Alden V. Loud

Morphometric study of early postnatal development in the left and right ventricular myocardium of the rat. II. Tissue composition, capillary growth, and sarcoplasmic alterations.

The absolute and differential growths of the capillary network and of myocyte cytoplasmic components in the left (L) and right (R) ventricular free walls were measured morphometrically from 1 to 5 days and from 5 to 11 days after birth. From 1 to 11 days, capillary length, luminal volume, luminal surface area, and endothelial cell volume each increased 2-3 times more rapidly than myocardial mass or myocyte mass in each ventricle. Mean intercapillary distance and the transverse crosssectional area of the average capillary decreased markedly. The mean number of capillaries across the ventricular walls increased from 16 to 79 (L) and 14 to 22 (R). Maturation of the cytoplasm of left and right ventricular myocytes from 1 to 11 days included increases in the volume percentage of myofibrils(1.2-fold), mitochondria (1.8-fold), and smooth endoplasmic reticulum (2.1-fold) and increases in mean mitochondrial size [1.9-fold (L), 1.2-fold (R)] and number per cell [2.7-fold (L), 3.6-fold (R)]. Despite a 2-fold greater overall left ventricular growth, the myocardial compositions of both ventricles were nearly indistinguishable at 1 and 11 days. Both subcellular and microvascular changes, however, were generally achieved more rapidly in the left ventricle from 1 to 5 days of age, demonstrating many structural differences and a lagging development in the right ventricle at 5 days. Thus, postnatal myocardial adaptation to the altered work demands on the left and right ventricles shortly after birth resulted in morphological changes that could be the basis for a transient disparity in ventricular functions at about 5 days of age. Circ Res 46: 503-512, 19S0

Morphometric study of early postnatal development in the left and right ventricular myocardium of the rat. I. Hypertrophy, hyperplasia, and binucleation of myocytes.

We reviewed the absolute amd differential growths of the myocyte populations in the left(L) and right (R) ventricular myocardium morphometrically from 1 to 5 days and from 5 to 11 days after birth. From 1 to 11 days hypertrophy of the average myocyte in the ventricles was (L) 2.7- and (R)2.4-fold, and myocyte proliferation was (L) 2.0- and (R) 1.2-fold. Mean cell volume, cell length, and percent binucleation of cardiac myocytes were similar in both ventricles at 1, 5, and 11 days of age. During this period, average myocyte length increased 2-fold (12 sarcomere lengths), and the percentage of binucleate myocytes increased approximately from 2.7 to 17 to 48%. Myocyte hypertrophy from 1 to 5 days resulted mainly from an increase in the volume of cytoplasm per nucleus and from 5 to 11 days from the accumulation of binucleate cells. No differences were observed in the characteristics of epicardial and endocardial myocytes in either ventricle up to 11 days of age. The 61% greater proliferation of myocytes in the left ventricle was the principal basis for the development of a 2-fold difference in ventricular weight gains: (L) 6.2- and (R) 3.4-fold. No increase in right ventricular midwall thickness was observed, in contrast to a 2.7-fold increase of the left ventricle. It was concluded that, asa result of the circulatory changes occurring shortly after birth, right ventricular growth is analogous to eccentric hypertrophy, whereas left ventricular growth represents a combination of eccentric and concentric hypertrophy. Circ Res 46: 495-502, 1980

Stereological measurement of cellular and subcellular hypertrophy and hyperplasia in the papillary muscle of adult rat.

Abstract Structural changes in the papillary muscle were examined following an induced growth of 51% by integrating tissue, cellular and subcellular morphometry. Comparison of the adaptive response in different cell populations by morphometric nuclear enumeration demonstrated 48% hyperplasia of endothelial cells, 35% for connective tissue cells and no change in the number of myocyte nuclei. Increases in the cell volume per nucleus, a measure of cellular hypertrophy, were 35% for endothelial cells, 64% for connective tissue cells and 53% for myocytes. No significant changes were found in the lengths of the papillary muscle, the average myocyte or the capillary network. Total capillary volume and the luminal and abluminal endothelial surfaces were significantly increased by 59, 38 and 43%, respectively. Each cell population in the papillary muscle showed varying increases in the volumes, surface areas and number of cytoplasmic organelles accompanying cellular hyperplasia and/or hypertrophy. Micropinocytotic vesicles in endothelial cells increased 73%; RER in fibroblasts: approximately 150%; SER and T-system in myocytes: each approximately 100%; and the numbers of mitochondria in endothelium, fibroblasts and myocytes: 54, 78 and 65%. As a result of a lateral duplication of normal sized components, the myofibrillar network in myocytes increased 67% in volume, 55% in surface area, 82% in the length of myofibril branches, and 86% in the overall number of its sarcomere units. These results provide a morphological basis for functional studies of normal and hypertrophied papillary muscle.

Morphometric study of early postnatal development of the thoracic aorta in the rat.

Tissue, cellular, and subcellular morphometry of the thoracic aorta in 1-, 5-and 11-day-old rats was used to quantify the cellular hypertrophy and proliferation of smooth muscle cells and the absolute increases in volume of elastic laminae and collagen during the early postnatal period. In the 1-to 5-day interval, total wall volume increased 2.9-fold, wall thickness and the number of smooth muscle cells doubled, mean cell volume increased by 40%, and the volumes of elastic laminae and collagen increased 3.7-and 3.3-fold, respectively. From 5 to 11 days, circumferential growth of the aortic wall, without further thickening, produced smaller and unequal growth increments of each of its component structures, with increases in collagen (3.2-fold) > elastic laminae (2.3-fold) > muscle cells (1.6-fold). In the overall growth of smooth muscle cells (4.6-fold) from 1 to 11 days, only the cytoplasmic volume fractions of glycogen aggregates and rough endoplasmic reticulum were altered significantly (−78%, +32%). Certain aspects of normal postnatal aortic growth paralleled the response of adult aorta to experimentally induced hypertension. Circ Res 47: 417−424, 1980

Postnatal development of the M-band in rat cardiac myofibrils.

Cardiac muscle fibers in rats at 1 and 5 days after birth showed little evidence of Mbands. An ultrastructural analysis of myofibrils failed to demonstrate dense M-band material in longitudinal sections or M-bridges in transverse sections of sarcomeres. M-bands began to increase in number after 5 days of postnatal life and were present in 60% of all sarcomeres at 11 days of age. Polypeptides with molecular weights of 190,000 (M«) and 175,000 (Mb) were obtained by polyacrylamide-sodium dodecyl sulfate (SDS) gel electrophoresis of myofibril preparations. These two proteins were found in both 1-and 11-day-old rats and were considered to be specific components of the M-band. The densitometric analysis demonstrated that M. and Mb polypeptides increased approximately 2-fold during the interval from 1 to 11 days after birth. These structural and biochemical changes of the M-band material in myocytes appear to be related to the maturation of contractile function in the young heart. Circ Res 48: 561-568, 1981

Morphometry of cardiac hypertrophy induced by experimental renal hypertension

Abstract A method for determining the mean volume of cells within a tissue has been applied to the measurement of endocardial and epicardial myocytes in the left ventricle of normal and hypertensive rats. The technique is based on nuclear counts per unit area in tissue slices of different known thicknesses. It measures the mean cell volume per nucleus and has been combined with electron microscopic morphometry. Compared with the epicardial regions, the normal endocardial regions contained 30 percent more myocytes, 27 percent less interstitial space, 48 percent less capillary volume, 17 percent less capillary surface and the same capillary length per unit of tissue volume. In terms of both the relative and absolute volumes and surface areas of their organelles, the cytoplasmic composition of normal endocardial and epicardial myocytes was nearly identical. After 1 to 4 weeks of hypertension induced by renal arterial constriction, endocardial myocytes enlarged from 10,370 ± 410 to 12,520 ± 490 μ m 3 whereas epicardial myocytes enlarged from 12,600 ± 1,600 to 17,300 ± 1, 100 μm 3 . The number of myocytes and the total length of capillaries remained constant. The epicardial region enlarged 37 percent with proportional increases of myocyte and interstitial volumes. In contrast, the endocardial enlargement was only 26 percent, consisting of 21 percent hypertrophy of myocytes and a 55 percent increase in interstitial components. Expansion of capillary lumens accounted for much of the interstitial enlargement throughout the myocardium. Hypertrophy of myocytes in the epicardial region was accompanied by a reduced mitochondria to myofibril ratio and disproportionately large increases (two- to three-fold) in both smooth endoplasmic reticulum and T system volume and surface area. On a cell basis the morphometric characteristics of myocytes from hypertensive rats are significantly different from normal, and significant differences occur between the inner and outer layers of the myocardium for practically every cytoplasmic component.

Quantitative radioautographic localization of newly synthesized protein in the postnatal rat heart

Abstract Electron microscopic radioautography following injection of 3 H-leucine was undertaken in the left ventricles of 30 g rats. The volume composition of cardiocyte sarcoplasm at this stage of postnatal development was measured by point counting to be 52% myofibrils, 30% mitochondria, 10% sarcoplasmic reticulum (SR) and 8% for the rest of the cell structures. The extra-cellular components of the myocardium constituted approximately 26% of the heart muscle. For analysis of electron microscope radioautographs a circle was drawn around each silver grain and the enclosed structures were identified as possible sources. The ratio of percent grain counts to volume percent in sarcoplasmic structures was 1.4 for myofibrils, 1.3 for mitochondria, 4.6 for SR and 1.4 for the combined other structures in the sarcoplasm. The developed grains associated with myofibrils revealed about 91% on the A band, 21% on the I band, 10% on the M band, 23% on the Z band, and 67% at the periphery of the myofibril. Comparing these percentages to values expected in a random distribution of labels it was particularly significant to note that the Z bands had a higher percentage of grains. These observations indicate that newly synthesized protein in cells undergoing myofibrillogenesis at this stage of development is selectively concentrated in regions of the Z bands and in the SR which abuts on the surface of the myofibrils. Developed grains associated with structures in the interstitial space are found predominantly in the endothelial cells and plasma.

Global myocardial hypertrophy in conscious dogs with chronic elevation of plasma norepinephrine levels.

Chronically elevated plasma norepinephrine is associated with many disease states in which myocardial hypertrophy is also found, yet whether the hypertrophy results from the hemodynamic actions of catecholamines or a trophic effect is still unknown. The goal of our study was to determine the extent of hypertrophy following 28 days of norepinephrine infusion and the role of altered hemodynamics as the stimulus for the hypertrophy in conscious dogs. In a retrospective study gross cardiac weights were examined in 25 control instrumented dogs (controls) and in 41 instrumented dogs with elevated norepinephrine (NE). In the NE dogs LV (94 +/- 2.8 g), septum (33 +/- 1.5 g) and total heart weights (172 +/- 4.5 g) were greater than in controls (85 +/- 5.0, 31 +/- 1.9, and 158 +/- 9.0 g, respectively). The LV (3.95 +/- 0.10 g/kg), RV (1.91 +/- 0.06), septum (1.38 +/- 0.06) and total weight (7.23 +/- 0.15) to body weight ratios were also significantly greater (3.32 +/- 0.12, 1.73 +/- 0.08, 1.24 +/- 0.06, and 6.28 +/- 0.23 g/kg). The dry/wet weight ratios were not different in the two groups. After cutting the hearts into 1 cm rings, the planimetered area of each ring showed that the wall thickness increased at all levels, e.g. at the base by 25 +/- 3.2%. The chamber internal diameter was significantly increased only near the apex in the NE dogs making the heart more cylindrical in shape. There was no obvious sign of fibrosis in any layer of the myocardium. In physiologic studies, no index of contractility was altered including: LV dP/dt, LV dD/dt, shortening, LV dWT/dt and there was also no change in preload. Heart rate was significantly reduced throughout the 28 days of study. Cardiac output was reduced and there were no significant changes in cardiac work. Thus in these dogs NE caused a 19% and 10% increase in LV and RV to body weight ratios, respectively, without any obvious hemodynamic stimulus.

EFFECTS OF INFLAMMATION ON SUBPOPULATIONS OF BLOOD LYMPHOCYTES

A method for quantitative cytochemistry has been used to study the surface properties of blood lymphocytes of guinea pigs 24 hr after the production of cell-mediated immunity or acute inflammation. Three subpopulations of small lymphocytes with different surface-binding characteristics for Thorotrast particles have been observed. The average number of particles per micron length of surface membrane, i.e., particle density, has been determined for each class of cells. Only two clusters separated by a distinct gap are apparent in each group of animals. There is a significant decrease in the mean particle density of the major subpopulation of lymphocytes in each group of animals with inflammatory reactions, thereby suggesting a reduction in surface charge. On the other hand, the particle density distribution of the minor population of cells does not differ from normal. The role of surface charge alterations on the circulation, margination and emigration of lymphocytes is discussed.

Protein synthesis in the atrioventricular node during acute experimental cardiac hypertrophy in the rat.

Abstract The relative concentration of autoradiographic grains was measured over the atrioventricular node of the adult rat heart 2 h after the intravenous injection of [3H]-leucine and 20 h after the induction of left ventricular cardiac hypertrophy by constriction of the abdominal aorta. In comparison with control tissues, the grain counts in equal area samples show a 32% greater concentration in the hypertrophying hearts. The results indicate a significantly increased rate of incorporation of amino acid into the proteins of nodal tissues accompanying early hypertrophic changes.