Diastolic dysfunction in hypertrophic cardiomyopathy Effect on active force generation during systole

Diastolic dysfunction in hypertrophic

cardiomyopathy. Effect on active force

generation during systole.

J K Gwathmey, … , W Grossman, J P Morgan

J Clin Invest.

1991;

87(3)

:1023-1031.

https://doi.org/10.1172/JCI115061

.

We tested the hypothesis that intracellular Ca++ [( Ca++]i) overload underlies the diastolic

dysfunction of patients with hypertrophic cardiomyopathy. Myocardial tissue was obtained at

the time of surgery or transplantation from patients with hypertrophic cardiomyopathy and

was compared with control myocardium obtained from patients without heart disease. The

isometric contractions and electrophysiologic properties of all myocardial specimens were

recorded by standard techniques and [Ca++]i was measured with the bioluminescent

calcium indicator aequorin. In contrast to the controls, action potentials, Ca++ transients,

and isometric contraction and relaxation were markedly prolonged in the hypertrophic

myocardium, and the Ca++ transients consisted of two distinct components. At 38 degrees

C and 1 Hz pacing frequency, a state of relative Ca++ overload appeared develop, which

produced a rise in end-diastolic [Ca++]i, incomplete relaxation, and fusion of twitches with a

resultant decrease in active tension development. We also found that drugs with increase

[Ca++]i, such as digitalis, exacerbated these abnormalities, whereas drugs that lower

[Ca++]i, such as verapamil, or agents that increase cyclic AMP, such as forskolin, prevented

them. These results may explain why patients with hypertrophic cardiomyopathy tolerate

tachycardia poorly, and may have important implications with regard to the pharmacologic

treatment of patients with hypertrophic cardiomyopathy.

Research Article

Find the latest version:

Diastolic

Dysfunction in Hypertrophic Cardiomyopathy

EffectonActive Force Generation During Systole

Judith K. Gwathmey, Sanford E. Warren, G. Maurice Briggs, LindaCopelas,Marc D.Feldman, Preston J. Phillips,

MarkCallahan, Jr., Frederick J. Schoen, William Grossman, and James P. Morgan

Fromthe CharlesA.Dana Research Institute and Harvard- Thorndike Laboratorv ofBeth Israel Hospital, the ConsolidatedDepartment

ofMedicine (Cardiovascular Divisions) and Departments ofPathology, Beth Israel and Brigham and U'omen 'sHospitals;the Division of CardiovascularSurgery, Brigham and Women's Hospital, Boston,MA02215;and the Departmentsof Pharmacology

and Medicine(Cardiovascular Division) and theDivisionof CardiothoracicSurgerY, MaYoAledical School

andMayo Graduate School ofMedicine, Rochester, Minnesota 55905

Abstract

We tested thehypothesis that intracellular Ca++

([Ca+Jli)

over-loadunderlies the diastolic dysfunction of patients with

hyper-trophic cardiomyopathy.Myocardialtissue was obtained at the time of surgeryortransplantation frompatientswith hypertro-phic cardiomyopathy and was compared with control myocar-diumobtainedfrompatientswithout heart disease. The

isomet-ric contractions and electrophysiologic properties of all myo-cardial specimens wererecorded by standard techniquesand

[Ca"],1

wasmeasured with the bioluminescent calcium indicator aequorin. In contrast to the controls, action potentials, Ca++ transients, andisometriccontraction and relaxation were mark-edly prolonged inthehypertrophicmyocardium,and theCa++ transients consisted oftwodistinct components. At38°C and1 Hzpacing frequency, a state of relative Ca++ overload appeared

todevelop, which produced a rise in end-diastolic

_[Ca"+Ji,

in-complete relaxation, and fusion of twitches with a resultant decrease in active tension development. We also found that drugs which increase

_ICa++Ii,

such as digitalis, exacerbated theseabnormalities, whereasdrugs that lower

_[Ca++Ii,

such as verapamil, or agents that increase cyclic AMP, such as forsko-lin, prevented them. These results may explain why patients withhypertrophic cardiomyopathy tolerate tachycardia poorly, and may haveimportant implications with regardtothe phar-macologic treatment of patients with hypertrophic cardiomyop-athy.(J. Clin. Invest. 1991.87:1023-1031.) Key words: cal-cium indicators * diastolicdysfunction-hypertrophic

cardiomy-opathy * sarcoplasmic reticulum * cyclic adenosine monophosphate

Introduction

Hypertrophic cardiomyopathy is a disease of unknown etiol-ogy that ischaracterizedanatomicallyby a smallleft ventricu-larcavity and marked hypertrophy of the myocardium with

myocardial fiber disarray(1-3). An intraventricular pressure

Portions of this workhave appeared in abstract form in 1986.

Circula-tion. 74(Suppl):169; 1986. Circulation. 74(Suppl):290; and 1989.J. Cell. Biochem. 13E(Suppl):171.

Address correspondence and reprint requests to Dr. James P. Mor-gan,Cardiovascular Division,Beth IsraelHospital, 330 Brookline Ave-nue,Boston, MA 02215.

Receivedforpublication 28 February 1990 and in revisedform 25

July1990.

gradientmay or may not be present (4-6).Amajor problemin

hypertrophic cardiomyopathy appears to be the severe hyper-trophy and associated decrease in left ventricular diastolic relax-ation andcompliance.

Clinical studies have shown that patientswithhypertrophic

cardiomyopathy typicallydisplay symptomsandsignsof

dia-stolic dysfunction, such as dyspnea and elevated pulmonary capillary wedge pressure, due to impaired ventricular filling and slowdecay of left ventricular pressure (4, 7-11).The basis

of these diastolic abnormalitiesisprobably multifactorial, but

hasbeen proposedtobe due inlarge parttochanges in intracel-lular Ca++ handling by hypertrophied myocytes (7, 12, 13).

Systolic dysfunction isnotbelieved toplayanimportant role until late in the disease when adilated cardiomyopathy may develop (14-16).

Since 198 1,wehave had the opportunitytostudy ventricu-lar muscle isolatedfromthe heartsof fivepatientswith hyper-trophic cardiomyopathy. Two of these patients underwent myomyectomy for intractable symptoms of outflowtract ob-struction; three underwentcardiac transplantation for

end-stage heart failure.Comparedwith preparations from control human hearts, musclesfromeach of the hypertrophic

cardio-myopathic hearts demonstrated both contraction and

relax-ation abnormalities. Usingthe Ca++ indicator aequorin, we

wereabletodirectlydeterminetherelationshipbetween con-tractiledysfunction and abnormal intracellular calcium han-dling in thehypertrophicmuscles.

Methods

Tissuewasobtained fromfivepatientsin whomhypertrophic cardio-myopathyhad beendiagnosedonthebasisofclinicalsigns and

symp-tomsandcharacteristicechocardiographicandhemodynamicfindings:

Patient 1. A 31-yr-old woman who underwentventricular myo-mectomyin May, 1981 forsymptomsofdynamic outflowtract

ob-structionunresponsivetomedical therapy. Medicationsatthetime of

surgery includedpropranolol, 100 mg, anddisopyramide, 100mg,

given four times daily.

Patient 2.A49-yr-oldmanwhounderwentcardiac transplantation in July, 1985 for end-stageheartfailure. Medicationsatthe timeof

surgeryincludeddigoxin, furosemide,and coumadin.

Patient3.A45-yr-oldwomanwho underwentcardiac transplanta-tioninJanuary, 1986for end-stageheartfailure. Medicationsatthe

time ofsurgeryincludedfurosemide, spironolactone, metolazone,and

coumadin.

Patient 4.A 19-yr-oldwomanwho underwentcardiac

transplanta-tioninNovember, 1987for end-stageheartfailure. Medicationsatthe

timeofsurgeryincludeddigoxin, chlorothiazide, coumadin, quinidine,

andcaptopril.

Patient 5. A 69-yr-old woman who underwent myomectomy in

January, 1989 forsymptoms of dynamic outflow tractobstruction J.Clin.Invest.

©TheAmericanSociety for Clinical Investigation,Inc.

0021-9738/91/03/1023/09 $2.00

Table I. Hemodynamic Values ofPatients with Hypertrophic Cardiomyopathy

Patient Days

No. PTS RA PA PCW LV,apex LV, base ASCaorta CI

mmHg mmHg mmHg mmHg mmHg mmHg liter/min per M

1 30 8 34 27 186/27 84/21 84/56 2.5

2 150 25 36 30 92/29 2.2

3 97 19 24 20 90/18 90/18 90/62 1.6

4 47 9 13 15 1.5

5 5 6 20 12 176/12 158/65 2.7

DaysPTS, daysprior to surgery; RA, right atrial pressure; PA, pulmonary artery pressure; PCW, pulmonary capillary wedge pressure; LV, left ventricular pressure; ASC aorta, ascending aorta pressure;CI,cardiacindex; LVEF, left ventricular ejection fraction; MR, mitral regurgitation; andWNL, within normallimits.

unresponsivetomedical therapy. Medicationsatthetimeof surgery includeddiltiazem, 60 mg givenfour times daily,disopyramide, 100 mggivenfour times,metoprolol, 50 mg giventwice,nifedipine, 10 mg given three times, isosorbide, and insulin.

Hemodynamic values for thesepatients before surgeryarelisted in TableI.Experimental preparations included thin trabeculae carneae (n

= 16)withmeanfiber diameters of 0.93±0.30 mm, andanaccessory papillary muscle (n= 1).

Resultsobtained with trabeculaecarneaewerecomparedwith those obtained with trabeculaecarneaeisolated from the hearts ofeight

brain-dead organ donors without cardiacdysfunction (meanfiber di-ameters, 1.03±0.05 mm;n=25).Inall cases,experimentaltissuewas

placed afterexcision into acontainerof oxygenated physiologicsalt solution (see composition below) at roomtemperature. 9 of the 16 musclesin the hypertrophiccardiomyopathicgroup and 10 of the 25 muscles in the control group wereobtained from the leftventricle;the remainderwereobtained from therightventricle. Duetothesimilarity

of results, left andright ventricularpreparationswereanalyzed

to-gether. We havepreviously reported that thereare nodifferences in contractileperformancein musclesobtained from the leftorright

ven-tricle (19). The muscle frompatientI wasexcludedfrom group analy-sis duetotheslightly differentcomposition of thesuperfusate. The

compositionof the solution usedtosuperfusemusclefrompatient I was asfollows (concentrations inmEq/liter): Na+, 140; K+, 5;

Ca",

2.25;

Mg",

2;ClI, 103.5;HCO-,24; HPO,2;SOi, 2;acetate-,20;

plus 10mM/literglucose. For allremainingstudies thecompositionof thesuperfusatewas asfollows(mM): Na+, 146.2; K+,5,9;

Ca",

2.5;

Mg++,2.4;Cl-, 133.3; HCO-,25;H2PO4, 1.2, plusglucose, 11.5mM/

liter. Although slightly different, both solutions contained bicarbonate buffers and similar concentrations of Ca++ and Mg++ (key ions). Solu-tionswerebubbledwith 95%02and 5%CO2toachieveapH of 7.4. Phosphatewasremovedfrom thesolution during performance of cal-ciumconcentration-responsecurvesin ordertopreventprecipitation of calcium phosphate. Muscleswere placed in organ bathsat30 or

38°C,connectedtotransducersforrecording tension,andwere

stimu-lated to contract at0.25or0.33Hz, through punctateelectrodes using voltagethat was . 10% above threshold with pulsedurations of 5 ms. Muscles weresuperfused in the bath for1-1.5 hduring which time they

werestretched to the length atwhich maximalisometric tension devel-oped.

Intracellularcalcium transientswererecordedwith aequorin,a bio-luminescentindicator that emits light when it combines with Ca++. Thepreparation ofaequorin for laboratoryuseand thekinetics of its reaction with Ca++ havebeendescribed in detail (17). Aequorinwas

loaded into hypertrophiccardiomyopathic muscles by microinjection

(17)orachemical technique(18).Wehave demonstrated that there is

qualitativelynodifference in the detected aequorinsignalwith either

technique (20). Light and tension responses wererecorded simulta-neously.Light signalswererecorded withaphotomultiplier (EMI 9635 qA) andsignalsaveragedtoobtainasatisfactory signal-to-noiseratio.

Action potentials were recorded with fine-tipped straight microelec-trodes madeof borosilicate glass. Electrodes were filled with KCI 3 M, and connected to an electrometer.Before making experimental record-ings several twitches were recorded under steady-state conditions in order to confirm stable impalement (21).

Histologic measurements were performed on experimental tissue stained with hemotoxylin and eosin, embedded in plastic, and sec-tioned at 1-2 Mm. The length and width of 10 fibers per trabecular strip were measured atthesite of a centrally located nucleus. Nuclear area was calculatedusing the formula (semiaxis Axsemiaxis B)xpi. Statis-ticalcomparisonsweremadebyStudents t test and P values < 0.05

wereconsideredsignificant.

Results

Sinceprolonged diastolic relaxation is themostfrequently

re-portedabnormality ofcontractile function in hypertrophic car-diomyopathy, we compared the time courses of isometric con-traction and relaxation in trabeculae carneae from control car-diac muscle versus cardiac muscle from patients with

hypertrophiccardiomyopathy.Table II shows that at an extra-cellularCa++ concentration

_([Ca`+].)'

of 1 or 16mM, the time

to50% relaxation from peak tension

(RT50)

and time to 80% relaxation from peak tension (RT80) in the hypertrophic car-diomyopathic trabeculae carneae were significantly prolonged compared with the controls. Moreover, hypertrophic cardio-myopathic muscles showed a progressive increase in the time course ofrelaxation as

_[Ca"+].

was increased that was much moremarked than in the controls.

Because thehypertrophiccardiomyopathictrabeculae de-scribedinTable IIwereremoved frompatientswithend-stage heartfailure, we were surprised to find that they were ableto

generate similar or greaterpeak isometric tension, as normal myocardium from the controls. It should be noted that the

valuesincluded inTable II wereobtainedunderrelatively hypo-thermic conditions (i.e., temperature = 30°C) and at a low frequencyof stimulation(i.e.,20twitches/min).To investigate whetherthisobservation mightbe an artifact oftemperature and stimulation rate we also studied musclesat38°Cand

physi-ologic frequenciesofstimulation.AsshowninFig.1at stimula-tionfrequencies of1 Hz orgreater,activetensiondevelopment

was less than atlowerfrequencies. This frequency-related de-crease inactive tensiondevelopment (Fig. I A)was associated

withacorrespondingincrease in end-diastolic tension(Fig. 1

1. Abbreviationsused in thispaper:

[Ca`+]j,0,

intracellular, extracellu-larCa++ concentration.

Table II. Amplitudes and Time Courses of Twitches in Control and Hypertrophic Cardiomyopathic Muscles

[Ca++J0 N PT TPT RT50 RTgo

M mN/mm2 ms ms ms

Control 1 22 3.7±1.5 317±20 258±16 435±33

16 24 12.6±5.4 348±12 300±35 485±60

HCM 1 8 9.1±3.2* 489±44* 350±45* 579±85*

16 8 21.8±6.5* 406±46* 475±62* 826±111*

Perfusate contained 1 or16 mM[Ca++]Oat 30°C and 3-sintervals of stimulation.N, numberof trabeculaestudied;HCM,hypertrophic

cardio-myopathy; PT, peak isometrictension; TPT, timetopeaktension;

_RT50,go,

timeto50, 80% relaxationfrom peak tension, respectively.Values are

mean±SE. *ForallHCM values,P <0.05comparedwith thecontrols.

B). Both the decrease inactivetension and increase in end-dias-tolictensionwereexacerbated by increases in

_[Ca"+]..

Similar abnormalitiesinactiveandend-diastolic tension development were seen athigher ratesofstimulation in muscles from all five patients with hypertrophic cardiomyopathy and were present

at30°Caswellas38°C.

We usedaequorin-loaded trabeculae carneae to testdirectly whether the systolic and diastolic abnormalities we observed were due to changes inintracellular Ca++

([Ca++]i)

handling.

Fig. 2shows thetensionandlight(i.e.,

[Ca`+]i)

recordingsin a trabecularstripfromapatientwithhypertrophic

cardiomyopa-thy (bottom) and a control trabecularstrip (top) at 2.5 mM

[Ca"+],.

Thefigure shows that the

[Ca++]i

transient in control muscleconsists of a single component thatrapidly rises to a peak and declines towards baseline. In contrast, the

_[Ca++]i

transient in hypertrophic cardiomyopathic muscle is greatly

prolonged andconsistsoftwocomponents, labeled

L,

andL2 in Fig. 2. Fig. 3 shows similar recordings in trabeculae at 16

mM

_[Ca"+]..

Note that in boththe control andhypertrophic cardiomyopathictrabeculae carneae, increasingthefrequency

A.

140

130

120

110

100 500

400 F

E 300

z

z

>

4 200

100

0

* co++ X2Co++

+ 4Ca++

o 8Co++

a16 Co++

of stimulation producedan increasein theamplitude ofthe Ca++ transient.However, incontrast tocontrolmuscles, higher frequencies ofstimulation of hypertrophic cardiomyopathic muscle resulted infusion of the Ca++ transients and twitches, andanincreaseinend-diastolic

_[Ca++]i

andtension relativeto

lowerfrequencies. Note that in the presenceofelevated

_[Ca"+].

(i.e., 16 mM

_[Ca"+].;

Fig. 3), active tension declined at the higher frequencies ofstimulation inhypertrophic

cardiomyo-pathicmuscles althoughtotal forcewasgreater(Fig. 3 A). This didnot occur incontrol muscles (Fig. 3 B).

Most positive inotropic agents, including increased

[Ca"+].,

digitalis, andbeta-adrenergic agonists,act toincrease theamplitude of the

_Ca"i

transient associated with contrac-tion.Fig. 4shows the effects ofisoproterenol on light and ten-sion recordingsin ahypertrophic cardiomyopathic muscle.A

Shows the effects ofisoproterenol on the amplitudes of the

Ca++i transient andisometric twitch; inB,signalshavebeen adjustedto equal amplitudesand superimposedso thattime

courses canbecompareddirectly. Beforeadministering drug,

theaequorin signal consisted oftwo components.

Isoproter-B.

X

2Co++

Figure1. Effects of

+ 4 C++O changes in stimulation

08Co++ frequency and[Ca++].,

a16_{Co++ onactive (A)}

and end-di-astolic (B) tension

devel-+ opmentinatrabecula

frompatient 3.

Tempera-ture= 38°C; tension

ex-pressedinmilligrams

*

(mg),

[Ca"+].

in

millimo-lar,andstimulation

fre-quency in hertz(Hz).

.01 .03 .1 .33 1.25 2 Propranolol, 6 x 10-' M

-& 90

0 80

z

'- 70 w

*s60

a

z 50

40

30

Hz inbath.

.01 .03 .1 .33 1.25 2

Hz

.1

.03

A

.33 1

,I

I5

,,

36.8nA

! l <,1.7g

_+ >_ <4 d}~~~~~~~~~~~~~~~~~140nA

500ms_____________ A .6g

500ms

Figure 2. Light and ten-sion responses to varying frequencies of stimulation from 0.03 to IHz in con-trol (A) and hypertrophic trabecula(B) from patient 3.Light intensity ex-pressed in nanoAmperes (nA) and tension in grams (g). Temperature=30°C; [Ca"+]0=2.5 mM. Note the two components of theCa++ transient in the hypertrophic

cardiomyo-pathicmuscle,labeledL, and L2 in bottom right panel. Deltas indicate rise inend-diastolic Ca++ or tension that occurs at fasterpacing frequencies compared with 0.03Hz.

enol, which increases intracellular cAMP concentrations, not

onlyincreased the amplitude of the Ca++ transient but

mark-edly abbreviated its timecourse. These changes were associated

withapositive inotropic effect; however, isoproterenol, in con-trast toelevations in

_[Ca++].

(Table II) and strophanthidin

(datanotshown), markedly abbreviated isometric contraction andrelaxation.

Fig.5shows the effects of a cardiotonic steroid

(acetylstro-phanthidin,aNa+-K+ATPase inhibitor) and an agent that

in-A

B

LL L

LUiW

UYJkfKNJ\

.33Hz .5Hz 1Hz

creasesintracellularcAMP

(forskolin,

anadenylate cyclase

ac-tivator)onthe

frequency-response

relationship ina

hypertro-phic

cardiomyopathicmuscle. Note thatacetylstrophanthidin mxacerbated, and forskolin attenuated the decrease in active tension and increaseinend-diastolic tension that occurred at

higher frequencies of stimulationin 16 mM

[Ca"+]J.

Fig. 6 shows the effects ofthecalcium channel-blocking agent, verapamil,on thelightandtension responsesinanother hypertrophic cardiomyopathic muscle. Verapamil decreased

3OnA

500mg

7OnA

Figure 3. Light and tension responsesto

varying frequencies of stimulation from 0.33to 1 Hzinahypertrophic trabecular

strip(A) from patient 3 andacontrol trabecularstrip (B). Light intensity 1 80mg expressedinnanoAmperes (nA), tension in

milligrams (mg). Temperature =_30°C;

[Ca"+]0

= _{16 mM.}

iso.

3xl0-7'M

1.3k

PAp

I

128 mg

B. iso.

3xI0-7M 1.3k

cont.

0.3k

iso.

3xl*7M

128mg

cont.

60mg

kL

1 1--100 ms

theamplitudeof both components of the aequorin light signal but did notsignificantlyabbreviate its timecourse or the

dura-tion of the isometric twitch.Verapamil prevented therise in

end-diastolic tension from occurringathigher frequenciesof

stimulation(Fig.7B),andproducedanegative inotropiceffect

(Fig.

7

_A).

Fig. 8 shows theeffects of caffeineonthelight and tension

responses ofa

hypertrophic

cardiomyopathicmuscle. Note that caffeineproducedadose-relatednegative inotropiceffect under the conditions of this experimentthat wasassociated

witha marked decreasein the

amplitude

of L1; incontrast,the amplitude ofL2wasdiminishedto alesserextentin responseto caffeine.

A.

Figure 4. Light and ten-sion responses to isopro-terenolof a papillary musclefrom patient 1. A Showseffects on ampli-tudesof light and tension responses. In B, ampli-tudes have been adjusted

tobeapproximately equal

sothattime courses can be compared directly. Absoluteamplitude is in-dicated to the right of eachpanel. Stimulus fre-quency=0.25 Hz; 38°C. Light is recorded with photon counter and ex-pressedin kilocounts (k) per second (s);

_[Ca"+].

=2.25 mM. Stimulus artifact indicated beneath each panel.

Resting membrane potentials and the peakamplitude of the action potentialswere notdifferent in the control versus

hypertrophic muscles. However, thetime to90%

repolariza-tion

(ADP90)

and theplateau phase of the actionpotentialwere prolonged in the hypertrophic muscles

_(ADPgo

=650±0.0ms; n=₂₎versusthe controls(529±39ms;n =_3).

Inordertodeterminethedegreeofhypertrophyinthe mus-cles of thepresent series, we measured myocytediametersand calculated nuclear areas for thehypertrophic cardiomyopathic preparations and controls.The muscle, frompatient 1 was not

available forexamination, but it isimportanttonotethat all heartsshowedevidence ofsignificant hypertrophyonthe basis

ofelectrocardiographic and

echocardiographic

findings.

Val-o S-MCe4+

* .5Co++p.PFORSl3.i0-0M

* ISSC.++Diu$AS4.10-?m

01 .03 .1 .33 L25 2

Figure S. Effects of fors-kolin(FORSK)and

ace-tylstrophanthidin (AS)on

thefrequency-response relationship andactive

andend-diastolic tension development ina

trabec-ularstripfrompatient3.

Valuesfor active tension expressedaspercentage change from baseline de-finedat0.03Hz.

Tem-perature=38°C;

[Ca"].

=16mM;propranolol,

6x 10-'Mpresentin

bath.

A.

loe",

Li

L2

PRE-DRUG

B. to

0

s

I -10

I

-240

too[

1-,.0

fAO40

r 140 I 20

I

100

a so

20 40

-GoL

-L

Li

L₂

_PRE-DRUG

-_k

-PRE-DRUG verap.

,i,/

DRUG Ix

10-6

M

L.U

~60

c/s

-ml cont.

-|

1W_

130C/S

--I

1-

lOOMs

L

_L2

DRUG

PRE-[

verap.

IX10-6M

130c/s

200 mg

verap. DRUG

_IxlO-6

M

80 mg

cont.

200 mg

Figure6.

_Light

and ten-sionresponsesto

vera-pamilofapapillary mus-cle frompatient 1. A Showseffectson

ampli-tudes of

light

andtension responses.InB,

ampli-tudes have been

adjusted

tobe

_{approximately equal}

sothattimecoursescan becompared

directly.

Absoluteamplitudeis in-dicatedtotherightof eachpanel.Stimulus

fre-quency=0.25Hz;38°C. Lightis recorded with photoncounterand

ex-pressedincounts/second

(c/s);tensionin

milli-grams(mg);

[Ca"+].

=2.25mM.Stimulus

ar-tifactindicatedbeneath eachpanel.

uesforthe control (n= 18) andhypertrophiccardiomyopathic (n = 8) groups were, respectively, fiber diameter

(,um)

= 14.6±0.36 vs.

28.7±0.36,

P<0.001; and nuclear area

_(Atm2)

= 58.1±2vs. 82.3±6.2, P .0.001.

Discussion

Heartfailurecan occur as a resultofsystolic dysfunction, dia-stolic dysfunction, or a combination of both. In this study, working myocardium from patients with hypertrophic cardio-myopathy demonstrated marked abnormalities of diastolic

re-laxationcomparedwithcontrols, under a variety ofconditions.

Systolic function(i.e.,active tensiondevelopment)wasnormal

orsupranormalatlowerfrequencies of stimulationwhen suffi-cient timeelapsedbetweentwitchesforcomplete relaxationto occur(Table II). However,at

faster,

more

physiologic

ratesof

stimulation, fusion of theCa++ transientsandcorresponding mechanicalresponsesoccurred, leadingto anincreasein

end-diastolic

_[Ca++]i

andanassociated increaseinend-diastolic ten-sion and a decrease in active tension development. This

oc-curredin cardiacmuscle from hypertrophic cardiomyopathy

patientswith and without clinical evidence of heartfailure,but

notin controls. It is importanttonotethat theseabnormalities mayreflect purely diastolic dysfunction with normal intrinsic systolic function, in whichcasethe decrease in active tensionat

high end-diastolic tensions would reflect the expected

length-tension relationship. Whetherornotsystolic function isfully

"normal" or to somedegree impaired is uncertain;however,

the diastolic abnormalitiesare clearlythe moreimportant of thetwo.These resultssuggestthathypertrophic

cardiomyopa-thyisapathophysiologicstatein which diastolic

dysfunction,

in partduetocytosolic Ca++ overload,canmarkedlydepress

activeforce generation duringsystole,anobservation that has important therapeuticimplications.

The force percross-sectional areagenerated bythe hyper-trophic preparationswassignificantlygreater than thecontrol

atnormal and maximally activating

[Ca"+]0,

asshown in Table

II. The force generated by these muscles is also significantly

greaterthan theaveragefor muscles from the heartsofpatients undergoing transplantation for end-stagecardiomyopathy,

which we have found to be similar to that of controls at a

0.33-Hz stimulation frequency, 30°C, 2.5 or 16 mM

_[Ca"+].

.01 .03 0.1

STIMULUS FREOUENCY (Hz)

Figure7. Effects of

vera-pamil onthe

frequency-responserelationshipand active(A)and end-dia-stolic(B)tension

devel-opmentin a trabecular

stripfrompatient3.

Pro-pranolol,6 x

_10-'

M

presentin thebath;

[Ca"+].

= 2.5mM;30°C.

A.

B.

A.

*CONTROL

A IxI0 VERAPAMIL

oIxIO5VERAPAMIL

B.

5

23

z

2

2.0 r

* CONTROL A I l1O0-VERAPAMIL

OIxIo-5 VERAPAMIL

_ 1.5

zU

u 1.0

Ca fIO.

L

F

F-N

.01 .03 0.1

STIMULUS FREOUENCY (Hz)

0.3 1.25

1028 Gwathmeyetal.

0

Li

/

CONTROL 0.6 mM CAFFEINE

AmM CAFFFINIF

C llwlv -|%14 Vll" 1L s-liv V

V-Ilv-290c/s

58mg

A|-lOOms

-.1

1--

lOOMs

(21, 22). Thereasonforthisapparentincrease in force

generat-ingcapacity is unclear, but does notappeartobe due tothe hypothermic conditions ofourexperiments and isnotmanifest

athigher pacingrateswherediastolic dysfunction develops (see Results). Individualmyocytediameterwassignificantlygreater

inthehypertrophic muscle of thepresentseries compared with those from control hearts or hearts from patients with

end-stage dilatedcardiomyopathy (see Results and reference 21).

Since the myofibrillar content of hypertrophied fibers is in-creased, it is possible that the favorable energeticstateand

ade-quaterestitutiontimefor

[Ca2J]i

that exist under the basal

con-ditionofourexperiments allow this increase in contractile

pro-teincontent tobecome functionally manifestas anincrease in

peak and maximally activated twitch force.

Asshown in Table II and Figs. 1 and3, contractile

abnor-malitiesof thehypertrophic cardiomyopathicmuscleswere

ex-acerbatedby increasing

[Ca"+]0,

which indicatesaninabilityto

maintainintracellular Ca++homeostasis(21, 23). Digitalis (i.e., strophanthidin andacetylstrophanthidin)exacerbated systolic anddiastolic dysfunction (Fig. 5) by increasing

[Ca+J]i

via transsarcolemmalNa+-Ca++ exchange, whichoccurs as aresult

ofNa+-K+ ATPase inhibition (24). In contrast, although

iso-proterenoland forskolin also increased

[Ca++]i,

asjudged from

the amplitude of the aequorin light signal (Fig. 4), diastolic

dysfunctioninhypertrophic cardiomyopathicmuscleswas

re-versed towards normal (Fig. 5). This apparentlyoccurredas a

resultof thepositive lusitropic actions of thesedrugs, which increase [cAMP], and enhance the ability ofthe sarcoplasmic

reticulum to sequester calcium and lower

[Ca+J]i

(25, 26).

Thesedatasuggestthat drugs with positive lusitropic actions

maybe usefulagentsfor correcting the cellular abnormalities

thatunderlie myocardial dysfunction of hypertrophic

cardio-myopathy,and thatagentswith negative lusitropic actions

shouldbeavoided. Inclinical practice, however,thesefactors

mustbebalancedagainst the demonstrated utility ofdrugs with

negative inotropic and chronotropic properties, such asthe

beta-adrenoceptor antagonists and verapamil, to reverse

hy-Figure 8. Effects of caffeine on the light and tension responses of a

papillary muscle from patient 1. In each panel, upper trace = light in photon counts/second

_(cl

s); middle trace=tension inmilligrams (mg); the lower trace is the stimulus

artifact.Dosesof caffeine expressed in millimolar; stimulusfrequency

=0.25Hz;

[Ca++].

=2.25

mM.

percontractile function and increase diastolic filling time in patients with hypertrophic cardiomyopathy (27, 28). In addi-tion, drugs that increase cAMP also increase transsarcolemmal

Ca++entryandmight, undersomecircumstances, exacerbate

[Ca+J]i

overload. Although verapamil didnotsignificantly ab-breviate the timecourseof theCa++ transientorisometric

con-traction, it decreased the amplitude of bothcomponentsof the Ca++ transient and prevented the increase in end-diastolic

ten-sionfromoccurringathigherfrequenciesofstimulation(Fig. 7 B). These resultsareconsistent withreportsinthe clinical

litera-turedocumenting hemodynamic improvement of patients with hypertrophic cardiomyopathy afteradministration of

Ca"

channel blockingagents (7, 29-32). Itmustalso be

re-membered that in clinically relevant doses, each of theseagents

hassignificant positive (i.e., isoproterenol)ornegative (i.e.,

car-diacglycosides, beta-adrenoceptor antagonists, and verapamil) chronotropic actions thatcanhaveimportant effects in view of

the frequency dependency of myocardial dysfunction in this

pathophysiologicstate.

Abnormal Ca++ handling may occurat severalcellular sites, including the sarcolemma, sarcoplasmic reticulum, mito-chondria, and troponin complex (33). Our previous studies haveindicated that the Ca++ transient recorded with aequorin incontrol human myocardium consists ofasinglecomponent

thatreflects therelease anduptake of Ca++ by the sarcoplasmic reticulum (34). Incontrast, theCa++ transient recorded in muscle from patients with hypertrophic cardiomyopathy, or

dilatedcardiomyopathy with significantcompensatory hyper-trophy, consists oftwodistinctcomponents,labeled

L,

andL2 inFig. 2.

_LI

appearstoreflectCa++ released from the sarcoplas-micreticulum andL2acombinationofCa++ release from the

sarcoplasmic reticulumandCa++ entering the cellvia voltage-dependent sarcolemmal Ca++ channels (21).

Tofurtherinvestigate the role of enhanced transsarcolem-malinflux of

Ca",

westudied theelectrophysiologic properties

ofcontrol andhypertrophicmuscles.Representativeaction

po-tentialsfrom controlandmyopathic muscles, includingoneof 9mM CAFFFINUF

thehypertrophic preparationsinthis report,areshown inFig.2

of reference 21. The prolonged repolarizationand plateau phaseofthe action potentials recorded from hypertrophic

mus-clessuggestthat theremaybe enhancedCa++influx across the

sarcolemma. Possible mechanisms mayinvolve an increase in

the number of calcium channelsasreportedfor the hypertro-phic cardiomyopathic Syrian hamster (35)orenhanced

con-ductancethrough existing channels.An increase in the number

ofdihydropyridine bindingsites has been demonstrated in

atrial tissuefrom patientswithhypertrophic cardiomyopathy, suggesting enhanced transsarcolemmalcalcium influxthrough voltage-dependent calcium channels (36). This concept has

re-cently been challenged (37), however.

Caffeineisadrugthat hasavarietyof cellularactions, in-cluding blockade of uptake, and subsequently release, of Ca++ from thesarcoplasmic reticulum,inhibition of phosphodiester-asewithaconsequent increase in

[cAMP]i,

and sensitization of the myofilaments to Ca++ (38-40). As shown in Fig. 8,

caf-feine'seffects on thesarcoplasmicreticulumappear predomi-nant,since the drug producedadose-relatednegative inotropic effect thatwasassociatedwithadecrease in theamplitudeof

_LI

andalesspronounceddecreasein theamplitudeofL2.These effectsaresimilartothosereported previouslyforryanodine, anagentthatselectivelyblocks release ofCa++fromthe sarco-plasmic reticulum (21). In contrast, caffeine and the related methylxanthine theophylline, produceapositive inotropic

ef-fectinnormal humanmyocardiumunder similar

experimen-talconditions(34). Similar negative inotropic actions of

caf-feine have been described inmuscle from patientswith end-stagedilatedcardiomyopathyand compensatoryhypertrophy

and may beareflection of deficientproduction ofcyclic

nu-cleotidesinhypertrophic cardiomyopathic muscle,which pre-vents the phosphodiesterase-inhibiting properties of caffeine

from becoming manifest (22). Since cAMP playsanimportant role in modulating the entry of Ca++ via voltage-dependent

sarcolemmalcalcium channels,the

uptake

of

Ca++ by

the

sar-coplasmic

reticulumand

Ca++ sensitivity

of the

troponin

com-plex(25), it is reasonabletospeculatethat abnormal

Ca++

han-dlingandcontractile

dysfunction

in

hypertrophic

cardiomyop-athymay be due in parttodeficientlevels of

cyclic

nucleotides. The corrective effects ofisoproterenol and forskolinsupport thishypothesis (Figs.4and 5).

We have reportedabnormal [Ca++]handling, mechanical,

and electricalactivity in musclefrom patients with end-stage dilated cardiomyopathy undergoing cardiac transplantation

(21).Ofinterest,the musclesinthatseriesall showedhistologic evidence ofsignificant hypertrophy compared with the

con-trols.Similarly, in this studywereportabnormalitiesin

excita-tion-contractioncouplingin muscles frompatients with hyper-trophic cardiomyopathy withandwithoutend-stageheart

fail-ure. Inthis study,meanfiber diametersand nuclearareas were

significantly

greater in muscles from

patients

with hypertro-phic cardiomyopathycompared with controls. Thesefindings raisetheimportant possibilitythatthecontractiledysfunction

weobserved may be areflectionof thehypertrophiedstateper

se(21).

Inconclusion,thesestudiesindicatethatatphysiologic

tem-peratures and heart rates,a stateof relative Ca++overload de-velops in ventricular muscle frompatients withhypertrophic cardiomyopathy. This calcium overload results in increased

end-diastolic

[Ca+J]i,

incomplete

relaxation and fusion of

twitches witharesultantincreaseinend-diastolic

tension,

and

adecrease in active tension development.This is a clear

exam-pleof a condition inwhich diastolicdysfunction canimpair active systolic forcegenerationand mayexplain why patients withhypertrophic cardiomyopathy poorlytolerate increases in heart rate.

Acknowledgments

The authorsgratefully acknowledgethecontributionstothis study of

Drs. LawrenceCohn,John J. Collins, andJamesMarshofBrigham

and Women'sHospital,Dr. JamesPluth ofthe Mayo Clinic and Foun-dation, the technical assistance ofMr.NormanLee, and themedical graphics of Mr. Al Brass.

This workwassupportedinpartby United States Public Health Service Grants HL-12186to J. R. Blinks of the Mayo Clinic, HL-07111, HL-31117, andaResearch CareerDevelopment Award (HL-01611)to Dr. Morgan, HL-139091, HL-07374,and HL-36797 to Dr.

GwathmeywhoisanEstablishedInvestigator supported in part by the

American Heart Association, aClinician-Scientist Awardfrom the

American HeartAssociation to Dr.Warren, a Fellowship from the

AmericanHeartAssociationto Dr.Phillips,andHL-07374 and a

Fel-lowship fromtheAmericanHeartAssociation to Dr.Briggs.

Publica-tioncosts are supportedinpart by theChristineAnne Bohannon

En-dowmentFund.

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