- Email: [email protected]
Influence of heart rate on metabolic and hemodynamic parameters in the Syrian hamster cardiomyopathy
Influence of heart rate on metabolic and hern~y~~~c parameters in the Syrian hamstw cardiomyopathy The effect of varying heart rate in 155- to 170-day-otd isolated, perfused cardiomyopathic Syrian hamster hearts was evaluated by 31P nuclear magnetic resonance spectroscopy, At a low paced heart rate of 170 bpm, cardromyopathic hearts did not differ from normal hearts except for a lower developed teft ventrkuiar pressure. As pacing rate was increased progressively to 270/min, cardromyopathk hearts showed prolongation of contraction, which led to a pronounced rise in diastolic pressure as the interstimulus interval shortened. This was accompanied by a marked decrease in energy-rich phosphorus compounds. By contrast, increasing heart rate in normal hearts did not change left ventrkutar pressure and caused only a mild reduction in energy-rich phosphorus compounds. intraceituiar pH of cardtomyopathtc animak paced at 270 bpm was signlfkaintly lower than in normal animals. Thus, indices reflecting mitochondriai function of 155- to 170-day-old cardiomyopathic hamsters appear adequate at low heart rate. increasing the heart rate unmasks latent mitochondrial dysfunction. (Am HEART J 1987;114:362.)
Walter Markiewicz,* Shao Wu,* Richard Sievers,* William W. Parmley,* Charles B. Higgins,* Thomas L. James,* Gaetan Jasmin,** and Joan Wikman-Coffelt.* Sun Francisco, Calif., and Mo~treul, Quebec, Cu~adu
The hereditary cardiomyopathic strain of Syrian hamsters, designated UM-X7.1, a derivative of the BIO 14.6 strain, has been used as a model of congestive heart failure.‘-* Four general phases in the cardiomyopathy have been defined: (1) a necrotic stage (30 to 100 days); (2) a fibrotic and healing stage (100 to 160 days); (3) a stage with hypertrophy and dilation, as well as an increase in scar calcification (160 to 200 days); and (4) a stage of con$inuing dilation and terminal heart failure (200 to 300 days). Heart failure is identified in the cardiomyopathic hamster by the presence of abdominal fluid and cirrhosis of the liver.3 Over 50% of the hamsters die by 250 days of age.3 Like any other heart failure models, the later stages demonstrate an imbalance of free calcium,3~8 a decrease in cyclic adenosine monophosphate (CAMP)? lo low actin-activated myosin ATPase activity,” intracellular acidity,7 a decrease in cardiac performance? defective mitoFrom the ‘Departments of Radiology, Medicine, Pharmaceutical Chemistry and the Cardiovascular Research Institute, University of California, San Francisco; and **University of Montreal. Supported in part by the George D. Smith Foundation, the Susan and Don Schleicher Fund, and by Medical Research Council (Canada). Received for publication Sept. 23, 1986; accepted Jan. 15, 1987. Reprint requests: Joan Wikman-Coffelt, Ph.D., Room M1186, University of California San Francisco, San Francisco CA 94143.
362
chondria,5 and a decrease in velocity of muscle shortening as well as peak force.” Recently,12 we used 3*P nuclear magnetic resonance (NMR) spectroscopy to study the metabolism of high-energy phosphorus compounds and intracellular pH (pHi) in the perfused, isolated heart of cardiomyopathic hamsters during the heart failure stage. 31P NMR spectroscopy has the advantage of allowing sequential, noninvasive correlations between metabolic and hemodynamic status under various experimental conditions.13* l4 Cardiac function in the Langendorff preparation is dependent upon factors such as heart rate, perfusion pressure, and contractility.15 These factors are complexly interrelated. Each can affect the production and utilization of energy-rich phosphorus compounds and therefore the 31PNMR spectroscopy pattern. In this study, we investigated the effects of varying heart rate on metabolic and hemodynamic parameters in the isolated, glucose-perfused cardiomyopathic hamster heart. METHODS isolated
perfused
heart
studies.
Syrian hamsters
of
the UlWX7.1 strain (a subline of BIO 14.6), between 155 and 170 days of age, were employed as experimental animals (n = 6). Age-matched healthy hamsters served as
Volums 114 Number 2
Table
Mitochondriat
function in cardiomyopathic
hamster
363
I. Hemodynamic parameters Heart rate (bpm-I) LV pressure (mm Hg)
170 220 250 270
A 170-270 A systolic pressure tl?O-270) A diastolic pressure (170-270) Coronary flow (ml/gm X min-‘)
170
AV oxygen difference (mm Hg)
170
Oxygen consumption wt X min,-‘)
270 170 270
270
(pmol/gm dry
Normal 207.5 221.7 203.3 195.5
(n = 6) + 47.2 * 48.8 + 50.5
Cardiomyopathic 148.3 115.4 83.4 58.2
+ 56.0
-12.7 k 31.2 -9.0 t 24.6 3.7 + 9.4 24.7 k 7.6 22.5 f 5.8 171.8 t 63.0 246.7 f 71.1 29.5 rf: 9.9 39.3 zk 10.9
-90.2 -25.0 63.3 16.7 12.4 200.5 175.8 27.4 17.4
(n = 6)
t x3.7* i 45.2f 1: 58.2t f 32.5t f 16.4j. z?r38.5 I 41.57 rt 4.6 iz 5.8t rt 26.4* t 27.3* t- 7.6 rt 8.47
Values are given as mean f SD. LV = left ventricle;
AV =
arteriovenous;
*p < 0.05; tp < 0.01. Statistical
significance
A = difference
between values at 170 and 270 bpm-‘. refers to the difference between normal and cardiomyopathic
controls (n = 6). The isolated beating heart was perfused by a modified Langendorff method,6 with a perfusion pressure of 110 mm Hg. The perfusate contained the following (in mmob‘L): 117 NaCl, 4.3 KCI, 1.2 MgC& 0.1 K2HP04, 25 NaHCO,, 2.5 CaCl,, 0.5 Na EDTA, and 10 U/L of insulin in 15 mmol glucose. The medium was mixed with 95 % 0, and 5% CO,. Perfusate temperature was maintained at 35* C by means of counter-current heat exchangers,“j and a thermostat regulated circulating water bath. Pacing leads designed to prevent noise artefact were inserted at the base of the right ventricle and were connected to a Medtronic pulse generator (model 5320, Medtronic Inc., Minneapolis, Minn.) for pacing of the heart. A polyethylene cannula (internal diameter = 1.07 mm, length = 1.3 cm) was inserted through the left atrium and mitral valve into the left ventricle and was then sutured into place. The cannula was connected to a Statham P23Db pressure transducer (Gould Inc. Cardiovascular Products, Oxnard, Calif.) via a 100 cm long polyethylene tube (internal diameter = 1.67 mm) for pressure recording on a four-ch~nel Beckman dynograph (Beckman Instruments Inc., Brea, Calif.). All pressure measurements were performed with the heart positioned in an NMR tube inserted into the magnet bore (described later) so that hem~~amic and metabolic parameters could be measured simultaneously. Thus, precise determination of the zero pressure level was impossible. Therefore, pressure measurements were expressed as developed left ventricular pressure (i.e., the difference between the highest systolic and lowest diastolic pressure). Arterial and venous oxygen samples were taken before and after placing the heart in the magnet. “Arterial” samples were aspirated from the aortic chamber and “venous” samples were drawn from a catheter introduced into the right ventricular outflow tract for oxygen measurements (Model 165/2 gas analyzer, Corning Glass Works, Corning, N.Y.) Coronary flow was measured by collecting the effluent from the right ventricle in a volumetric container.
hamsters
at a given heart
rate.
Myocardial oxygen consumption was calculated as indicated elsewhere.17 31P NMR spectroscopy. 31PNMR spectra of the beating isolated perfused heart were obtained on a 5.6 Tesla vertical 76 mm bore spectrometer. The home-built spectrometer was connected to a 1180 Nicolet computer (Nicolet Instruments, Madison, Wise.), a Nicolet 2938 pulse programmer, and a high-resolution 20 mm broad band probe. 31P spectra were obtained at 97.3 MHz without proton decoupling. Pulse angle was 75 degrees, recycle time was 2.25 seconds, and spectral width was r+_4000Hz. Chemical shifts are referred to the resonance position of phosphocreatine (PCr) at zero parts per million (ppm). Transients were accumulated for 20 minutes for each spectrum. For each spectrum the characteristic peaks of inorganic phosphate (Pi), PCr, and phosphate groups of adenosine triphosphate (ATP) were identified.13*” The area of each peak was integrated and expressed as “mole fraction” by dividing the integrated value for Pi, PCr, and ATP by the sum of the integrated values for all three peaks. The mole fraction does not give an absolute value and is not normalized for tissue weight. The ATP/Pi and Per/Pi ratios are also given. Intracellular pH was estimated from the chemical shift of the pH-dependent peak of Pi relative to the peak of Pcr.ls*‘* Procedure. The hearts were perfused for 20 minutes in the magnet bore before data collection. This allowed the beating heart to reach steady state with the perfusate.” Pacing rate was set at 170 bpm. This heart rate was slightly higher than the spontaneous beating frequency so that ventricular capture was constant and regular. Following accumulation of NMR spectra and recording of hemodynamic parameters, the pacing rate was increased to 220 bpm for 1 minute under constant pressure recording. Heart rate was then increased to 250 bpm for 1 minute and then to 270 bpm for 20 minutes. Pacing threshold was increased as required. Following this interval, spectra were again accumulated and all hemodynamic parameters
364
Markiewicz et al.
American
Table
August 1887 Heart Journal
II. 31PNMR spectroscopy parameters Heart
r&e (bm) Pi (mole
fraction,
%)
170 210
PCr (mole
fraction,
%)
170 270
ATP (mole
fraction,
%)
170 270
P&/Pi (ratio)
170 270
ATP,fPi (ratio)
170 270
PH
170 270
UJ)
~~rrn~~ (n = 6) 22.8 28.8 5.9 39.5 36.2 -3.3 37.7 35.0 -2.7 1.79 1.34 -0.45 1.69 1.25 -0.44 7.15 7.10 -0.05
t + f zk 2 + i r -rf k + _t k f + * k
Car~~ornyopff~~~c (n = 6) 4.2 5.2 4.5 4.9 6.7 3.3 3.0 2.1 2.1 0.45 0.55 0.41 0.31 0.22 0.34 0.05 0.02 0.01
27.3 43.3 16.0 33.4 24.9 -8.5 39.3 33.1 -6.1 1.61 0.68 -0.93 1.82 0.89 -0.93 7.10 7.04 -0.07
f It + f 4 rtr 2 i k rt + 2 t -13z * * *
13.2 13.7* 8.5* 9.8 8.6* 7.9 5.1 7.1 2.6* 0.98 0.41* 0.64 1.06 0.52 0.58 0.06 0.06 0.05
Pi = inorganic phosphate; PCr = phosphocreatine. Mole fractions (in percent) are crtlcutated by dividing the integrated values for Pi, PCr, or ATP resonances by the sum of the integrated values for all three peaks. ‘p < 0.05.
-5
-10
-15
PPM Fig. 1. 31P nuclear magnetic resonance spectra of a healthy hamster. Heart rate is increased from 170 bpm (upper trace) to 270 bpm (lower trace). Integration of the individual resonances showed that Pi mole fraction rose from 20% to 30%, whereas PCr fell from 37% to 33% and -ATP fell from 43% to 37%. were recorded. Wet and dry weights were measured at the end of the experiment for calculation of the dry weight/ wet weight ratio. Statistical analysis. Results are given as mean + SD. Differences in the same animal under different pacing conditions were analyzed by paired t test. Differences between healthy and myopathic animals were analyxed by one-way analysis of variance. RESULTS Comparison between healthy and cardiomyopathic hamsters. Developed left ventricular pressure was
significantly lower in cardiomyopathic hamsters at a rate of 170 bpm (148.3 + 18.7 vs 207.5 _+ 47.2 mm Hg, p < 0.05). No other sign&ant difference between the two groups was noted (Tables I and II).
The rate of rise of left ventricular pressure was low and the duration of contraction was prolonged in cardiomyopathic hamsters when compared to healthy animals. Marked differences between the two groups were noted at a heart rate of 270 bpm. Myopathic hamsters had significantly lower deveioped left ventricular pressure (58.2 + 32.5 vs 195.5 + 56.0 mm Hg, p < O.Ol), markedly lower coronary flow (12.4 -r 5.8 vs 22.5 t 5.8 ml/ gm X min-‘, p < O.Ol), wider arteriovenous difference in oxygen partial pressure (p < 0.05), and lower myocardial oxygen ~ons~ption (17.4 f 8.4 vs 39.3 Z!Z10.9 firno1 O&m wet weight X min-l, p < 0.01) (Table I). Cardiomyopathic hamsters had higher Pi (43.3 zk 13.7 vs 28.8 + 5.2%) p < 0.051, lower PCr (24.9 f 8.6 vs 36.2 +- 6.7%, p < 0.05), lower PCr/Pi (0.68 f 0.41 vs 1.34 + 0.55, p < 0.05), and lower pHi (7.04 + 0.06 vs 7.10 + 0.02, p < 0.05) (Table II). Values for ATP and ATP/Pi were lower in the c~diomyopat~c animals but not si~i~c~tly so. The dry weight/wet weight ratio was slightly lower in cardiomyopathic animals (0.171 + 0.006 vs 0.183 z!z0.008, p < 0.05). Healthy hamsters. As heart rate increased from 170 to 270 bpm in normal hamsters, developed left ventricular pressure fell slightly (207.5 + 47.2 to 195.5 rt 56.0 mm Ha;, NS) (Table I). Systolic pressure fell 9.0 -+ 24.6 mm Hg, whereas diastolic pressure rose 3.7 rt: 9.4 mm Hg. Coronary flow was
Volume 114 Number 2
unchanged. The arteriovenous oxygen difference widened si~ific~tly (p < O-05), indicating increased myocardial extraction of oxygen and a significant rise in myocardial oxygen consumption (29.5 rt 9.9 to 39.3 + 10.9 pmol OJgm dry weight x min-*, p < 0.05). There was a significant rise in Pi (22.8 f 4.2 to 28.8 + 5.2 % , p < 0.05) and a decrease in ATP (37.7 + 3.0 to 35.0 rt 2.1%) p < 0.05), PCr/Pi (1.79 + 0.45 to 1.34 + 0.55, p <0.05), and ATP/Pi (1.69 + 0.31 to 1.25 it 0.22, p < 0.05) (Table II). pHi fell from 7.15 +- 0.05 to 7.10 t 0.02, p < 0.10) in normal hamsters. Example of a 31PNMR spectrum in a normal heart is shown in Fig 1. Cardiomyopathic hamsters. Developed left ventricular pressure fell in the cardiomyopathi~ hamsters from 148.3 + 18.7 mm Hg at a heart rate of 170 bpm to 115.4 + 45.2 mm Hg at 220 bpm (NS), 83.4 + 58.2 mm Hg at 250 bpm (p < 0.05 vs baseline), and 58.2 + 32.5 mm Hg at 270 bpm @ < 0.01 vs baseline) in ~diomyopathic hamster hearts (Table I). Prolonged contraction persisted at high heart rate, leading to a marked reduction in relaxation time. In going from 170 to 270 bpm, diastolic pressure rose 65.0 & 38.7 mm Hg (Table I), while at the same time coronary flow fell from 16.7 f 4.6 to 12.4 +- 5.8 ml/gm x min-’ (p < 0.05). The arteriovenous difference in oxygen fell slightly (though nonsignificantly) so that myocardial oxygen consumption fell significantly from 27.4 t 7.6 to 17.4 + 8.4 pm01 OJgm dry weight X min-‘, p < 0.01. Pi increased markedly (27.3 -+ 13.2 to 43.3 + 13.7%, p < O.Ol), whereas PCr (33.4 4 9.8 to 24.9 f 8.6%, p < 0.05), ATP (39.3 + 5.1 to 33.1 -+ 7-l%, p
31P NMR spectroscopy is a new technique used with increasing frequency in the analysis of heart disease in the animal and in the clinical setting.13p14 With the use of 31P NMR spectroscopy, analyses of phosphorus metabolites and pHi in the heart have been obtained in isolated perfused hear&,18 in openchest animals,‘s and even in the intact human heart.20 Ultimately, it might be possible to detect
~ito~hondria~
function in cardio~yoputhi~
I
I
I
I
5
0
-5
-10
hamster
366
I
-15
PPM Fig. 2. 31Pnuclear magnetic resonance spectra of a cardiomyopathic hamster heart. Heart rate is increased from 170 bpm (upper trace) to 270 bpm (lower trace). Integration of the individual resonances showed that Pi mole fraction rose from 27 % to 58 % whereas PCr fell from 38% to 15% and -ATP fell from 34% to 26%.
noninvasively disturbances in metabolism occurring in specific areas of the human heartzl An impor~nt advantage of the technique is the ability to evaluate heart metabolism noninvasively and sequentially. Thus, the influence of interventions on metabolite levels and pHi can be assessed and correlated with hemodynamic function. The mole fraction of cellular energy-rich phosphorus compounds as well as inorganic phosphate at a given time reflects the equilibrium between supply and demand of energy. Heart rate is an impost determinant of myocardial oxygen consumption, i.e., of the metabolic rate of the heartz2 Furthermore, heart rate can influence other determinants of heart function such as myocardial con~acti~ty and chamber size. Thus ‘it is important to consider the chronotropic status of the heart when evaluating the
366
Markiewicz et al.
American
Pi
PCr
ATP
PCr/Pi
August 1987 Heart Journal
ATPlPi
+120 +lOO +80 w
8
0 HH B CDMH I
+60
U
-20 -40 -60 -80 3. Percent change of phosphorus compounds as heart rate is increased from 170 to 270 bpm. Statistical significance shown refers to the difference between groups of healthy and cardiomyopathic hamsters.
Fig.
metabolic status of normal and abnormal hearth, by 31P NMR spectroscopy. In this study, we varied heart rate in glucoseperfused hamster heart preparations. The high perfusion pressure (110 mm Hg) used in these studies imposes chemical work on the heart and increases coronary flow.% Results obtained in normal hamsters were then compared with findings in 155- to 170-day-old cardiomyopathic Syrian hamsters of the UM-X7.1 strain, a myopathic strain of hamsters that is well characterized.23-25 Our study shows that at a paced heart rate of 170 bpm, ~diomyopathic hamsters developed significantly lower left ventricular pressure but otherwise did not differ significantly from normal hamsters with any other parameter evaluated. Thee-~din~ suggest that mitochondrial function was adequate to maintain normal levels of energy-rich compounds in the presence of reduced developed left ventricular pressure, i.e., reduced metabolic requ~emen~.~ As heart rate was increased to ‘220,250, and 270 BPM, developed left ventricular pressure remained statistically unchanged in healthy animals. Increasing heart rate caused a mild but significant rise in Pi and a drop in PCr/Pi (an index of mitochondrial function)27 and ATP/Pi, indicating that the requirements for energy-rich phosphorus compounds at bigher heart rate increased faster than the ability of the heart to synthesize them. ATP is not repleted during the shortening phase of the cardiac cycle because aden-
osine diphospha~ (ADP) is bound during this time.17 An increase in heart rate thus reduces recovery time, causing a new steady-state in ATP synthesis. This new steady-stats did not cause any significant change in developed left ventricul~ pressure in healthy hamsters. Myocardial oxygen consumption rose, pointing to the increased metabolic rate of the heart at higher pacing rates,22 A similar relationship between work output and the steady-state oapability of oxidative phosphorylation has been noted in the exercising human forearm analyzed by 31P NMR S~CtFOSCOpy.27
The hemodynamic response of cardiomyopathic hamsters to an increase in heart rate was markedly different, The fall in developed left ventricular pressure was dramatic and occurred mostly as a result of a marked rise in diastolic pressure. COFOnary flow and myocardial oxygen consumption both decreased significantly. Thus, the major hemodynamic response to increasing heart rate in the myopathic hamster is a severe rise in diastolic pressure, reflecting primarily the inability of the heart to relax during diitole. Qualitative evaluation of pressure recordings showed a major prolongation of contraction, which led to a rise in diastolic pressure as the interstimulus interval shortened. Roth the level of energy-rich phosphorus compounds and pHi fell markedly and were si~i~c~tly lower than in healthy animals paced at the same rapid heart rate. Despite diminished demand for
Volume 114 Number 2
Mitochondrial
function in cardiomyopathic
hamster
367
nomenon, occurring as a result of an abnormal mitochondrial function, so that the cell is unable to maintain the calcium gradient across the sarcolemmal membrane. The initial defect causing mitochondrial dysfunction has not been identified, but may be due to depressed enzymes in the sarcolemmal membrane.@ The adverse effects of cellular calcium overload are well documented41 and probably play an important role in causing an abnormality of relaxation. When heart rate is low, relaxation of the cardiomyopathic hamster heart still appears to be adequate. Raising the heart rate further increases the amount of cytosolic calcium,37 overwhelming the ability of the sarcoplasmic reticulum to sequestrate calcium and thus to initiate the active relaxation process.41 The low ATP level may also be an important contributing cause for the marked impairment in ventricular relaxation noted at high heart rate in the cardiomyopathic hamster.35s36 Intracellular pH is an important parameter of ly. The reason for the abnormal mechanical function cellular function. At a heart rate of 170 bpm, pHi was lower in cardiomyopathic hamsters, but not seen in cardiomyopathic hamster myocardium has statistically different from values in healthy aninot been elucidated.‘j, lo The changes in contractility mals. At the end of 20 minutes of pacing at a rate of have been well characterized and follow a course similar to changes in mitochondrial function.5s 24,25 270 bpm, pHi was significantly lower in cardiomyopathic than in healthy animals. The increased conReduced mitochondrial function is probably important in causing depressed contractile function.6* lo,l2 centration of intracellular hydrogen ions as heart rate is raised in the glucose-perfused cardiomyoOther factors such as reduced pHi,12 alteration in the pathic hamster heart may be due to the substrate, components of the contractile protein system,31 and glucose, contributing to an inefficient production of abnormality in the intracellular concentration or mitochondrial substrate, namely pyruvate, as transport of calcium may also be involved.2,11s21*32-34 described in an earlier study.12 A decrease in developed pressure, dpldt, and high Our results show the importance of heart rate in energy phosphates also occurs in vivo in the cardiodetermining the hemodynamic and metabolic promyopathic hamster.34 file of normal and cardiomyopathic hearts. ImporThe abnormal relaxation noted in the cardiomyopathic hamster heart at high pacing rate might be tant defects may not be apparent when heart rate is related to a combination of low ATP and high low, but are unmasked when heart rate is increased. Pacing at a fast heart rate may be required when intracellular calcium35-37 and may also be responsible interventions improving myocardial metabolism are for the depressed developed pressure observed in assessed. Conversely, pacing at a lower heart rate situ.34 The mechanism for the markedly increased may be advantageous when the examined intervenintracellular calcium, first noted during the necrotic tion is expected to cause deterioration. phase, is controversial. One hypothesis invokes a primary genetic defect in the sarcolemmal membrane, causing increased calcium influx into the REFERENCES cell8 Increased intracellular calcium initially 1. Angelakos ET, Carballo LC, Daniels JB, King MP, Bajusz E. Adrenergic neurohumors in the heart of hamsters with heredincreases myocardial contractility and therefore the itary myopathy during cardiac hypertrophy and failure. consumption of ATP. Mitochondrial calcium overRecent Adv Stud Card Struct Metab 1972;1:262. load soon occurs, followed by mitochondrial dys2. Jasmin G, Proscher L. Calcium, and myocardial cell injury. function and reduced production of ATP.38 The cell An appraisal in the cardiomyopathic hamster. Can J Physiol Pharmacol 1984;62:891. becomes unable to maintain its calcium homeosta3. Jasmin G, Proscher L. Hereditary polymyopathy and cardiosis, causing further influx of calcium across the myopathy in the Syrian hamster. I. Progression of heart and skeletal muscle lesions in the UM-X7.1 line. Muscle Nerve sarcolemma. Cellular necrosis and cell death eventu1982;5:20. ally occur.38 4. Jasmin G, Proscher L, Cailloux MF. Congestive cardiomyopOther authors2*3s have suggested that the increase athy in the Syrian hamster. Possible role of catecholamines in in intracellular calcium might be a secondary pheits pathogenesis. In: Goodwin JF, Hjalmarson A, Olsen EGJ,
energy, the level of energy-rich phosphorus compounds fell, indicating several mitochondrial dysfunction at a high pacing rate. The latter may partly reflect the reduced glycolysis12 and the depressed fatty acid and acetate oxidation28 noted in cardiomyopathic hamsters. The reduction in coronary flow may have reflected the reduced energy requirements, or this reduction in flow occurred as a result of increased coronary vascular resistance secondary to impaired relaxation.2s Since the extraction of oxygen by the myocardium was not increased, the reduction in coronary flow probably did not cause the reduction in energy supply noted in this study. The possibility that increased diastolic tension produced localized areas of ischemia or was responsible for the focal abnormality in microcirculation without vessel structural abnormality, as noted in cardiomyopathic anima1s,30 cannot be excluded but appears unlike-
August
366
5.
6. 7. 8.
9.
10.
11.
12.
13. 14.
15. 16. 17. 18. 19. 20.
21.
22.
Markiewicz et al.
editors. Congestive cardiomyopathy. Siidertiilje, Sweden: AB Astra, 1981;113. Proschek L, Jasmin G. Hereditary polyopathy and cardiomyopathy in the Syrian hamster. II. Development of heart necrotic changes in relation to defective mitochondrial function. Muscle Nerve 1982;5:26. Sievers R, Parmley WW, James T, Wikman-Coffelt J. Energy levels at systole vs diastole in normal hamsters vs myopathic hamster hearts. Circ Res 1983;53:759. Sievers R, Parmley WW, Wikman-Coffelt J. Energy reserve at systole versus diastole in myopathic hamster hearts (abstr). Circulation 1983;68:267. Wrogeman K, Jacobson B, Blanchaer MC. On the mechanism of calcium-associated defect of oxidative phosphorylation in progressive muscular dystrophy. Arch Biochem Biophys 1973;159:267. Harrow JAC, Singh JN, Jasmin G, Dhalla NS. Studies on adenylate cyclase-cyclic AMP system of the myopathic hamster (UM-X7.1) skeletal and cardiac muscle. Can J Biochem 1975;53:1122. ’ Wikman-Coffelt J, Sievers R, Coffelt RJ, Parmley WW. Biochemical and mechanical correlates at peak systole in myopathic Syrian hamster. In: Jacob R, editor. Cardiac adaptation to hemodynamic overload, training and stress. Dar-mstadt, FRG: Steinkopff Verlag, 1983:197. Rouleau JL. Chuck HS. Hollosi G. Kidd P. Sievers R. Wikman-CofIelt J, Parmley WW. Verapamil preserves myo: cardial contractility in the hereditary cardiomyopathy of the Syrian Hamster. Circ Res 1982;50:405. Wikman-Coffelt J, Sievers R, Parmley WW, Jasmin G. Cardiomyopathic and healthy acidotic hamster hearts. Mitochondrial activity may regulate cardiac performance. Cardiovast Res 1986;20:471. Ingwall JS. Phosphorus nuclear magnetic resonance spectroscopy of cardiac and skeletal muscles. Am J Physiol 1982; 242:H729. James TL. In vivo nuclear magnetic resonance spectroscopy. In: Moss AA, Ring EJ, Higgins CB, editors. NMR, CT and interventional radiology. San Francisco: Radiology Research Educational Fund, 1984;235. Braunwald E, Sonnenblick EH, Ross J Jr. Contraction of the normal heart. In: Braunwald E, editor. Heart disease. Philadelphia: W.B. Saunders, Second edition. 1984;409:446. Wikman-Coffelt J, Coffelt RJ. Flexible tube: Counter-current heat exchanger. Rev Sci Instrum 1985;56:165. Wikman-Coffelt J, Sievers R, Coffelt RJ, Parmley WW. The cardiac cycle: Regulation and energy oscillations. Am J Physiol 1983;245:H354. Garlick PB, Radda GK, Seeley PJ, Chance B. Phosphorus NMR studies on perfused heart. Biochem Biophys Res Commun 1977;74:1256. Grove TH, Ackerman JJH, Radda GK, Bore PJ. Analysis of rat heart in vivo by phosphorus nuclear magnetic resonance. Proc Nat1 Acad Sci USA 1980;77:299. Whitman GJR, Chance 3, Bode H, Maris J, Hasselgrove J, Kelley R, Clark BJ, Harken AH. Diagnosis and therapeutic evaluation of a pediatric case of cardiomyopathy using phosphorus-31 nuclear magnetic resonance spectroscopy. J Am Co11 Cardiol 1985;5:745. Stein PD, Goldstein S, Sabbah HN, Liu Z, Helpern JA, Ewing JR. Lakier JB. Chonn M. LaPenna WF. Welch WF. In vivo evaluation of ‘intraclllular pH and high-energy phosphate metabolites during regional ischemia in cats using 3’P nuclear magnetic resonance. Magn Reson Med 1986;3:262-269. Berglund E, Borst HG, Duff F, Schreiner GL. Effect of heart rate on cardiac work, myocardial oxygen consumption and coronary blood flow in the dog. Acta Physiol Stand 1958; 42185.
American
lS87
Heart Journal
23. Jasmin G, Proschek L. Hereditary polymopathy and cardiomyopathy in the Syrian hamster. I. Progression of heart and skeletal muscle lesions in the UM-X7.1 line. Muscle Nerve 1982;5:20-25. 24. Forman R, Parmley WW, Sonnenblick EH. Myocardial contractility in relation to hypertrophy and failure in myopathic Svrian hamsters. J Mol Cell Cardiol 1972:4:203. 25. Lochner A, Brink AJ, Van der Walt JJ. The significance of biochemical and structural changes in the development of the cardiomyopathy of the Syrian hamster. J Mol Cell Cardiol 1970;1:47. 26. Neely JR, Liebermeister H, Battersby EJ, Morgan HE. Effect of pressure development on oxygen consumption by isolated rat heart. Am J Physiol 1967;212:804. 27. Chance B, Eleff S, Leigh JS, Jr, Sokolow D, Sapega AA. Mitochondrial regulation of phosphocreatine/inorganic phosphate ratios in exercising human muscle: A Rated 31-P NMR study. Proc Nat1 Acad Sci 1981;78:6714. 28. Kako KJ. Thornton MJ. Heaweit A. Denressed fattv acid and acetate oxidation and other metabolic defects in homogenates from hearts of hamsters with hereditary cardiomyopathy. Circ Res 1974;34:570. 29. Apstein CS, Mueller M, Hood WB Jr. Ventricular contracture and compliance changes with global ischemia and reperfusion and compliance changes with global ischemia and reperfusion, and their effect on coronary resistance in the rat. Circ Res 1977;41:206. 30. Spector SM, Minase T, Cho S, Dominitz R, Sonnenblick EH. Microvascular spasm in the cardiomyopathic Syrian hamster: A preventable cause of focal myocardial necrosis. Circulation 1982;66:342. 31. Malhotra A, Karell M, Scheuer J. Multiple cardiac contractile protein abnormalities in myopathic Syrian hamsters (Bio 5358). J Mol Cell Cardiol 1985;17:95. 32. Lossnitzer K, Janke J, Hein B, Stauch M, Fleckenstein F. Distributed myocardial calcium metabolism: A possible pathogenic factor in the hereditary cardiomyopathy of the Syrian hamster. In: Fleckenstein A, Rona G, editors. Recent advances in studies on cardiac structure and metabolism. Baltimore: Universitv Park Press, 1975:6:207. 33. Jasmin G, Solymoss B. Prevention of hereditary cardiomyopathy in the hamster by verapamil and other agents. Proc Sot Exp Biol Med 1975;149:193. J, Sievers R, Parmley WW, Jasmin G. 34. Wikman-Coffelt Verapamil preserves adenine nucleotide pool in cardiomyopathic Syrian hamster. Am J Physiol 1986,250:H22. 35. Nayler WG, Williams A. Relaxation in heart muscle: Some morphological and biochemical considerations. Eur J Cardiol 1978;7(suppl):35. 36. Hearse DJ, Garlick PB, Humphrey SM. Ischemic contracture of the myocardium: Mechanisms and prevention. Am J Cardiol 1977;39:986. 37. Henry PD, Shuchle R, Davis BJ, Weiss ES, Sobel BE. Myocardial contracture and accumulation of mitochondrial calcium in ischemic rabbit heart. Am J Physiol 1977; 233:H677. 38. Fleckenstein A. Calcium antagonism in heart and smooth muscle. New-York: John Wiley & Sons, 1982113. 39. Nayler WG. Calcium and cell death. Eur Heart J 1983;4(suppl C):33. 40. Panagia V, Singh JN, Anand-Srivastava MB, Pierce GN, Jasmin G, Dhalla NS. Sarcolemmal alterations during the development of genetically determined cardiomyopathy. Cardiovasc Res 1984;18:567. 41. Brink AJ, Lochner A. Contractility and tension development of the myopathic hamster (BIO 14.6) heart. Cardiovasc Res 1969:3:453. --
I
Walter Markiewicz,* Shao Wu,* Richard Sievers,* William W. Parmley,* Charles B. Higgins,* Thomas L. James,* Gaetan Jasmin,** and Joan Wikman-Coffelt.* Sun Francisco, Calif., and Mo~treul, Quebec, Cu~adu
The hereditary cardiomyopathic strain of Syrian hamsters, designated UM-X7.1, a derivative of the BIO 14.6 strain, has been used as a model of congestive heart failure.‘-* Four general phases in the cardiomyopathy have been defined: (1) a necrotic stage (30 to 100 days); (2) a fibrotic and healing stage (100 to 160 days); (3) a stage with hypertrophy and dilation, as well as an increase in scar calcification (160 to 200 days); and (4) a stage of con$inuing dilation and terminal heart failure (200 to 300 days). Heart failure is identified in the cardiomyopathic hamster by the presence of abdominal fluid and cirrhosis of the liver.3 Over 50% of the hamsters die by 250 days of age.3 Like any other heart failure models, the later stages demonstrate an imbalance of free calcium,3~8 a decrease in cyclic adenosine monophosphate (CAMP)? lo low actin-activated myosin ATPase activity,” intracellular acidity,7 a decrease in cardiac performance? defective mitoFrom the ‘Departments of Radiology, Medicine, Pharmaceutical Chemistry and the Cardiovascular Research Institute, University of California, San Francisco; and **University of Montreal. Supported in part by the George D. Smith Foundation, the Susan and Don Schleicher Fund, and by Medical Research Council (Canada). Received for publication Sept. 23, 1986; accepted Jan. 15, 1987. Reprint requests: Joan Wikman-Coffelt, Ph.D., Room M1186, University of California San Francisco, San Francisco CA 94143.
362
chondria,5 and a decrease in velocity of muscle shortening as well as peak force.” Recently,12 we used 3*P nuclear magnetic resonance (NMR) spectroscopy to study the metabolism of high-energy phosphorus compounds and intracellular pH (pHi) in the perfused, isolated heart of cardiomyopathic hamsters during the heart failure stage. 31P NMR spectroscopy has the advantage of allowing sequential, noninvasive correlations between metabolic and hemodynamic status under various experimental conditions.13* l4 Cardiac function in the Langendorff preparation is dependent upon factors such as heart rate, perfusion pressure, and contractility.15 These factors are complexly interrelated. Each can affect the production and utilization of energy-rich phosphorus compounds and therefore the 31PNMR spectroscopy pattern. In this study, we investigated the effects of varying heart rate on metabolic and hemodynamic parameters in the isolated, glucose-perfused cardiomyopathic hamster heart. METHODS isolated
perfused
heart
studies.
Syrian hamsters
of
the UlWX7.1 strain (a subline of BIO 14.6), between 155 and 170 days of age, were employed as experimental animals (n = 6). Age-matched healthy hamsters served as
Volums 114 Number 2
Table
Mitochondriat
function in cardiomyopathic
hamster
363
I. Hemodynamic parameters Heart rate (bpm-I) LV pressure (mm Hg)
170 220 250 270
A 170-270 A systolic pressure tl?O-270) A diastolic pressure (170-270) Coronary flow (ml/gm X min-‘)
170
AV oxygen difference (mm Hg)
170
Oxygen consumption wt X min,-‘)
270 170 270
270
(pmol/gm dry
Normal 207.5 221.7 203.3 195.5
(n = 6) + 47.2 * 48.8 + 50.5
Cardiomyopathic 148.3 115.4 83.4 58.2
+ 56.0
-12.7 k 31.2 -9.0 t 24.6 3.7 + 9.4 24.7 k 7.6 22.5 f 5.8 171.8 t 63.0 246.7 f 71.1 29.5 rf: 9.9 39.3 zk 10.9
-90.2 -25.0 63.3 16.7 12.4 200.5 175.8 27.4 17.4
(n = 6)
t x3.7* i 45.2f 1: 58.2t f 32.5t f 16.4j. z?r38.5 I 41.57 rt 4.6 iz 5.8t rt 26.4* t 27.3* t- 7.6 rt 8.47
Values are given as mean f SD. LV = left ventricle;
AV =
arteriovenous;
*p < 0.05; tp < 0.01. Statistical
significance
A = difference
between values at 170 and 270 bpm-‘. refers to the difference between normal and cardiomyopathic
controls (n = 6). The isolated beating heart was perfused by a modified Langendorff method,6 with a perfusion pressure of 110 mm Hg. The perfusate contained the following (in mmob‘L): 117 NaCl, 4.3 KCI, 1.2 MgC& 0.1 K2HP04, 25 NaHCO,, 2.5 CaCl,, 0.5 Na EDTA, and 10 U/L of insulin in 15 mmol glucose. The medium was mixed with 95 % 0, and 5% CO,. Perfusate temperature was maintained at 35* C by means of counter-current heat exchangers,“j and a thermostat regulated circulating water bath. Pacing leads designed to prevent noise artefact were inserted at the base of the right ventricle and were connected to a Medtronic pulse generator (model 5320, Medtronic Inc., Minneapolis, Minn.) for pacing of the heart. A polyethylene cannula (internal diameter = 1.07 mm, length = 1.3 cm) was inserted through the left atrium and mitral valve into the left ventricle and was then sutured into place. The cannula was connected to a Statham P23Db pressure transducer (Gould Inc. Cardiovascular Products, Oxnard, Calif.) via a 100 cm long polyethylene tube (internal diameter = 1.67 mm) for pressure recording on a four-ch~nel Beckman dynograph (Beckman Instruments Inc., Brea, Calif.). All pressure measurements were performed with the heart positioned in an NMR tube inserted into the magnet bore (described later) so that hem~~amic and metabolic parameters could be measured simultaneously. Thus, precise determination of the zero pressure level was impossible. Therefore, pressure measurements were expressed as developed left ventricular pressure (i.e., the difference between the highest systolic and lowest diastolic pressure). Arterial and venous oxygen samples were taken before and after placing the heart in the magnet. “Arterial” samples were aspirated from the aortic chamber and “venous” samples were drawn from a catheter introduced into the right ventricular outflow tract for oxygen measurements (Model 165/2 gas analyzer, Corning Glass Works, Corning, N.Y.) Coronary flow was measured by collecting the effluent from the right ventricle in a volumetric container.
hamsters
at a given heart
rate.
Myocardial oxygen consumption was calculated as indicated elsewhere.17 31P NMR spectroscopy. 31PNMR spectra of the beating isolated perfused heart were obtained on a 5.6 Tesla vertical 76 mm bore spectrometer. The home-built spectrometer was connected to a 1180 Nicolet computer (Nicolet Instruments, Madison, Wise.), a Nicolet 2938 pulse programmer, and a high-resolution 20 mm broad band probe. 31P spectra were obtained at 97.3 MHz without proton decoupling. Pulse angle was 75 degrees, recycle time was 2.25 seconds, and spectral width was r+_4000Hz. Chemical shifts are referred to the resonance position of phosphocreatine (PCr) at zero parts per million (ppm). Transients were accumulated for 20 minutes for each spectrum. For each spectrum the characteristic peaks of inorganic phosphate (Pi), PCr, and phosphate groups of adenosine triphosphate (ATP) were identified.13*” The area of each peak was integrated and expressed as “mole fraction” by dividing the integrated value for Pi, PCr, and ATP by the sum of the integrated values for all three peaks. The mole fraction does not give an absolute value and is not normalized for tissue weight. The ATP/Pi and Per/Pi ratios are also given. Intracellular pH was estimated from the chemical shift of the pH-dependent peak of Pi relative to the peak of Pcr.ls*‘* Procedure. The hearts were perfused for 20 minutes in the magnet bore before data collection. This allowed the beating heart to reach steady state with the perfusate.” Pacing rate was set at 170 bpm. This heart rate was slightly higher than the spontaneous beating frequency so that ventricular capture was constant and regular. Following accumulation of NMR spectra and recording of hemodynamic parameters, the pacing rate was increased to 220 bpm for 1 minute under constant pressure recording. Heart rate was then increased to 250 bpm for 1 minute and then to 270 bpm for 20 minutes. Pacing threshold was increased as required. Following this interval, spectra were again accumulated and all hemodynamic parameters
364
Markiewicz et al.
American
Table
August 1887 Heart Journal
II. 31PNMR spectroscopy parameters Heart
r&e (bm) Pi (mole
fraction,
%)
170 210
PCr (mole
fraction,
%)
170 270
ATP (mole
fraction,
%)
170 270
P&/Pi (ratio)
170 270
ATP,fPi (ratio)
170 270
PH
170 270
UJ)
~~rrn~~ (n = 6) 22.8 28.8 5.9 39.5 36.2 -3.3 37.7 35.0 -2.7 1.79 1.34 -0.45 1.69 1.25 -0.44 7.15 7.10 -0.05
t + f zk 2 + i r -rf k + _t k f + * k
Car~~ornyopff~~~c (n = 6) 4.2 5.2 4.5 4.9 6.7 3.3 3.0 2.1 2.1 0.45 0.55 0.41 0.31 0.22 0.34 0.05 0.02 0.01
27.3 43.3 16.0 33.4 24.9 -8.5 39.3 33.1 -6.1 1.61 0.68 -0.93 1.82 0.89 -0.93 7.10 7.04 -0.07
f It + f 4 rtr 2 i k rt + 2 t -13z * * *
13.2 13.7* 8.5* 9.8 8.6* 7.9 5.1 7.1 2.6* 0.98 0.41* 0.64 1.06 0.52 0.58 0.06 0.06 0.05
Pi = inorganic phosphate; PCr = phosphocreatine. Mole fractions (in percent) are crtlcutated by dividing the integrated values for Pi, PCr, or ATP resonances by the sum of the integrated values for all three peaks. ‘p < 0.05.
-5
-10
-15
PPM Fig. 1. 31P nuclear magnetic resonance spectra of a healthy hamster. Heart rate is increased from 170 bpm (upper trace) to 270 bpm (lower trace). Integration of the individual resonances showed that Pi mole fraction rose from 20% to 30%, whereas PCr fell from 37% to 33% and -ATP fell from 43% to 37%. were recorded. Wet and dry weights were measured at the end of the experiment for calculation of the dry weight/ wet weight ratio. Statistical analysis. Results are given as mean + SD. Differences in the same animal under different pacing conditions were analyzed by paired t test. Differences between healthy and myopathic animals were analyxed by one-way analysis of variance. RESULTS Comparison between healthy and cardiomyopathic hamsters. Developed left ventricular pressure was
significantly lower in cardiomyopathic hamsters at a rate of 170 bpm (148.3 + 18.7 vs 207.5 _+ 47.2 mm Hg, p < 0.05). No other sign&ant difference between the two groups was noted (Tables I and II).
The rate of rise of left ventricular pressure was low and the duration of contraction was prolonged in cardiomyopathic hamsters when compared to healthy animals. Marked differences between the two groups were noted at a heart rate of 270 bpm. Myopathic hamsters had significantly lower deveioped left ventricular pressure (58.2 + 32.5 vs 195.5 + 56.0 mm Hg, p < O.Ol), markedly lower coronary flow (12.4 -r 5.8 vs 22.5 t 5.8 ml/ gm X min-‘, p < O.Ol), wider arteriovenous difference in oxygen partial pressure (p < 0.05), and lower myocardial oxygen ~ons~ption (17.4 f 8.4 vs 39.3 Z!Z10.9 firno1 O&m wet weight X min-l, p < 0.01) (Table I). Cardiomyopathic hamsters had higher Pi (43.3 zk 13.7 vs 28.8 + 5.2%) p < 0.051, lower PCr (24.9 f 8.6 vs 36.2 +- 6.7%, p < 0.05), lower PCr/Pi (0.68 f 0.41 vs 1.34 + 0.55, p < 0.05), and lower pHi (7.04 + 0.06 vs 7.10 + 0.02, p < 0.05) (Table II). Values for ATP and ATP/Pi were lower in the c~diomyopat~c animals but not si~i~c~tly so. The dry weight/wet weight ratio was slightly lower in cardiomyopathic animals (0.171 + 0.006 vs 0.183 z!z0.008, p < 0.05). Healthy hamsters. As heart rate increased from 170 to 270 bpm in normal hamsters, developed left ventricular pressure fell slightly (207.5 + 47.2 to 195.5 rt 56.0 mm Ha;, NS) (Table I). Systolic pressure fell 9.0 -+ 24.6 mm Hg, whereas diastolic pressure rose 3.7 rt: 9.4 mm Hg. Coronary flow was
Volume 114 Number 2
unchanged. The arteriovenous oxygen difference widened si~ific~tly (p < O-05), indicating increased myocardial extraction of oxygen and a significant rise in myocardial oxygen consumption (29.5 rt 9.9 to 39.3 + 10.9 pmol OJgm dry weight x min-*, p < 0.05). There was a significant rise in Pi (22.8 f 4.2 to 28.8 + 5.2 % , p < 0.05) and a decrease in ATP (37.7 + 3.0 to 35.0 rt 2.1%) p < 0.05), PCr/Pi (1.79 + 0.45 to 1.34 + 0.55, p <0.05), and ATP/Pi (1.69 + 0.31 to 1.25 it 0.22, p < 0.05) (Table II). pHi fell from 7.15 +- 0.05 to 7.10 t 0.02, p < 0.10) in normal hamsters. Example of a 31PNMR spectrum in a normal heart is shown in Fig 1. Cardiomyopathic hamsters. Developed left ventricular pressure fell in the cardiomyopathi~ hamsters from 148.3 + 18.7 mm Hg at a heart rate of 170 bpm to 115.4 + 45.2 mm Hg at 220 bpm (NS), 83.4 + 58.2 mm Hg at 250 bpm (p < 0.05 vs baseline), and 58.2 + 32.5 mm Hg at 270 bpm @ < 0.01 vs baseline) in ~diomyopathic hamster hearts (Table I). Prolonged contraction persisted at high heart rate, leading to a marked reduction in relaxation time. In going from 170 to 270 bpm, diastolic pressure rose 65.0 & 38.7 mm Hg (Table I), while at the same time coronary flow fell from 16.7 f 4.6 to 12.4 +- 5.8 ml/gm x min-’ (p < 0.05). The arteriovenous difference in oxygen fell slightly (though nonsignificantly) so that myocardial oxygen consumption fell significantly from 27.4 t 7.6 to 17.4 + 8.4 pm01 OJgm dry weight X min-‘, p < 0.01. Pi increased markedly (27.3 -+ 13.2 to 43.3 + 13.7%, p < O.Ol), whereas PCr (33.4 4 9.8 to 24.9 f 8.6%, p < 0.05), ATP (39.3 + 5.1 to 33.1 -+ 7-l%, p
31P NMR spectroscopy is a new technique used with increasing frequency in the analysis of heart disease in the animal and in the clinical setting.13p14 With the use of 31P NMR spectroscopy, analyses of phosphorus metabolites and pHi in the heart have been obtained in isolated perfused hear&,18 in openchest animals,‘s and even in the intact human heart.20 Ultimately, it might be possible to detect
~ito~hondria~
function in cardio~yoputhi~
I
I
I
I
5
0
-5
-10
hamster
366
I
-15
PPM Fig. 2. 31Pnuclear magnetic resonance spectra of a cardiomyopathic hamster heart. Heart rate is increased from 170 bpm (upper trace) to 270 bpm (lower trace). Integration of the individual resonances showed that Pi mole fraction rose from 27 % to 58 % whereas PCr fell from 38% to 15% and -ATP fell from 34% to 26%.
noninvasively disturbances in metabolism occurring in specific areas of the human heartzl An impor~nt advantage of the technique is the ability to evaluate heart metabolism noninvasively and sequentially. Thus, the influence of interventions on metabolite levels and pHi can be assessed and correlated with hemodynamic function. The mole fraction of cellular energy-rich phosphorus compounds as well as inorganic phosphate at a given time reflects the equilibrium between supply and demand of energy. Heart rate is an impost determinant of myocardial oxygen consumption, i.e., of the metabolic rate of the heartz2 Furthermore, heart rate can influence other determinants of heart function such as myocardial con~acti~ty and chamber size. Thus ‘it is important to consider the chronotropic status of the heart when evaluating the
366
Markiewicz et al.
American
Pi
PCr
ATP
PCr/Pi
August 1987 Heart Journal
ATPlPi
+120 +lOO +80 w
8
0 HH B CDMH I
+60
U
-20 -40 -60 -80 3. Percent change of phosphorus compounds as heart rate is increased from 170 to 270 bpm. Statistical significance shown refers to the difference between groups of healthy and cardiomyopathic hamsters.
Fig.
metabolic status of normal and abnormal hearth, by 31P NMR spectroscopy. In this study, we varied heart rate in glucoseperfused hamster heart preparations. The high perfusion pressure (110 mm Hg) used in these studies imposes chemical work on the heart and increases coronary flow.% Results obtained in normal hamsters were then compared with findings in 155- to 170-day-old cardiomyopathic Syrian hamsters of the UM-X7.1 strain, a myopathic strain of hamsters that is well characterized.23-25 Our study shows that at a paced heart rate of 170 bpm, ~diomyopathic hamsters developed significantly lower left ventricular pressure but otherwise did not differ significantly from normal hamsters with any other parameter evaluated. Thee-~din~ suggest that mitochondrial function was adequate to maintain normal levels of energy-rich compounds in the presence of reduced developed left ventricular pressure, i.e., reduced metabolic requ~emen~.~ As heart rate was increased to ‘220,250, and 270 BPM, developed left ventricular pressure remained statistically unchanged in healthy animals. Increasing heart rate caused a mild but significant rise in Pi and a drop in PCr/Pi (an index of mitochondrial function)27 and ATP/Pi, indicating that the requirements for energy-rich phosphorus compounds at bigher heart rate increased faster than the ability of the heart to synthesize them. ATP is not repleted during the shortening phase of the cardiac cycle because aden-
osine diphospha~ (ADP) is bound during this time.17 An increase in heart rate thus reduces recovery time, causing a new steady-state in ATP synthesis. This new steady-stats did not cause any significant change in developed left ventricul~ pressure in healthy hamsters. Myocardial oxygen consumption rose, pointing to the increased metabolic rate of the heart at higher pacing rates,22 A similar relationship between work output and the steady-state oapability of oxidative phosphorylation has been noted in the exercising human forearm analyzed by 31P NMR S~CtFOSCOpy.27
The hemodynamic response of cardiomyopathic hamsters to an increase in heart rate was markedly different, The fall in developed left ventricular pressure was dramatic and occurred mostly as a result of a marked rise in diastolic pressure. COFOnary flow and myocardial oxygen consumption both decreased significantly. Thus, the major hemodynamic response to increasing heart rate in the myopathic hamster is a severe rise in diastolic pressure, reflecting primarily the inability of the heart to relax during diitole. Qualitative evaluation of pressure recordings showed a major prolongation of contraction, which led to a rise in diastolic pressure as the interstimulus interval shortened. Roth the level of energy-rich phosphorus compounds and pHi fell markedly and were si~i~c~tly lower than in healthy animals paced at the same rapid heart rate. Despite diminished demand for
Volume 114 Number 2
Mitochondrial
function in cardiomyopathic
hamster
367
nomenon, occurring as a result of an abnormal mitochondrial function, so that the cell is unable to maintain the calcium gradient across the sarcolemmal membrane. The initial defect causing mitochondrial dysfunction has not been identified, but may be due to depressed enzymes in the sarcolemmal membrane.@ The adverse effects of cellular calcium overload are well documented41 and probably play an important role in causing an abnormality of relaxation. When heart rate is low, relaxation of the cardiomyopathic hamster heart still appears to be adequate. Raising the heart rate further increases the amount of cytosolic calcium,37 overwhelming the ability of the sarcoplasmic reticulum to sequestrate calcium and thus to initiate the active relaxation process.41 The low ATP level may also be an important contributing cause for the marked impairment in ventricular relaxation noted at high heart rate in the cardiomyopathic hamster.35s36 Intracellular pH is an important parameter of ly. The reason for the abnormal mechanical function cellular function. At a heart rate of 170 bpm, pHi was lower in cardiomyopathic hamsters, but not seen in cardiomyopathic hamster myocardium has statistically different from values in healthy aninot been elucidated.‘j, lo The changes in contractility mals. At the end of 20 minutes of pacing at a rate of have been well characterized and follow a course similar to changes in mitochondrial function.5s 24,25 270 bpm, pHi was significantly lower in cardiomyopathic than in healthy animals. The increased conReduced mitochondrial function is probably important in causing depressed contractile function.6* lo,l2 centration of intracellular hydrogen ions as heart rate is raised in the glucose-perfused cardiomyoOther factors such as reduced pHi,12 alteration in the pathic hamster heart may be due to the substrate, components of the contractile protein system,31 and glucose, contributing to an inefficient production of abnormality in the intracellular concentration or mitochondrial substrate, namely pyruvate, as transport of calcium may also be involved.2,11s21*32-34 described in an earlier study.12 A decrease in developed pressure, dpldt, and high Our results show the importance of heart rate in energy phosphates also occurs in vivo in the cardiodetermining the hemodynamic and metabolic promyopathic hamster.34 file of normal and cardiomyopathic hearts. ImporThe abnormal relaxation noted in the cardiomyopathic hamster heart at high pacing rate might be tant defects may not be apparent when heart rate is related to a combination of low ATP and high low, but are unmasked when heart rate is increased. Pacing at a fast heart rate may be required when intracellular calcium35-37 and may also be responsible interventions improving myocardial metabolism are for the depressed developed pressure observed in assessed. Conversely, pacing at a lower heart rate situ.34 The mechanism for the markedly increased may be advantageous when the examined intervenintracellular calcium, first noted during the necrotic tion is expected to cause deterioration. phase, is controversial. One hypothesis invokes a primary genetic defect in the sarcolemmal membrane, causing increased calcium influx into the REFERENCES cell8 Increased intracellular calcium initially 1. Angelakos ET, Carballo LC, Daniels JB, King MP, Bajusz E. Adrenergic neurohumors in the heart of hamsters with heredincreases myocardial contractility and therefore the itary myopathy during cardiac hypertrophy and failure. consumption of ATP. Mitochondrial calcium overRecent Adv Stud Card Struct Metab 1972;1:262. load soon occurs, followed by mitochondrial dys2. Jasmin G, Proscher L. Calcium, and myocardial cell injury. function and reduced production of ATP.38 The cell An appraisal in the cardiomyopathic hamster. Can J Physiol Pharmacol 1984;62:891. becomes unable to maintain its calcium homeosta3. Jasmin G, Proscher L. Hereditary polymyopathy and cardiosis, causing further influx of calcium across the myopathy in the Syrian hamster. I. Progression of heart and skeletal muscle lesions in the UM-X7.1 line. Muscle Nerve sarcolemma. Cellular necrosis and cell death eventu1982;5:20. ally occur.38 4. Jasmin G, Proscher L, Cailloux MF. Congestive cardiomyopOther authors2*3s have suggested that the increase athy in the Syrian hamster. Possible role of catecholamines in in intracellular calcium might be a secondary pheits pathogenesis. In: Goodwin JF, Hjalmarson A, Olsen EGJ,
energy, the level of energy-rich phosphorus compounds fell, indicating several mitochondrial dysfunction at a high pacing rate. The latter may partly reflect the reduced glycolysis12 and the depressed fatty acid and acetate oxidation28 noted in cardiomyopathic hamsters. The reduction in coronary flow may have reflected the reduced energy requirements, or this reduction in flow occurred as a result of increased coronary vascular resistance secondary to impaired relaxation.2s Since the extraction of oxygen by the myocardium was not increased, the reduction in coronary flow probably did not cause the reduction in energy supply noted in this study. The possibility that increased diastolic tension produced localized areas of ischemia or was responsible for the focal abnormality in microcirculation without vessel structural abnormality, as noted in cardiomyopathic anima1s,30 cannot be excluded but appears unlike-
August
366
5.
6. 7. 8.
9.
10.
11.
12.
13. 14.
15. 16. 17. 18. 19. 20.
21.
22.
Markiewicz et al.
editors. Congestive cardiomyopathy. Siidertiilje, Sweden: AB Astra, 1981;113. Proschek L, Jasmin G. Hereditary polyopathy and cardiomyopathy in the Syrian hamster. II. Development of heart necrotic changes in relation to defective mitochondrial function. Muscle Nerve 1982;5:26. Sievers R, Parmley WW, James T, Wikman-Coffelt J. Energy levels at systole vs diastole in normal hamsters vs myopathic hamster hearts. Circ Res 1983;53:759. Sievers R, Parmley WW, Wikman-Coffelt J. Energy reserve at systole versus diastole in myopathic hamster hearts (abstr). Circulation 1983;68:267. Wrogeman K, Jacobson B, Blanchaer MC. On the mechanism of calcium-associated defect of oxidative phosphorylation in progressive muscular dystrophy. Arch Biochem Biophys 1973;159:267. Harrow JAC, Singh JN, Jasmin G, Dhalla NS. Studies on adenylate cyclase-cyclic AMP system of the myopathic hamster (UM-X7.1) skeletal and cardiac muscle. Can J Biochem 1975;53:1122. ’ Wikman-Coffelt J, Sievers R, Coffelt RJ, Parmley WW. Biochemical and mechanical correlates at peak systole in myopathic Syrian hamster. In: Jacob R, editor. Cardiac adaptation to hemodynamic overload, training and stress. Dar-mstadt, FRG: Steinkopff Verlag, 1983:197. Rouleau JL. Chuck HS. Hollosi G. Kidd P. Sievers R. Wikman-CofIelt J, Parmley WW. Verapamil preserves myo: cardial contractility in the hereditary cardiomyopathy of the Syrian Hamster. Circ Res 1982;50:405. Wikman-Coffelt J, Sievers R, Parmley WW, Jasmin G. Cardiomyopathic and healthy acidotic hamster hearts. Mitochondrial activity may regulate cardiac performance. Cardiovast Res 1986;20:471. Ingwall JS. Phosphorus nuclear magnetic resonance spectroscopy of cardiac and skeletal muscles. Am J Physiol 1982; 242:H729. James TL. In vivo nuclear magnetic resonance spectroscopy. In: Moss AA, Ring EJ, Higgins CB, editors. NMR, CT and interventional radiology. San Francisco: Radiology Research Educational Fund, 1984;235. Braunwald E, Sonnenblick EH, Ross J Jr. Contraction of the normal heart. In: Braunwald E, editor. Heart disease. Philadelphia: W.B. Saunders, Second edition. 1984;409:446. Wikman-Coffelt J, Coffelt RJ. Flexible tube: Counter-current heat exchanger. Rev Sci Instrum 1985;56:165. Wikman-Coffelt J, Sievers R, Coffelt RJ, Parmley WW. The cardiac cycle: Regulation and energy oscillations. Am J Physiol 1983;245:H354. Garlick PB, Radda GK, Seeley PJ, Chance B. Phosphorus NMR studies on perfused heart. Biochem Biophys Res Commun 1977;74:1256. Grove TH, Ackerman JJH, Radda GK, Bore PJ. Analysis of rat heart in vivo by phosphorus nuclear magnetic resonance. Proc Nat1 Acad Sci USA 1980;77:299. Whitman GJR, Chance 3, Bode H, Maris J, Hasselgrove J, Kelley R, Clark BJ, Harken AH. Diagnosis and therapeutic evaluation of a pediatric case of cardiomyopathy using phosphorus-31 nuclear magnetic resonance spectroscopy. J Am Co11 Cardiol 1985;5:745. Stein PD, Goldstein S, Sabbah HN, Liu Z, Helpern JA, Ewing JR. Lakier JB. Chonn M. LaPenna WF. Welch WF. In vivo evaluation of ‘intraclllular pH and high-energy phosphate metabolites during regional ischemia in cats using 3’P nuclear magnetic resonance. Magn Reson Med 1986;3:262-269. Berglund E, Borst HG, Duff F, Schreiner GL. Effect of heart rate on cardiac work, myocardial oxygen consumption and coronary blood flow in the dog. Acta Physiol Stand 1958; 42185.
American
lS87
Heart Journal
23. Jasmin G, Proschek L. Hereditary polymopathy and cardiomyopathy in the Syrian hamster. I. Progression of heart and skeletal muscle lesions in the UM-X7.1 line. Muscle Nerve 1982;5:20-25. 24. Forman R, Parmley WW, Sonnenblick EH. Myocardial contractility in relation to hypertrophy and failure in myopathic Svrian hamsters. J Mol Cell Cardiol 1972:4:203. 25. Lochner A, Brink AJ, Van der Walt JJ. The significance of biochemical and structural changes in the development of the cardiomyopathy of the Syrian hamster. J Mol Cell Cardiol 1970;1:47. 26. Neely JR, Liebermeister H, Battersby EJ, Morgan HE. Effect of pressure development on oxygen consumption by isolated rat heart. Am J Physiol 1967;212:804. 27. Chance B, Eleff S, Leigh JS, Jr, Sokolow D, Sapega AA. Mitochondrial regulation of phosphocreatine/inorganic phosphate ratios in exercising human muscle: A Rated 31-P NMR study. Proc Nat1 Acad Sci 1981;78:6714. 28. Kako KJ. Thornton MJ. Heaweit A. Denressed fattv acid and acetate oxidation and other metabolic defects in homogenates from hearts of hamsters with hereditary cardiomyopathy. Circ Res 1974;34:570. 29. Apstein CS, Mueller M, Hood WB Jr. Ventricular contracture and compliance changes with global ischemia and reperfusion and compliance changes with global ischemia and reperfusion, and their effect on coronary resistance in the rat. Circ Res 1977;41:206. 30. Spector SM, Minase T, Cho S, Dominitz R, Sonnenblick EH. Microvascular spasm in the cardiomyopathic Syrian hamster: A preventable cause of focal myocardial necrosis. Circulation 1982;66:342. 31. Malhotra A, Karell M, Scheuer J. Multiple cardiac contractile protein abnormalities in myopathic Syrian hamsters (Bio 5358). J Mol Cell Cardiol 1985;17:95. 32. Lossnitzer K, Janke J, Hein B, Stauch M, Fleckenstein F. Distributed myocardial calcium metabolism: A possible pathogenic factor in the hereditary cardiomyopathy of the Syrian hamster. In: Fleckenstein A, Rona G, editors. Recent advances in studies on cardiac structure and metabolism. Baltimore: Universitv Park Press, 1975:6:207. 33. Jasmin G, Solymoss B. Prevention of hereditary cardiomyopathy in the hamster by verapamil and other agents. Proc Sot Exp Biol Med 1975;149:193. J, Sievers R, Parmley WW, Jasmin G. 34. Wikman-Coffelt Verapamil preserves adenine nucleotide pool in cardiomyopathic Syrian hamster. Am J Physiol 1986,250:H22. 35. Nayler WG, Williams A. Relaxation in heart muscle: Some morphological and biochemical considerations. Eur J Cardiol 1978;7(suppl):35. 36. Hearse DJ, Garlick PB, Humphrey SM. Ischemic contracture of the myocardium: Mechanisms and prevention. Am J Cardiol 1977;39:986. 37. Henry PD, Shuchle R, Davis BJ, Weiss ES, Sobel BE. Myocardial contracture and accumulation of mitochondrial calcium in ischemic rabbit heart. Am J Physiol 1977; 233:H677. 38. Fleckenstein A. Calcium antagonism in heart and smooth muscle. New-York: John Wiley & Sons, 1982113. 39. Nayler WG. Calcium and cell death. Eur Heart J 1983;4(suppl C):33. 40. Panagia V, Singh JN, Anand-Srivastava MB, Pierce GN, Jasmin G, Dhalla NS. Sarcolemmal alterations during the development of genetically determined cardiomyopathy. Cardiovasc Res 1984;18:567. 41. Brink AJ, Lochner A. Contractility and tension development of the myopathic hamster (BIO 14.6) heart. Cardiovasc Res 1969:3:453. --
I