J Mel
Cell
Cardiol
23,
695-704
(1991)
~ectofSodiumDeprivationonCardiacHypertrophyinSpontaneously Hypertensive Rats: Influence Subha
Sen and David
of Aging
Young
Department of Heart and Hypertension Research, Research Institute, The Cleveland Clinic Foundation, One Clinic Center, 9500 Euclid Avenue, Cleveland, OH 44195-5071, USA (Received 29 November 1990, accepted in revisedform 25 Jamwry 1991) S. SEN AND D. YOUNG. Effect of Sodium Deprivation on Cardiac Hypertrophy in Spontaneously Hypertenstve Rats: Influence of Aging.Joumal of Molecular and Cellular Cardiolqy (1991) 23,695-704. Sodium has been identified as a causal factor in the development of hypertension in experimental animal models%as well as in clinical human subjects. Sodium is also known to play a role in modulating myocardial mass and its pattern of myosin isozyme distribution. In the rodent model, the accumulation ofV, myosin isozyme (MI), due to the modulating influence of sodium, has been shown to be associated with persistent cardiac hypertrophy and heart failure. In this paper, we have examined the effect of the restriction of dietary sodium on blood pressure, ventricular weight and myosin isoforms in spontaneously hypertensive rats (SHR) and the relationship of these parameters with age. In lo- to 11 -week-old SHR, dietary sodium restriction for 14 weeks resulted in a significant reduction in ventricular mass associated with systolic shifting of myosin isoform from V, type to V, type with no change in systolic blood pressure level; dietary sodium restriction also showed a significant reduction in body weight. When the effect of dietary sodium restriction (for 8 weeks) was studied in relation to age (in ll-, 16- and 24-week-old rats) a significant shift in myosin isoform from the V, to the V, type was noted in the 11-week-old rats; a slight but significant shift was noted in 16-week-old rats, and no change in myosin isoform distribution was noted in the 24-week-old SHR. The alteration in myosin isoform and myocardial mass in the ll- and 16-week-old rats was independent of changes in systolic blood pressure. This study demonstrates that sodium plays an important role not only in modulating myocardial mass but also in changing the biochemical composition of the heart. This study also suggests that in genetic hypertension, the restriction of sodium at a very young age may fully prevent the development of hypertension and hypertrophy. However, the mechanism by which the sodium ion modulates myocardial mass and the expression of either V, or V, myosin genes is unknown; the question of how sodium affects the cardiac function also remains. Some evidence suggests that sympathetic outflow may play an important role, but further studies are needed to validate this. KEY
WORDS:
Sodium;
Hypertension;
Hypertrophy;
Myocardium;
Introduction Sodium has been identified as a causal factor in the development of hypertension in experimental animal models as well as in clinical cases of human hypertension [6]. Although some work has been done to describe the role of sodium in the development of hypertension, in particular its mediation through the sympathetic nervous system, very little is known about the role of sodium in the modulation of cardiac hypertrophy [I, 231. Cardiac hypertrophy associated with systemic hypertension does not appear to be simply a functional response of the myocardium to the 00222828/91/060695
+ 10 $03.00/O
Myosin
isozymes.
mechanical stress of increased afterload. Our studies and those of other researchers have previously shown that a dissociation exists between the degree of cardiac hypertrophy and the level of blood pressure in animal models as well as in humans [2, 4, 20, 23, 261. These observations have led to speculation that factors other than blood pressure control may regulate the development and regression of myocardial hypertrophy, and, in particular, that the sympathetic nervous system appears to be an important modulating factor. Recently, Lindpaintner and Sen [7j have shown that in the two-kidney-one-clip renal hypertensive rat :5) 1991
Academic
Press
Limited
696
S. Sen and D. Young
model, exposure to low sodium after hypertension had developed caused a regression of hypertrophy without a substantial change in hypertension, again demonstrating a dissociation between blood pressure and heart weight in the model of renovascular hypertension. Sen and Young [ 181 have shown that attenuation of the normal cardiac myosin isozyme distribution pattern (the shifting of V, to V,, a change believed to be associated with the velocity of cardiac muscle shortening) can be achieved by dietary sodium restriction. However, in spontaneously hypertensive rats (SHR), exposure of the adult SHR to low sodium resulted in a moderate reduction in blood pressure associated with a regression of cardiac hypertrophy [19]. There is substantial evidence in the literature suggesting that sodium is a modulatory factor in the development of hypertension in experimental animal models [27, 281. The present paper describes a detailed study of the effect of sodium restriction on hypertension, myocardial mass, and the myosin isozyme distribution pattern in SHR of various age groups. Materials and Methods All rats used in this study were obtained commercially (Taconic Farms, German Town, New York). Male SHR of various age groups (11, 16 and 24 weeks of age) and the corresponding age- and sex-matched normal controls were kept three rats per cage. All rats had free access to water and food. The sodium content of the diet given to the control group was 144mEq/kg, and the sodium restriction diet contained 7 mEq/kg. All rats were given deionized water ad libitum. The experimental protocol for sodium deprivation was as follows: In the first series, dietary sodium was restricted in lo- to ll-week-old male SHR and in male Wistar Kyoto rats (WKY) for 14 weeks. In the second series of experiments ll-, 16- and 24-week-old SHR were given low-sodium diets for 8 weeks. In addition, this paper also describes the effect of sodium restriction on American normal Wistar compared to WKY and the effect of thyroxine treatment on myocardial mass and myosin isoforms. To determine the effect of thyroxine on myosin isozyme and ventricular weight, 24-week-old SHR were treated with thyroxin (0.5pg/day, IM) for 7 days.
The effectiveness of dietary sodium restriction was confirmed by both urinary sodium excretion and a significant increase in plasma renin activity. Blood pressure was determined in all animals twice a week by the same person at the same time of day using the tail-cuff method [ 161.
Analytical
method
During the entire experimental procedure, blood pressure was monitored at least two times a week by the tail-cuff method [16]. In the last week of the experiment, three consecutive readings were taken before the experiment was terminated. The rats were killed by decapitation, and heart weight, body weight, and myosin isozyme patterns were determined.
Preparation of myosin Approximately 300 mg of the bottom of the left ventricle were removed and flash-frozen in liquid nitrogen. The tissue was minced with small sharp scissors and washed in phosphatebuffered saline (40mM NaCl,SmM Na2P0,, pH 7.0 at 2°C). The tissue was then homogenized in 7ml of saline buffer using a Kontes ground-glass tissue grinder for a period of 90s (three 30s homogenization with a 30s rest interval between each). The homogenate was then centrifuged at 3000 r/min for 10 min in a Sorvall RC5B centrifuge at 2’C. The supernatant was discarded and the pellet again washed with 7ml of the phosphate-buffered saline and centrifuged at 3000r/min for 10 min. The supernatant was again discarded. The pellet was resuspended in 3 ml of an extracting solution containing 100 mM Na.+P207, 5 mM EGTA and 5 mM dithiothreitol, pH 8.6. The homogenate was shaken in an ice bath in a walk-in cold room (4°C) for 60 min, and then centrifuged at 20 000 r/min in a Sorvall Model RC5B using a SM-24 rotor for 2 h at 2%. The supernatant was collected and mixed with an equal volume of ice-cold glycerol and stored at - 20%.
Preparation of the gels Polyacrylamide gel electrophoresis using 4 % gels was done according to the method of Hoh et al. [5]. Two gels consisted of 1 ml of an
Sodium
Deprivation
in Hypertension
acrylamide stock solution (200 g/l acrylamide, 6.2 g/l of N,n’-methylene-bis-acrylamide), 3.1 ml of a pryrophosphate stock solution Na4P207 (27 mM), 13.4% v/v [PH 8.8, glycerol], and 22 ~1 of N,N,N’ ,N’-tetramethylethylene-diamine. Polymerization was initiated by adding 35~1 of freshly prepared ammonium persulphate solution (125 g/l). Gel tubes were 9.5 cm long and 5 mm internal diameter (the gels polymerized in 20min and were left at room temperature for a further 20 min before use).
Separation of myosin isozymes by electrophoresis Electrophoresis was carried out in a Pharmacia gel electrophoresis apparatus (Model GE2/4; Piscataway, NJ, USA) sufficient to accommodate 20 gel tubes. The electrophoresis buffer contained 20mM Na4P,07 and 10% (V/V) glycerol, pH 8.8 at 2%. The buffer was recirculated during the run by pumping it from the lower to the upper reservoir. This was done to neutralize the products of electrolysis formed during the run. The temperature of the electrophoresis buffer was maintained at 2’C by a refrigeration unit that circulated coolant through the coils of the electrophoresis apparatus. A 60min pre-run was carried out before myosin was applied to the gel using a current of 2mA per gel. Two microlitres of myosin [2.65 f 1.38~1 in 50% (v/v) glycerol] was loaded directly on top of the gel. The current of 2 mA per gel was maintained for 5 h, and then the gels were run under constant voltage supplied by a constant power supply (Model 3-1500; Buchler, Fort Lee, NJ, USA).
Staining of the gels The gels were stained for protein in 10ml of Coomassie Brilliant Blue R solution (0.3g/l) for 2 h at room temperature. The stain was dissolved first in 500ml of methanol, then 100 ml of acetic acid was added and the volume brought up to 11 with distilled water. Gels were then destained in a 7% (v/v) acetic acid and 30% (v/v) methanol solution in a bio-Rad model 171A gel electrophoresis diffusion destainer.
Quantitation The
myosin
of myosin isozymes
isozymes
were
quantified
by
and Hypertrophy
densitometric of a Helena densitometer.
697
scanning of the gels with the use Laboratories quick-scan R & D
Determination
of ventricular wkght
At the end of each experiment, each rat was killed by decapitation, the heart was quickly removed and washed thoroughly with saline to remove blood, and then the aorta was cut flush with the ventricular surface removing the atria and large vessels and weighed in an analytical Mettier balance. The data are expressed as ventricular weight/body weight ratio.
Determination
of urinary sodium
Urinary sodium excretion was measured to demonstrate the effectiveness of dietary sodium restriction. Animals were housed singly for several days in metabolic cages, which allow collection of urine uncontaminated with food or faeces. During these studies, the rats received their respective diets in pulverized form. The urinary sodium excretion was determined from recorded daily urine volumes and urinary sodium concentrations that were assayed on a flame photometer (Instrument Laboratory Model # 343).
Statistical analysis The statistical analyses were calculated from mean values and standard errors of the mean (s.E.M.) from each group using the PROPHET computer system. Analysis of variance was then performed (PROPHET Statistics Manual, 1980). Comparison between individual groups are based on the Student’s t test, Neuman-Keul’s multiple range test or the Kruskal-Wallis test, as appropriate. For comparisons of ventricular mass, both absolute weight and ratio of ventricular to heart weight was obtained by calculating the ratio of heart weight to body weight. For statistical analysis, the blood pressure data are expressed as the mean of average systolic blood pressure during the mean of the last four readings. All data are expressed as mean f S.E.M. Statistical significance was defined as PcO.05. Lack of a P value indicates that no significance difference exists.
698
S. Sen and
Efect
Results deprivation rats
of sodium
in SHR
and
D. Young
in the sodium-deprived group showed a significant regression of hypertrophy (3.4 mg/g vs 3.1 mg/g, P (0.05). The myosin isoform distribution pattern due to sodium restriction showed a significant change in its distribution. There was a significant increase in per cent distribution of VI from 54% to 67% in the sodium-deprived group associated with a significant reduction in V, from 22% to 13% (Table 1, P
Cell
Cardiol
23,
695-704
(1991)
~ectofSodiumDeprivationonCardiacHypertrophyinSpontaneously Hypertensive Rats: Influence Subha
Sen and David
of Aging
Young
Department of Heart and Hypertension Research, Research Institute, The Cleveland Clinic Foundation, One Clinic Center, 9500 Euclid Avenue, Cleveland, OH 44195-5071, USA (Received 29 November 1990, accepted in revisedform 25 Jamwry 1991) S. SEN AND D. YOUNG. Effect of Sodium Deprivation on Cardiac Hypertrophy in Spontaneously Hypertenstve Rats: Influence of Aging.Joumal of Molecular and Cellular Cardiolqy (1991) 23,695-704. Sodium has been identified as a causal factor in the development of hypertension in experimental animal models%as well as in clinical human subjects. Sodium is also known to play a role in modulating myocardial mass and its pattern of myosin isozyme distribution. In the rodent model, the accumulation ofV, myosin isozyme (MI), due to the modulating influence of sodium, has been shown to be associated with persistent cardiac hypertrophy and heart failure. In this paper, we have examined the effect of the restriction of dietary sodium on blood pressure, ventricular weight and myosin isoforms in spontaneously hypertensive rats (SHR) and the relationship of these parameters with age. In lo- to 11 -week-old SHR, dietary sodium restriction for 14 weeks resulted in a significant reduction in ventricular mass associated with systolic shifting of myosin isoform from V, type to V, type with no change in systolic blood pressure level; dietary sodium restriction also showed a significant reduction in body weight. When the effect of dietary sodium restriction (for 8 weeks) was studied in relation to age (in ll-, 16- and 24-week-old rats) a significant shift in myosin isoform from the V, to the V, type was noted in the 11-week-old rats; a slight but significant shift was noted in 16-week-old rats, and no change in myosin isoform distribution was noted in the 24-week-old SHR. The alteration in myosin isoform and myocardial mass in the ll- and 16-week-old rats was independent of changes in systolic blood pressure. This study demonstrates that sodium plays an important role not only in modulating myocardial mass but also in changing the biochemical composition of the heart. This study also suggests that in genetic hypertension, the restriction of sodium at a very young age may fully prevent the development of hypertension and hypertrophy. However, the mechanism by which the sodium ion modulates myocardial mass and the expression of either V, or V, myosin genes is unknown; the question of how sodium affects the cardiac function also remains. Some evidence suggests that sympathetic outflow may play an important role, but further studies are needed to validate this. KEY
WORDS:
Sodium;
Hypertension;
Hypertrophy;
Myocardium;
Introduction Sodium has been identified as a causal factor in the development of hypertension in experimental animal models as well as in clinical cases of human hypertension [6]. Although some work has been done to describe the role of sodium in the development of hypertension, in particular its mediation through the sympathetic nervous system, very little is known about the role of sodium in the modulation of cardiac hypertrophy [I, 231. Cardiac hypertrophy associated with systemic hypertension does not appear to be simply a functional response of the myocardium to the 00222828/91/060695
+ 10 $03.00/O
Myosin
isozymes.
mechanical stress of increased afterload. Our studies and those of other researchers have previously shown that a dissociation exists between the degree of cardiac hypertrophy and the level of blood pressure in animal models as well as in humans [2, 4, 20, 23, 261. These observations have led to speculation that factors other than blood pressure control may regulate the development and regression of myocardial hypertrophy, and, in particular, that the sympathetic nervous system appears to be an important modulating factor. Recently, Lindpaintner and Sen [7j have shown that in the two-kidney-one-clip renal hypertensive rat :5) 1991
Academic
Press
Limited
696
S. Sen and D. Young
model, exposure to low sodium after hypertension had developed caused a regression of hypertrophy without a substantial change in hypertension, again demonstrating a dissociation between blood pressure and heart weight in the model of renovascular hypertension. Sen and Young [ 181 have shown that attenuation of the normal cardiac myosin isozyme distribution pattern (the shifting of V, to V,, a change believed to be associated with the velocity of cardiac muscle shortening) can be achieved by dietary sodium restriction. However, in spontaneously hypertensive rats (SHR), exposure of the adult SHR to low sodium resulted in a moderate reduction in blood pressure associated with a regression of cardiac hypertrophy [19]. There is substantial evidence in the literature suggesting that sodium is a modulatory factor in the development of hypertension in experimental animal models [27, 281. The present paper describes a detailed study of the effect of sodium restriction on hypertension, myocardial mass, and the myosin isozyme distribution pattern in SHR of various age groups. Materials and Methods All rats used in this study were obtained commercially (Taconic Farms, German Town, New York). Male SHR of various age groups (11, 16 and 24 weeks of age) and the corresponding age- and sex-matched normal controls were kept three rats per cage. All rats had free access to water and food. The sodium content of the diet given to the control group was 144mEq/kg, and the sodium restriction diet contained 7 mEq/kg. All rats were given deionized water ad libitum. The experimental protocol for sodium deprivation was as follows: In the first series, dietary sodium was restricted in lo- to ll-week-old male SHR and in male Wistar Kyoto rats (WKY) for 14 weeks. In the second series of experiments ll-, 16- and 24-week-old SHR were given low-sodium diets for 8 weeks. In addition, this paper also describes the effect of sodium restriction on American normal Wistar compared to WKY and the effect of thyroxine treatment on myocardial mass and myosin isoforms. To determine the effect of thyroxine on myosin isozyme and ventricular weight, 24-week-old SHR were treated with thyroxin (0.5pg/day, IM) for 7 days.
The effectiveness of dietary sodium restriction was confirmed by both urinary sodium excretion and a significant increase in plasma renin activity. Blood pressure was determined in all animals twice a week by the same person at the same time of day using the tail-cuff method [ 161.
Analytical
method
During the entire experimental procedure, blood pressure was monitored at least two times a week by the tail-cuff method [16]. In the last week of the experiment, three consecutive readings were taken before the experiment was terminated. The rats were killed by decapitation, and heart weight, body weight, and myosin isozyme patterns were determined.
Preparation of myosin Approximately 300 mg of the bottom of the left ventricle were removed and flash-frozen in liquid nitrogen. The tissue was minced with small sharp scissors and washed in phosphatebuffered saline (40mM NaCl,SmM Na2P0,, pH 7.0 at 2°C). The tissue was then homogenized in 7ml of saline buffer using a Kontes ground-glass tissue grinder for a period of 90s (three 30s homogenization with a 30s rest interval between each). The homogenate was then centrifuged at 3000 r/min for 10 min in a Sorvall RC5B centrifuge at 2’C. The supernatant was discarded and the pellet again washed with 7ml of the phosphate-buffered saline and centrifuged at 3000r/min for 10 min. The supernatant was again discarded. The pellet was resuspended in 3 ml of an extracting solution containing 100 mM Na.+P207, 5 mM EGTA and 5 mM dithiothreitol, pH 8.6. The homogenate was shaken in an ice bath in a walk-in cold room (4°C) for 60 min, and then centrifuged at 20 000 r/min in a Sorvall Model RC5B using a SM-24 rotor for 2 h at 2%. The supernatant was collected and mixed with an equal volume of ice-cold glycerol and stored at - 20%.
Preparation of the gels Polyacrylamide gel electrophoresis using 4 % gels was done according to the method of Hoh et al. [5]. Two gels consisted of 1 ml of an
Sodium
Deprivation
in Hypertension
acrylamide stock solution (200 g/l acrylamide, 6.2 g/l of N,n’-methylene-bis-acrylamide), 3.1 ml of a pryrophosphate stock solution Na4P207 (27 mM), 13.4% v/v [PH 8.8, glycerol], and 22 ~1 of N,N,N’ ,N’-tetramethylethylene-diamine. Polymerization was initiated by adding 35~1 of freshly prepared ammonium persulphate solution (125 g/l). Gel tubes were 9.5 cm long and 5 mm internal diameter (the gels polymerized in 20min and were left at room temperature for a further 20 min before use).
Separation of myosin isozymes by electrophoresis Electrophoresis was carried out in a Pharmacia gel electrophoresis apparatus (Model GE2/4; Piscataway, NJ, USA) sufficient to accommodate 20 gel tubes. The electrophoresis buffer contained 20mM Na4P,07 and 10% (V/V) glycerol, pH 8.8 at 2%. The buffer was recirculated during the run by pumping it from the lower to the upper reservoir. This was done to neutralize the products of electrolysis formed during the run. The temperature of the electrophoresis buffer was maintained at 2’C by a refrigeration unit that circulated coolant through the coils of the electrophoresis apparatus. A 60min pre-run was carried out before myosin was applied to the gel using a current of 2mA per gel. Two microlitres of myosin [2.65 f 1.38~1 in 50% (v/v) glycerol] was loaded directly on top of the gel. The current of 2 mA per gel was maintained for 5 h, and then the gels were run under constant voltage supplied by a constant power supply (Model 3-1500; Buchler, Fort Lee, NJ, USA).
Staining of the gels The gels were stained for protein in 10ml of Coomassie Brilliant Blue R solution (0.3g/l) for 2 h at room temperature. The stain was dissolved first in 500ml of methanol, then 100 ml of acetic acid was added and the volume brought up to 11 with distilled water. Gels were then destained in a 7% (v/v) acetic acid and 30% (v/v) methanol solution in a bio-Rad model 171A gel electrophoresis diffusion destainer.
Quantitation The
myosin
of myosin isozymes
isozymes
were
quantified
by
and Hypertrophy
densitometric of a Helena densitometer.
697
scanning of the gels with the use Laboratories quick-scan R & D
Determination
of ventricular wkght
At the end of each experiment, each rat was killed by decapitation, the heart was quickly removed and washed thoroughly with saline to remove blood, and then the aorta was cut flush with the ventricular surface removing the atria and large vessels and weighed in an analytical Mettier balance. The data are expressed as ventricular weight/body weight ratio.
Determination
of urinary sodium
Urinary sodium excretion was measured to demonstrate the effectiveness of dietary sodium restriction. Animals were housed singly for several days in metabolic cages, which allow collection of urine uncontaminated with food or faeces. During these studies, the rats received their respective diets in pulverized form. The urinary sodium excretion was determined from recorded daily urine volumes and urinary sodium concentrations that were assayed on a flame photometer (Instrument Laboratory Model # 343).
Statistical analysis The statistical analyses were calculated from mean values and standard errors of the mean (s.E.M.) from each group using the PROPHET computer system. Analysis of variance was then performed (PROPHET Statistics Manual, 1980). Comparison between individual groups are based on the Student’s t test, Neuman-Keul’s multiple range test or the Kruskal-Wallis test, as appropriate. For comparisons of ventricular mass, both absolute weight and ratio of ventricular to heart weight was obtained by calculating the ratio of heart weight to body weight. For statistical analysis, the blood pressure data are expressed as the mean of average systolic blood pressure during the mean of the last four readings. All data are expressed as mean f S.E.M. Statistical significance was defined as PcO.05. Lack of a P value indicates that no significance difference exists.
698
S. Sen and
Efect
Results deprivation rats
of sodium
in SHR
and
D. Young
in the sodium-deprived group showed a significant regression of hypertrophy (3.4 mg/g vs 3.1 mg/g, P (0.05). The myosin isoform distribution pattern due to sodium restriction showed a significant change in its distribution. There was a significant increase in per cent distribution of VI from 54% to 67% in the sodium-deprived group associated with a significant reduction in V, from 22% to 13% (Table 1, P
