Direct contact between sympathetic neurons and rat cardiac myocytes in vitro increases expression of functional calcium channels

Direct contact between sympathetic neurons

and rat cardiac myocytes in vitro increases

expression of functional calcium channels.

S Ogawa, … , T W Smith, J D Marsh

J Clin Invest. 1992;89(4):1085-1093. https://doi.org/10.1172/JCI115688.

To test the hypothesis that direct contact between sympathetic neurons and myocytes

regulates expression and function of cardiac Ca channels, we prepared cultures of neonatal rat ventricular myocytes with and without sympathetic ganglia. Contractile properties of myocytes were assessed by an optical-video system. Contractility-pCa curves showed a 60% greater increase in contractility for innervated myocytes compared with control cells at 6.3 mM [Ca]0 (n = 8, P less than 0.05). Cells grown in medium conditioned by growth of ganglia and myocytes were indistinguishable physiologically from control cells. [Bay K 8644]-contractility curves revealed a 60 +/- 10% enhancement of the contractility response at 10(-6) M for innervated cells compared with control cells. The increased response to Bay K 8644 was not blocked by alpha- or beta-adrenergic antagonists. Moreover, increased efficacy of Bay K 8644 was maintained for at least 24 h after denervation produced by removal of ganglia from the culture. Dihydropyridine binding sites were assessed with the L channel-specific radioligand 3[H]PN200-110. PN200-110 binding sites were increased by innervation (51 +/- 5 to 108 +/- 20 fmol/mg protein, P less than 0.01), with no change in KD. Peak current-voltage curves were determined by whole-cell voltage clamp techniques for myocytes contacted by a neuron, control myocytes, and myocytes grown in conditioned medium. Current density of L-type Ca channels was significantly higher in […]

Research Article

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Direct

Contact

between

Sympathetic

Neurons and Rat Cardiac

Myocytes

In Vitro

Increases Expression of Functional Calcium Channels

ShunichiOgawa,JoeyV.Bamett, Luyi Sen,Jonas B.Galper,ThomasW. Smith,and James D. Marsh DepartmentofMedicine, Brigham and Women's Hospital and Harvard Medical School, Boston, Massachusetts02115

Abstract

To test the hypothesis that direct contact betweensympathetic

neurons and myocytes regulates expression and function of

car-diacCa channels,weprepared cultures of neonatalrat ventricu-larmyocytes with and without sympatheticganglia. Contractile

properties of myocytes were assessed by an optical-video sys-tem.Contractility-pCacurvesshoweda60% greater increase in

contractility for innervated myocytes compared with control cells at63mM

_[Cab

(n=8,P <0.05).Cells grown in medium conditioned bygrowthofganglia and myocytes were

indistin-guishable physiologically from control cells.

[Bay

K

86441-contractilitycurvesrevealeda60±10%enhancement ofthe

con-tractilityresponseat106Mfor innervated cellscomparedwith controlcells. The increased response to BayK8644 wasnot

blockedbya- or

,-adrenergic

antagonists. Moreover, increased

efficacyof BayK8644wasmaintained foratleast 24hafter denervationproduced by removal of ganglia from the culture. Dihydropyridine binding sites were assessed with the L

chan-nel-specific radioligand

3[HJPN200-110.

PN200-110 binding sites were increased by innervation(51±5to108±20fmol/mg

protein, P<0.01),with no change in KD. Peak current-voltage

curvesweredetermined by whole-cell voltage clamptechniques

for myocytes contacted by a neuron, control myocytes, and myocytes grown in conditioned medium. Currentdensityof

L-type Ca channelswas significantly higherininnervated myo-cytes (10.5±0.4 pA/pF, n = 5) than in control myocytes

(5.9±03 pA/pF,n=8,P <0.01)ormyocytes grown in condi-tioned medium(6.2±0.2pA/pF,n=10,P <0.01).Thus, physi-cal contact between asympathetic neuron andpreviously unin-nervated neonatal rat ventricular myocytes increases expres-sion of functional L-type calcium channels as judged by contractile responses to

Cao

and Bay K8644, as well as by electrophysiological and radioligand binding properties. (J. Clin.Invest.1992.89:1085-1093.) Keywords:cardiomyocyte

cellculture *dihydropyridine* ion channel

Introduction

Cardiac L-type calcium channels playacrucial role in excita-tion-contraction coupling. Modulation of L-type calcium channel expression may influence the inotropic state of the heart. However, littleis known about molecular and cellular

Address reprint requests to Dr. Marsh, Cardiovascular Division,

Brigham and Women's Hospital, 75 Francis Street, Boston, MA

02115.

Receivedfor publication 22 February 1991 and in revisedform 22 October 1991.

mechanisms that regulate the abundance of calcium channels. The L-typecalcium channelisaheteromericproteinwitha1,

a2,

_f,

y,and 5 subunits (1). The

_a,

subunitis believed to pro-vide the pore forCa2'entry and contains the binding site for

dihydropyridine calcium channel antagonistsandagonists.

Re-cently,Barnettetal.(2) developedamethod for thestudyofthe

effect ofco-cultureofciliary gangliaandembryonicchick heart cells on theparasympatheticresponse of the heart. Togainan

enhancedunderstandingof fundamental mechanisms that reg-ulate the number of cardiac calcium channels present on a

myocyte, we adaptedthismethod to testthehypothesesthat the presence ofsympatheticneuronsincreases abundance of L-typecalciumchannels in myocytes invitro,andthatan in-crease in calcium channel number is physiologically

impor-tant.

Itis clear that thefunction of existingcalcium channels can bemodulatedbyneurotransmittersandhormones(3).Agonist bindingto

_{f3-adrenergic}

receptors leads tophosphorylation of calciumchannelsandanincreaseincalciumchannel current (3, 4). However, theeffect ofthe processofsympathetic

inner-vation on the expression of the L-type calcium channel

_al

sub-unitgeneproduct is unknown.Severallines ofevidencesuggest thatcation channelexpression mightbe alteredby innervation.

Forexample, Marsh and Allen (5)foundthatduringontogeny

oftheavianheart, shortly after ingrowth ofsympatheticnerves, there was a marked increase in numberofdihydropyridine (DHP)1 bindingsites. OffordandCatterall (6) found that

elec-tricalactivity andcyclicAMPcan regulate mRNA-encoding sodiumchannelasubunitsin muscle cells.

The in vitromodel system for co-culture ofsympathetic gangliaandventricularmyocytes used inthisstudypermitted selection ofthe cell type under study andexamination ofthe role ofcell contactanddenervation inregulation of calcium

channelexpression.Ourfindingsdemonstrate thatdirect

con-tact with myocytes by sympathetic neurons does lead to an

increaseinexpressionof functional L-type calcium channels.

Methods

Cultureofneonatalratventricularmyocytes.Neonatal rats(1 d old) were deeplyanesthetized with ether and killed by decapitation. Hearts were removed, trimmed ofcormectivetissue, and minced into frag-mentsof 3mm3. Myocytesweredissociatedbycyclic trypsinization

(0.25% trypsin solution)in Hanks' balanced salt solution. After centrif-ugation at 800 g for 5 min, cells were suspended in Dulbecco's modi-fiedessential mediumcontaining7%fetalcalfserum (FCS) and antibi-otics (gentamicin, streptomycin, penicillin) and were preplated (1 X 106cells/ml, 50-mm culture dishes) for 60minin ahumidified 5%

C0J95%air atmosphere at370C.Cells wereplated at 1 X106cells/ml

1. Abbreviations used in this paper: DHP, dihydropyridine; I-V, current-voltage.

J.Clin. Invest.

© TheAmericanSociety for Clinical Investigation,Inc.

in 35-mm culture dishes on 12-mm circular coverslips in the same culturemedium. Cell cultures were maintained in a humidified 5%

C0J95% airatmosphere(370C).

Co-cultureofneonatal rat supracervicalsympathetic ganglia with ventricular myocytes. Cultures were prepared by amodification of the

methodof AtkinsandMarvin(7) and by amodification ofthe method

ofBarnettandGalper (2). After sacrifice of l-d-oldneonatal rats,

su-pracervical sympathetic gangliawere removedasepticallyand placedin culturedishes thatcontainedHanks' saltsolution. The ganglia were

dissected free from associatedtissue and cut into 1-mm3 fragments.

Thegangliawere plated on 12-mm circular,collagen-coated (typeI, from adultrattail tendons) coverslips in35-mmculture dishes.

Gan-gliawereincubated witha small volumeofculturemedium ina

humidi-fied5%C0J95% air atmosphereat370Cfor 2-3hduring which time

theganglia attachedtothe substrate.Atthis timefreshly dissociatedrat

ventricularmyocytes(1X 106cells/ml) were added to the culturedishes

containing ganglia. Myocyte-ganglion co-cultureswereincubated as

above.

Assessmentofneuroeffector transmission. Neuroeffector transmis-sion ofsympatheticneurons wasassessed physiologicallybydirect elec-trode stimulation of ganglia with axons terminating on ventricular

myocytes. Aplatinumelectrode was placeddirectlyonaganglionand shortunipolar pulses (3-4Hz, 3 ms induration)weredelivered. Myo-cytes werecontinuously superfused with Hepes-buffered solution

con-taining (mM):Hepes,5.0;CaCl2, 0.9; KCI, 4.0; NaCl, 140; MgCl2, 0.5.

Thefrequencyof contractions of themyocytescontactedbyaxons was recorded by anoptical videosystem aspreviously describedindetail (8),and asignalindicating electrical stimulationwasrecorded

concur-rently.

Measurement ofcontractile responsiveness ofmyocytes. Contractile

responses toCa2'and(±)BayK8644weremeasuredafter 3or4 din

culture. The cells wereelectrically stimulated (2.5 Hz),and were stabi-lized foratleast15 minbeforeexposure tograded,increasing

concen-trationsofCa2"(0.3, 0.6, 0.9, 1.8, 3.6, 5.4, 6.3 mM),or(±)Bay K 8644

(10-9to10-6M).For eachcell, contractileresponse toBay K8644was

assessed in Hepesbuffer containing theECOconcentration ofCa2".

Maximalamplitude ofcontractionwasmeasuredforeachcellusingthe

EC1oo

concentrationofCa2'.Mediumwassuperfusedat1.0ml/min.

Theanalog voltageoutputfromthevideo motiondetector wasfiltered

at6Hzand wascalibratedtoindicateactualmicronsof motion.

Am-plitudeandvelocity of contractionwasrecorded on astripchart re-corder.Contractility measurements weremade ononlyonecell per

coverslip. Changes in amplitude andvelocityof cell motionwereused

toquantitatetheeffects of interventionsoncellcontractileproperties.

Themethods usedhave beenestablished andextensivelyvalidated in this laboratory (8, 9). Whileamplitudeandvelocityof contraction of singlemyocytesarerecordedassurrogatesforactualforce of

contrac-tion,amplitudeandvelocityof contraction for isotonic contraction of lightlyloaded muscleclosely tracksdeveloped force(8, 10),and

there-foreserveasuseful indices ofacellinotropicstate.

PN200-110 binding assay. Ligand binding experiments using [3H](+)-PN200-110wereconductedonmembranespreparedfrom

ven-tricularmyocytesormyocytes grown in co-culture withgangliaafter 3 d ofgrowth.Formyocytes grown inco-culture,gangliawereremoved

aseptically by microscopic dissection after3 dofgrowth;8 h later dener-vated cells werestudied. Before ligandbindingexperiments, culture

mediumwasaspirated,andcells were washed twice inbufferA

[con-taining (mM): Tris, 20; EDTA-Na, 2.0;EGTA,0.1;sucrose, 250(pH

7.0)], andscrapedfrom culture dishes intobufferA. The cellswere

frozenandthawedtwice followed by12strokesofDounce

homogeniza-tionusingatight-fitting pestle.Thecrudehomogenatewascentrifuged

at19,000g for 40 min and theresultingmembraneswereresuspended

in Hepesbuffer containingthe

_EC5o

concentration ofCa2",identicalto

thebufferforcontractility experiments.Theentirepreparationwas at 4°C.400

_,d

of membranesuspensionwasaddedtoglasstubes

contain-ing gradedconcentrationsof(3HJ(+)-PN200-110(50-1,500 pM) with

orwithout 50 Ml of

IO-'

Munlabeled(+)-PN200-110todefine

nonspe-cificbinding.Proteinconcentrationwas0.15-0.8mg/ml.Incubation

was at370C for 30min(5).Bindingwasterminated byvacuum

filtra-tionusingacell harvester (Brandel Co.,Gaithersburg, MD). Filters

(GF/B, Whatman, Inc., Clifton,NJ)weredriedunderhighvacuum andwereplacedinscintillationcountingvialsforassessmentofbound

[3H](+-PN200-110.

Equilibriumbindingdata wereanalyzedusing the interactive

non-linear least-squaresmethodofMunsonand Rodbard(a modification oftheLIGANDprogram) withaMicroVaxcomputer(Digital

Equip-mentCorp., Maynard, MA)asdescribed (11). Thisprogramutilizes initial estimates for agonistandantagonist KD values, maximum bind-ing(B.),andnonspecificbinding for each ligandtosolve aseries of linearequationsthatcompletely describeanequilibriumbinding

iso-therm.

Eachpointwasassayed intriplicate,andeachindividualbinding

curve wasreplicatedatleastfour times.Allequilibriumdissociation

constants areexpressedasthemean±SEM.

Whole-cellpatch-clamp.Calciumcurrents wereobtainedusingthe whole cellpatch clamptechnique.Currentswererecordedwithan

Ax-opatch-IC patchclampamplifier (Axon Instruments, Inc.,FosterCity, CA;cutofffrequency IKHz)andanalyzedon anIBM-AT computer

usingthepciAmpprogram. Standardprocedureswereused forthe

patch clamp recordings and isolation of calcium channelcurrents.

Whole cellrecordingswereobtained withthe externalsolution con-taining (mM):20 Ba acetate; 135tetraethylammoniumaspartate; 10

Hepes; 30 MMtetrodotoxin;pH7.5.Theinternal solutioncontained

135 Csaspartate; 10EGTA; 10Hepes;4ATP;5MgCl2; pH7.5.The averagecellcapacitancewas53±10pFininnerverted cells and 49±13 pFincontrol cells.Restingmembranepotentialwastypically-75mV andnoconsistent differencewasnoted between noninnervated and innervatedmyocytes.Series resistancecompensationwas0.55Mgout

ofatotalpipetteresistance of0.65Mg.Stimulationfrequencywas0.2 Hz. ForrecordingofL-typecalcium channelcurrentsthe_holding

po-tentialwas-50mVtoinactivateT-typechannels. Allrecordingswere obtainedat220C.Afterpatchbreaktheunfiltered transient inresponse

toapulsefrom-80to-75mVwasrecordedandintegratedtoobtain

cellcapacitance.

Materials. The (+)enantiomerof

[3H]PN200-1

1O was obtained

fromNewEngland Nuclear, Boston,MA.(±)BayK8644was agiftof MilesLaboratories,WestHaven,CT.Theunlabeled(+)enantiomerof PN200-110was agiftfromSandozLtd., Basel,Switzerland.

Proprano-lol andphentolaminewerefromSigmaChemicalCompany,St.Louis,

MO.Dihydropyridinesweredissolved in 70% ethanolat a

concentra-tion of

lo-'

M,protectedfromlight,storedamaximum of 3wk,and subsequentlydiluted inbuffer beforeuse. Control

experiments

con-firmedthatthelowfinal ethanol concentrationbyitselfhadno

measur-ableeffect.

Statisticalanalysis. Contractilityand_{current-voltage}curveswere compared byanalysisofvariance(ANOVA).Bindingparameterswere compared by unpaired t test. Data are _{expressed throughout} as

means±SEM. A P valueof<0.05waschosentoindicatea

_{statistically}

significantdifference.

Results

Myocyteinnervation in vitro.After 3 d of

growth,

neonatalrat

ventricular myocytes developed a stellate

configuration

and

spontaneously

contractedat103±1

contractions/min (n

=

_60).

When

sympathetic

ganglia

were_{co-cultured with the myocytes}

for the initial 72 h in

culture,

axons were

_readily

apparent

(Fig.

1,aand

b).

Individualaxonsmadedirectcontactwith 20-50% ofmyocytes in each culture

(Fig.

1

c). Directly

innervated myocytes contracted at 125±4

contractions/min

(n

=

_55,

P

<

0.001).

For some

experiments, ganglia

were removedand

axons

permitted

to

degenerate.

8hafter

ganglia

removal,

most axons appearedto degenerate;a few remained visible inthe

cultures

_(Fig.

1

_d).

By

24 hafter removal of

_ganglia,

virtually

all

dener-vation could be accomplished in vitroby thisco-culture

ap-proach.

To determine whethertrue functional innervation could

occurbetween theganglia andmyocytes, amicroelectrodewas

placedadjacentto aganglion and the motion ofaninnervated

myocyte wastracked.Aganglionwasstimulated andas canbe

seeninFig. 2, stimulation ofaganglion producedanincreased frequency ofcontraction of the innervated cell, with 1:1

cou-pling. Ganglion-myocyte coupling could be maintained forat

least5min. Ifstimulationwasstoppedfor5minthenrestarted, ganglion-myocyte coupling againwasevident. If stimulation

wasstopped and the cellssuperfusedwith1 uMpropranolol for

Figure1.Morphologyofmyocytes, myocytes cultured with

sympa-thetic ganglia, anddenervated myocytes.(a)Neonatal ratventricular

myocytes growninserum-containingmedium for 72 h.(b)Myocytes growninthe presenceofsympatheticganglia. At the lower left a gan-glionisseen.Thefine linearprocesses are axons. Cells andganglia have been in culturefor72 h. Allphotomicrographswerewitha phase-contrastmicroscope (IM, Carl Zeiss, Inc., Thornwood, NY),

X100for a andb. (c) Contact of a myocyte by an axon. Arborization

ofaxon andmultiple pointsof contact by the axon on a myocyte are

evident.Thehighlyrefractile,rounded elements are celldebrisin the culturemedium.X320.(d)Same cell and axon as c, 8 hafterremoval

oftheganglion.Most axons hadcompletelydisappeared by this time.

The axon inthe centerofthefieldappears to bedegeneratingand hasabnormal morphology.(e) Same myocytes as in c andd,24 hafter

removalofganglia.Itis evidentthat the axon hasentirelydegenerated atthispoint.

5 min, ganglion-myocyte couplingwas abolished in eachof

seven experiments. However, in thepresenceofpropranolol,

eventhoughganglionstimulation couldnotproducemyocyte

contractions, ifmyocytes weredirectlystimulated byan elec-trodeplaced closetothecell,acontractile responsecould be

easilyproduced. Thus, itappearslikelythat there isa

stimula-tory neurotransmitter (presumablynorepinephrine)being re-leasedfromthe axonwithphysiologicalresponsebythe myo-cyte.

Response to

[Ca2+]0

and calciumchannel

effectors.

The con-tractileresponsesof threetypesofmyocytepreparationswere

Stimulus

Ampliu

Control Propranolol

_m~I

58 18

Figure2. Contractileresponseofamyocyte tostimulation ofa

sym-pathetic ganglion. A symsym-pathetic ganglionwasstimulated and

con-tractile amplitude ofamyocytecontacted byanaxon wasrecorded. The ganglionwasstimulatedat3.5 Hz, 2-ms pulse, 5 V, 370C, Stim-ulusrecord (top tracing) and amplitude of contraction ofmyocyte

(lower tracing)areshown. Control indicatesresponsein control physiological buffer. Stimulation andresponsecontinued for5mi,

thenthe stimulatorwasturned off for 5min. When stimulationwas

restarted after 5min ofrest,myocytecontraction immediately

re-sumed inanidentical fashion (not shown). The stimulationwas

haltedand the monolayerwassuperfused with 1jIMpropranololfor

5min,then stimulationwasrestarted (panel labeled PropranoloO.

Under these conditions, contractileresponse wasabolished.

cytescultured for 72 h in theabsence of sympathetic ganglia (control cells). The secondpreparationconsisted ofmyocytes

culturedfor 72 h inthepresenceofsympathetic ganglia. Specifi-cally, cells that werein contact with theaxons were studied (innervated myocytes). The third preparation constituted

myo-cytesonculture dishes where ganglia andmyocytesweregrown

inco-culture. However, the thirdtypeofmyocytedidnothave

directphysicalcontactwithan axon.Inthisway,the

environ-mentwasidenticaltothat of the directly innervatedmyocytes,

savefor direct neuronalcontact. These myocyteswere

there-fore considered to be grown in medium "conditioned" by growthofganglia.

The pCa-contractile amplitude relationship was deter-mined for control myocytes, innervatedmyocytes, and

myo-cytesgrowninconditioned medium(Fig. 3). Myocytes directly innervated showedasignificantlygreaterincreaseinamplitude

ofcontractioninresponsetoincreasing

[Ca2+]0

thandid

con-trol myocytes or myocytes grown in conditioned medium. With the contractileresponsein0.9mM

[Ca2`]0

takenas con-trol,innervated myocytes increased theiramplitudeof

contrac-tion to 200% of control in response to increasing [Ca2+]0, whereascontrolmyocytesincreasedtheiramplitude of

contrac-tiontoonly 140%of control.

Therearemanypotentialaspectsofexcitation-contraction couplingwherein innervationmightleadtomodulationof the

pCa-contractileresponserelationship. Asanapproachto

de-terminingwhethercalciumchannel functionmight contribute

tothe alteredresponseto[Ca2+]0,thecontractileresponseto

the DHP calcium channelagonist BayK8644wasdetermined. Myocytes innervated by sympatheticneuronshadamarkedly increased response to graded concentrations ofBay K 8644 comparedtocontrolmyocytesorto myocytesgrownin condi-tionedmediumbutlacking directcontactbyan axon(Fig.4a).

When the[BayK8644]-contractileresponserelationshipwas

replottedwith normalizationto 100% responseofeach

myo-cyte,curvesfor the threetypesofpreparationswere superim-posable(Fig. 4 b), indicatingnochangein the

EC50

forBAYK 8644. These data indicate that sympathetic innervation in-creasesthe contractileresponsetoacalcium channelagonist, possibly duetoanincreasein L channel calciumcurrent.

In theforegoing studies,myocyteswerestimulatedto

con-tractbyaplatinum wireelectrode placed closetothemyocyte

of interest. With thistechniquethereisapossibilitythat the electrical stimulation of themyocytecouldcause norepineph-rine release from the nerveterminalsnearthemyocytes, en-hancing the contractileresponses.Toexaminethispossibility, theresponsetoBayK8644 andtoelevated[Ca2+]0were exam-ined in thepresenceofpropranolol(10-6M)toblock

fl-adren-ergicreceptorsorinthepresenceof phentolamine(10-6M)to

blocka-adrenergicreceptors.Ascanbeseenin Fig.5,aandb, aand

ft

blockade didnotdiminish the contractileresponseto

Bay K 8644, making incidental norepinephrine release

un-likely as amechanism for the enhanced response to Bay K 8644. The response to

_[Ca2+]0

in the presence ofa and A blockerswasalso unaffected (datanot

shown).

Thefindings described above indicatethatsympathetic in-nervation producedone or morealterationsin directly inner-vated myocytes such thattheir response to

[Ca2eJ0

andto a

DHPcalcium channel agonistwasaugmented.Among possi-blechanges produced by innervationarealterations in number orgating of L-type calcium channels, alterations in contractile protein isoforms, oralterations in ion pumps orexchangers suchasthe Na-Ca exchanger, the sarcoplasmic reticulum

cal-ciumATPase,orthesarcolemmal calcium ATPase. To deter-mineifthe effect of innervationonCachannel function per-sisted afterdenervation, the ganglia whereremoved by

micro-0

Ls

c

0

0

F-z

0o

200

150

100

50

0

4 3

pCa

2

Figure 3. pCa-contractileresponserelationships. Myocyteswere

di-rectly stimulatedtocontractand amplitudeof contractionwas

re-cordedinresponsetograded[Ca2+]0. 100%amplitudeofcontraction

wasdefinedasthatpresentin0.9 mM[Ca2+]O.(o)Contractile re-sponseof control myocytes(n= 8); (C) contractileresponseof

inner-vatedmyocytes(n=8); (v) contractileresponseofmyocytesgrown

inconditioned medium (n=8). Analysis of variance revealed that

thecurvefor the innervated myocytes differedsignificantlyfromthat of control myocytes(P<0.05).Thecurvefor myocytesgrownin

conditioned mediumwasindistinguishablefrom thecurvefor control

myocytes.Error bars indicate standarderrorsof themean.Where

errorbarsareomitted,theerrorbaris smaller than thesymbol.The

EC5ofor control myocyteswas0.55±0.05 mM andwas0.92±0.01 mM for innervated myocytes(P<0.01).

-~~~~~~~~~~~T

*

Al

A!

-

*

0

-

s-o 25(

z 0 0

'0 201 vx

j ₁₅₁

0 af z 0_o

10

) 100

E

' 100

E

25

x

E 75

¢7

50

a::

z0

CONTROL 10 9 10-8 10-7 1o6 HIGH-Ca++ BAYK8644(M)

0 I *

CONTROL 10-9 10 8 10-7 1o-6

BAYK 8644 (M)

Figure 4. [Bay K86441-contractileresponserelationship.The

con-tractileresponsetoBay K8644wasstudied in the myocyte

prepara-tions. Eachpreparationwasstudiedinits

_EC5

concentration of [Ca2]0. Ordinate: amplitude of contraction.Controlbuffercontained

noadded Bay K8644. (a) Thecontractileresponse toBay K8644 for innervatedmyocyteswassignificantlygreaterthanthatforcontrol myocytes.ANOVA revealedanoveralldifferencebetween the curves (P<0.05).Thereisnodifferenceincontractileresponsebetween control myocytes and myocytes grown inconditioned medium.The

high-[Ca2+]0response wasthecontractileresponseofeachpreparation

toits

_EC10o

calcium concentration (n=10foreachexperiment).(.) Innervated myocytes; (o) control myocytes;(v)myocytes grown in conditionedmedium.(b)Data inFig.4areplotted with 100%defined

asthemaximumresponseforeach myocyte. The curves are-statisti-callyindistinguishable, indicatingnochange insensitivitytoBay K 8644.

dissectionand 8 and 24 h later cellsweretestedforCa channel

function.8 hafterganglia removal,most axons were nolonger visible,andthose that were visible appeared to be degenerating (Fig. 1 d).Whenstudied8h after removal ofganglia,myocytes

that hadpreviouslybeeninnervated maintained their robust contractile response to BayK8644andto [Ca2+]0(Fig. 6 a).

Cells weremaintained inculturefor upto 24 hafter

dener-vation. Contractile responses of previously innervated myo-cyteswasagainassessed(Fig. 6b).The contractile responseto

BayK8644 remained augmented andindistinguishablefrom myocytes that had not undergone denervation. Thus, the fac-tor(s) that account for the enhancedcontractile response to

extracellular calcium and toacalcium channel agonist

subse-quent toinnervationappear to turn overslowly andtoremain functionally unchanged foratleast 24 h after denervation. In-nervation may be producing irreversible commitmentof the

cellto analteredphenotypethatis morehighlydifferentiated.

Longer-term observations after sympatheticdenervation will

be necessarytoresolvethispossibility.

DHPbindingsites. Todeterminewhethersympathetic in-nervation alters the number of L-typecalcium channels as

de-terminedby DHPbinding, equilibrium binding experiments were performed with the DHP antagonist ligand

3H(+)-PN200-110. Assays were performed on membranes from con-trol myocytes and from myocytesco-cultured withgangliafor

72 hfollowed by denervation for 8 h. Denervation was per-formedbefore theassayto ensurethat DHPbindingsites on

neurons were not being detected (Fig. 7). PN200-110boundto

membranes with a singleaffinitystate and withacceptably low

nonspecificbinding (nonspecific binding was<25%of total at theKD); bindingwassaturable. Computer analysisof

equilib-I--,

0 a: z 0

0

1-C) a: HY z 0 CD

l-0

O

c)-a: z 0

0

P::

z 0 0)

250

200

150

100

200

150

100

T

0

a~~~

la

_ml<

CONTROL 10 9 10o8 10-7 10 6 HIGH-Ca++

BAY K 8644 (M)

I

T i

I

T

El

1"//

_ .

0

CONTROL 10 9 10o8 10 7 10 6 HIGH-Ca++

BAY K 8644 (M)

Figure 5.Effect ofaand dblockadeoncontractileresponsestoBay K 8644.(a) ContractileresponsetoBayK8644 in the presenceof106

Mpropranolol is plotted. Drugwas

supeffused

5minbeforethe

ex-periment.Forcomparison,cells from thesameculturenotexposed

topropranololalso hadtheir contractile response measured under control conditions and in responseto10-6MBayK8644 andtothe

EC1oo

concentration of calcium. Propranolol didnotalter the

con-tractile response of innervated myocytes.(a)Presence ofpropranolol

(n=5);(-) absence of propranolol (n=4).(b)Theprotocolwasthe

same asin a, except that thea-adrenergicblockerphentolaminewas

present. Theablockadedidnotalter the responsetoBayK8644for innervated myocytes.(a)Presenceofphentolamine (n=4);

(.)

° 250

a-z 0 C)

0 200

- 150

z 0 100

C-200

150

100

CONTROL 10-9 10-8 1o-7 10i6 HIGH-Ca++

BAY K 8644 (M)

2~~~~~~~~

11/

I

b

CONTROL 10i9 10-8 l i716 HIGH-Ca++ BAY K 8644 (M)

Figure 6.Effect of denervationoncontractileresponsestoBay K 8644.(a) 8 h after denervation, the contractileresponsetoBay K8644

wasdeterminedand comparedtothatofmyocytesthat hadnot

un-dergone denervation. (o) Denervatedmyocytes(n=4); (.)innervated

myocytes(n=4). (b) ContractileresponsetoBay K8644 24 h after denervation.(0) Denervatedmyocytes(n =6) (e) innervated

myo-cytes(n =6). Denervation didnotalter the contractileresponseto

Bay K8644 eitherat8or24 h.

rium binding isothermsindicated thatasingle affinitystatefor the DHP antagonist waspresent. Goodness-of-fit ofbinding

data to a two-site model was compared to fit to a one-site

model.Innocasedidthe two-site model offerabetter fit than

thesingle-site model(Ftest,P> 0.05). When computer-der-ivedbindingparametersfor control myocytes and innervated myocyteswerecompared,nodifferenceinKDwasfound,buta significantdifference in the number ofDHPbindingsiteswas

evident(Table I).Control membraneshad51±5fmol/mg

pro-tein PN200-110bindingsites whereas membranesfrom

inner-vatedmyocyteshad 108±20fmol/mg protein bindingsites(P <0.01). Thus,concordant with the increased physiologic re-sponsetoacalcium channel agonist, therewas atwofold

in-creaseinthe number ofDHPbindingsitesoninnervated

myo-cytescomparedwith control cells.

Whole-cell voltage clamp data. Controlmyocytesand

myo-cytesindirectcontactwithasympatheticneuronwerestudied in the whole cell voltage clamp configuration. The L-type

channelcurrentcarriedby Ba2+at-50mVholding potential

with -40 to +50-mV test potentials was recorded. Current

density-voltage (I- V)curves weredetermined for control

myo-cytes, innervated myocytes and myocytes grown in condi-tioned medium. Calcium currents were measured as peak in-ward currentin response to zero current. As an index ofcurrent

density, weused cell currentdivided by cell capacitance (pi-coamperes perpicofarad)tocorrectfor variance in cell size. Care was taken tostudysinglemyocytesthat were not in con-tactwith othermyocytes.Compared to control myocytes, in-nervated myocytesshowedasignificantlygreater peak current

density (Fig. 8).The peak of the I-Vcurve was shifted slightly, from 0 mV for control myocytes to -10 mV for innervated myocytes.The I- V curve for myocytes grown in conditioned

mediumwasindistinguishable fromthatfor control myocytes. The currentdensity ofL-typecalcium channels is summarized

inFig. 8c.Onaverage,the currentdensityatVm=-10 mV for innervated myocytes was 10.5±0.4 pA/pF, whereas for control myocytes it was 4.5±0.4 pA/pF (P < 0.01). Inactivation of L-typecalciumcurrents wasinvestigated(Fig. 9). There was a

significant differenceinvoltageforhalf-inactivation ofcurrent

from innervatedand control myocytes (P < 0.01). Cells grown

inconditioned mediumwereindistinguishable from noninner-vated cells. Thus, at a given holding potential the innernoninner-vated myocytes had morecalciumchannels available for activation.

Discussion

Theprincipal finding of thisstudyisthatphysical contact

be-tween asympatheticneuronandventricularmyocytesinvitro

produced increased expression of functional L-type calcium channels. This directphysicalcontactbyasympatheticnerve

axon(physical innervation)appears to beessential;cells grown

in medium conditioned by growth ofmyocytesand

sympa-theticganglia didnotdemonstrateincreasedexpression of L-typecalciumchannels.

There arethree linesof evidenceto supporttheconclusion thatinnervation increasedL-typecalcium channel expression. First, innervatedmyocytesshowedamarkedlyincreased

con-tractileresponseto[Ca2+]0. While thiseffect is consistent with increased calcium channel expressionand anincrease in cal-cium channel-dependent calcium flux leadingto greater

re-OnO 5

-1

0-0550 100 15

BOUND(fmol/mg)

Figure7. [3H](+)-PN200-110binding. Computer-derivedScatchard plotof

[3H](+)-PN200-l

10bindingtocell membranesfor represen-tativeexperiments. [Boundligand]isindicated on the abscissa and

bound/freeis indicatedontheordinate.

(.)

Membranes from inner-vatedmyocytes(KD = 124pM,B, =69

fmol/mg

protein);(o)

membranesfromcontrol myocytes(KD= 156 pM,

_B.,

= 157fmol/

mgprotein).

0

Ho

z

0 C-)

4

0

H

z

TableI. [3H](+)PN200-JIO BindingParameters

n KD B

pM fmol/mgprotein

Control myocytes 8 138.9±15.9 51.2±5.4

Innervated

_myocytes*

5

_145.0±12.7

_108.0±20.1$

*_{Innervated myocytes underwentdenervation 8 h before ligand}

binding experiments.

*P<0.01 comparedtocontrol_myocytes.

lease ofcalcium from the sarcoplasmic reticulum, other mecha-nismsininnervated myocytes are also possible. Indeed, there is no reason to suspect that innervation would alter calcium channel abundance or function without other effects on the cell.Therefore,a morespecificprobeofL-type calcium

chan-nel function was utilized, the DHP calcium channel agonist Bay K 8644.This ligandspecifically interacts with the L-type calcium channel, favors a moreprolonged open state of the channel,andincreases the inward calcium current (12). Inner-vated myocytes demonstrated a markedly increased maximal contractile response to this specific calcium channel agonist, withnochange insensitivity. Thesefindingssupport the view

-,+40

L~~~~~~~~~~~~~~~~~~~~~-i

(I,-

ai-Q En

z

LLI

0

z tYI

1

thatL-type calcium channel abundance increased after inner-vation withnostructural change inthe ligand-binding domain of the a, subunit that might affect ligand affinity. We didnot

observea change in sensitivitytoBay K 8644 in innervated cells. Withaneffector-response system ascomplex asligand bindingto achannelaltering excitation-contraction coupling, it is not possible to predictapriori how concentration-effect curves may shift(13). Moreover, it is distinctly possible that

morethanone componentof the excitation-contraction

cou-pling pathway is altered by innervation.

Second,to test moredirectly the hypothesis that innerva-tion produced an increase in expression of L-type calcium channels, ligand binding studieswereconducted.Membranes frommyocytes growninthepresenceofgangliashoweda

two-foldincrease in DHPbindingsitescomparedtocontrols.There

was nochange inKDfordihydropyridine binding consistent withnochange in

_EC50

forBAYK8644 contractileresponse.

Quantitation of the increase in DHP binding sites is subjectto

certain limitations. First,axonsmadedirect contact withonly 20-50% of the cells in a culture plate. Thus,ifdirectcontact

was necessary to produce an increase in calcium channel

_a,

subunit expression, thedirectly contacted cellsmighthave a

much greater increaseinDHPbinding site number than that determined by assayingtheentire population of cells. Typi-cally, each innervated myocyte appears to becoupled

physi-4.

-4

-8

b

-12

4--80

0_.

T/ /

O-i

7/

/

-60 -40 -20 0 20 40 60 80

MEMBRANEVOLTAGE (mV)

U-Q \ Q

_0

CL

a_

200 pA 30 rns

15 [

5

0

C

- INNERVATEDMYOCYTES CM MYOCYTES

CO MYOCYTESwith CONDIONED MEDIUM

Figure 8. I-Vrelationship of_L-typecalciumchannels. (a) Current tracing for representative control and innervatedmyocytes.Membrane po-tentialwassteppedfromholding potentialof-50 mVtoindicatedsteppotential.Duration ofstep nottoscale.Panel1showscurrenttracings

foracontrolmyocyte;panel2foraninnervatedmyocyte.(b) TheI-Vcurveforcontrolmyocytesis showninopencircles(n=8), andfor

in-nervatedmyocytesinfilled circles (n=5).Myocyteswereselectedforstudy thatwerenotincontactwith othermyocytes.(c) Peakcurrent

densityofL-typecalcium channels. Thepeakcurrentdensity for innervatedmyocytesis shown in the closedbar, for controlmyocytesin the hatchedbar, and formyocytesgrowninconditioned mediuminthe cross-hatched bar. Thenvaluesare asinb. Thecurrentdensitywas

signifi-cantlygreater for innervatedmyocytesthan forcontrolmyocytes(P<0.01).

2

a

-r

ll:zm

0\₀

4

1

a _C)

_0

N

0

E

L.

0

z

0.0I

-50 -60 -40 -20 0 20

Holding Potential

(mV)

Figure 9.Inactivation oftype calcium current. Inactivation of L-typecalciumcurrentwasstudied fornoninnervated cells (o) and in-nervated cells (X) bymeasuringCa2+-activatedcurrent at +20 mV

from various holding potentials.TheCa2` current wasnormalized topeak currentelicited fromaholding potentialof -80 mV. The re-sults wereplotted against holding potentialandfittedto the Boltz-mannequation.Atagivenmembranepotential, more L-type calcium

channelswereavailable for activationin control than ininnervated

myocytes. Forinnervatedmyocytes,current was half inactivated at -31.2±2.1 mV, comparedwith-23.7±0.9 mV (P < 0.01) for nonin-nervated myocytes.

cally

andelectricallywithtwo tothreeadjacentmyocytes.Ifthe

factor(s) that lead to increased calcium channel expression

cross gapjunctions, it is possible that not only the directly contactedmyocytebutclosely coupledmyocytes aswellmight

demonstrateanincreaseincalciumchannelexpression.Thus, itislikelythat thetwofold increase inDHPbinding sites ob-servedisaminimalestimatefortheincreaseinDHP receptors onacellthatisdirectly innervated.Weconsider it unlikelythat

ligand binding studieswereconfoundedby thepresenceof L-type calcium channels onresidual neural elements. Cultures underwent removal ofganglia 8 h before topreparation of membranes. By 8 hours,axons werealmostentirelyabsentor

degenerated.

Thethird line ofevidence supporting the postulate that

direct innervationincreases L-type calcium channelexpression is derived from thewhole-cell voltage clamp recordings. For these

experiments,

onlysinglemyocytesthatwere not

contact-ingother cellssaveforthesympatheticnerve axonwere

stud-ied. Calcium channelcurrentdensitywasclearly increasedin the single, innervatedmyocytes. ThatI-V curves were being

recordedfrom L-type calcium channelswasconfirmed by

typi-cal voltage dependence and sensitivity to Bay K 8644 (12).

Therewas asmallshifttotheleft ofthe voltage for peak inward

currentforinnervatedcells. Thebiologicalimportance of this shift and theunderlyingmechanism remaintobedetermined.

Itis possible that innervation may altereither sarcoplasmic reticulumfunctionor

[Ca2+]i,

either of whichCohen and

Le-derer(14)have showncanalter

Ic~.

Taken together, these three sets ofexperiments

demon-stratethatdirectsympatheticinnervation increasesexpression

offunctionalL-typecalcium channels. Evidence isnow

con-vincing thattheDHP receptordomainis within the

_a1I

subunit oftheL-type calciumchannel(15),sothatligand

binding

stud-ies with aDHP ligand and patch clamp studies provide comple-mentarydata on the same structures.

Themodel system for the in vitro innervation experiments deserves comment (2, 7, 16). Ventricular myocytes were

cul-turedfrom l-d postnatal rat hearts. Although autonomic neu-rons arepresent in the rat heart during embryonic cardiac

or-ganogenesis, functional innervation does not develop until after birth (17). Functional adrenergic transmission in the rat heart can be detectedconsistently 1 d after birth, but develop-mentofmatureadrenergic innervationis further delayed until 21 dafter gestation(17). Thus, the studies reported here were

conducted onmyocytes that hadatmost abriefexposure to

sympatheticneurotransmitters invivo.

Apossibleconfoundingfactor for in vitro innervation stud-iesisthat upon prolongedtissueculture, ratsympathetic neu-rons are known to develop the ability to synthesize and accu-mulateacetylcholineand losetheirabilitytosynthesize norepi-nephrine (18). However, it takes 2-4 wk in vitro for the

transition fromsympathetictoparasympathetic propertiesto occur.Ourstudieswereconductedat72-96 h invitro,making this effect unlikely. Moreover, we were able to demonstrate

(Fig. 2) thatstimulation ofganglia producedan increase in frequency of contractionthatisatypical sympathetic

neuro-transmitterresponse.

While itisevidentthat co-cultureof sympathetic ganglia with myocytes produces physical association (Fig. 1),with a

concomitant changein spontaneous beating rate and an

in-creaseincalciumchannelexpressionandfunction,wecannot

beabsolutelycertain in this in vitro system functional

sympa-thetic neuroeffector transmission has beenachieved. Clearly

neuron-myocyte contactproduces important changes in the myocyte. Theresponsible effector(s)arebeing sought.

Adistinctadvantageofthe neuron-myocyte co-culture

sys-tem is that the response ofspecificmyocytescould be

deter-mined after innervationordenervation.Intheintact ventricle, myocytescompriseanumericalminority ofcells, so thatligand binding studies orphysiologic studies reflectthe responsesof

neural, vascular, and connective tissue elements as well as ven-tricular myocytes. In vitro sympathetic innervation models have beenreported by several laboratories (7, 16, 19).A particu-lar advantage ofour approach using relatively large ganglia

ratherthanfinelyminced neuraltissuein cultureis that it

per-mitsthe examination of denervation in vitro by allowing re-movalordegradationofvirtuallyall neuraltissue (2).

An additionalimportant finding of this study isthatafter denervation foratleast 24

h,

anincreaseinfunctional L-type calcium channels can still be found.

Although

the available

data do not

permit

conclusions

regarding

themechanisms

by

which the sustained increase in calcium channel number

oc-curs,possibilitiesinclude innervation

increasing transcription

rates forgenes forone or moreofthe fivesubunitsoftheL-type

calcium channel.

Sympatheticinnervationofmyocytesproduced alterations

incontractile properties thatare notreadily

explained

byan

increase in number of L-type calcium channels alone.

Innerva-tionwasassociatedwithasmall but definitedeclinein

contrac-tilesensitivityto

[Ca2+]0.

One

possible explanation

is that in-nervation alters the expression of several genes in a pattern similartothat whichoccursduringmaturation.We

previously

reported (5)thatin the

developing

chick heart there isa

de-creaseincontractile

sensitivity

to

[Ca2+J0,

andother

changes

in

structures

modulating

calciumhomeostasishavebeen

_reported

with innervationand/or development (8, 20-22). Thus,

inner-vation-induced events

analogous

to those

occurring

during normal maturationof neonatal myocytes isa

unifying

hypoth-esis that

might explain

many of these observations

(23,

24).

Itis ofnotethat myocytes grown in conditioned medium didnotdemonstrate increased numbers of functional calcium channels. Directcontact

by

a

sympathetic

neuronappearstobe

required

for this effect. This observationsuggests that the tro-phicinfluence of the neural element is confinedtothecell(s)in

contactwith

it,

orthat the

factor(s)

releasedbythesympathetic

nervedoesnotdiffuse beyondthesynapticcleftintothe culture medium inconcentrations sufficienttoproducea trophic

ef-fect on other myocytes. What

signal might

be produced by directcontactwiththe

sympathetic

nerve toproduce increased

expression

of L-type calcium channels? Neurotransmitter(s)

thatinduce differentiation (22, 25-27) orthatmightalter

G-protein expression

(2, 19, 28)arestrongcandidates.Giventhe

alterationsincontractile

properties

ofinnervatedmyocytes,it is

unlikely

that the

signal

produced by innervationonly alters

L-typecalcium channel

expression;

ratheritismorelikelythat the

signaling

process hasabroadereffectonthe cells'genetic

program. Thedependence ofCa channel induction on very closeassociation ordirectcontact betweennerveand muscle suggestsa

paracrine

mechanismof growth factor inductionor

interaction ofmembrane-bound componentsofthe muscle andnerve(29, 30).Therewas a21%differencein spontaneous

rate ofcontraction of control and directly innervated myo-cytes.This

might

produceachangeinrateof deformationof the cell membraneorin

cytosolic

ionconcentrationsthat could

conceivably

contributetoalteration in abundanceof calcium

channelsaswell.Additional studieswill be necessarytodiscern

theeffect of

beating

ratealoneon

expression

offunctional ion

channels.

In

conclusion, sympathetic

innervation in vitro of

previ-ously

uninnervated neonatal rat ventricular myocytes pro-duces an increase in

expression

of functional

L-type

calcium

channelsas

judged by ligand binding

and

electrophysiological

andcontractileresponse criteria.

Subsequent

denervationdoes

not reduce calcium channel number to control levels for at

least24h. Theexperimental findings,takentogether,are

con-sistent with the

hypothesis

that

sympathetic

innervation in-duces

changes

in cultured neonatal rat ventricular myocytes

analogous

tothose thatoccur

during

normalmaturation.

Acknowledgments

WearegratefultoMs.MichelleSomersforexcellentsecretarial

assis-tance.

Thisstudywassupportedin partbygrants HL35781andHL36 141 fromtheNational Institutesof Health.

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