Is the ATP decline a signal for stimulating protein synthesis in isoproterenol-induced cardiac hypertrophy?

J&ma1

of Molecular

and Cellular

Cardiology

(1980)

Is the ATP Decline a Signal in Isoproterenol-induced

for

12, 42 l-426

Stimulating Protein Cardiac Hypertrophy?

Synthesis

Among the mechanisms that have been proposed to be involved in the stimulation of protein synthesis associated with the development of cardiac hypertrophy [I, 6, 7, 8, 9, 11, 12, 14, 1.51 the hypothesis of Meerson and Pomoinitsky [9] appears to be generally applicable. According to this hypothesis the ATP decline which occurs during the initial phase in almost every type of experimentally induced myocardial hypertrophy leads to activation of the genetic apparatus of the cell. As a consequence, the synthesis of nucleic acids and proteins becomes enhanced resulting in the development of hypertrophy. Essentially two kinds of experimental approach were utilized to test this hypothesis in the model of isoproterenol-induced cardiac hypertrophy. (1) The timedependent changes in ATP content and in protein synthesis were compared to examine whether there is a strictly inverse relationship between these two parameters. (2) The alleged trigger, the ATP decline, was removed, and cardiac protein synthesis was then measured to see whether and in which direction the isoproterenol-induced increase in protein synthesis may be altered. This approach is based on the ability of ribose to accelerate the biosynthesis of adenine nucleotides in the isoproterenol-stimulated myocardium over a long period of time to such an extent that the well-known diminution of ATP [3] is prevented [19, ZZ]. The experiments were done on female Sprague-Dawley rats (200 to 220 g body weight) maintained on a diet of Altrominn with free access to water. Isoproterenol (from Fluka GmbH, Neu-Ulm) was injected subcutaneously as a single dose of 25 mg/kg. This dose has previously been shown to induce a rapid and considerable increase in cardiac protein synthesis [ZZ]. The animals then received constant intravenous infusion of either 0.9:/, NaCl or ribose (200 mg/kg/h, infusion rate: 5 ml/kg/h) for 24 h [ZO]. When cardiac adenine nucleotide and protein synthesis should be measured, the animals were injected into the tail vein with lJ4C-glycine (0.25 mCi/kg, obtained from Amersham-Buchler, Braunschweig, with a specific activity of 58 mCi/mM) at the beginning of the This study was supported by grants Zi 199/l and Zi 199/3 from the Deutsche Forschungsgemeinschaft. Address for correspondence: Priv. Doz. Dr H.-G. Zimmer, Department of Physiology, University of Munich, Pettenkoferstr. 12,800O Miinchen 2, Germany. 0022-2828/80/04042

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$02.00/O

0

1980 Academic

Press (London)

Limited

422

H.-G.

ZIMMER

ET

AL.

last 60 min of the 24 h infusion period. After the exposure to 1-14C-glycine, the hearts were rapidly excised in ether anesthesia and quickly frozen in liquid nitrogen. Rates of adenine nucleotide biosynthesis were determined from the total radioactivity of cardiac adenine nucleotides due to the stoichiometric incorporation of lJ4C-glycine and the mean specific activity of the intracellular gIycine [17, 181. Relative rates of lJ4C-glycine incorporation into total cardiac proteins were calculated by relating the radioactivity of proteins to the mean specific activity of glycine [16]. In experiments designed to assay the ATP level at different time periods after administration of isoproterenol, the rats were anesthetized with ether and tracheotomized, The hearts were rapidly removed and immediately immersed in Freonn at a temperature of -156°C. The analytical procedures

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FIGURE 1. Effect of a single dose of isoproterenol (25 mg/kg, subcutaneously applied) on the ATP level (a), on the biosynthesis of adenine nucleotides (b) and on protein synthesis (c) in rat hearts in uivo over a period of 24 h. The respective control valves are indicated by the hatched areas. Mean values &s.E.M. number of experiments in parentheses.

ISOPROTERENOL-INDUCED

CARDIAC

423

HYPERTROPHY

for measuring the ATP content were essentially those described previously [4]. Figure 1 shows the effect of isoproterenol on the ATP level and on the rates of adenine nucleotide and protein synthesis in rat hearts within the first 24 h after administration. There was an immediate fall in the ATP level [Figure l(a)] which became more pronounced with time and persisted throughout the entire observation period. On the other hand, adenine nucleotide biosynthesis [Figure 1 (b)] increased steeply within the first 5 h. It declined thereafter, but remained elevated over the base line level. Protein synthesis [Figure 1 (c)] was enhanced after 12 h and subsequently fell off in a similar manner as did adenine nucleotide biosynthesis. Continuous intravenous infusion of ribose for 24 h resulted in an exaggeration of the isoproterenol-induced enhancement of cardiac adenine nucleotide biosynthesis (Figure 2). Under these conditions, the ATP decline caused by isoproterenol did not occur. The isoproterenol-induced increase in protein synthesis was not altered when ATP was kept at a normal level. Likewise, cardiac protein synthesis measured after 5 h of constant intravenous infusion of ribose in isopro(data not terenol-treated rats revealed no difference in the extent of stimulation

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FIGURE 2. Cardiac protein synthesis, adenine nuclcotide (AN) biosynthesis and ATP levels in rats under control conditions and 24 h after subcutaneous injection of isoproterenol (25 mg/kg) with constant intravenous infusion of either 0.9% NaCl or ribose (ZOO mg/kg/h). All data are mean values ~s.E.M. number of experiments in parentheses.

424

H.-G.

ZIMMER

ET AL.

shown). Moreover, constant intravenous infusion of ribose alone for 24 h did not affect myocardial protein synthesis as compared to the controls. From these results it appears that the increase in protein synthesis in this particular model of experimentally induced myocardial hypertrophy is only transitory [,?I]. In fact, protein synthesis declined already after 24 h despite the persisting diminution of the ATP level. If the ATP decline would serve as a trigger, protein synthesis should be expected not to fall off in the presence of a diminished level of ATP. Previous studies on cardiac hypertrophy induced by constriction of the abdominal aorta in rats also revealed discrepancies: In the initial phase of the development of hypertrophy in this model, myocardial protein synthesis was first diminished concomitantly with the reduction in the ATP pool. It then recovered reaching the base line level and started to increase after 48 h [16], whereas the ATP level was low throughout the entire period of time [Z, 12, 161. These discrepancies, however, can only be regarded as indirect and suggestive evidence against the alleged trigger role of the ATP decline for stimulating protein synthesis. A more direct and rigorous approach to test this hypothesis was possible by eliminating the incriminated trigger. This was achieved by constant intravenous infusion of ribose for 24 h. With this procedure the stimulation of adenine nucleotide biosynthesis was of such an extent that it can account for preventing the isoproterenol-induced ATP decline (Figure 2). Despite the normal ATP level, the enhancement of protein synthesis turned out to be of the same magnitude as that under the influence of isoproterenol alone. It thus appears that the ATP decline does not serve a trigger function for the enhancement of protein synthesis at least in this particular model. It seems appropriate, however, to discuss some possible reservations when interpreting this finding. It is not possible at the present time with the available methodology to determine precisely the site in the myocardium where the isoproterenol-induced ATP fall and the stimulation of adenine nucleotide biosynthesis actually take place [5]. Our recent morphologic studies, however, suggest, that both processes may occur within the myocardial cell, since focal myocardial cell lesions that always occur when isoproterenol is applied in high doses [13] are reduced appreciably when the ATP level is kept normal by constant intravenous infusion of ribose [ZZ]. On the other hand, only the synthesis of total cardiac proteins has been measured in these studies. It cannot be ruled out that the synthesis of specific proteins such as myosin, actin and the regulatory proteins may be affected differently when adenine nucleotide biosynthesis is stimulated by ribose. However, it has been shown previously that total cardiac protein synthesis appears to be a reliable indicator at least for myosin synthesis [ 101. In conclusion, the results of our studies demonstrate that there is no consistent correlation between ATP decline and stimulation of protein synthesis in isoproterenol-as well as in pressure-induced hypertrophy and that cardiac protein synthesis is enhanced in isoproterenol-caused hypertrophy when the fall in ATP

ISOPROTERENOL-INDUCED

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HYPERTROPHY

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425 concept

ofprotein

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the

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H.

KOSCHINE

hypertrophy. H.-G.

ZIMMER,

G.

STEINKOPFF,

H.

IBEL

AND

Defiartment of Physiology, University of Munich, Pettenkoferstr. 12, 8000 Mtinchen 2 West Germany

REFERENCES 1.

2.

3. 4.

5.

6.

7. 8.

9.

10.

11. 12.

13.

CALDARERA, C. M., ORLANDINI, G., CASTI, A. & MORUZZI, G. Polyamine and nucleic acid metabolism in myocardial hypertrophy of the overloaded heart. Journal of Molecular and Cellular Cardiology 6,95-104 ( 1974). FIZEL, A. & FIZELOVA, A. Cardiac hypertrophy and heart failure: Dynamics of changes in high-energy phosphate compounds, glycogen and lactic acid. Journal of Molecular and Cellular Cardiology 2, 187-192 (197 1). FLECKENSTEIN, A. Pathophysiologische kausalfaktoren bei myokardnekrose und infarkt. Wiener
H.-G. ZIMMER

426 14.

RUSSELL, D. H.,

SHIVERICK, K. T., HAMRELL, B. B. & ALPERT, N. R. Polyamine initial phases of stress-induced cardiac hypertrophy. American Journal ofPhysiology 221, 1287-1291 (1971). SCHREIBER, S. S., ORATZ, M., ROTHSCHILD, M. A. & REFF, F. Effect of hydrostatic pressure on isolated cardiac nuclei: stimulation of RNA polymerase II activity. Cardiovas lar Research 12, 165-168 (1978). ZIMMER, IfI .-G., STEINKOPPF, G. & GERLACH, E. Changes of protein synthesis in the hypertrophying rat heart. PJliigers Archiv. European Journal of Physiology 336, 3 1 l-325 (1972). ZIMMER, H.-G., TRENDELENBURG, C. & GERLACH, E. Acceleration of adenine nucleot de synthesis de novo during development of cardiac hypertrophy. Journal of Molecular and Cellular Cardiology 4, 279-282 (1972). ZIMMER, H.-G., TRENDELENBURG, C., KAMMERMEIER, H. & GERLACH, E. De novo synthesis of myocardial adenine nucleotides in the rat. Acceleration during recbvery from oxygen deficiency. Circulation Research 32, 635-642 (1973). ZIMMER, H.-G. & GERLACH, E. Stimulation of myocardial adenine nucleotide biosynthesis by pentoses and pentitols. Pjiigers Archiv. European 3ournnl of Physiology 376, 223-227 (1978). ZIMMER, H.-G. & IBEL, H. Effects of isoproterenol and dopamine on the myocardial hexose monophosphate shunt. Experientia 35, 5 10-5 11 (1979). ZIMMER, H.-G. & IBEL, H. Studies on the mechanism for the isoproterenol-induced stimulation of cardiac glucosed-phosphate dehydrogenase. FEBS Letters 106, 335-337 (1979). ZIMMER, H.-G., IBEL, H., STEINKOPFF, G. & KORB, G. Reduction of the isoproterenolinduced alterations in cardiac adenine nucleotide content and morphology by ribose. Science (In press). synthesis

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16.

17.

18.

19.

20. 21. 22.

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