Progression of Myocardial Abnormalities inExperimental Alcoholism
GEORGE THOMAS, MD'BUNYAD HAIDER, MD, FACCHENRY A. OLDEWURTELMICHAEL M. LYONS, MDCHIEN K . YEH, PhDTIMOTHY J. REGAN, MD, FACC
Newark, New Jersey
From the Department of Medicine, College ofMedicine and Dentistry of New Jersey MedicalSchool, Newark, New Jersey . This investigationwas partially supported by Research GrantAA00242 from the National Institute on AlcoholAbuse and Alcoholism, and Postgraduate TrainingGrantFL 05510 from the National Heart, Lung, andBlood Institute, Bethesda, Maryland . Manuscriptreceived October 17, 1979 ; revised manuscriptreceived December 28, 1979, accepted March 5,1980 .
' Presented at the American College of Car-diology Young Investigators' Awards Competition,1978 .
Address for reprints : Timothy J. Regan, MD,College of Medicine and Dentistry of New Jersey,New Jersey Medical School, 100 Bergen Street,Newark, New Jersey 07103 .
To determine those characteristics of left ventricular functional andmetabolic alterations In chronic ethanolism that may be tine-dependent,up to 36 percent of total daily calories as ethanol was led to dogs for anaverage of 16 months (study group 1) or 52 months (study group 2) . Theshort- and long-term study groups were fed the same diet with vitaminsupplements and were compared with simultaneously studied controlanimals. Left ventricular function was assessed In the intact anesthetizeddogs using the thermodllution method for end-diastolic volume and strokevolume determinations. During preload Increments with saline solution,a significantly greater increase in end-diastolic pressure was observedIn both groups receiving ethanol as compared with the control animals ;this Increase was associated with reduced end-diastolic and stroke vol-ume. However, the responses were similar in the short- and long-termstudy groups. Increased left ventricular collagen was the apparent basisfor the compliance abnormality, but neither variable differed in the groupsreceiving ethanol .
In contrast, the first derivative of ventricular pressure (dP/dt) nor-malized for preload and afterload, an Index of left ventricular contractility,and the velocity of the contractile element (Vce) were significantly re-duced only in the long-term study group while tissue calcium was normal .When chromium-51-EDTA was used as an extracellular marker, accu-mulation of water and sodium In myocardial cells was observed only Inthe long-term study group, without a reduction of cell potassium . In viewof the dilatation of sarcoplasmic reticulum observed on electron mi-croscopy, It Is postulated that distortion of the tubular membranes maylimit the rate of calcium availability to contractile protein and thus diminishcontractile function In chronic alcoholism .
In human alcoholics various degrees of cardiac functional abnormalityhave been identified before the appearance of heart failure . 1-3 Alteredventricular function during increments of afterload as well as a declinein an index of contractility have been observed in patients with normalheart size . I However, ethanol has not been established as the sole etio-logic factor. In a previous attempt to eliminate some of the variables thatexist in human beings, a group of young adult male dogs were maintainedin a relatively normal nutritional state while receiving up to 36 percentof calories as ethanol. After an average of 18 months these dogs dem-onstrated enhanced diastolic stiffness of the left ventricle without alteredindexes of contractility, 4 in agreement with a prior report . 5
To determine whether altered left ventricular compliance and indexesof contractility are time-dependent, we undertook a more prolongedexperiment to compare animals fed ethanol for either 18 or 52 monthswhile they were maintained in an adequate state of nutrition . At ter-mination of the experiment the left ventricular end-diastolic pressure-volume relation was assessed and related to accumulation of collagen
August 1980 The American Journal of CARDIOLOGY Volume 46
233
ETHANOL-INDUCED CARDIAC ABNORMALITIES-THOMAS ET ALt
in the left ventricle . Because impaired regulation ofcation transport after long-term use of ethanol wasobserved in other tissue,6 intracellular cation and watercomposition was assessed and related to indexes ofcontractility .
Methods
Long-Term Animal Model
Healthy male mongrel dogs, aged 18 to 24 months, wereselected for this study. One half of each group was observedfor an average of 18 months (group 1) and the other half for52 months (group 2) . All were vaccinated against distemper,hepatitis and leptospirosis, and were housed in individualcages . They were free of intestinal parasites and clinical evi-dence of disease for a period of 6 to 8 weeks of observationbefore admission to the study . Blood samples were negativefor heart worms and both hematocrit and serum protein levelswere initially normal.
Diet: Both the control and experimental animals were fedapproximately 28 calories/0 .5 kg body weight, consisting ofa mixture of Blue Ribbon Beef Dog Food (Deep Rung PackingCo., Dublin, Pennsylvania) and Kibble (Old Mother HubbardDog Food Co., Inc., Gloucester, Massachusetts) in a 1 :3 ratio .The control animals received a diet sufficient to maintainbody weight in the adult animal, - consisting of 26 percent ofcalories as protein, 12 percent as fat and 62 percent as carbo-hydrate . The corresponding values in the alcoholic groupswere 16.6 percent of calories as protein, 7 .7 percent as lipid and39.7 percent as carbohydrate, which met the minimal standardfor maintaining a normal nutritional state.. All animals re-ceived Folbesyn (Lederle Laboratories, Pearl River, NewYork) a vitamin supplement, intramuscularly, twice weekly .The total vitamin dosage/week included thiamine hydro-chloride, 5 mg ; riboflavin, 5 mg ; niacin, 35 mg; pyridoxinehydrochloride, 2.5 mg; ascorbic acid, 150 mg ; B 12, 2µg; sodiumpantothenate, 5 mg and folic acid, 1 .5 mg .
Ethanol administration: Because of the difficulties offeeding by gastric tube for a prolonged period, the ethanol(USP, acquired from U .S. Industrial Chemicals, Division ofNational Distillers and Chemical Corp ., New York, New York)was administered in drinking water up to a final concentrationof 40 percent . The actual intake per animal was estimated atbiweekly intervals by measuring the residual ethanol solutionof the previous day, and correcting for evaporation from aseparate container. After 4 weeks of progressively increasingdoses, those animals that spontaneously consumed substantialamounts of the ethanol provided were admitted to the ex-perimental group. This process of self-selection was necessaryto examine the effects of intoxicating amounts of long-termethanol on the myocardium . It was presumed that these ef-fects did not differ from the myocardial responses in the re-jected animals had they ingested the same quantities . Theanimals fed ethanol for a long period were given 36 percentof calories as ethyl alcohol for 6 days/week . On the 7th day,the caloric composition was the same as that in the controlgroup. The intake of each animal varied between two thirdsand all of the administered dose, and was often associated withsome degree of locomotive incoordination and occasional shortperiods of sleep . We have previously reported 4 that this eth-anol regimen effected maximal blood concentrations of 213t 7 mg/100 ml .Blood determinations: In both the short- and long-term
groups of control and experimental animals, two venous bloodsamples were taken 4 weeks apart at the onset of the study andat intervals of 2 months thereafter, for determination of
234 August 1980 The American Journal of CARDIOLOGY Volume 46
plasma lipids, hematocrit and plasma constituents includingtotal protein,' albumin ,9 glucose 10 and the lipid classes ." 14
Blood was placed in chilled tubes containing EDTA and afterseparation in a refrigerated centrifuge, stored at -20 0 beforeanalysis .Terminal study: The terminal study was performed 18 and
52 months, respectively, after the onset of ethanol feeding inthe short- and long-term groups. Although studies in the on-anesthetized state would have been preferable, long term in-strumentation of these animals for left ventricular functionalstudies was not feasible . To minimize the acute influence ofthe last administered dose, the experimental groups did notreceive ethanol for the previous 32 hours . This arbitrary timewas chosen as an interval when acetaldehyde and ethanol fromthe last dose would be dissipated . Each dog was carefully ob-served for signs of tremor, convulsions or other evidence ofcentral nervous system irritability. None of these were present,a finding in accord with a prior study of nonhuman pri-mates . 15
Hemodynamic Studies
Experimental preparation: Each animal was provided,with its regular diet on the afternoon before it was killed, andfeeding was completed by 6 p .m. After induction of anesthesiawith 3 mg/kg of morphine and 12 mg/kg of Nembutal®, thetrachea was intubated and the animals were ventilated on aHarvard respiratory pump to maintain arterial oxygen satu-ration and pH in the normal range . Ejection fraction levelswere unusually reduced after this anesthesia's to approxi-mately 50 percent of those levels found in unanesthetizeddogs."- However, a relatively steady hemodynamic state ap-peared to be present approximately 3 hours after inductionof anesthesia when our studies were undertaken, since furtherdecline of cardiac output after pentobarbital was usually notobserved," Because ventricular function was assessed incontrol and experimental animals at approximately the sametime, variations due to anesthesia would presumably affectboth control and experimental groups similarly .
Hemodynamic data were obtained with the chest intact andthe animals maintained in the left lateral position . Catheterswere placed in the pulmonary artery, left ventricle and rootof the descending aorta and maintained patent by infusion orintermittent flushing with small volumes of degassed salinesolution . The Goodale-Lubin SF catheters, 50 cm in length,were connected directly to Statham strain gauge transducers(model P23Gb, Statham Instruments, Inc., Oxnard, Califor-nia). Photographic recordings were made from a multichanneloscilloscope recorder (Electronics for Medicine, White Plains,New York) . The frequency of this recording system was linearfrom 0 to 30 cycles/s . Catheters of 50 cm dimensions have beencompared with the Millar catheter-tip micromanometer andhave given equivalent ventricular systolic and end-diastolicpressures as well as the positive first derivative, in accord witha prior study ."Hemodynamic measurements: The first derivative of the
left ventricular pressure pulse (dP/dt) was computed con-tinuously by a resistance-capacitance differentiating circuitand converted into millimeters of mercury per second . Theamplitude of dP/dt was a linear function of frequency to 70cycles/s . Ventricular diastolic pressure was recorded at suf-ficient sensitivity so that 1 mm Hg equaled 5 mm in the trac-ing. All measurements were made at the end expiration phaseof the respiratory cycles ; an average of six separate determi-nations were taken .
Cardiac output and left ventricular volumes were deter-mined with the thermal indicator-dilution method 2 0 '21 Car-
diac -output values obtained with this technique have beenfound to correlate well with those obtained with the dye-dilution technique and end-diastolic volume measurementsobtained with the indicator method correlated with the valuesobtained with the angiographic method. 22 -24 A Swan-Ganzthermodilution catheter tip was placed in the main pulmonaryartery and 10 ml of normal saline solution at room tempera-ture was injected as a bolus dose through the proximal lumenin the right atrium. The temperature change was detected bythe thermistor in the pulmonary artery, displayed on the re-corder through a Wheatstone bridge and the curve area wascomputed manually for cardiac output calculation . Five mlof cold saline solution was injected as a bolus dose into theinflow site of the ventricle, and the left ventricular washoutcurves obtained just above the aortic valve were used to cal-culate the left ventricular ejection fraction . End-diastolicvolume was calculated from the ratio of stroke volume andejection fraction and expressed per kilogram of body weightbecause a linear relation to body weight has been reported ina wide range of mammals.25 Stroke work index in grams perkilogram was calculated from the product of stroke volumeper kilogram and the mean left ventricular systolic pressureminus end-diastolic pressure X 0.0136 .
Contractility indexes : Contractility was assessed from anindex expressing the end isometric force-velocity relationnormalized for initial fiber length . This contractility indexexhibits a relatively narrow range in normal ventricles, in-creases in a predictable fashion with positive inotropic in-terventions and is depressed in patients with left ventriculardisease. The formulation includes (dP/dt max/MIP)/tar,where dP/dt max is the maximal rate of rise of left ventricularpressure in millimeters of mercury per second, MIP themaximal isovolumetric pressure in millimeters of mercury and27rr the end-diastolic left ventricular circumference in centi-meters. The units of contractility are muscle lengths per sec-ond per centimeter of initial circumferential fiber length .26The left ventricular radius was calculated from end-diastolicvolume on the assumption that the ventricle was a sphere atthe end of the isovolumetric period, and circumferential fiberlength was calculated as tar . In addition, the velocity of thecontractile element (Vce) at peak dP/dt was calculated as theratio of dP/dt max to simultaneous left ventricular pressure,assuming a uniform series elastic constant27
Left ventricular function was further assessed duringvolume expansion . A second catheter was introduced into theleft ventricle and saline solution was infused at a rate of 48ml/min. During the infusion of saline solution, end-diastolicpressure was continuously recorded at high sensitivity and thecardiac output and ejection fraction were determined imme-diately after 3 minutes of infusion when the animals were ina steady state as judged from heart rate, aortic pressure andventricular end-diastolic pressure .
Myocardial ischemia during atrial pacing : To evaluatethe potential for myocardial ischemia when oxygen require-ments are enhanced, a bipolar electrode catheter was posi-tioned at the junction of the superior vena cava and rightatrium through the jugular vein to accelerate heart rate anda Goodale-Lubin catheter placed in the coronary sinus . Aftersimultaneous collection of arterial and coronary sinus bloodsamples for estimation of lactate 2s and pyruvate29 in thecontrol state, atrial pacing was initiated with the use of aMedtronic model 5737 pacemaker. A 50 to 60 beat/min in-crement over the control state was achieved with atrial pacing.At 3 minutes of steady state pacing simultaneous arterial andcoronary sinus blood samples were obtained in duplicate forlactate and pyruvate determinations . The left ventricular and
ETHANOL-INDUCED CARDIAC ABNORMALITIES-THOMAS ET AL .
aortic pressures were continuously monitored just before andduring atrial pacing .
Myocardial Composition and Morphology
Cell cation distribution: To determine cell cation distri-bution extracellular space was measured with chromium-51-EDTA, a marker that has been described as a reliablemethod of measuring extracellular apace . 3D We have comparedsucrose and chromium-51-EDTA during pulse injections andfound the calculated extracellular space in myocardium at 19percent of tissue space to be essentially the same with bothindicators. 31 For the purpose of this study, we used a sustained60 minute infusion of the isotope because equilibrium betweenthe vascular and interstitial space is more readily sustainedas judged by the constancy of serial venous plasma samplestaken from the coronary sinus . 32 These were taken at 1 minuteintervals in the last 10 minutes of the infusion .Myocardial samples : At the conclusion, the chest was
rapidly opened and the heart arrested with iced Ringer'slactate. A transmural sample was taken from the left ven-tricular free wall and lipid extracted by the Folch procedurefor determination of triglyceride, cholesterol and phospho-lipid . 12-14 For calcium analyses, a left ventricular transmuralsample was homogenized in 0 .3N hydrochloride using a Pol-ytron PT-10. After standing 24 hours, the homogenate wascentrifuged at 600 X g and the supernatant analyzed for cal-cium by the calcium o-cresolplethalein complexone-com-plexometric reaction33 using an automated analyzer. Eight-hydroxy-quinoline was included in the reaction to eliminateinterference from magnesium . 34 Septal and free walltranamural samples were also taken ; the latter were dividedinto outer, middle and inner thirds . Trichloracetic extractsof these samples were analyzed for hydroproline to calculatecollagen content."
Another transmural sample was rapidly obtained and theepicardium carefully trimmed to remove perivascular adiposetissue. Samples were homogenized in a volume of distilledwater three times the tissue weight and electrolytes were ex-tracted for 48 hours . Calculation of cell and extracellular waterand electrolyte distribution was done according to Manery .'6Potassium and sodium levels were determined on an AutoAnalyzer system with flame attachment . A portion was takenfor analysis of Chromium-51-EDTA in a Nuclear-Chicagoscintillation counter . Assays of plasma cations and isotope incoronary venous blood were also performed. Separate sampleswere taken to determine wet and dry weights by heating in anoven at 100°C for 18 hours .
Histology: Sections were taken from the left ventricularmyocardium for histochemical examination . These includedtrichrome staining and Alcian Blue at three acid pH levels
'37 -18
Also, specimens for electron microscopy were fixed in coldglutaraldehyde buffered with phosphate . The tissue was thenwashed, postfixed in osmium, exposed to lead and uranyl ac-etate and imbedded in epon .
Results
The eight chronic alcoholic animals were divided intotwo groups on the basis of the duration of alcohol con-sumption . There were four animals in each control andexperimental group . A small weight gain occurred at theconclusion of the observation period that was similarin both alcoholic and normal control animals . Hemat-ocrit, serum albumin and fasting blood glucose in theunanesthetized state were initially within normal limitsin the alcoholic and control groups and were essentially
August 1980 The American Journal of CARDIOLOGY Volume 46
235
N of m a 8
C = preinfusion control;
I = during infusion
; LV
EDP
= le
ft v
entr
icul
ar e
nd-d
iast
olic
pre
ssur
e; LVEDV = left ventricular end-diastolic volume;
n = number ;
N = normal group;
NS = not significant
; p
= pr
obab
ilit
y; SEM = standard error of the mean; 2xr = circumferential fiber length
(cm)
.
G M m m O Sm a 3 Cn m a e 9
TABLE
I
Left
Ven
tric
ular
Res
pons
eto
Inf
usio
n of Saline Solution
Dog
(n)
Hear
t Ra
te
Mean
Aort
icPressure
(mm
Hg)
LVEDP
(mm Hg)
LVEDV
(ml/
kg)
Stroke
Volume
(ml/kg)
dP/d
t ma
x(mm Hg/s)
C
21rr
Contrac-
tility
Index
CI
C
IC
IC
IC
IC
IC
I
n sNo
rmal
Gro
up (
n =
8)a 0
Mean
139
144
131
134
5.13
8.25
2.78
4.28
0.79
1.26
2230
2345
17.3
18.2
1.15
1.18
0SEM
86
1.9
2.3
0.82
0.93
0.21
0.20
0.1
30.
1213
0
118
0.53
0.25
0.13
.07
Grou
p 1
(sho
rt-t
erm)
1156
156
115
116
4
73.
84.
41.
081.
1221
60
2252
17.3
17.9
1.17
1.15
c2
150
148
144
159
8
222.
52.
90.
760.
8816
40
1713
15.9
16.8
0.85
0.88
03
130
128
114
123
11
19..
...
...
.15
003
4120
130
124
143
8
213.
63.
71
11.
22014
2120
16.4
16.7
1.13
1.07
0Mean
139
140
124
135
7.7
17.3
3.3
3.66
0.98
1.07
1828
2028
16.6
17.1
1.05
1 .03
mSE
M8.
46.
86.
99.
71.
4
3.5
0.4
0.43
0.11
0.09
155
162
0.41
0.38
0.1
0.08
pC
vs. I
NSNS
NSNS
NSNS
NS<0.0
30
vs. N
NSNS
NSNS
<0.0
5NS
NSNS
NSNS
NSNS
NSNS
NS
Grou
p 2
(lon
g-te
rm)
5120
122
145
152
716
4 .3
5.1
1.36
1.47
1804
1948
18.6
19.2
0.78
0.74
6158
162
128
133
411
5 .8
5.9
1.08
1.06
1520
1760
20.0
20.1
0.64
0.72
7170
170
142
146
1025
2,5
2.8
0.73
0.86
1690
1945
15.7
16.3
0.84
0.86
8122
120
123
124
814
3.0
3.4
0.87
1.02
1554
1680
17.7
18.4
0.81
0.83
Mean
142
143
134
138
7.2
16.5
3.9
4.3
1.01
1.10
1642
1833
18.1
18.5
0.767
0.787
SEM
12.6
135.
36.
31,
33.
00.
740.
720.
140.1
365
670.
90.
80.
040.
03p
C vs
. 1NS
NS<0.0
3NS
NSNS
NSNS
2 vs
. 1
NSNS
NSNS
NS
0.04
NS_
NSNS
NS<0
.01
<0.0
2NS
<0.0
3<0
.01
1 vs
. 2
NSNS
NSNS
NS
NSNS
NSNS
NSNS
NSNS
NS<0
.04
<0.0
3
unchanged at termination. Because animals in theshort- and long-term control groups did not exhibitdifferences in left ventricular function or composition,they were pooled for comparison with the alcoholicgroups .
Hemodynamic StudiesHemodynamic records were measured without
knowledge of the type of animal . Values for heart rateand mean aortic pressure at rest in the control animalswere not different from those of the alcoholic groups(Table I) . Left ventricular end-diastolic pressure andend-diastolic volume at rest were also similar in thethree groups. Although the control group showed asignificant increase of stroke volume after infusion ofsaline solution neither alcoholic group had a significantstroke output increment . Ejection fraction responseswere similar in the alcoholic groups and not significantlydifferent from those of control animals . Saline infusionwas associated with a significantly higher rise of end-diastolic pressure in both short- and long-term alcoholicgroups than in the control groups, without a significantdifference in the end-diastolic volume responses .
To assess the dependence of the filling pressure re-sponse on the initial end-diastolic volume in the twogroups of alcoholic animals, the four with larger vol-umes were compared with the three with smaller vol-umes. The former, at a mean end-diastolic volume of4.37 ± 0.5 ml/kg exhibited an end-diastolic pressureincrease from 5 .8 ± 0.0 mm Hg to 13 .8 ± 3.0. Those an-imals with the lower initial end-diastolic volume at 2 .66± 0.16 ml/kg responded to infusion of saline solutionwith an end-diastolic pressure increment from 8.7 10.7mm Hg to 20.3 ± 3.2 mm Hg, a response that did notdiffer from the animals with larger initial volumes .These contrast with a 3.1 mm Hg increase in the normalcontrol animals, despite a similar level of end-diastolicvolume during infusion of saline solution . Thus thefilling pressure response in the alcoholic animals ap-pears to be largely independent of volume and as indi-
FIGURE 1. The index of contractility (right panel) wassignificantly lower than the control value only in thelong-term alcoholic group . Similarly, the velocity ofthe contractile element (Vce) (tall panel) was alsoreduced in the long-term group . MIP = maximal Iso-volumic pressure; p = probability.
ETHANOL.NDUCED CARDIAC ABNORMALITIES-THOMAS ET AL .
cated later is compatible with enhanced myocardialstiffness .
The index of contractility in the resting state wassignificantly lower in the long-term (group 2) than in theshort-term (group 1) study group or in the control group(Fig. 1, Table I) . This finding was attributable to a sig-nificant difference in the left ventricular dP/dt max . Inaddition, Vce was also significantly reduced in thelong-term study group. However, ejection fraction atrest was similar in the three groups (control animals 28 .3± 2.0 percent; group 1, 30 .1 f 84 percent; and Group 2,27.0 f 2.9 percent) .
A potential ischemic basis for the abnormalities ofleft ventricular performance was assessed duringcardiac pacing . Arterial blood lactate and pyruvatewere relatively constant before and during atrial pacingand there was no evidence of lactate production duringatrial pacing. The arterial-coronary sinus difference was0.39 f 0.04 ml/liter in the control state and 0 .47 ± 0 .05during pacing. Pyruvate extraction was also unal-tered .
Myocardial CompositionCollagen concentration : Because the observed
decrease in ventricular compliance could be related toalterations of myocardial composition, the latter wasanalyzed for collagen concentrations (Table II) . Therewas significant accumulation of collagen in most layersof myocardium in the alcoholic dogs compared withnormal animals. However, there was no difference be-tween alcoholic groups . By histologic examination withAlcian blue stain this accumulation was observed in theinterstitium between myofibers as well as at perivas-cular loci . Combined left ventricular and septal weightswere 4.5 ± 0.27 g/kg body weight in the control animals,4.1 ± 0.31 in group 1 and 4.3± 0.26 in group 2 . Becausethe weight of the left ventricle and septum did not ex-ceed that of control animals, hypertrophy did not ap-pear to contribute to the functional alterations of theventricle in the alcoholic animals .
LP<0.0
CONTRACTILITY INDEX1.5 -
dP/df MI
1.0 P
21rr(ML /sec/cm)
0.5
0
O CONTROLS
SHORT TERM
0LONG TERM
August 1980 The American Journal of CARDIOLOGY Volume 46
237
VCE30
25
20dP/dt/MIP
15(ML/sec)
f0
5
0
ETHANOL-INDUCED CARDIAC ABNORMALITIES-THOMAS ET AL .
TABLE II
Left Ventricular Collagen'
Normal group (N) 4.64
3.94
4.25
4.60(n = 8)
±0.17
10.17
±0.22
±0.35Groups
5.93
4.91
5.17
5.561 and 2
±0.43
±0.24
±0.34
±0.27(n = 8)
p vs . N
<0.03
<0.02
<0.07
<0.05
238
µg/mg of dry weight .
Cell water and cations : Left ventricular calciumconcentration was 9 .2 ± 1 .8 mEq/kg dry weight in thecontrol group, 9.7 ± 0.6 in the short-term group and 10 .6± 1.5 in the long-term group . Determination of myo-cardial extracellular volume did not indicate any dif-ference from the control value in the alcoholic groups(Fig. 2, Table III) . However, there was a significant in-
O CONTROLSSHORT TERMLONG TERM
CELL CATIONS
No +
40
20
r
0
0mEg/kg CELL WATER
30
10
140
120
100
BO
60
40
20
0mEq/kg CELL WATER
Outer t/3
Mid 1/3
Inner 1/3
Septum
3
2
1
0
WATER
CELLULAR
P < 0 .05
ml/kg x10 2
EXTRACELLULARt
ml/kg x10 2
tESTIMATED BY 51 Cr EOTACONSTANT INFUSION TECHNIQUE
FIGURE 2. Cell sodium (Na+) and water were significantly enhancedin the long-term study group without an associated potassium (K+) loss .There were no significant changes in the short-term group .
August 1980 The American Journal of CARDIOLOGY Volume 46
crease of cell water and sodium ion concentration in thelong-term study group. Despite these alterations cellpotassium concentration was not reduced in the alco-holic animals . As the only time-dependent alterationof myocardial composition observed, the increments incell sodium and water may relate to the time-dependentchange in contractility indexes .
Electron microscopy revealed dilatation of the sar-coplasmic reticulum that appeared to be more promi-nent in the long-term study group (Fig. 3), but thischange was not assessed quantitatively . Both alcoholicgroups exhibited some areas of swollen subsarcolemmalregions and dilatation of the undifferentiated portionof the intercalated disc . There were no evident abnor-malities in the appearance of myofibrils .
Myocardial lipids : Analysis of the myocardial lipidsshowed significant triglyceride accumulation as the onlylipid alteration in the myocardium. Concentrations were5.83 ± 0.75 µcool/g in group 1 and 5 .39 ± 0.59 in group2 and were two-fold above control levels of 2 .6 ± 0.37 .Plasma lipids were not significantly changed in anygroup .
Discussion
Because heart failure has not been produced in ananimal model fed ethanol for a long period, the questionhas been raised as to whether the cardiomyopathy ob-served in alcoholic patients is solely attributable toethanol intake. An ingestion period of many years isapparently required for the development of clinicaldisease in human beings . 39Compliance and collagen: This study has indicated
that a long-term intake of ethanol for approximately 4years is not associated with significant progression ofthe diminished compliance seen in shorter-term ex-periments. Because left ventricular weight in bothgroups of alcoholic animals did not differ from that ofcontrol animals, in contrast to circumstances with col-lagen increments associated with enhanced afterload, 40hypertrophy did not appear to contribute to the func-tional abnormalities. In both groups of alcoholic ani-mals, collagen increments in the myocardium were lo-cated in perivascular loci, penetrating between myofi-bers as judged from Alcian blue histochemical studies .Elasticity of muscle has been attributed predominantlyto extracellular structures 41 and accumulation of ex-tracellular fibrous protein can diminish diastolic com-pliance as seen in the early stages of amyloid heart dis-ease42,43 Although alteration of other myocardial ele-ments such as sarcolemma could contribute to thechange in compliance in alcoholic animals, the findingthat the increase in collagen and alterations in compli-ance were not progressive within these time limitssupports this view .
Collagen deposition in myocardium occurs relativelyearly during ethanol feeding, analogous to the hepaticalterations . 44 .45 Lack of progression in the animals fedethanol for 4 years suggests that after early accumula-tion, synthesis and degradation of this protein are rel-atively equal. Larger increments may depend on a
P<0.0004
7
6
5
4
3
2
ETHANOL-INDUCED CARDIAC ABNORMALITIES-THOMAS ET AL .
longer exposure to ethanol ; genetic or dietary factorsthat have not yet been delineated .
Altered contractility : A prior study 46 of a caninealcoholic model concluded from analysis of glycerinatedheart muscle preparations that the force-velocity rela-tion (Vmax) was significantly reduced . The duration ofethanol feeding was approximately one half of that in
Normal Group (N) (n = 8)
our long-term study grou , which may explain the ab-
118.3 4:4
12.6 4:2
157.7±9 590.8±8sence of changes in maximal developed tension, maxi-mal rate of tension rise and time to peak tension in thatstudy. That the diminished indexes of contractilityobserved after 4 years of ethanol feeding has a parallelin human alcoholism is noteworthy . In subjects withoutsigns or symptoms of cardiac abnormality and normalhemodynamics at rest, contractility was reduced to alevel intermediate between those of patients with heartfailure and normal persons.' Ejection fraction in ourlong-term groups was slightly, but not significantly,reduced and did not reflect the diminished contractilityat rest. This was presumably related to the larger pre-vailing end-diastolic volume compared with that ofcontrol animals which would be expected to raise a re-
47duced ejection fraction just as reduced afterload may
Concentrations in mEq/kg cell water .be associated with a normal ejection fraction even when
TABLE IIICell Callous and Water Distribution In Left Ventricle
Water (ml/kg)Extra-
Dog
Potassium*
Sodium*
cellular
Cell
FIGURE 3. Electron micrograph from the left ventricular myocardlum of a long-term alcoholic animal . Arrow at upper right indicates a portion ofthe dilated sarcoplasmic reticuium system . At the lower lea Is a lipid body, and the arrow In the middle Indicates a mitochondrion that appearsto be within the limits of normal . Glycogen particles are diffusely distributed .
August 1980 The American Journal of CARDIOLOGY Volume 46
Mean 110.7 35.8 164.1 637.1SEM ±5.8 3.8 13 .9 6 .6p vs . N NS <0.006 NS <0.005
ETHANOL-INDUCED CARDIAC ABNORMALITIES-THOMAS ET AL .
contractility is depressed . 48 Alcoholic subjects withoutheart failure who had moderate impairment of thecontractility index did not have a significant reductionof ejection fraction (Ahmed et al ., unpublished obser-vations) .
Pathogenesis of altered contractility: The mech-anism of altered contractility in the long-term alcoholicgroup is not clear. High energy phosphate levels do notappear to be diminished 46 and total calcium concen-trations in the left ventricle were not reduced in ourlong-term group, although an intracellular redistribu-tion has been suggested . 46 Impaired fluxes of calciumin sarcoplasmic reticulum have been observed in achronic alcoholic model and dilatation of this organellewas particularly prominent in the animals that con-sumed ethanol for 4 years . Assuming that the significantincrease of cardiac cell water and sodium in this groupwas reflected in the sarcoplasmic reticulum, resultantdistortion of the membranes in this system may be re-sponsible for altered calcium transport and contractilityafter prolonged feeding of ethanol . A direct effect oflong-term use of ethanol on contractile protein and itsregulatory enzymes is not excluded.
Inhibition of sodium potassium adenosine triphos-phatase (ATPase) has been described in several organsas a result of the long-term feeding of ethanol . 6 How-ever, the gain of sodium by the myocardium was notassociated with potassium loss in contrast to the situa-tion in which myocardial ATPase is inhibited by digi-talis . 49 In addition, accumulation of water contrasts withthe typical response to inhibition of this enzyme . Pre-vious observations have indicated an altered fatty acidincorporation 4 as well as composition of phospholipid50in animals fed ethanol for a long period . Thus, one of themembrane properties limiting permeability of the cellto sodium and water may be affected by the phospho-lipid alteration without affecting the normal transcel-lular gradient of potassium .
Increased levels of cell sodium and water do not ap-pear to represent a nonspecific consequence of dimin-
1 . Regan TJ, Levison GE, Oldewurtel HA, et al . Ventricular functionIn noncardiacs with alcoholic fatty liver: the role of ethanol in theproduction of cardiomyopathy . J Clin Invest 1969;48:397-407 .
2 . Gould L, Sham M, DILleto M. Cardiac hemodynamics in alcoholicpatients with chronic liver disease and presystolic gallop . J GinInvest 1969 ;48:860-8.
3 . Spodlck DH, Plgott VM, Chills R. Precllnical cardiac malfunctionIn chronic alcoholism . Comparison with matched normal controlsand with alcoholic cardiomyopathy . N Engl J MW 1972;287 :677-80 .
4 . Regan TJ, Khan MI, ERYger PO, Holder B, Lyons MM, OldewurtelHA. Myocardial function and lipid metabolism in the chronic al-coholic animal . J Clin Invest 1974 ;54:740-52 .
5 . Pachinger OM, TIBnwnns H, Mao JC . Fauvel JM, Sing RJ. Theeffect of prolonged administration of ethanol on cardiac metabolismand performance in the dog . J Clin Invest 1973 ;52:2690-6 .
6 . Israel Y, Roeemwm E, Heln S, Colombo 0, Canewa-Flecher M .Effects of alcohol on the nerve cell . In : Israel Y, Mardones J, ads .Biological Basis of Alcoholism . New York : Wiley-Interscience,1971:53 .
240
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August 1980 The American Journal of CARDIOLOGY Volume 46
ished contractility . In experimental pressure overloadwith aortic banding for up to 9 months cell sodium wasfound to be diminished without an apparent change incell water when cardiac decompensation was present .'1Before this time cell cations remained normal . More-over, there is no clear evidence of a generalized disorderof sodium and water metabolism after long-term use ofethanol in contrast to acute administration . 52 In rela-tively shorter long-term experiments, renal excretionof sodium as well as plasma levels of the cation were notaltered significantly . A role for enhanced secretion ofaldosterone is possible but no systematic change inplasma levels of this hormone has been observed in al-coholism." Moreover, one would anticipate a changein the extracellular space of myocardium with enhancedactivity of this hormone and such was not present in ourstudy .
Progression to heart failure: These experimentssupport the view that long-term use of ethanol in sub-stantial quantities can produce myocardial alterationsthat may be considered as antecedent to heart failure,analogous to the preclinical cardiomyopathy in humanalcoholics . ,' Whether development of heart failure in theanimal model depends on a longer period of ethanolingestion, cigarette use common to alcoholism in humanbeings, physical activity or subtle nutritional abnor-malities remains to be elucidated . Gender is one factorthat may modify the long-term cardiac response in al-coholism, because young female alcoholics failed todemonstrate a preclinical cardiac abnormality that waspresent in a matched male group 54 Intensification ofinterstitial collagen accumulation and the intracellularcation abnormalities thought to be related to dimin-ished contractility may both he critical for progressionto cardiac decompensation .
AcknowledgmentWe appreciate the expert technical assistance provided by
Bess Jenkins, Carol Eaddy and Remy Torres and the secre-tarial services rendered by Anne Binetti and AudreyBrown.
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