Mol
Cell
Effects
Cardiol
23,
1177-1190
of Age! on Right
Masao
Kmroha,
(1991)
Ventricular Hypertrophic Overload in Rats
Shogen
Isoyama,
First Department of Internal Medicine,
Nob&o
Response
Ito and Tamotsu
to Pressure-
Takishima
Tohoku University School of Medicine, Sendai, Japan
(Received 2 November 1990, accepted in revisedform 30 May 1991) M. KUROHA. S. ISOYAMA, N. ITO AND T. TAKISHIMA. Effects of Age on Right Ventricular Hypertrophic Response to Pressure-Overload in Rats. Journal of Molecular and Cellular Cardiology (1991) 23, 1177-1190. To examine the effects ofage on hypertrophic response to pressure-overload in the right ventricle, we determined the rate and extent of hypertrophy in three age groups of Wistar rats: 2, 7 and 18 months. We created pulmonary artery constriction so that increases in right ventricular pressure were similar in those groups. One or 3 weeks after pulmonary artery constriction, the rats were sacrificed and hypertrophic response was estimated from right ventricular weight, myocyte width and protein content. In the 2-month-old rats, significant hypertrophy was observed 1 and 3 weeks after pulmonary artery constriction in terms of right ventricular weight/body weight (161% of controls at 1 week and 209% at 3 weeks), right ventricular weight/tibial length (161 and 251%. respectively) and right ventricular weight/left ventricular weight (174 and 211%. respectively). In the 7-month-old rats. significant hypertrophy was observed only at 3 weeks, but was not observed even at 3 weeks in the 18-month-old rats. The age-associated decrease and delay in hypertrophic response was also observed at cellular (myocyte width) and biochemical (protein content) levels. Thus, there is an age-associated diminution in the rate and extent of myocardial hypertrophy in the right ventricle. KEY WORDS:
Aging;
Pulmonary
artery
constriction;
Introduction Hemodynamic overload causes muscle hypertrophy in the left and right ventricles. The capacity for myocardial hypertrophy diminishes with age in pressure-overloaded left ventricles (Isoyama et al., 1987; 1991) and volume-overloaded left ventricles (Isoyama et al., 1988) and hearts (Walford et al., 1988). In the right ventricle, however, there are only a few data available concerning the effects of age on myocardial hypertrophic response. The effects of age on myocardial hypertrophic response in the right ventricle may differ from that in the left ventricle for the following reasons. First, the functional loads of the left and right ventricles grow (Peterson et al., 1989) and age in a different manner. Right ventricular growth is analogous to eccentric hypertrophy, whereas left ventricular growth represents a combination of eccentric and concentric hypertrophy during development after Please address all correspondence to: Tamotsu School of Medicine, l-l Seiryo-machi, Aobaku, 0022-2828/91/101177
+ 14$03.00/O
Takishima, Sendai980,
Myocyte
width;
Protein
synthesis.
birth (Anversa et al., 1980). In addition, physiologic aging after maturation causes different myocardial changes between the two ventricles. In the senescent human or animals, the left ventricle gains weight, but the right ventricle does not do so (Lakatta, 1979; Yin et al., 1980; 1982; Lakatta and Yin, 1982; Wei et al., 1984; Isoyama et al., 1987; 1988). Cellular hypertrophy and scar formation in the aged left ventricle are more prominent than those in the right ventricle (Anversa et al., 1986b; 1989). Second, aging shifts myosin heavy chain from the (Y- to the /3-isoform (Effron et al.. 1987; Boluyt et al., 1989), and causes functional changes: prolongation in contraction duration and decreases in shortening velocity (Rumberger and Timmerman, 1976; Yin et al., 1980; Capasso et al., 1982; Wei et al., 1984; 1986; Effron et al., 1987; Walford et al., 1988; Anversa et al., 1989). However, Anversa et al. reported that these age-related (1989) First Department Japan
of Internal
Medicine,
Tohoku
0 1991 Academic
University
Press Limited
1178
M.
Kuroha
functional changes were more pronounced in the left ventricle than in the right ventricle. Third, differences exist in the capacity to react and adapt to an increased work load or hormonal stimulation. For example, the extent of myocardial hypertrophy in response to running exercise (Anversa et al., 1982; 1983; 1986a), aortocaval shunt (Flaim et al., 1979) or thyroid hormone administration (Gerdes et al., 1987; Zierhut and Zimmer, 1989) was greater in the right ventricle than in the left ventricle. Thus, growing and aging after maturation do not always cause the same structural and functional changes in the right and left ventricles. Therefore, we attempted to study the course of right ventricular growth and to examine the effects of age on hypertrophic response produced by pressure-overload in the right ventricle. For this purpose, we determined the rate and extent of right ventricular myocardial hypertrophic response at the organ, cellular and biochemical levels after pulmonary artery constriction in rats of three age groups. Methods We used three different age groups of male Wistar rats: 2 months, 7 months and 18 months old, which correspond to developmental, young-adult and old stage of rats, respectively. In the first experiment, to estimate stress of the right ventricular free wall we studied right ventricular growth in the three age groups of rats (n = 6 in P-month-old rats; n = 7 in 7-month-old rats; n = 6 in 18-monthold rats). The heart was isolated, and the right ventricular weight and geometry were measured. In the second experiment, rats (n = 21 in 2-month-old rats; n = 20 in 7-monthold rats; n = 21 in 18-month-old rats) were divided into the three subgroups: a control group, a group of rats with pulmonary artery constriction for 1 week and a group of rats with pulmonary artery constriction for 3 weeks. In some of each subgroup of rats (n = 10 in 2-month-old rats; n = 11 in 7-month-old rats; n = 9 in l-month-old rats), while continuously monitoring right ventricular pressure, we created the pulmonary artery constriction after opening the thorax so as to obtain the same right ventricular pressure increase
et al.
immediately after pulmonary artery constriction. We examined the degree of right ventricular hypertrophic response to the same degree of pressure-overload at the organ, cellular and biochemical levels in the three age groups. In the experiment, rats were treated in accordance with the Declaration of Helsinki and the Guiding Principles in the Care and Use of Animals. Also, the experimental and animal care protocol was approved by the Animal Care Committee of the Tohoku University School of Medicine. Right ventricular growth and pressure-volume relationship Each rat was anesthetized with pentobarbital sodium (50mg/kg, i.p.). The thorax was opened, and the heart was arrestd with KC1 solution. The heart was removed and immersed in cold saline. A latex balloon with a double lumen polyethylene cannula (Polyethylene Tubing, SP-31, 0.50mm i.d., 0.80 mm o.d., Natsume Instrument Co., Ltd., Tokyo, Japan) was inserted into the right ventricle through a right atria1 incision and tied tightly at the atrioventricular groove with a silk thread. One lumen was used to measure the right ventricular pressure with a strain gauge transducer (Model TP-300T, Nihon Kohden Co., Ltd., Tokyo). Another lumen was used to change the volume of the right ventricle. Before the infusion of cold saline, the right ventricular pressure was made negative by manual compression, and the balloon was emptied. Cold saline was infused with stepwise increments of 0.05 ml up to 25 mmHg of the ventricular pressure. The presright sure-volume relationship was obtained three times, and the last relationship was measured, because the second and last relationships were reproducible. Right ventricular free wall was removed, and the surface area, weight and wall thickness were measured immediately after measurements of pressure-volume relationships. The surface area was measured planimetrically after extending the free wall without force on a flat plate. The thickness of the right ventricular free wall was calculated based on the assumption that specific gravity of the myocardial tissue is 1.06 (Mendez and Keys, 1960).
Aging
meet
on Right
Ventricular
Operative procedures for pulmonary artery constriction Each rat was anesthetized as described above and intubated endotracheally under direct visualization, as was done in our previous studies (Isoyama et al., 1987; 1989; 1991). In some rats of each group, a polyethylene cannula (Polyethylene Tubing, SP-31) was inserted into the right ventricular cavity through the right jugular vein to measure right ventricular pressure with a strain gauge transducer. Under controlled ventilation with room air (model 141, Princeton Medical Instruments Inc., Natick, MA, USA), the thorax was opened at the third intercostal space. The pulmonary artery was exposed and dissected free from the surrounding tissues. A surgical silk thread (3-O) was drawn under the pulmonary artery. A rigid tube was placed alongside the pulmonary artery. The rigid tube and the pulmonary artery were tied tightly together and the tube was rapidly removed. To create the same degree of right ventricular pressure increase in the three age groups we used rigid tubes of the following three different outer diameters: 1.4 mm for the 2-month-old rats, 2.Omm for the 7-month-old rats and 2.2mm for the 18-month-old rats. In a pilot study, we ascertained that right ventricular pressure increase immediately after pulmonary constriction was approximately 15 mmHg in each age group. We inflated the lung under 5 cmH*O of positive end-expiratory pressure to expel the air in the thorax, and the thorax was closed with a silk thread. Rats were housed in cages and were allowed free access to tap water and standard rat pellet chow. At either 1 or 3 weeks after pulmonary constriction, the rat was anesthetized again as described above and sacrificed. The heart was rapidly isolated. Fat and atria were removed, and the ventricles were divided into a right ventricular free wall portion and a left ventricular free wall and septal portion. Each portion was cut into two pieces after measurements of right and left ventricular weights. Each piece of the right and left ventricles was stored at - 8O’C until chemical analysis. Myocardial tissue protein was measured by the method of Lowry et al. (1951). Other pieces of the right and left ventricles were fixed in 10% formaline for histological examination. We also measured body weight and tibia1 length to
Hypertrophy
1179
normalize the right ventricular weight by body size (Yin et al., 1980; 1982; Lakatta and Yin, 1982; Wei et al., 1984; Isoyama et al., 1987; 1988; 1991). Frequency response of the pressure measurement system was estimated by transient response to a stepwise pressure change. The damping coefficient and undamped natural frequency were 0.56 and 41 Hz, respectively.
Histological
examination
Samples of right and left ventricles were cut parallel and perpendicular to the apex-base line. Sections were processed conventionally for histological examination (dehydrated in graded alcohol and embedded in paraffin), cut in 5 Frn sections, and stained with hematoxylin and eosin, as was done in previous studies (Rakusan et al., 1978; Isoyama et al., 1987; 1991). We chose longitudinally oriented myocytes in which the nucleus was positioned in the center of the myocyte, and measured widths of 50 myocytes in each heart. The mean values in each heart were averaged and compared between control and experimental rats of each age group. Statistical analysis Variables measured presented as are mean it: S.E.M. Analysis of variance was used to determine the effect of age or pulmonary artery constriction on the variables tested. Subsequent multiple t-test with Bonferroni’s corrections was used to determine specific effects of age or pulmonary artery constriction. Unpaired t-test was employed for statistical analysis between the two mean values before and after pulmonary artery constriction. A Pvalue less than 0.05 was taken as the level of significance (Snedecor and Cochran, 1980).
Results
Dimension
and pressure-volume relationship right ventricle
in the
Figure 1 shows the relationship between right ventricular pressure and volume (upper panel), and the relationship between pressure and volume normalized by right ventricular
1.
6 7 6
n
17.3 f .6 24.1 k .3** 24.0 + .4**
b (mm) 127k12 272k 9’: 299 + 14**
RVW (mg)
free wall length from the apex to RVW = right ventricular weight; and 7-month-old rats or between
139 * 12 273+12** 263+ 5**
R VSA (mm’)
geometry in the three age groups
11.6* .6 15.4 + .8** 16.1 k .3**
(mm)
a
Right ventricular
Values are mean + SEM. Abbreviations:a = right ventricular base line; RVSA = surface area of right ventricular free wall; *P
Cell
Effects
Cardiol
23,
1177-1190
of Age! on Right
Masao
Kmroha,
(1991)
Ventricular Hypertrophic Overload in Rats
Shogen
Isoyama,
First Department of Internal Medicine,
Nob&o
Response
Ito and Tamotsu
to Pressure-
Takishima
Tohoku University School of Medicine, Sendai, Japan
(Received 2 November 1990, accepted in revisedform 30 May 1991) M. KUROHA. S. ISOYAMA, N. ITO AND T. TAKISHIMA. Effects of Age on Right Ventricular Hypertrophic Response to Pressure-Overload in Rats. Journal of Molecular and Cellular Cardiology (1991) 23, 1177-1190. To examine the effects ofage on hypertrophic response to pressure-overload in the right ventricle, we determined the rate and extent of hypertrophy in three age groups of Wistar rats: 2, 7 and 18 months. We created pulmonary artery constriction so that increases in right ventricular pressure were similar in those groups. One or 3 weeks after pulmonary artery constriction, the rats were sacrificed and hypertrophic response was estimated from right ventricular weight, myocyte width and protein content. In the 2-month-old rats, significant hypertrophy was observed 1 and 3 weeks after pulmonary artery constriction in terms of right ventricular weight/body weight (161% of controls at 1 week and 209% at 3 weeks), right ventricular weight/tibial length (161 and 251%. respectively) and right ventricular weight/left ventricular weight (174 and 211%. respectively). In the 7-month-old rats. significant hypertrophy was observed only at 3 weeks, but was not observed even at 3 weeks in the 18-month-old rats. The age-associated decrease and delay in hypertrophic response was also observed at cellular (myocyte width) and biochemical (protein content) levels. Thus, there is an age-associated diminution in the rate and extent of myocardial hypertrophy in the right ventricle. KEY WORDS:
Aging;
Pulmonary
artery
constriction;
Introduction Hemodynamic overload causes muscle hypertrophy in the left and right ventricles. The capacity for myocardial hypertrophy diminishes with age in pressure-overloaded left ventricles (Isoyama et al., 1987; 1991) and volume-overloaded left ventricles (Isoyama et al., 1988) and hearts (Walford et al., 1988). In the right ventricle, however, there are only a few data available concerning the effects of age on myocardial hypertrophic response. The effects of age on myocardial hypertrophic response in the right ventricle may differ from that in the left ventricle for the following reasons. First, the functional loads of the left and right ventricles grow (Peterson et al., 1989) and age in a different manner. Right ventricular growth is analogous to eccentric hypertrophy, whereas left ventricular growth represents a combination of eccentric and concentric hypertrophy during development after Please address all correspondence to: Tamotsu School of Medicine, l-l Seiryo-machi, Aobaku, 0022-2828/91/101177
+ 14$03.00/O
Takishima, Sendai980,
Myocyte
width;
Protein
synthesis.
birth (Anversa et al., 1980). In addition, physiologic aging after maturation causes different myocardial changes between the two ventricles. In the senescent human or animals, the left ventricle gains weight, but the right ventricle does not do so (Lakatta, 1979; Yin et al., 1980; 1982; Lakatta and Yin, 1982; Wei et al., 1984; Isoyama et al., 1987; 1988). Cellular hypertrophy and scar formation in the aged left ventricle are more prominent than those in the right ventricle (Anversa et al., 1986b; 1989). Second, aging shifts myosin heavy chain from the (Y- to the /3-isoform (Effron et al.. 1987; Boluyt et al., 1989), and causes functional changes: prolongation in contraction duration and decreases in shortening velocity (Rumberger and Timmerman, 1976; Yin et al., 1980; Capasso et al., 1982; Wei et al., 1984; 1986; Effron et al., 1987; Walford et al., 1988; Anversa et al., 1989). However, Anversa et al. reported that these age-related (1989) First Department Japan
of Internal
Medicine,
Tohoku
0 1991 Academic
University
Press Limited
1178
M.
Kuroha
functional changes were more pronounced in the left ventricle than in the right ventricle. Third, differences exist in the capacity to react and adapt to an increased work load or hormonal stimulation. For example, the extent of myocardial hypertrophy in response to running exercise (Anversa et al., 1982; 1983; 1986a), aortocaval shunt (Flaim et al., 1979) or thyroid hormone administration (Gerdes et al., 1987; Zierhut and Zimmer, 1989) was greater in the right ventricle than in the left ventricle. Thus, growing and aging after maturation do not always cause the same structural and functional changes in the right and left ventricles. Therefore, we attempted to study the course of right ventricular growth and to examine the effects of age on hypertrophic response produced by pressure-overload in the right ventricle. For this purpose, we determined the rate and extent of right ventricular myocardial hypertrophic response at the organ, cellular and biochemical levels after pulmonary artery constriction in rats of three age groups. Methods We used three different age groups of male Wistar rats: 2 months, 7 months and 18 months old, which correspond to developmental, young-adult and old stage of rats, respectively. In the first experiment, to estimate stress of the right ventricular free wall we studied right ventricular growth in the three age groups of rats (n = 6 in P-month-old rats; n = 7 in 7-month-old rats; n = 6 in 18-monthold rats). The heart was isolated, and the right ventricular weight and geometry were measured. In the second experiment, rats (n = 21 in 2-month-old rats; n = 20 in 7-monthold rats; n = 21 in 18-month-old rats) were divided into the three subgroups: a control group, a group of rats with pulmonary artery constriction for 1 week and a group of rats with pulmonary artery constriction for 3 weeks. In some of each subgroup of rats (n = 10 in 2-month-old rats; n = 11 in 7-month-old rats; n = 9 in l-month-old rats), while continuously monitoring right ventricular pressure, we created the pulmonary artery constriction after opening the thorax so as to obtain the same right ventricular pressure increase
et al.
immediately after pulmonary artery constriction. We examined the degree of right ventricular hypertrophic response to the same degree of pressure-overload at the organ, cellular and biochemical levels in the three age groups. In the experiment, rats were treated in accordance with the Declaration of Helsinki and the Guiding Principles in the Care and Use of Animals. Also, the experimental and animal care protocol was approved by the Animal Care Committee of the Tohoku University School of Medicine. Right ventricular growth and pressure-volume relationship Each rat was anesthetized with pentobarbital sodium (50mg/kg, i.p.). The thorax was opened, and the heart was arrestd with KC1 solution. The heart was removed and immersed in cold saline. A latex balloon with a double lumen polyethylene cannula (Polyethylene Tubing, SP-31, 0.50mm i.d., 0.80 mm o.d., Natsume Instrument Co., Ltd., Tokyo, Japan) was inserted into the right ventricle through a right atria1 incision and tied tightly at the atrioventricular groove with a silk thread. One lumen was used to measure the right ventricular pressure with a strain gauge transducer (Model TP-300T, Nihon Kohden Co., Ltd., Tokyo). Another lumen was used to change the volume of the right ventricle. Before the infusion of cold saline, the right ventricular pressure was made negative by manual compression, and the balloon was emptied. Cold saline was infused with stepwise increments of 0.05 ml up to 25 mmHg of the ventricular pressure. The presright sure-volume relationship was obtained three times, and the last relationship was measured, because the second and last relationships were reproducible. Right ventricular free wall was removed, and the surface area, weight and wall thickness were measured immediately after measurements of pressure-volume relationships. The surface area was measured planimetrically after extending the free wall without force on a flat plate. The thickness of the right ventricular free wall was calculated based on the assumption that specific gravity of the myocardial tissue is 1.06 (Mendez and Keys, 1960).
Aging
meet
on Right
Ventricular
Operative procedures for pulmonary artery constriction Each rat was anesthetized as described above and intubated endotracheally under direct visualization, as was done in our previous studies (Isoyama et al., 1987; 1989; 1991). In some rats of each group, a polyethylene cannula (Polyethylene Tubing, SP-31) was inserted into the right ventricular cavity through the right jugular vein to measure right ventricular pressure with a strain gauge transducer. Under controlled ventilation with room air (model 141, Princeton Medical Instruments Inc., Natick, MA, USA), the thorax was opened at the third intercostal space. The pulmonary artery was exposed and dissected free from the surrounding tissues. A surgical silk thread (3-O) was drawn under the pulmonary artery. A rigid tube was placed alongside the pulmonary artery. The rigid tube and the pulmonary artery were tied tightly together and the tube was rapidly removed. To create the same degree of right ventricular pressure increase in the three age groups we used rigid tubes of the following three different outer diameters: 1.4 mm for the 2-month-old rats, 2.Omm for the 7-month-old rats and 2.2mm for the 18-month-old rats. In a pilot study, we ascertained that right ventricular pressure increase immediately after pulmonary constriction was approximately 15 mmHg in each age group. We inflated the lung under 5 cmH*O of positive end-expiratory pressure to expel the air in the thorax, and the thorax was closed with a silk thread. Rats were housed in cages and were allowed free access to tap water and standard rat pellet chow. At either 1 or 3 weeks after pulmonary constriction, the rat was anesthetized again as described above and sacrificed. The heart was rapidly isolated. Fat and atria were removed, and the ventricles were divided into a right ventricular free wall portion and a left ventricular free wall and septal portion. Each portion was cut into two pieces after measurements of right and left ventricular weights. Each piece of the right and left ventricles was stored at - 8O’C until chemical analysis. Myocardial tissue protein was measured by the method of Lowry et al. (1951). Other pieces of the right and left ventricles were fixed in 10% formaline for histological examination. We also measured body weight and tibia1 length to
Hypertrophy
1179
normalize the right ventricular weight by body size (Yin et al., 1980; 1982; Lakatta and Yin, 1982; Wei et al., 1984; Isoyama et al., 1987; 1988; 1991). Frequency response of the pressure measurement system was estimated by transient response to a stepwise pressure change. The damping coefficient and undamped natural frequency were 0.56 and 41 Hz, respectively.
Histological
examination
Samples of right and left ventricles were cut parallel and perpendicular to the apex-base line. Sections were processed conventionally for histological examination (dehydrated in graded alcohol and embedded in paraffin), cut in 5 Frn sections, and stained with hematoxylin and eosin, as was done in previous studies (Rakusan et al., 1978; Isoyama et al., 1987; 1991). We chose longitudinally oriented myocytes in which the nucleus was positioned in the center of the myocyte, and measured widths of 50 myocytes in each heart. The mean values in each heart were averaged and compared between control and experimental rats of each age group. Statistical analysis Variables measured presented as are mean it: S.E.M. Analysis of variance was used to determine the effect of age or pulmonary artery constriction on the variables tested. Subsequent multiple t-test with Bonferroni’s corrections was used to determine specific effects of age or pulmonary artery constriction. Unpaired t-test was employed for statistical analysis between the two mean values before and after pulmonary artery constriction. A Pvalue less than 0.05 was taken as the level of significance (Snedecor and Cochran, 1980).
Results
Dimension
and pressure-volume relationship right ventricle
in the
Figure 1 shows the relationship between right ventricular pressure and volume (upper panel), and the relationship between pressure and volume normalized by right ventricular
1.
6 7 6
n
17.3 f .6 24.1 k .3** 24.0 + .4**
b (mm) 127k12 272k 9’: 299 + 14**
RVW (mg)
free wall length from the apex to RVW = right ventricular weight; and 7-month-old rats or between
139 * 12 273+12** 263+ 5**
R VSA (mm’)
geometry in the three age groups
11.6* .6 15.4 + .8** 16.1 k .3**
(mm)
a
Right ventricular
Values are mean + SEM. Abbreviations:a = right ventricular base line; RVSA = surface area of right ventricular free wall; *P
