscle fibres, BiocBiophys. Acta 1241 (1995) 59–116.

[2] R.T. Dirksen, Bi-directional coupling between dihydropyridine rectors and ryanodine receptors, Front Biosci. 7 (2002) d659–d67

44 G. Avila, R.T. Dirksen / Cell Calcium 38 (2005) 35–44

[3] A.F. Dulhunty, C.S. Haarmann, D. Green, D.R. Laver, P.G. Board,M.G. Casarotto, Interactions between dihydropyridine receptors andryanodine receptors in striated muscle, Prog. Biophys. Mol. Biol. 79(2002) 45–75.

[4] M.G. Chelu, C.I. Danila, C.P. Gilman, S.L. Hamilton, Regulation ofryanodine receptors by FK506-binding proteins, Trends Cardiovasc.Med. 14 (2004) 227–234.

[5] X.H. Wehrens, A.R. Marks, Altered function and regulation of car-diac ryanodine receptors in cardiac disease, Trends Biochem. Sci.28 (2003) 671–678.

[6] A.B. Brillantes, K. Ondrias, A. Scott, et al., Stabilization of calciumrelease channel (ryanodine receptor) function by FK506-binding pro-tein, Cell 77 (1994) 513–523.

[7] M. Mayrleitner, A.P. Timerman, G. Wiederrecht, S. Fleischer, Thecalcium release channel of sarcoplasmic reticulum is modulated byFK-506-binding protein: effect of FKBP-12 on single channel activ-ity of the skeletal muscle ryanodine receptor, Cell Calcium 15 (1994)99–108.

[8] G.P. Ahern, P.R. Junankar, A.F. Dulhunty, Subconductance states insingle-channel activity of skeletal muscle ryanodine receptors afterremoval of FKBP12, Biophys. J. 72 (1997) 146–162.

[9] S.O. Marx, K. Ondrias, A.R. Marks, Coupled gating between indi-vidual skeletal muscle Ca2+ release channels (ryanodine receptors),Science 281 (1998) 818–821.

[10] T. Wagenknecht, R. Grassucci, J. Berkowitz, G.J. Wiederrecht, H.B.Xin, S. Fleischer, Cryoelectron microscopy resolves FK506-bindingprotein sites on the skeletal muscle ryanodine receptor, Biophys. J.70 (1996) 1709–1715.

[11] G. Avila, E.H. Lee, C.F. Perez, P.D. Allen, R.T. Dirksen, FKBP12binding to RyR1 modulates excitation–contraction coupling in mouseskeletal myotubes, J. Biol. Chem. 278 (2003) 22600–22608.

[ on–efi-

[13] A.P. Timerman, E. Ogunbumni, E. Freund, G. Wiederrecht, A.R.Marks, S. Fleischer, The calcium release channel of sarcoplasmicreticulum is modulated by FK-506-binding protein. Dissociationand reconstitution of FKBP-12 to the calcium release channel ofskeletal muscle sarcoplasmic reticulum, J. Biol. Chem. 268 (1993)22992–22999.

[14] G.D. Lamb, D.G. Stephenson, Effects of FK506 and rapamycin onexcitation–contraction coupling in skeletal muscle fibres of the rat,J. Physiol. 494 (1996) 569–576.

[15] G. Avila, J.J. O’Brien, R.T. Dirksen, Excitation–contraction uncou-pling by a human central core disease mutation in the ryanodinereceptor, Proc. Natl. Acad. Sci. U.S.A. 98 (2001) 4215–4220.

[16] G. Avila, R.T. Dirksen, Functional impact of the ryanodine receptoron the skeletal muscle L-type Ca2+ channel, J. Gen. Physiol. 115(2000) 467–480.

[17] G. Avila, K.M. O’Connell, L.A. Groom, R.T. Dirksen, Ca2+ releasethrough ryanodine receptors regulates skeletal muscle L-type Ca2+channel expression, J. Biol. Chem. 276 (2001) 17732–17738.

[18] S. Adachi-Akahane, L. Cleemann, M. Morad, BAY K 8644 modifiesCa2+ cross signaling between DHP and ryanodine receptors in ratventricular myocytes, Am. J. Physiol. 276 (1999) H1178–H1189.

[19] G.P. Ahern, P.R. Junankar, A.F. Dulhunty, Ryanodine receptors fromrabbit skeletal muscle are reversibly activated by rapamycin, Neu-rosci. Lett. 225 (1997) 81–84.

[20] A. Lo, M.F. Egidi, L.W. Gaber, A.O. Gaber, Observations on the useof sirolimus and tacrolimus in high-risk renal transplant recipients,Transplant Proc. 35 (Suppl. 3) (2003) 105S–108S.

[21] A.R. Marks, Cellular functions of immuophilins, Physiol. Rev. 76(1996) 631–649.

[22] S.N. Sehgal, Sirolimus: its discovery, biological properties, andmechanism of action, Transplant Proc. 35 (Suppl. 3) (2003) 7S–14S.

[ lton,har-

12] W. Tang, C.P. Ingalls, W.J. Durham, et al., Altered excitaticontraction coupling with skeletal muscle specific FKBP12 dciency, FASEB J. 18 (2004) 1597–1599.

23] S.H. Snyder, D.M. Sabatini, M.M. Lai, J.P. Steiner, G.S. HamiP.D. Suzdak, Neural actions of immunophilin ligands, Trends Pmacol. Sci. 19 (1998) 21–26.