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The effects of long-term pressure-overload and aging on the myocardium
Journal of Molecular and Cellular Cardiology (1981)
The
Effects
of Long-Term Pressure-Overload Aging on the Myocardium
Robert Defiartment
of
13, 471-488
J. Tomanek
and Jane
Anatomy and the Cardiouascular
Center,
and
M. Hovanec University of Iowa,
(Received 25 September 1980, accepted in revised form
13 January
U.S.A.
198 1)
R. J* TOMANEK AND J. M. HOVANEC. The Effects of Long-Term Pressure-Overload and Aging on the Myocardium. Journal of Molecular and Cellular CardioloQ (1981) 13, 471-488. The effects and interactions of aging and hypertension on the myocardium were studied in spontaneously hypertensive (SHR) and normotensive (WKY) rats. Cardiac hypertrophy developed in SHR during the first 7 months of life and was initially more marked in the subendocardium than in the subepicardium. Capillary density decreased in proportion to cellular enlargement but became normalized between 7 and 15 months when cell size no longer incqzased, suggesting a proliferation of capillaries during this time period. Between 15 and 22 to 23 months significant cellular enlargement occurred in both SHR and WKY despite the fact that blood pressure levels were not changed. Stereological analysis showed that mitochondria/myofibrils volume ratio in SHR decreased during the development of hypertrophy, continued to decrease as hypertrophy stabilized, but normalized during senescence. Residual bodies (lipofuscin granules), characteristic of aging in myocardial cells, accumulated more markedly in SHR than in WKY. Certain other focal alterations which also are associated with aging, i.e. cell membrane invaginations and increased numbers of caveolae, appeared earlier and became more widespread in SHR. In addition, long-term hypertension elicited alterations which were not associated with aging (honey-combed t-tubules, and mitochondrial and myofibril degeneration) . These data provide evidence for three condlusions regarding cardiac hypertrophy and hypertension, and aging in SHR. First, changes which are inevitable with aging are accentuated. Second, certain ultrastructural changes appear to be related to long-term hypertension rather than to the natural aging process. Finally, the relationship between *the volumes of contractile and energy producing organelles is modified by factors which are not clearly related to either aging or hypertrophy and hypertension. KEY WORDS: Hypertension;
Mitochondria; Spontaneously
Myofibrils; hypertensive
Ultrastructure; rat.
Capillary
density;
Hypertrophy;
Introduction Cardiac hypertrophy due to pressure overload is associated with changes which may contribute to heart failure. It has been suggested that some of the changes akin to cardiac hypertrophy are also present in the aged and constitute a complex of “myocardial wear” [21]. Both long term [6 to 7 months] hypertrophy and physiological aging in rats are characterized by parallel alterations, i.e., decreases in RNA concentrations, rates of RNA and protein synthesis, and rates of RNA and protein degradation [22]. Moreover, contraction time is prolonged [12, 16, 361 and endogenous catecholamine content is reduced [JO, 161 in sensescent rats. Similar alterations also occur during pressure-induced cardiac hypertrophy [30]. These data lead to the supposition that the persistence of pressure-overIoad, as evidenced in hypertensive disease, may accentuate aging of the myocardial cell and contribute to a deterioration in function. 0022-2828/81/050471+
18 $02.00/O
0
1981 Academic
Press Inc.
(London)
Limited
472
R. J. Tomanek
and J. M. Hovanec
The spontaneously hypertensive rat (SHR) re p resents an appropriate model for the study of myocardial aging with reference to long-term hypertrophy, since hypertension develops early in life and is akin to essential hypertension in man. Previous studies have shown that cardiac enlargement in SHR is characterized by decreases in mitochondria/myofibrils volume ratio and capillary density [17, 321, conditions which may contribute to a decline in cardiac performance. This communication includes data on the myocardial cell and its capillary bed from SHR and normotensive rats of various ages representing the entire life-span. The aim of the study was to identify specific cellular changes characteristic of longterm cardiac hypertrophy and to determine whether such changes contribute to aging of the myocardium. Quantitative methods, i.e., morphometry and stereology, as well as qualitative approaches, were utilized. In addition to age group comparisons, we compared the subepi- and subendo-cardial regions of the myocardium since previous work indicated that the subendocardium is prone to more marked changes in SHR [32, 331. Materials
and
Methods
Data were obtained from male spontaneously hypertensive (SHR) and Wistar Kyoto normotensive (WKY) rats aged 1,4, 7, 15 and 22 to 23 months. From the time of weaning all animals were maintained in the same room with a 12 h light cycle, and received Purina rat chow and water ad libitum. Arterial blood pressures were determined by the tail-cuff method using a Technilab Instruments apparatus and a Beckman Dynogram recorder. The weekly recordings were obtained during the early afternoon and the median of at least six measurements was recorded for each session. The mean value for the 3 weeks preceding the animal being killed was calculated and was used to compute the group means reported in this paper. Tissue preparation At the time of being killed the animal was anesthetized with sodium pentobarbital (Nembutal), 50 mg/kg (i.p.), and ventilated via a tracheal cannula connected to a Harvard Respirator. Following cardiac arrest with Procaine (10 mg), the coronary vasculature was flushed with Locke’s solution and the heart fixed by retrograde perfusion via the ascending aorta with a 1.5% glutaraldehyde solution buffered with sodium cacodylate. Details of the perfusion procedure have been previously described [32, 341. After the heart and ventricles were weighed, specimens from the subepi- and subendo-cardium were excised, postfixed in 1 o/0 OsO,, dehydrated, and embedded in Epon. Cell size and capillary
density
Myocyte cross-sectional area measurements and capillary density were determined from projected images of cross-sectional fields of l-pm sections stained with Richardson’s solution [32, 331. Outlines of myocytes which contained a nucleus were traced and subsequently their cross-sectional areas measured with a Talos digitizer interfaced with a Monroe calculator. The number of capillaries lying in a prescribed area was determined in several fields. Myocyte cross-sectional areas were measured in at least 25 cells; capillary density was based on fields which included approximately 400 capillaries.
Hypertension,
Aging
and Myocardium
Ultrastructural
173
analysis
Cross-sections from two specimens from each myocardial region were used for morphometric data. The relative cell volumes of mitochondria, myofibrils, and sarcoplasm (all other intracellular components) were estimated by point-counting using a line grid with intercepts every 6 mm [32, 331. At least nine cells from each specimen were photographed and analyzed at a final magnification of x 18 000 to 21 000. This method has been shown to be highly reproducible, with an error of <4%. Mitochondrial surface/volume ratios were determined by measuring the length of the outer mitochondrial membrane and the area of the mitochondrial profile directly from electron micrographs with the aid of the digitizer. Based on the assumption of random average orientation in space, the mitochondrial surface to volume ratio (S,,/V,it) can be obtained from two dimensional micrographs according to the relationship: S/V = L/A, where L is the length of the outer mitochondrial membrane and A is the area of the mitochondrial profile. This is similar to the relationship used in point counting stereology [18], but does not necessitate adjustments for distance between grid lines. Qualitative data were based on micrographs of both longitudinally and transversely sectioned specimens. Statistical Analysis of variance (P < 0.05) between
analysis
and the Bonferroni t test were employed to determine the various experimental and control groups.
significance
Results Ventricular
mass and capillary density
Blood pressure and left ventricular weight data are illustrated in Figure 1. At 6 weeks of age systolic blood pressure in SHR, while significantly higher than that of WKY normotensives, had not as yet attained hypertensive levels. A gradual rise in blood pressure was evident up to about 4 months, with most SHR reaching hypertensive levels between 8 and 10 weeks. Left ventricular mass in SHR, as demonstrated by left ventricular weight/body weight (LVWjBW) ratio, preceded the attainment of hypertensive blood pressure levels. The differences between SHR and WKY for this index were 170/, at 1 month and 379/o at 4 months. A comparison between the changes in left ventricular weights (Figure 2) indicates that absolute left ventricular weight was significantly higher in SHR than in WKY at all ages except 15 months. The data indicate that in both normotensive and hypertensive rats, stabilization of growth of the left ventricle characterized the period between the seventh and 15th month of life. The magnitude of cardiac hypertrophy in the subepiand subendo-cardial regions of SHR are contrasted in Figure 3, where the effect of mean cross-sectional area of myocytes on capillary density is illustrated. The data are summarized as follows: (1) the major increase in cell size during the first 4 months of life in SHR occurred in the subendocardium; (2) preferential hypertrophy of the subepicardial myocytes between 4 and 7 months nearly equated cell size in the two regions; (3) myocyte growth ceased in SHR between 7 and 15 months, after which time a marked cellular hypertrophy developed; (4) capillary density was, in general
474
R. J. Tomanek
and
J. M.
Hovanec
(6)
Age (months)
FIGURE 1. Systolic blood pressure and left ventricular weight (mg)/body weight (g) in spontaneously hypertensive (SHR) and normotensive Wistar Kyoto (WKY) rats. Values are means + S.E.M. Number of animals is indicated in parentheses. All differences between SHR and WKY of similar age are significant (P I 0.05). Note: The earliest blood pressure data are from 1.5-month-old-rats (tail cuff recordings cannot be obtained from smaller rats), while the earliest LVW/BW data are based on l-month-old animals.
related inversely to cell size, except at 15 months when this parameter became normalized in SHR and corresponded to a period of stabilized myocardial hyperto trophy (i.e., no further increase in cell size). The data on cell size, in addition supporting the LVW/SW data, show that mean fiber area increased throughout life in WKY normotensive animals. The increase was particularly marked during the late period of life when cellular growth paralleled that of SHR. As indicated by the data in Table 1, mean fiber area and capillary density are not statistically different in l-month-old SHR and WKY. Therefore, most of the accelerated increased in fiber cross-sectional area and the decrement in capillary density occurred after the prehypertensive period. Ultrastructural
alterations
Relative myocardial cell fractions were similar in l-month-old SHR and WKY (Table 1). Therefore, the changes evident in SHR at 4 months occurred during the development of hypertension. The effects of hypertension on relative myocardial cell fractions were most notable in the subendocardial layer of the myocardium
Hypertension,
Aging
and Myocardium
475
(Table 2). An increase in relative myofibril volume and a decrease in relative mitochondrial volume occurred by the fourth month in SHR and persisted until 15 months. These changes were most marked in the 15-month-old group at which time they were also evident in the subepicardium. Aging did not appear to influence these cellular fractions, since relative myofibril and mitochondrial volumes in WKY were similar at all ages. Since the alterations in these organelle volumes were most
P(6)
out, I
,
,
4
7
,
,
‘2.
,
15
22-
Age (months)
FIGURE
2. Body weight and absolute left ventricular weight in WKY and SHR rats of various are means f S.E.M. Number of animals is indicated in parentheses. Significant differences between WKY and SHR at a given age are indicated by an asterisk.
ages. Values (P < 0.05)
TABLE
1. Myocardial WKY rats
cell
and
capillary
data
from
prehypertensive
(l-month-old)
WKY Subepicardium
Mean fiber Capillary
area
134 f
(pm2)
5069
density
194
34.8 & 52.6 * 12.6 & 0.66 f
1.5 1.3 0.5 0.04
and
SHR Subendocardium
10
&
SHR
135 f 4903
+
28 61
Subepicardium 137 f 4914
f
Subendocardium
6
149 f
126
4638
5
16 177
(number/mmt) Cell volume Mitochondria Myofibrils Sarcoplasm
(%)
Mitochondria/myofibrils volume ratio All v&~es are means i differences between WKY M.C.C.
S.E.M.
and
and SHR
are based on are statistically
35.9 * 1.7 51.1 f 1.2 13.0 & 2.7
32.4 & 0.9 52.3 + 0.9 14.5 f 0.8
36.0 f 1.8 54.3 + 0.8 11.3 f 1.8
0.65
0.62
0.63
i
0.02
four WKY significant
f
and five SHR (P > 0.05).
0.02
animals.
None
+ 0.01
of the
R
476
R. J. Tomanek
650
and
J. M.
Hovanec
-
El 04
I 7
15
22-23
4
7
15
22-23
Age (months) FIGURE 3. Mean fiber cross-sectional area and capillary density in spontaneously hypertensive (SHR) and normotensive Wistar Kyoto (WKY) rats. The subepicardial (left) and subendocardial (right) regions were evaluated separately. Values arc means & S.E.M. Number of animals is indicated in parentheses. All differences between SHR and WKY of similar age are significant (P IO.05) except capillary density in 15-month-old animals. (ma) SHR; ( HA) WKY.
pronounced at 15 months, despite the fact that myocyte growth was stabilized between 7 and 15 months, the data indicate that the relative volumes of mitochondria and myofibrils were not directly influenced by hypertrophy per se. Moreover, further cellular growth between 15 and 22 to 23 months was accompanied by an increase in relative mitochondrial volume compared to the 15-month group, while in WKY the mitochondrial cell fraction remained normal despite a substantial cellular growth, The quantitative relationship between these organelles is illustrated by the ratio: mitochondria/myofibrils volume (Figure 4). In the subendocardium of 4- and 7-month-old animals the ratio was sign’ificantly lower in SHR compared to WKY. At 15 months of age the decrement in this ratio was most marked, i.e., a 30% and 20% decline in the subendocardium and the subepicardium respectively. In contrast, in the oldest SHR the ratio was virtually normalized in the subendocardium and increased in the subepicardium to near-normal values. These data suggest that mitochondria/myofibrils volume ratio was affected by factors other than hypertrophy. Neither hypertension nor aging significantly altered mitochondrial density, i.e., number of mitochondria/lOO pm2 (Table 3), indicating that mitochondrial proliferation was proportional to the increase in cell mass. Surface to volume ratio,
(a)
Group
WKY (8) SHR (7) P value
months
of aging
effects
are means
& S.E.M.
WKY (5) SHR (7) P value
22 to 23 months
WKY (7) SHR (6) P value
15 months
WKY (7) SHR (7) P value
7 months
4
2. The
All values
TABLE
N.S.
* *
N.S.
0.4 0.7
f 0.9 rfi- 0.6
31.8 f- 1.2 28.7 & 0.8 < 0.025
30.6 f 1.2 25.6 & 0.6 < 0.002
30.3 29.4
30.7 28.5
Mitochondria
on myocardial
(%
N.S.
-& 0.8 f 1.0
N.S.
-+ 1.0 f 1.4
62.5 61.1 N.S.
* $
1.5 1.3
60.7 & 1.3 64.9 f 0.7 to.01
60.5 61.9
63.1 63.3
MyoJibrils
Subepicardium
cell fractions
of total
5.8 f 0.9 10.1 * 0.8 < 0.002
N.S.
8.7 i 1.0 9.5 & 0.9
N.S.
9.2 5 0.7 8.7 & 0.6
N.S.
6.2 & 1.2 8.5 4 1.2
Sarcoplasm
cell volume)
31.2 29.8
N.S.
f f
0.9 1.4
30.9 f 0.8 25.0 & 0.8 < 0.0001
29.9 f 1.0 27.2 + 0.6 < 0.04
32.7 f 1.3 29.5 f 0.4 < 0.04
Mitochondria
in spontaneously
(SHR)
61.1 61.1
N.S.
f *
1.1 1.6
59.9 * 0.9 67.5 f 0.9 < 0.0001
59.2 f 0.6 64.2 & 0.9 < 0.001
61.8 f 1.5 66.5 4 0.6 < 0.04
Myofibrils
Subendocardium
hypertensive
and
0.8 0.7
1.0 0.7
N.S.
7.7 f 1.1 9.1 + 0.5
N.S.
8.2 f 7.5 f
N.S.
10.9 f 8.6 +
N.S.
5.4 -& 1.0 4.5 + 1.0
Sarcoplasm
normotensive
(WKY)
rats
478
R. J. Tomanek
and
J. M.
Hovanec
0.46
5
0.40
2
0.46 0.44 0.42 0.40 0.36 0.36 4
15
7
22-23
Age (months) FIGURE 4. Mitochondria/myofibrils and subendocardium (bottom). parentheses. Significant differences asterisks.
volume ratio in cells comprising the subepicardium of animals is indicated Values are means k S.E.M. Number (P S 0.05) between SHR and WKY of similar age are indicated
II
I 4
I
I 15
I 7 Age
FIGURE myocardial the l-month
5. Mean values cells. All differences age group.
(&
s.E.M.)
between
(top) in by
1 22-
3
(mocths)
for the numbers SHR and WKY
of residual bodies in cross are statistically significant
sectional fields of (P I 0.05) except
Hypertension,
Aging
and
Myocardium
479
on the other hand, was higher in the oldest SHR than in their age-corresponding WKY. The increase in this ratio may be due to conformational changes in the organelles. Lipofuscin granules (residual bodies) were seen in the perinuclear region of myocytes from all age groups. They became more evident with age in nonperinuclear regions of both WKY and SHR. A numerical increase with age is illustrated in Figure 5. These data are based on counts/cross-sectional cell areas obtained from electron micrographs. It is evident that residual bodies accumulated at a higher rate in SHR than in WKY. In SHR residual bodies increased substantially in the two oldest groups (Figures 6 to 8). Thus, the hearts of 22- to 23-month-old SHR contained numerous residual bodies (Figure 7) throughout the cell, while such structures in similarly aged WKY were less common. We also observed these structures in capillary endothelial cells in the two oldest groups of hypertensive and normotensive animals (Figure 8). Again, their occurrence was much more common in SHR, particularly in the oldest group studied. Some focal alterations, while infrequent, appeared to be related to aging as evidenced by their appearance in the oldest WKY rats. Myelin figures, invaginations of the cell membrane, dilatations of the non-specialized junctions of the intercalated disc and adjacent t-tubules, and increased numbers of subsarcolemmal vesicles could occasionally be found in the oldest WKY animals. These focal alterations were detected earlier in SHR. The enhancement of subsarcolemmal vesicles was usually limited to the oldest animals while myelin figures occurred earlier. We also noted structural modifications in the two oldest SHR groups which might be attributed to long-term hypertension and hypertrophy. These alterations were extremely rare or absent in the oldest WKY rats. Mitochondrial changes included occasional large spherical inclusions (Figure 9) and focal mitochondrial degeneration (Figure 10). The latter was restricted to a few adjacent mitochondria which were associated with lysosomes. In both 15- and 22- to 23-month-old SHR “honeycombed” t-tubules (Figure 11) were common, but rare in WKY. While irregular Z lines were not uncommon in younger SHR with developed hypertension and
TABLE
3. Comparisons tensive (SHR)
of mitochondrial and normotensive
indices in young (WKY) rats
Number/l00
pm2
and
P
old
spontaneously
Surface
( t*m2)
Volume
( ym3)
hyper-
P
Subepicardium 4 month 4 months
WKY SHR
(5) (6)
22 to 23 month 22 to 23 month
WKY SHR
(5) (6)
101 & 6 92 5 + 91 5 5 97 5 9
N.S.
7.46 8.13 6.54 8.85
& + f &
0.44 0.26
N.S.
0.26 0.62
0.47 0.67
N.S.
Subendocardium 4 month 4 month
WKY SHR
(5) (6)
22 to 23 month 22 to 23 month
All values
are means
WKY SHR
(5) (7)
& S.E.M. Number
101 + 8 98 & 8
N.S.
7.69 8.20
91 * 5 103 & 8
N.S.
7.19 8.70
of animals
is indicated
in parentheses.
5 + & i
0.47 0.38
to.02
480
R. J. Tomanek
and
J. M.
Hovanec
1) WKY illustrating a relatively normal morphology. FIGURE 6. Micrograph from an old (23-montl of faint N bands (arrows) are Occasional membrane fragments in t-tubules (T) and the appearance here) are common. x 20 000. observed in old normotensive WKY. Residual bo dies (not shown (23-month SHR). These structures, FIGURE 7. Four residual bodies (arrows) are seen in the field throughout the cell with aging and limited to the perinuclear region in young anim als, accumulate are common in hypertensive animals. most markedly in the SHR. Irregular Z lines in SC,me sarcomeres x 11 000. cell from a 15-month-old SHR. FIGURE 8. Residual bodies (arrows) withil n an endothelial x 42 000.
Hypertension,
Aging
and Myocardium
481
hypertrophy, Z line streaming and focal myofibrillar disruption occurred only in the two oldest SHR groups (Figure 12). A focal fibrosis was characteristic of the oldest SHR group (Figure 13), but also could be found to a lesser extent in the 15-month-SHR. Discussion This study provides evidence for three conclusions regarding the myocardial cell in the spontaneously hypertensive rat. First, changes characteristic of aging appear earlier and are accentuated. Second, other ultrastructural abnormalities appear to be the consequences of long-term hypertrophy and hypertension rather than events of the natural aging process. Finally, the relationship between the volumes of contractile and energy producing organelles is modified by factors which are not clearly related to either aging or hypertension and hypertrophy. While left ventricular mass was greater in l-month-old SHR than in their age group WKY, cell size was not altered. Moreover, capillary density and the relative cell fractions of mitochondria, myofibrils and sarcoplasm were similar to WKY. We therefore concluded that the larger left ventricular mass in SHR at 1 month is not due to cellular hypertrophy, but more likely is the consequence of a greater number of cells. Accordingly, the changes (with the exception of LVW) observed at subsequent ages developed during the hypertensive period. Our data demonstrate that the marked increase in myocardial cell size between the first and seventh months of life in SHR is not accompanied by an adequate increase in the absolute number of capillaries. Thus, a decrement in capillary’ density characterizes this stage of developing hypertrophy. Since cell size became stabilized and capillary density normalized by the 15th month, it is evident that capillary growth was sufficient to overcome the decrement characteristic of developing hypertrophy. On the other hand, cellular growth in WKY was slow during this time and capillary density was maintained at a consistent level. Both the data on cell size and absolute left ventricular weight support the conclusion that hypertrophy became stabilized between 7 and 15 months. In contrast to the stage of developing hypertrophy, the cellular hypertrophy which occurred during senescence may be an age-associated phenomenon since the increase in cell size was almost parallel in both SHR and WKY. The sharp decrease in capillary density in SHR at this time, which reduced this parameter to levels typical of the earlier stage of hypertrophy, indicates an inadequate proliferation of capillaries. Since capillary density remained constant in the subepicardium of WKY, it would appear that age was not a factor in limiting the growth of the vascular bed. The increase in cell size during senescence, however, was not accompanied by a significant increase in absolute ventricular weight, a finding which suggests the possibility of a decrease in cell number.
Aging We noted consistent alterations in the oldest SHR and WKY which are similar to those found in senescent Sprague-Dawley rats [34]. The accumulation of residual bodies (lipofuscin), generally considered an aging phenomenon in non-dividing cells, was conspicuous and clearly increased with age. This accumulation may be due to concomitant increases in the production of primary lysosomes and their transformation to residual bodies as a consequence of enhanced autophagy [35].
482
R. J. Tomanek
and
J. M.
Hovanec
FIGURE 9. A large dense spherical mitochondrial inclusion (arrow) represents an occasional finding in old SHR. Several osmophilic bodies and a lysosome-like structure (ly) are also seen between myofibrils. x 16 000. FIGURE 10. Focal degeneration of mitochondria (arrows) in old SHR (23-month) occurs in conjunction with lysosomes (1~). Note that a lysosomal figure also appears in the capillary (ca) endothelial cell. Mitochondria adjacent to those which show degenerative changes appear normal. x 14 500. FIGURE 11. “Honeycombed” appearance of transverse tubule (between arrows), common in old SHR. Such structures occur between myofibrils near Z lines and show evidence of a glycocalyx. x 30 000.
Hypertension,
Aging
and
Myocardium
483
Whether the induction of enhanced lysosomal activity is due to a feedback system from digested material or to a genetically programmed phenomenon has not been established. However, the former appears to be more likely since lysosomal activation and residual bodies are particularly occurs in various pathological conditions, numerous in diseased hearts [14]. 0 ur data clearly indicate that residual bodies accumulate more rapidly in SHR than in WKY myocardial cells, which suggests that this aging characteristic is related to factors which are also present during hypertension and/or cardiac hypertrophy. An increase in the accumulation of residual bodies and membrane fragments (including myelin figures) in myocardial cells of SHR appears to be consistent with the idea of “wear and tear” relating to enhanced cellular work [,?I]. It is not clear if exocytosis of residual bodies is limited and allows for the accumulation of these products. However, it has been shown that residual bodies undergo dissolution and exocytosis after dimethylaminoethyl p-chlorophenoxyacetate treatment and that pigment residuals are incorporated into phagocytic cells and the capillary wall and lumen. While our observations indicate the presence of residual bodies and dense bodies in capillary endothelial cells of old animals, it is not certain whether they originate in endothelial or myocardial cells. Other age-associated alterations are also consistent with those found in senescent Sprague-Dawley rats [34]. These structural modifications involve membrane systems and are, for the most part, similar to those which we found in both normotensive and hypertensive rats exposed to chronic altitude hypoxia [27]. Cell membrane invaginations and an increase in the number of caveolae (subsarcolemmal vesicles), characteristic of senescent hearts, were also typical of all animals subjected to hypoxia. While membrane irregularities were found in degenerating myocytes of human hypertrophied hearts, they usually occurred in conjunction with other degenerative changes [8]. In contrast, the surface alterations described here were usually characteristic of cells which did not show any degenerative changes. Since cellular hypoxia in old animals has not been demonstrated, the reasons for such structural adaptations are elusive. It is, however, clear that these membrane alterations, as well as those involving the intercalated disc, have an earlier onset and become accentuated in SHR. Long-term
hypertrophy
In contrast to age-associated changes, we found evidence of structural abnormalities which appeared only after long-term pressure-overload and cardiac hypertrophy, and were absent or rare in the oldest WKY. Such alterations were also absent in senescent Sprague-Dawley rat hearts [34]. T-tubule proliferation giving rise to a honey combed (labyrinthine) appearance, typical of the two oldest SHR groups, has also been reported in numerous disorders of skeletal muscle [19]. We concluded that these structures are in fact t-tubules because of the presence of a glycocalyx. The formation of such membrane networks in skeletal muscle has been considered to provide a membrane source for autophagic vacuoles [7]. Although the stimulus for these modifications in hearts of old SHR has yet to be elucidated, they may be related to degenerative events. Focal alterations of myofibrils also appear to correspond to the long-term effects of functional overload. Since they do not characterize hearts of younger SHR, these alterations appear to be related to the persistence of the increased afterload imposed by elevated blood pressure.
R. J. Tomanek
and
J. M.
Hovanec
12. Myofibrillar disruption with streaming of Z line substance (between arrows) in an 1FIGURE (22-month). Such regions are usually characterized by occasional polyribosomes and sn la11 old SHR profiles. mi tochondrial x 16 000. 13. Collagen deposition near a myocardial cell and capillary in a 23-month-old SH R. F ‘IGURE fibroblast processes and collagen fibrils (co) are evident. A narrow process of a myocard NIlI merous ial numerous subsarcolemmal vesicles (arrows) is seen at the bottom of the field. cell with x 8000.
Hypertension,
Aging
and
Myocardium
485
These changes are not regarded as general characteristics of various types of hypertrophy. Ultrastructural changes in patients with ventricular hypertrophy are focal, with only 20% of the specimens showing degenerative changes [ZO]; more bizarre arrangements of muscle cells may be related to specific types of hypertrophy, e.g. idiopathic hypertrophic subaortic stenosis [9]. While widened and irregular Z lines and altered intercalated discs are considered characteristic of experimental myocardial hypertrophy [3], it appears the other qualitative differences between normal growth and hypertrophy are not marked and consistent. The present study suggests that certain ultrastructural changes become characteristic in the SHR myocardial cell only after long-term hypertrophy. Since they become evident during a period of stabilized myocyte growth, they are not related to the magnitude of hypertrophy. Moreover, their absence or rare occurrence in sensescent WKY as well as Sprague-Dawley rats [34] suggests that they do not characterize the aging processes. While it appears that certain ultrastructural alterations are associated with long-term hypertension, one should be cautious in extrapolating these findings based on a model of genetic hypertension to other models of cardiac hypertrophy. Mitochondrial
and myojibril
volumes
Anversa et al. [Z] noted that the most significant changes in moderate cardiac hypertrophy are quantitative rather than qualitative. The decrease in mitochondrial volume in hypertrophic hearts of SHR is consistent with a number of studies on pressure overload in rats [17, 2.5, 32, 371, rabbits [Z, II]. and dogs [4]. Our work, however, provides evidence that the quantitative relationship between mitochondrial and myofibrillar volumes is related to factors other than cell mass. During the first 7 months of life, mitochondria/myofibrils ratio appears to reflect the magnitude of hypertrophy, as indicated by the data on the subepi- and subendo-cardium, but fails to its lowest value at 15 months despite the stabilization of hypertrophy. Thus, the further decline in this ratio cannot be attributed to cellular growth, but rather appears to be related to the turnover rates of the two organelles. It has been suggested that these organelles are regulated by separate mechanisms [27]. While these mechanisms are not clear, the findings in the 15- and 22- to 23-month-old SHR are not so surprising, since a variety of chemicaf and physiological factors may come into play during aging and a given stage of hypertrophy. We considered the possibility that aging might limit mitochondrial growth. Yet mitochondria/myofibrils volume ratio became normalized during senescence even though further cellular hypertrophy occurred. One possible explanation for the normalization of mitochondrial volume during old age may be the loss of some contractiIe material as suggested by our observations. Thus, the degradation of myofilaments, although less than the synthesis of these organelles, could offset the imbalance between mitochondrial and myofibril growth. The alternative explanation is that mitochondrial growth is accelerated during this late phase of hypertrophy. The increase in surface/volume ratio of mitochondria in old SHR may be an adaptation aimed at enhanced energy production as noted in studies on pressure-overload in rabbits [Z] and dogs [3]. As shown by our data on normotensive WKY, as well as by another study on rats [15], mitochondrial volume is not appreciably altered by age. In contrast the relative volume of this organelle has been reported to decrease in senescent mice [13] and increase in Syrian hamsters [28]. Such inconsistencies could be due to differences in age or species variation.
486
R. J. Tomanek
and
J. M.
Hovanec
General discussion In spite of the fact that left ventricular mass was slightly larger in SHR than in WKY aged 1 month, cellular hypertrophy and a decrement in capillary density were not evident at this time. Thus, our data suggest that hypertrophy develops primarily during the period when blood pressure becomes elevated. Yet the reasons underlying the various responses of the myocardial cell during different stages of life are not apparent. Increased afterload due to the development of hypertension is not the sole determinant of cardiac hypertrophy, because pressure-overload in SHR can be prevented without normalizing cardiac mass [29]. Since blood pressure levels did not differ significantly in SHR once the peak hypertension levels were attained, neither the stabilization of the hypertrophy nor the additional growth which occurred late in life can be explained exclusively by pressure-overload. In addition, the data on cell size in old normotensive rats indicates a significant myocardial growth in the absence of blood pressure changes. Consistent with these findings is the evidence that modification of cardiac hypertrophy in SHR is related to drug specificity rather than to the hypotensive effects of the agents [32, 331. This rationale leads to the consideration of neural or humoral factors which might be altered during senescence. While catecholamines are known to induce cardiac hypertrophy [23, 241, cardiac enlargement during old age can hardly be attributed to increases in norepinephrine since the endogenous content of this catecholamine declines in senescent rats [5, 101. M oreover, sympathetic neural activity does not appear to contribute directly to hypertrophy in SHR since neonatal immunosympathectomy does not prevent its development [6]. However, recent evidence suggests that neural influences play a selective role in myocyte growth. Cellular enlargement in immunosympathectomized SHR, in contrast to SHR with intact sympathetic nerves, is characterized by a proportional growth of mitochondria and myofibrils [26]. While cardiac hypertrophy in the senescent SHR cannot be explained by decreases in endogenous norepinephrine levels, the normalization of mitochondria/myofibrils ratio, as noted in the subendocardium, may be related to a decline in this catecholamine. Whether the restoration of this ratio to control (normotensive) values is of functional significance is uncertain, since other ultrastructural alterations are considerable and may contribute to the decline in the intrinsic mechanical properties of the heart. Acknowledgements
The authors appreciate the technical assistance of MS B. Carroll and MS Shirley Velle. This work was supported by grant HL 18629 from the National Institutes Health.
La of
REFERENCES 1.
2.
3.
ANVERSA, P., LOUD, A. V. & VITALI-MAZZA, L. Morphometry and radiography of early hypertrophic changes in the ventricular myocardium of aduIt rat. An electron microscopic study. Laboratory Investigation 35, 475-483 (1976). ANVERSA, P., VITALI-MAZZA, L., VISIOLI, 0. & MARCHETTI, G. Experimental cardiac hypertrophy: A quantitative ultrastructural study in the compensatory state. Journal of Molecular and Cellular Cardiology 31, 2 13-2 2 7 ( 197 1) . BISHOP, S. P. Structural alterations of the myocardium induced by chronic work overload. In Comparative Pathophvsiolopy of Circulatory Disturbances. C. M. Bloor, Ed., pp. 289-314. New York: Plenum Press (1972).
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6.
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10. 11.
12. 13. 14.
15. 16.
17. 18. 19. 20.
21. 22.
23. 24.
25.
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and
Myocardium
487
BISHOP, S. P. & COLE, C. R. Ultrastructural changes in the canine myocardium with right ventricular hypertrophy and congestive heart failure. Laboratory Investigation 20, 219-229 (1969). BURKARD, W. P., GEY, K. F. & PLETSCHER, A. Alteration of the catecholamine metabolism of the rat heart in old age. Proceedings of the 7th International Congress on Gerontology, pp. 237-239. 26 June to 2 July (1966). CUTILLETTA, A. F., ERINOFF, L., HELLER, A., Low, J. & OPARIL, S. Development of left ventricular hypertrophy in young spontaneously hypertensive rats after peripheral sympathectomy. Circulation Research 40, 428-434 (1977). ENGEL, A. G. & MACDONALD, R. D. Ultrastructural reactions in muscle disease and their light-microscopic correlates. In Muscle Diseases, J. N. Walton, N. Caral & 6. Scarlato, Eds, pp. 71-78. Amsterdam: Excerpta Medica (1970). FERRANS, V. J., BUJA, L. M. & MARON, B. J. Sarcolemmal alterations in cardiac hypertrophy and degeneration. Recent Advances in Studies on Cardiac Structure and Metabolism 9, 395-419 (1976). FERRANS, V. J., MORROW, A. G. & ROBERTS, W. C. Myocardial ultrastructure in idiopathic hypertrophic subaortic stenosis. A study of operatively excised left ventricular outflow tract muscle in 14 patients. Circulation 95, 769-792 (1972). GEY, K. F., BURKARD, W. P. & PLETSCHER, A. Variation of the norepinephrine metabolism of the rat heart with age. Gerontologia 11, l-l 1 (1965). GOLSTEIN, M. A., SORDAHL, L. A. & SCHWARTZ, A. Ultrastructural analysis of left ventricular hypertrophy in rabbits. Journal of Molecular and Cellular Cardiology 6, 265-273 (1974). HELLER, L. J. & WHITEHORN, W. V. Age-associated alterations in myocardial contractile properties. American Journal of Physiology 222, 1613-1619 (1972). HERBENER, G. H. A morphometric study of age-dependent changes in mitochondrial populations of mouse liver and heart. Journal of Gerontology 31, 8-12 (1976). HIBBS, R. G., FERRANS, V. J., WALSH, J. J. & BURGH, G. E. Electron microscopic observations on lysosomes and related cytoplasmic components of normal and pathological cardiac muscle. Anatomical Record 153, 173-185 (1965). KMENT, V. A., LEIBETSEDER, J. & BURGER, H. Gerontologische Untersuchungen an Rattenherzmitochondrien. Gerontologia 12, 193-199 (1966). LAKATTA, E. G., GERSTENBLITH, G., ANGELL, C. S., SHOCK, N. W. & WEISFELDT, M. L. Prolonged contraction duration in aged myocardium. Journal of Clinical Investigation 55, 61-68 (1975). LUND, D. D. & TOMANEK, R. J. Myocardial morphology in spontaneously hypertensive and aortic-constricted rats. American Journal of Anatomy 152, 141-152 (1978). MCCALLISTER, L. P. & PAGE, E. Effects of thyroxin on ultrastructure of rat myocardial cells : A stereological study. Journal of Ultrastructure Research 42, 136-I 55 (1973). MAIR, W. G. P. & TOME, F. M. S. Atlas of the Ultrastructure of Diseased Human Muscle. Baltimore : Williams and Wilkins (1972). MARON, B. J., FERRANS, V. J. & JONES, M. The spectrum of degenerative changes in hypertrophied human cardiac muscle cells: An ultrastructural study. Recent Advances in Studies on Cardiac Structure Metqbolism 8, 447466 (1975). MEERSON, F. Z. The myocardium in hyperfunction, hypertrophy, and heart failure. Circulation Research 24-25 (Suppl II) 1-163 (1969). MEERSON, F. Z., JAVICH, M. P. & LERMAN, M. I. Decrease in the rate of RNA and protein synthesis and degradation in the myocardium under long-term compensatory hyperfunction and on aging. Journal of Molecular and Cellular Cardiology 10, 145-159 (1978). MUELLER, R. A. & AXELROD, J. Abnormal cardiac norepinephrine storage in isoproterenol-treated rats. Circulation Research 23, 77 l-778 (1968). MUELLER, E. A., GRIFFIN, W. S. T. & WILDENTHAL, K. Isoproterenol-induced Cardiomyopathy: Changes in cardiac enzymes and protection by methylprednisolone. Journal of Molecular and Cellular Cardiology 9, 565-578 (1977). PAGE, E. & MCCALLISTER, L. P. Quantitative electron microscopic description of heart muscle cells. Application to normal, hypertrophied and thyroxin-stimulated hearts. American Journal of Cardiology 31, 172 (1973).
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27. 28. 29. 30.
31.
32.
33.
34. 35. 36.
37.
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and J. M. Hovanec
PAGE, E. & OPARIL, S. Effect of peripheral sympathectomy of left ventricular ultrastructure in young spontaneously hypertensive rats. Journal of Molecular and Cellular Cardiology 10, 301-315 (1978). RABINOWITZ, M. Overview on pathogenesis of cardiac hypertrophy. Circulation Research 34-35 (Suppl II) 3-11 (1974). SACHES, H. G., COLGAN, J. A. & LAZARUS, M. L. Ultrastructure of the aging myocardium: A morphometric approach. American Journal of Anatomy 150, 63-72 (1978). SEN, S., TARAZI, R. C., KHARALLAH, P. A. & BIJMPUS, F. M. Cardiac hypertrophy in spontaneously hypertensive rats. Circulation Research 35, 775-781 (1974). SPANN, JR, J. I;., BUCCINO, R. A., SONNENBLICK, E. H. & BRAUNWALD, E. Contractile state of cardiac muscle obtained from cats with experimentally produced ventricular hypertrophy and heart failure. Circulation Research 21, 341-354 (1967). SPOERRI, P. E., GLEES, P. & CHAZZAW, E. E. Accumulation of lipofuscin in the myocardium of senile guinea-pigs: dissolution and removal of lipofuscin following dimethylamninoethyl p-chlorphenoxyacetate administration. An electron microscope study. Mechanisms of Aging and Development 3, 3 1 l-32 1 (1974). TOMANEK, R. J. The role of prevention or relief of pressure overload on the myocardial cell of the spontaneously hypertensive rat. A morphometric and stereologic study. Laboratory Investigation 40, 83-91 (1979). TOMANEK, R. J., DAVIS, J. W. & ANDERSON, S. C. The effects of alpha-methyldopa on cardiac hypertrophy in spontaneously hypertensive rats: Ultrastructural, stereological and morphometric analysis. Cardiovascular Research 13, 173-182 (1979). TOMANEK, R. J. & KARLSSON, U. L. Myocardial ultrastructure of young and senescent rats. Journal of Ultrastructural Research 42, 201-220 (1973). TRAVIS, D. F. & TRAVIS, A. Ultrastructural changes in the left ventricular rat myocardial cells with age. Journal of Ultrastructural Research 39, 124-148 (1972). WEISFELDT, M. L., WRIGHT, J. R., SHREINER, D. P., LAKATTA, E. & SHOCK, N. W. Coronarv flow and oxygen extraction in the perfused heart of senescent male rats. Journal of Afiplied Physioio~ 30, 4449 (197 1). WENDT-GALLITELLI, M. F. & JACOB, R. Time course of electron microscopic alterations in the hypertrophied myocardium of Goldblatt rats. Basic Research in Cardiology 72,222-227 (1979).
The
Effects
of Long-Term Pressure-Overload Aging on the Myocardium
Robert Defiartment
of
13, 471-488
J. Tomanek
and Jane
Anatomy and the Cardiouascular
Center,
and
M. Hovanec University of Iowa,
(Received 25 September 1980, accepted in revised form
13 January
U.S.A.
198 1)
R. J* TOMANEK AND J. M. HOVANEC. The Effects of Long-Term Pressure-Overload and Aging on the Myocardium. Journal of Molecular and Cellular CardioloQ (1981) 13, 471-488. The effects and interactions of aging and hypertension on the myocardium were studied in spontaneously hypertensive (SHR) and normotensive (WKY) rats. Cardiac hypertrophy developed in SHR during the first 7 months of life and was initially more marked in the subendocardium than in the subepicardium. Capillary density decreased in proportion to cellular enlargement but became normalized between 7 and 15 months when cell size no longer incqzased, suggesting a proliferation of capillaries during this time period. Between 15 and 22 to 23 months significant cellular enlargement occurred in both SHR and WKY despite the fact that blood pressure levels were not changed. Stereological analysis showed that mitochondria/myofibrils volume ratio in SHR decreased during the development of hypertrophy, continued to decrease as hypertrophy stabilized, but normalized during senescence. Residual bodies (lipofuscin granules), characteristic of aging in myocardial cells, accumulated more markedly in SHR than in WKY. Certain other focal alterations which also are associated with aging, i.e. cell membrane invaginations and increased numbers of caveolae, appeared earlier and became more widespread in SHR. In addition, long-term hypertension elicited alterations which were not associated with aging (honey-combed t-tubules, and mitochondrial and myofibril degeneration) . These data provide evidence for three condlusions regarding cardiac hypertrophy and hypertension, and aging in SHR. First, changes which are inevitable with aging are accentuated. Second, certain ultrastructural changes appear to be related to long-term hypertension rather than to the natural aging process. Finally, the relationship between *the volumes of contractile and energy producing organelles is modified by factors which are not clearly related to either aging or hypertrophy and hypertension. KEY WORDS: Hypertension;
Mitochondria; Spontaneously
Myofibrils; hypertensive
Ultrastructure; rat.
Capillary
density;
Hypertrophy;
Introduction Cardiac hypertrophy due to pressure overload is associated with changes which may contribute to heart failure. It has been suggested that some of the changes akin to cardiac hypertrophy are also present in the aged and constitute a complex of “myocardial wear” [21]. Both long term [6 to 7 months] hypertrophy and physiological aging in rats are characterized by parallel alterations, i.e., decreases in RNA concentrations, rates of RNA and protein synthesis, and rates of RNA and protein degradation [22]. Moreover, contraction time is prolonged [12, 16, 361 and endogenous catecholamine content is reduced [JO, 161 in sensescent rats. Similar alterations also occur during pressure-induced cardiac hypertrophy [30]. These data lead to the supposition that the persistence of pressure-overIoad, as evidenced in hypertensive disease, may accentuate aging of the myocardial cell and contribute to a deterioration in function. 0022-2828/81/050471+
18 $02.00/O
0
1981 Academic
Press Inc.
(London)
Limited
472
R. J. Tomanek
and J. M. Hovanec
The spontaneously hypertensive rat (SHR) re p resents an appropriate model for the study of myocardial aging with reference to long-term hypertrophy, since hypertension develops early in life and is akin to essential hypertension in man. Previous studies have shown that cardiac enlargement in SHR is characterized by decreases in mitochondria/myofibrils volume ratio and capillary density [17, 321, conditions which may contribute to a decline in cardiac performance. This communication includes data on the myocardial cell and its capillary bed from SHR and normotensive rats of various ages representing the entire life-span. The aim of the study was to identify specific cellular changes characteristic of longterm cardiac hypertrophy and to determine whether such changes contribute to aging of the myocardium. Quantitative methods, i.e., morphometry and stereology, as well as qualitative approaches, were utilized. In addition to age group comparisons, we compared the subepi- and subendo-cardial regions of the myocardium since previous work indicated that the subendocardium is prone to more marked changes in SHR [32, 331. Materials
and
Methods
Data were obtained from male spontaneously hypertensive (SHR) and Wistar Kyoto normotensive (WKY) rats aged 1,4, 7, 15 and 22 to 23 months. From the time of weaning all animals were maintained in the same room with a 12 h light cycle, and received Purina rat chow and water ad libitum. Arterial blood pressures were determined by the tail-cuff method using a Technilab Instruments apparatus and a Beckman Dynogram recorder. The weekly recordings were obtained during the early afternoon and the median of at least six measurements was recorded for each session. The mean value for the 3 weeks preceding the animal being killed was calculated and was used to compute the group means reported in this paper. Tissue preparation At the time of being killed the animal was anesthetized with sodium pentobarbital (Nembutal), 50 mg/kg (i.p.), and ventilated via a tracheal cannula connected to a Harvard Respirator. Following cardiac arrest with Procaine (10 mg), the coronary vasculature was flushed with Locke’s solution and the heart fixed by retrograde perfusion via the ascending aorta with a 1.5% glutaraldehyde solution buffered with sodium cacodylate. Details of the perfusion procedure have been previously described [32, 341. After the heart and ventricles were weighed, specimens from the subepi- and subendo-cardium were excised, postfixed in 1 o/0 OsO,, dehydrated, and embedded in Epon. Cell size and capillary
density
Myocyte cross-sectional area measurements and capillary density were determined from projected images of cross-sectional fields of l-pm sections stained with Richardson’s solution [32, 331. Outlines of myocytes which contained a nucleus were traced and subsequently their cross-sectional areas measured with a Talos digitizer interfaced with a Monroe calculator. The number of capillaries lying in a prescribed area was determined in several fields. Myocyte cross-sectional areas were measured in at least 25 cells; capillary density was based on fields which included approximately 400 capillaries.
Hypertension,
Aging
and Myocardium
Ultrastructural
173
analysis
Cross-sections from two specimens from each myocardial region were used for morphometric data. The relative cell volumes of mitochondria, myofibrils, and sarcoplasm (all other intracellular components) were estimated by point-counting using a line grid with intercepts every 6 mm [32, 331. At least nine cells from each specimen were photographed and analyzed at a final magnification of x 18 000 to 21 000. This method has been shown to be highly reproducible, with an error of <4%. Mitochondrial surface/volume ratios were determined by measuring the length of the outer mitochondrial membrane and the area of the mitochondrial profile directly from electron micrographs with the aid of the digitizer. Based on the assumption of random average orientation in space, the mitochondrial surface to volume ratio (S,,/V,it) can be obtained from two dimensional micrographs according to the relationship: S/V = L/A, where L is the length of the outer mitochondrial membrane and A is the area of the mitochondrial profile. This is similar to the relationship used in point counting stereology [18], but does not necessitate adjustments for distance between grid lines. Qualitative data were based on micrographs of both longitudinally and transversely sectioned specimens. Statistical Analysis of variance (P < 0.05) between
analysis
and the Bonferroni t test were employed to determine the various experimental and control groups.
significance
Results Ventricular
mass and capillary density
Blood pressure and left ventricular weight data are illustrated in Figure 1. At 6 weeks of age systolic blood pressure in SHR, while significantly higher than that of WKY normotensives, had not as yet attained hypertensive levels. A gradual rise in blood pressure was evident up to about 4 months, with most SHR reaching hypertensive levels between 8 and 10 weeks. Left ventricular mass in SHR, as demonstrated by left ventricular weight/body weight (LVWjBW) ratio, preceded the attainment of hypertensive blood pressure levels. The differences between SHR and WKY for this index were 170/, at 1 month and 379/o at 4 months. A comparison between the changes in left ventricular weights (Figure 2) indicates that absolute left ventricular weight was significantly higher in SHR than in WKY at all ages except 15 months. The data indicate that in both normotensive and hypertensive rats, stabilization of growth of the left ventricle characterized the period between the seventh and 15th month of life. The magnitude of cardiac hypertrophy in the subepiand subendo-cardial regions of SHR are contrasted in Figure 3, where the effect of mean cross-sectional area of myocytes on capillary density is illustrated. The data are summarized as follows: (1) the major increase in cell size during the first 4 months of life in SHR occurred in the subendocardium; (2) preferential hypertrophy of the subepicardial myocytes between 4 and 7 months nearly equated cell size in the two regions; (3) myocyte growth ceased in SHR between 7 and 15 months, after which time a marked cellular hypertrophy developed; (4) capillary density was, in general
474
R. J. Tomanek
and
J. M.
Hovanec
(6)
Age (months)
FIGURE 1. Systolic blood pressure and left ventricular weight (mg)/body weight (g) in spontaneously hypertensive (SHR) and normotensive Wistar Kyoto (WKY) rats. Values are means + S.E.M. Number of animals is indicated in parentheses. All differences between SHR and WKY of similar age are significant (P I 0.05). Note: The earliest blood pressure data are from 1.5-month-old-rats (tail cuff recordings cannot be obtained from smaller rats), while the earliest LVW/BW data are based on l-month-old animals.
related inversely to cell size, except at 15 months when this parameter became normalized in SHR and corresponded to a period of stabilized myocardial hyperto trophy (i.e., no further increase in cell size). The data on cell size, in addition supporting the LVW/SW data, show that mean fiber area increased throughout life in WKY normotensive animals. The increase was particularly marked during the late period of life when cellular growth paralleled that of SHR. As indicated by the data in Table 1, mean fiber area and capillary density are not statistically different in l-month-old SHR and WKY. Therefore, most of the accelerated increased in fiber cross-sectional area and the decrement in capillary density occurred after the prehypertensive period. Ultrastructural
alterations
Relative myocardial cell fractions were similar in l-month-old SHR and WKY (Table 1). Therefore, the changes evident in SHR at 4 months occurred during the development of hypertension. The effects of hypertension on relative myocardial cell fractions were most notable in the subendocardial layer of the myocardium
Hypertension,
Aging
and Myocardium
475
(Table 2). An increase in relative myofibril volume and a decrease in relative mitochondrial volume occurred by the fourth month in SHR and persisted until 15 months. These changes were most marked in the 15-month-old group at which time they were also evident in the subepicardium. Aging did not appear to influence these cellular fractions, since relative myofibril and mitochondrial volumes in WKY were similar at all ages. Since the alterations in these organelle volumes were most
P(6)
out, I
,
,
4
7
,
,
‘2.
,
15
22-
Age (months)
FIGURE
2. Body weight and absolute left ventricular weight in WKY and SHR rats of various are means f S.E.M. Number of animals is indicated in parentheses. Significant differences between WKY and SHR at a given age are indicated by an asterisk.
ages. Values (P < 0.05)
TABLE
1. Myocardial WKY rats
cell
and
capillary
data
from
prehypertensive
(l-month-old)
WKY Subepicardium
Mean fiber Capillary
area
134 f
(pm2)
5069
density
194
34.8 & 52.6 * 12.6 & 0.66 f
1.5 1.3 0.5 0.04
and
SHR Subendocardium
10
&
SHR
135 f 4903
+
28 61
Subepicardium 137 f 4914
f
Subendocardium
6
149 f
126
4638
5
16 177
(number/mmt) Cell volume Mitochondria Myofibrils Sarcoplasm
(%)
Mitochondria/myofibrils volume ratio All v&~es are means i differences between WKY M.C.C.
S.E.M.
and
and SHR
are based on are statistically
35.9 * 1.7 51.1 f 1.2 13.0 & 2.7
32.4 & 0.9 52.3 + 0.9 14.5 f 0.8
36.0 f 1.8 54.3 + 0.8 11.3 f 1.8
0.65
0.62
0.63
i
0.02
four WKY significant
f
and five SHR (P > 0.05).
0.02
animals.
None
+ 0.01
of the
R
476
R. J. Tomanek
650
and
J. M.
Hovanec
-
El 04
I 7
15
22-23
4
7
15
22-23
Age (months) FIGURE 3. Mean fiber cross-sectional area and capillary density in spontaneously hypertensive (SHR) and normotensive Wistar Kyoto (WKY) rats. The subepicardial (left) and subendocardial (right) regions were evaluated separately. Values arc means & S.E.M. Number of animals is indicated in parentheses. All differences between SHR and WKY of similar age are significant (P IO.05) except capillary density in 15-month-old animals. (ma) SHR; ( HA) WKY.
pronounced at 15 months, despite the fact that myocyte growth was stabilized between 7 and 15 months, the data indicate that the relative volumes of mitochondria and myofibrils were not directly influenced by hypertrophy per se. Moreover, further cellular growth between 15 and 22 to 23 months was accompanied by an increase in relative mitochondrial volume compared to the 15-month group, while in WKY the mitochondrial cell fraction remained normal despite a substantial cellular growth, The quantitative relationship between these organelles is illustrated by the ratio: mitochondria/myofibrils volume (Figure 4). In the subendocardium of 4- and 7-month-old animals the ratio was sign’ificantly lower in SHR compared to WKY. At 15 months of age the decrement in this ratio was most marked, i.e., a 30% and 20% decline in the subendocardium and the subepicardium respectively. In contrast, in the oldest SHR the ratio was virtually normalized in the subendocardium and increased in the subepicardium to near-normal values. These data suggest that mitochondria/myofibrils volume ratio was affected by factors other than hypertrophy. Neither hypertension nor aging significantly altered mitochondrial density, i.e., number of mitochondria/lOO pm2 (Table 3), indicating that mitochondrial proliferation was proportional to the increase in cell mass. Surface to volume ratio,
(a)
Group
WKY (8) SHR (7) P value
months
of aging
effects
are means
& S.E.M.
WKY (5) SHR (7) P value
22 to 23 months
WKY (7) SHR (6) P value
15 months
WKY (7) SHR (7) P value
7 months
4
2. The
All values
TABLE
N.S.
* *
N.S.
0.4 0.7
f 0.9 rfi- 0.6
31.8 f- 1.2 28.7 & 0.8 < 0.025
30.6 f 1.2 25.6 & 0.6 < 0.002
30.3 29.4
30.7 28.5
Mitochondria
on myocardial
(%
N.S.
-& 0.8 f 1.0
N.S.
-+ 1.0 f 1.4
62.5 61.1 N.S.
* $
1.5 1.3
60.7 & 1.3 64.9 f 0.7 to.01
60.5 61.9
63.1 63.3
MyoJibrils
Subepicardium
cell fractions
of total
5.8 f 0.9 10.1 * 0.8 < 0.002
N.S.
8.7 i 1.0 9.5 & 0.9
N.S.
9.2 5 0.7 8.7 & 0.6
N.S.
6.2 & 1.2 8.5 4 1.2
Sarcoplasm
cell volume)
31.2 29.8
N.S.
f f
0.9 1.4
30.9 f 0.8 25.0 & 0.8 < 0.0001
29.9 f 1.0 27.2 + 0.6 < 0.04
32.7 f 1.3 29.5 f 0.4 < 0.04
Mitochondria
in spontaneously
(SHR)
61.1 61.1
N.S.
f *
1.1 1.6
59.9 * 0.9 67.5 f 0.9 < 0.0001
59.2 f 0.6 64.2 & 0.9 < 0.001
61.8 f 1.5 66.5 4 0.6 < 0.04
Myofibrils
Subendocardium
hypertensive
and
0.8 0.7
1.0 0.7
N.S.
7.7 f 1.1 9.1 + 0.5
N.S.
8.2 f 7.5 f
N.S.
10.9 f 8.6 +
N.S.
5.4 -& 1.0 4.5 + 1.0
Sarcoplasm
normotensive
(WKY)
rats
478
R. J. Tomanek
and
J. M.
Hovanec
0.46
5
0.40
2
0.46 0.44 0.42 0.40 0.36 0.36 4
15
7
22-23
Age (months) FIGURE 4. Mitochondria/myofibrils and subendocardium (bottom). parentheses. Significant differences asterisks.
volume ratio in cells comprising the subepicardium of animals is indicated Values are means k S.E.M. Number (P S 0.05) between SHR and WKY of similar age are indicated
II
I 4
I
I 15
I 7 Age
FIGURE myocardial the l-month
5. Mean values cells. All differences age group.
(&
s.E.M.)
between
(top) in by
1 22-
3
(mocths)
for the numbers SHR and WKY
of residual bodies in cross are statistically significant
sectional fields of (P I 0.05) except
Hypertension,
Aging
and
Myocardium
479
on the other hand, was higher in the oldest SHR than in their age-corresponding WKY. The increase in this ratio may be due to conformational changes in the organelles. Lipofuscin granules (residual bodies) were seen in the perinuclear region of myocytes from all age groups. They became more evident with age in nonperinuclear regions of both WKY and SHR. A numerical increase with age is illustrated in Figure 5. These data are based on counts/cross-sectional cell areas obtained from electron micrographs. It is evident that residual bodies accumulated at a higher rate in SHR than in WKY. In SHR residual bodies increased substantially in the two oldest groups (Figures 6 to 8). Thus, the hearts of 22- to 23-month-old SHR contained numerous residual bodies (Figure 7) throughout the cell, while such structures in similarly aged WKY were less common. We also observed these structures in capillary endothelial cells in the two oldest groups of hypertensive and normotensive animals (Figure 8). Again, their occurrence was much more common in SHR, particularly in the oldest group studied. Some focal alterations, while infrequent, appeared to be related to aging as evidenced by their appearance in the oldest WKY rats. Myelin figures, invaginations of the cell membrane, dilatations of the non-specialized junctions of the intercalated disc and adjacent t-tubules, and increased numbers of subsarcolemmal vesicles could occasionally be found in the oldest WKY animals. These focal alterations were detected earlier in SHR. The enhancement of subsarcolemmal vesicles was usually limited to the oldest animals while myelin figures occurred earlier. We also noted structural modifications in the two oldest SHR groups which might be attributed to long-term hypertension and hypertrophy. These alterations were extremely rare or absent in the oldest WKY rats. Mitochondrial changes included occasional large spherical inclusions (Figure 9) and focal mitochondrial degeneration (Figure 10). The latter was restricted to a few adjacent mitochondria which were associated with lysosomes. In both 15- and 22- to 23-month-old SHR “honeycombed” t-tubules (Figure 11) were common, but rare in WKY. While irregular Z lines were not uncommon in younger SHR with developed hypertension and
TABLE
3. Comparisons tensive (SHR)
of mitochondrial and normotensive
indices in young (WKY) rats
Number/l00
pm2
and
P
old
spontaneously
Surface
( t*m2)
Volume
( ym3)
hyper-
P
Subepicardium 4 month 4 months
WKY SHR
(5) (6)
22 to 23 month 22 to 23 month
WKY SHR
(5) (6)
101 & 6 92 5 + 91 5 5 97 5 9
N.S.
7.46 8.13 6.54 8.85
& + f &
0.44 0.26
N.S.
0.26 0.62
0.47 0.67
N.S.
Subendocardium 4 month 4 month
WKY SHR
(5) (6)
22 to 23 month 22 to 23 month
All values
are means
WKY SHR
(5) (7)
& S.E.M. Number
101 + 8 98 & 8
N.S.
7.69 8.20
91 * 5 103 & 8
N.S.
7.19 8.70
of animals
is indicated
in parentheses.
5 + & i
0.47 0.38
to.02
480
R. J. Tomanek
and
J. M.
Hovanec
1) WKY illustrating a relatively normal morphology. FIGURE 6. Micrograph from an old (23-montl of faint N bands (arrows) are Occasional membrane fragments in t-tubules (T) and the appearance here) are common. x 20 000. observed in old normotensive WKY. Residual bo dies (not shown (23-month SHR). These structures, FIGURE 7. Four residual bodies (arrows) are seen in the field throughout the cell with aging and limited to the perinuclear region in young anim als, accumulate are common in hypertensive animals. most markedly in the SHR. Irregular Z lines in SC,me sarcomeres x 11 000. cell from a 15-month-old SHR. FIGURE 8. Residual bodies (arrows) withil n an endothelial x 42 000.
Hypertension,
Aging
and Myocardium
481
hypertrophy, Z line streaming and focal myofibrillar disruption occurred only in the two oldest SHR groups (Figure 12). A focal fibrosis was characteristic of the oldest SHR group (Figure 13), but also could be found to a lesser extent in the 15-month-SHR. Discussion This study provides evidence for three conclusions regarding the myocardial cell in the spontaneously hypertensive rat. First, changes characteristic of aging appear earlier and are accentuated. Second, other ultrastructural abnormalities appear to be the consequences of long-term hypertrophy and hypertension rather than events of the natural aging process. Finally, the relationship between the volumes of contractile and energy producing organelles is modified by factors which are not clearly related to either aging or hypertension and hypertrophy. While left ventricular mass was greater in l-month-old SHR than in their age group WKY, cell size was not altered. Moreover, capillary density and the relative cell fractions of mitochondria, myofibrils and sarcoplasm were similar to WKY. We therefore concluded that the larger left ventricular mass in SHR at 1 month is not due to cellular hypertrophy, but more likely is the consequence of a greater number of cells. Accordingly, the changes (with the exception of LVW) observed at subsequent ages developed during the hypertensive period. Our data demonstrate that the marked increase in myocardial cell size between the first and seventh months of life in SHR is not accompanied by an adequate increase in the absolute number of capillaries. Thus, a decrement in capillary’ density characterizes this stage of developing hypertrophy. Since cell size became stabilized and capillary density normalized by the 15th month, it is evident that capillary growth was sufficient to overcome the decrement characteristic of developing hypertrophy. On the other hand, cellular growth in WKY was slow during this time and capillary density was maintained at a consistent level. Both the data on cell size and absolute left ventricular weight support the conclusion that hypertrophy became stabilized between 7 and 15 months. In contrast to the stage of developing hypertrophy, the cellular hypertrophy which occurred during senescence may be an age-associated phenomenon since the increase in cell size was almost parallel in both SHR and WKY. The sharp decrease in capillary density in SHR at this time, which reduced this parameter to levels typical of the earlier stage of hypertrophy, indicates an inadequate proliferation of capillaries. Since capillary density remained constant in the subepicardium of WKY, it would appear that age was not a factor in limiting the growth of the vascular bed. The increase in cell size during senescence, however, was not accompanied by a significant increase in absolute ventricular weight, a finding which suggests the possibility of a decrease in cell number.
Aging We noted consistent alterations in the oldest SHR and WKY which are similar to those found in senescent Sprague-Dawley rats [34]. The accumulation of residual bodies (lipofuscin), generally considered an aging phenomenon in non-dividing cells, was conspicuous and clearly increased with age. This accumulation may be due to concomitant increases in the production of primary lysosomes and their transformation to residual bodies as a consequence of enhanced autophagy [35].
482
R. J. Tomanek
and
J. M.
Hovanec
FIGURE 9. A large dense spherical mitochondrial inclusion (arrow) represents an occasional finding in old SHR. Several osmophilic bodies and a lysosome-like structure (ly) are also seen between myofibrils. x 16 000. FIGURE 10. Focal degeneration of mitochondria (arrows) in old SHR (23-month) occurs in conjunction with lysosomes (1~). Note that a lysosomal figure also appears in the capillary (ca) endothelial cell. Mitochondria adjacent to those which show degenerative changes appear normal. x 14 500. FIGURE 11. “Honeycombed” appearance of transverse tubule (between arrows), common in old SHR. Such structures occur between myofibrils near Z lines and show evidence of a glycocalyx. x 30 000.
Hypertension,
Aging
and
Myocardium
483
Whether the induction of enhanced lysosomal activity is due to a feedback system from digested material or to a genetically programmed phenomenon has not been established. However, the former appears to be more likely since lysosomal activation and residual bodies are particularly occurs in various pathological conditions, numerous in diseased hearts [14]. 0 ur data clearly indicate that residual bodies accumulate more rapidly in SHR than in WKY myocardial cells, which suggests that this aging characteristic is related to factors which are also present during hypertension and/or cardiac hypertrophy. An increase in the accumulation of residual bodies and membrane fragments (including myelin figures) in myocardial cells of SHR appears to be consistent with the idea of “wear and tear” relating to enhanced cellular work [,?I]. It is not clear if exocytosis of residual bodies is limited and allows for the accumulation of these products. However, it has been shown that residual bodies undergo dissolution and exocytosis after dimethylaminoethyl p-chlorophenoxyacetate treatment and that pigment residuals are incorporated into phagocytic cells and the capillary wall and lumen. While our observations indicate the presence of residual bodies and dense bodies in capillary endothelial cells of old animals, it is not certain whether they originate in endothelial or myocardial cells. Other age-associated alterations are also consistent with those found in senescent Sprague-Dawley rats [34]. These structural modifications involve membrane systems and are, for the most part, similar to those which we found in both normotensive and hypertensive rats exposed to chronic altitude hypoxia [27]. Cell membrane invaginations and an increase in the number of caveolae (subsarcolemmal vesicles), characteristic of senescent hearts, were also typical of all animals subjected to hypoxia. While membrane irregularities were found in degenerating myocytes of human hypertrophied hearts, they usually occurred in conjunction with other degenerative changes [8]. In contrast, the surface alterations described here were usually characteristic of cells which did not show any degenerative changes. Since cellular hypoxia in old animals has not been demonstrated, the reasons for such structural adaptations are elusive. It is, however, clear that these membrane alterations, as well as those involving the intercalated disc, have an earlier onset and become accentuated in SHR. Long-term
hypertrophy
In contrast to age-associated changes, we found evidence of structural abnormalities which appeared only after long-term pressure-overload and cardiac hypertrophy, and were absent or rare in the oldest WKY. Such alterations were also absent in senescent Sprague-Dawley rat hearts [34]. T-tubule proliferation giving rise to a honey combed (labyrinthine) appearance, typical of the two oldest SHR groups, has also been reported in numerous disorders of skeletal muscle [19]. We concluded that these structures are in fact t-tubules because of the presence of a glycocalyx. The formation of such membrane networks in skeletal muscle has been considered to provide a membrane source for autophagic vacuoles [7]. Although the stimulus for these modifications in hearts of old SHR has yet to be elucidated, they may be related to degenerative events. Focal alterations of myofibrils also appear to correspond to the long-term effects of functional overload. Since they do not characterize hearts of younger SHR, these alterations appear to be related to the persistence of the increased afterload imposed by elevated blood pressure.
R. J. Tomanek
and
J. M.
Hovanec
12. Myofibrillar disruption with streaming of Z line substance (between arrows) in an 1FIGURE (22-month). Such regions are usually characterized by occasional polyribosomes and sn la11 old SHR profiles. mi tochondrial x 16 000. 13. Collagen deposition near a myocardial cell and capillary in a 23-month-old SH R. F ‘IGURE fibroblast processes and collagen fibrils (co) are evident. A narrow process of a myocard NIlI merous ial numerous subsarcolemmal vesicles (arrows) is seen at the bottom of the field. cell with x 8000.
Hypertension,
Aging
and
Myocardium
485
These changes are not regarded as general characteristics of various types of hypertrophy. Ultrastructural changes in patients with ventricular hypertrophy are focal, with only 20% of the specimens showing degenerative changes [ZO]; more bizarre arrangements of muscle cells may be related to specific types of hypertrophy, e.g. idiopathic hypertrophic subaortic stenosis [9]. While widened and irregular Z lines and altered intercalated discs are considered characteristic of experimental myocardial hypertrophy [3], it appears the other qualitative differences between normal growth and hypertrophy are not marked and consistent. The present study suggests that certain ultrastructural changes become characteristic in the SHR myocardial cell only after long-term hypertrophy. Since they become evident during a period of stabilized myocyte growth, they are not related to the magnitude of hypertrophy. Moreover, their absence or rare occurrence in sensescent WKY as well as Sprague-Dawley rats [34] suggests that they do not characterize the aging processes. While it appears that certain ultrastructural alterations are associated with long-term hypertension, one should be cautious in extrapolating these findings based on a model of genetic hypertension to other models of cardiac hypertrophy. Mitochondrial
and myojibril
volumes
Anversa et al. [Z] noted that the most significant changes in moderate cardiac hypertrophy are quantitative rather than qualitative. The decrease in mitochondrial volume in hypertrophic hearts of SHR is consistent with a number of studies on pressure overload in rats [17, 2.5, 32, 371, rabbits [Z, II]. and dogs [4]. Our work, however, provides evidence that the quantitative relationship between mitochondrial and myofibrillar volumes is related to factors other than cell mass. During the first 7 months of life, mitochondria/myofibrils ratio appears to reflect the magnitude of hypertrophy, as indicated by the data on the subepi- and subendo-cardium, but fails to its lowest value at 15 months despite the stabilization of hypertrophy. Thus, the further decline in this ratio cannot be attributed to cellular growth, but rather appears to be related to the turnover rates of the two organelles. It has been suggested that these organelles are regulated by separate mechanisms [27]. While these mechanisms are not clear, the findings in the 15- and 22- to 23-month-old SHR are not so surprising, since a variety of chemicaf and physiological factors may come into play during aging and a given stage of hypertrophy. We considered the possibility that aging might limit mitochondrial growth. Yet mitochondria/myofibrils volume ratio became normalized during senescence even though further cellular hypertrophy occurred. One possible explanation for the normalization of mitochondrial volume during old age may be the loss of some contractiIe material as suggested by our observations. Thus, the degradation of myofilaments, although less than the synthesis of these organelles, could offset the imbalance between mitochondrial and myofibril growth. The alternative explanation is that mitochondrial growth is accelerated during this late phase of hypertrophy. The increase in surface/volume ratio of mitochondria in old SHR may be an adaptation aimed at enhanced energy production as noted in studies on pressure-overload in rabbits [Z] and dogs [3]. As shown by our data on normotensive WKY, as well as by another study on rats [15], mitochondrial volume is not appreciably altered by age. In contrast the relative volume of this organelle has been reported to decrease in senescent mice [13] and increase in Syrian hamsters [28]. Such inconsistencies could be due to differences in age or species variation.
486
R. J. Tomanek
and
J. M.
Hovanec
General discussion In spite of the fact that left ventricular mass was slightly larger in SHR than in WKY aged 1 month, cellular hypertrophy and a decrement in capillary density were not evident at this time. Thus, our data suggest that hypertrophy develops primarily during the period when blood pressure becomes elevated. Yet the reasons underlying the various responses of the myocardial cell during different stages of life are not apparent. Increased afterload due to the development of hypertension is not the sole determinant of cardiac hypertrophy, because pressure-overload in SHR can be prevented without normalizing cardiac mass [29]. Since blood pressure levels did not differ significantly in SHR once the peak hypertension levels were attained, neither the stabilization of the hypertrophy nor the additional growth which occurred late in life can be explained exclusively by pressure-overload. In addition, the data on cell size in old normotensive rats indicates a significant myocardial growth in the absence of blood pressure changes. Consistent with these findings is the evidence that modification of cardiac hypertrophy in SHR is related to drug specificity rather than to the hypotensive effects of the agents [32, 331. This rationale leads to the consideration of neural or humoral factors which might be altered during senescence. While catecholamines are known to induce cardiac hypertrophy [23, 241, cardiac enlargement during old age can hardly be attributed to increases in norepinephrine since the endogenous content of this catecholamine declines in senescent rats [5, 101. M oreover, sympathetic neural activity does not appear to contribute directly to hypertrophy in SHR since neonatal immunosympathectomy does not prevent its development [6]. However, recent evidence suggests that neural influences play a selective role in myocyte growth. Cellular enlargement in immunosympathectomized SHR, in contrast to SHR with intact sympathetic nerves, is characterized by a proportional growth of mitochondria and myofibrils [26]. While cardiac hypertrophy in the senescent SHR cannot be explained by decreases in endogenous norepinephrine levels, the normalization of mitochondria/myofibrils ratio, as noted in the subendocardium, may be related to a decline in this catecholamine. Whether the restoration of this ratio to control (normotensive) values is of functional significance is uncertain, since other ultrastructural alterations are considerable and may contribute to the decline in the intrinsic mechanical properties of the heart. Acknowledgements
The authors appreciate the technical assistance of MS B. Carroll and MS Shirley Velle. This work was supported by grant HL 18629 from the National Institutes Health.
La of
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