YuuL A. Farber, D.D.S., Ph.D.” Lowell A. Glasgow, M.D.

Rochester, N. Y.

ng the diseases which may affect the eart, viral myocarditis remains one

of the most poorly understood.l XIyocar- ditis often occurs as a secondary manifes- tation of virus infection and may assume a subclinical course. However, damage to cardiac tissue can result from these mild infections. Modifying factors such as hor- monal or physiologic stress play an im- portant role in determining the extent of myocardial involvement during experimen- tal virus infection.2 During pregnancy, there are striking changes in cardiovascular dynamics, In humans, the heart rate as well as the cardiac output increases. There is also an increase in blood volume due to a 20 per cent increase in plasma volume.3z4 Such stresses placed on cardiac function may serve to predispose this organ to damage during a virus infection.

Recently, we have demonstrated that during encephalomyocarditis (ElMC) virus infection in pregnant CD-I mice there was an increased multiplication of virus in the heart5 which, however, was not associated with any histologic evidence of myocarditis. This report describes EMC virus infection in pregnant mice of another strain, the MLM-1 mouse, in which EMC virus infec-

tion is characterized by ?XflallWd virus replication in cardiac tissue which results in severe myocarditis.

Mice of the BiLM-1 strain (derived from an ICR strain), 12 to 14 days pregnant, were obtained from the Western New York Animal Resources, Ontario, N. Y. Female mice of the same age and strain were used as controls. These mice were housed in air- conditioned quarters with constant lighting conditions giving 12 hours of light and 12 hours of darkness.

Encephalomyocarditis virus was origi- nally derived from a large plaque mutant obtained from Dr. K. K. Takemoto at the National Institutes of Health. It had under- gone several mouse brain passages followed by two passages in L cells. The stock preparation of EMC virus utilized for these experiments contained 107 plaque-forming units (PFU) per milliliter when assayed in 3 cells and was composed of a mixed popu- lation of large and small plaque variants with small plaque predominating in ratio of 1OO:l. Assay of tissues for virus was the same as described previously.5

For electrocardiograms, mice were anes-

From the Departments of Dental Research, Microbiology, and Pediatrics. University of Rochester School of Medicine and Dentistry. Rochester, N. I’.

Supported by Training Grant 2Tl DE00687 from the lYational Institute of Dental Researeb and Grant AI06388 from the National Institute of Allergic and Infectious Diseases, United States Public Health Service.

Received for publication Sept. 15, 1969. *Dr. Farber’s present address and address for reprints: Department of Microbiology. Temple University, School oi

Dentistry. Philadelphia, Pa. 19140.

96 American Heart Journal July, $970 Vol. 80, No. 1, pp. 96-10.2

Viral myocurdilis during prvegnuncy 9:

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Fig. 1’. Mortality following EMC infection in pregnant and nonpregnant MLM-1 mice.

Gg. 2. Multiplication of EMC in brains of pregnant and nonpregnant MLM-1 mice.

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HOURS AFTER CHALLENGE

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Fig. 3. Multiplication of EMC in hearts of pregnant aud nonpregnant Mi,M-1 mice.

thetized by intraperitoneal administration of pentobarbital, The dose necessary to produce anesthetic threshold was 0.045 nag. per gram of body weight after the method of Pilgrim and DeOme.6 Electrocardio- grams were made using a Grass recorder, Model SC (Grass Instrument Co., Quincy, Mass.). Bipolar safety-pin electrodes were utilized, one pinned to the skin directly over the sternum, the other to the back, to the left of midline. The tracings were recorded at a paper speed of 100 mm. per second, with the recorder calibrated to 0.5 mv. per centimeter.

Results

Groups of 12 to 13 twelve-day pregnant and control female mice were inoculated intraperitoneally with 0.2 ml. of stock EMC virus which contained lo4 PFU. The result- ing mortality rate is illustrated in Fig. 1. As shown in previous studies,5 pregnant mice exhibited enhanced susceptibility to this virus. Eighty-four per cent of the preg- nant mice succumbed by 14 days as opposed to only 8 per cent of the nonpregnant con- trols. During the course of infection, the fetuses also became infected and no live births occurred even in the survivors. EMC virus infection in mice is characterized by

encephalitis and less frequently by myo- carditis and pancreatitis. Virus assays of the brain and heart were performed at various times following infection to deter- mine whether differences existed between pregnant and nonpregnant mice. The or- gans from two infected animals of each group were pooled and tested at the time periods specified in the figures. Virus multi- plication in the brain is shown in Fig. 2. For the three periods measured, 48, 96, and 120 hours, there was little difference be- tween pregnant and nonpregnant mice. Virus replication in the heart (Fig. 33, how- ever, was significantly higher in pregnant mice. Histologic sections of the hearts of nonpregnant mice revealed no significant pathology (Fig. 4) while infection in preg- nant animals resulted in a diffuse inflam- matory infiltrate with extensive necrosis of myocardial fibers (Fig. 5)- Another indica- tion of the degree of myocardial involve- ment in pregnant mice was liver congestion. Gross evidence of congestion (“nutmeg liver”) was found as early as 12 hours after infection. Such changes in liver appearance were not seen in infected controls. Histo- logically there was stasis of blood in central veins and sinusoids of pregnant mice (Fig. 6). Microscopic examination of the pan-

Fig. 4. Myocardium from nonpregnant mouse 6 days after EMC infection. (Hematoxylin and eosin; X220.

Fig. 5. Myocardium from pregnant mouse 6 days after EMC infection. (Hematoxylin and eosin; X220.)

Fig. 6. Liver of pregnant mouse 4 days after EMC infection. (Hematoxylin and eosin; X220.)

cress revealed no inflammation in either group.

Electrocardiograms were performed on infected mice to determine if there was any derangement in cardiac function which might account for the higher mortality rate seen in pregnant animals. An attempt was also made to correlate the differential path- ologic changes in the hearts of those mice with electrophysiologic alterations. Using the procedure as outlined, it was possible to obtain some indication of heart function by comparing electrocardiograms of normal animals with those of EMC-infected mice. Despite the fact that electrode placement was different for each animal, wave forms were fairly constant in uninfected mice and there was some degree of reproducibility. The three wave forms, P, the QRS corn- plex, and the T, can be distinguished. The T wave is usually superimposed on the end of the &KS complex so that it represents a gentle slope rather than a distinct peak. This is due to the rapid heart rate in mice Lthere repolarization occurs too rapidly to be detected with this method.

Readings were taken on the same animals

at 1 to 3 day intervals. Individual varia- tions due to electrode placement and depth of anesthesia can be seen with the control animals (Fig. 7). No notching or gross alterations of wave patterns were seen up co 144 hours after infection In the preg- nant animals, a change in wave pattern was seen in Animal No. 1 by 72 hours after inoculation. In the second animal repre- sented, a more severe change in wave form was evident by 120 hours. The pattern becomes quite distorted by 144 hours with bizarre and widespread QRS complexes reflecting aberrant intraventricular con- duction. Some semblance of normal pattern returned by 168 hours, at which time the animal was moribund. Both pregnant ani- mals had myocarditis on histologic exami- nation after death, Animal No. 2 showing the more severe changes. The control ani- mals were put to death and microscopic examination of the myorardium revealed ilo significant. patltology.

Evidence of abnormal electrocardio- gra~ris, liver ~~origestiori, and elevated virus levels associated with cardiac inflammation suggest that viral myocarditis is the major

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NON- PREGNANT

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Viral rnyocarditis during pvcgnuncy 101

PREONAM

Fig. 7. Electrocardiograms of pregnant and nonpregnant MLM-1 mice infected with EMC virus.

factor in the enhanced susceptibility of MLM-1 mice to EMC virus during preg- nanc y.

Discussion

The extreme susceptibility of pregnant mice to EMC virus infection has been attributed to the hormonal changes which occur during parturition, particularly the increased secretion of estrogens and corti- costerone.5 While the two mouse strains, CD-31 and MLM-1, are equally susceptible to EMC during pregnancy, the pathogene- sis of infection is quite different for each. CD-;1 mice had increased virus multiplica- tion in the heart without the production of overt myocarditis or any significant altera- tion in the electrocardiograms.7 Another difference between these two mouse strains was .the degree of pancreatic involvement. CD-J. pregnant mice evidenced a severe pancreatitis while none was seen in preg- nant MLM-1.

It has been inferred from several experi-

mental studies that increased demand placed on cardiac function may enhance viral myocarditis. Lerner2zg demonstrated that myocardial hypertrophy induced in mice by enforced exercise was associated with increased Coxsackie virus isolation from the heart. Pearce9 investigated myo- carditis produced by Virus III in rabbits and demonstrated that stresses placed on the heart by treatment with drugs such as epinephrine, digitalis, and Pitressin in- creased the incidence and severity of infec- tion. He attributed the enhancing quality of these drugs to their ability to reduce the supply of oxygen to the heart. Procedures that damaged the heart but did not de- crease oxygen availability did not increase the susceptibility of the heart to this virus infection. Decreased oxygen availability could, he stated, also be brought about by excessivel>s strong myocardial contractions which caused impingement of the coronary arteries.

In humans, heart disease may accompany

pregnancy, particularly in the last trimester and in the early puerperium. This associa- tion between heart failure and pregnancy is not entirely fortuitous and represents an important obstetrical problem.rOJr Mead- owsr” has estimated the incidence of post- partum heart disease at between one in every 1,300 to 4,000 births. Benchimol and co-workersI have suggested that the hemo- dynamic changes which occur during preg- nancy and persist until shortly after deliv- ery may be important factors in post- partum heart disease. In most cases, the etiology has not been well defined although virus infection has been implicated.rO Thus, during pregnancy, the heart may be jeop- ardized not only by hormonal modification of host resistance but also by the stress resulting from the increased work load. The present report provides a model sys- temic virus infection which should permit further investigation of the modification of host resistance to viral infection and the enhancement in susceptibility of the myo- cardium during pregnancy.

Summary

The enhanced susceptibility of pregnant MLM-1 mice to EMC was studied. Virus multiplication in the brain was comparable in both pregnant and nonpregnant controls EMC virus multiplied to higher levels in the hearts of pregnant mice. This was accom- panied by myocarditis which was demon- strated histologically. Electrocardiograms of infected pregnant mice evidenced de- rangement in cardiac function. These find-

ings suggest taac involvement of the myo- cardium during ERIC infection might account for the higher mortality rate during pregnancy.

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REFERENCES

Burch, G. E., and DePasquale, N. P.: Viral myocarditis, Ciba Symposium on Cardiomyop- athies, 1965, p. 376. Lerner, A. M.: An experimental approach to virus myocarditis, Progr. Med. Viral. 7:97, 1965. Metcalfe, J., and Ueland, D.: in Hurst, J. W., and Logue, R. B., editors: The heart, arteries, and veins, New York, 1966, McGraw-Hill Book Company, p. 1094. Wade, 0. L., and Bishop, J. M.: Cardiac output and regional bloodflow, Philadelphia, 1960, F. A. Davis Company, p. 48. Farber, P. A., and Glasgow, L. A.: Factors mod- ifying host resistance to virus infection. II. Enhanced susceptibility of mice to encepha- lomyocarditis virus infection during pregnancy, &%mer. 1. Path. 53:463. 1968. Pilgrim”, H. I., and DeOme, K. B.: Intraperi- toneal pentobarbital anesthesia in mice, Exp. Med. Surg. 13:401, 1955. Farber, P. A.: Ph.D. Thesis, University of Rochester, 1967. Tilles, J. G., Eison, II., Lerner, A. M., and Finland, M.: Effects of exercise on Coxsackie *As myocarditis in adult mice, Proc. Sot. Exp. Biol. Med. II7:777, 1964. Pearce, J. M.: Heart disease and filterable viruses, Circulation 21:448, 1960. Meadows, W. R.: Idiopathic myocardial failure in the last trimester of pregnancy and the puerperium, Circulation 15:903, 1957. Meadows, W. R.: Postpartum heart disease, Amer. J. Cardiol. 6:78&, 1960. Benchimol, A. B., Carneiro, R. B., and Schles- inger, P.: Postpartum heart disease, Brit. Heart Jo 21:89, 1959.