Karel Rakusan

Morphometry of human coronary capillaries during normal growth and the effect of age in left ventricular pressure-overload hypertrophy.

BackgroundIn adults, acquired pressure-overload left ventricular hypertrophy can result in myocardial ischemia, which may be due in part to insufficient capillary growth during development of hypertrophy. The coronary microvascular response to congenital pressure-overload hypertrophy in children has not been previously characterized. Methods and ResultsAverage capillary density and heterogeneity of capillary spacing were measured in 63 postmortem human hearts with left ventricular hypertrophy and control hearts without heart disease. Pathology specimens were chosen that had left ventricular hypertrophy caused by 1) congenital isolated aortic valve stenosis in infants <1 year old at death, children 9–14 years old, and adults 15–30 years old; 2) congenital isolated coarctation of the aorta in adults 15–39 years old; and 3) acquired aortic stenosis in adults 51–86 years old. Major findings of the study were: 1) Human left ventricular capillary density and heterogeneity of capillary spacing are similar to other mammalian species. 2) Capillary density is higher in infants (3,315±85 capillaries per square millimeter), decreases with increasing heart weight during normal growth in early childhood (children, 2,388±75 capillaries per square millimeter, p<0.05), and thereafter remains relatively constant. 3) Capillary density with left ventricular hypertrophy is dependent on the age of onset. Congenital aortic stenosis and coarctation are characterized by an increase in capillary supply proportional to myocyte volume, maintaining capillary density similar to control hearts. Adults with acquired aortic stenosis have decreased capillary density (1,671±66 capillaries per square millimeter, p<0.01 versus control). ConclusionsPressure-overload left ventricular hypertrophy in children demonstrates proportional capillary angiogenesis, whereas in adults, hypertrophy appears to be associated with failure of compensatory angiogenesis.

Transmural Channels Can Protect Ischemic Tissue Assessment of Long-term Myocardial Response to Laser- and Needle-Made Channels

BACKGROUND We previously found that transmural laser channels failed to acutely increase myocardial blood flow. Nevertheless, this method is being used to treat patients with coronary artery disease who are unable to undergo angioplasty or bypass graft surgery and in cases in which previous surgery has failed. To reconcile the lack of an acute increase in blood flow with beneficial effects claimed in patients, our hypothesis was that the channel-making process might, over time, stimulate a protective effect, possibly by the growth of new vessels linking channels to the existing circulation. We tested this hypothesis in rat hearts, which have little native collateral circulation. METHODS AND RESULTS We made six transmural channels in the left ventricle of each heart using a 400-micrometer-diameter optic fiber coupled to a holmium:yttrium-aluminum-garnet laser or a 400-micrometer-diameter syringe needle. Two months after the channels were made, rats were randomized to either an infarct-size study or analysis of myocardial capillary density. We challenged any induced protective mechanism by acutely occluding the left coronary artery for 90 minutes, followed by 4.5 hours of reperfusion. The artery was then reoccluded, and pigment was injected into the circulation to delineate tissue perfused by the occluded vessel and to detect perfusion via the channels. We used triphenyltetrazolium staining to determine the amount of muscle necrosis and the location of muscle protection. Infarct size in needle-treated hearts was smaller than in controls (15 +/- 6% versus 40 +/- 3% of the left ventricle, P<.01). Infarct size in laser-treated hearts (27 +/- 5%) did not differ significantly from controls; however, all eight laser-treated hearts showed evidence of muscle protection in areas adjacent to channels. We found that the laser-made channels were associated with more fibrosis than the needle-made channels (mean width of fibrosis 430 +/- 50 versus 180 +/- 30 micrometer, P<.0001), and, in tissue remote from channels, fibrosis was increased more in laser-treated hearts (3.6 +/- 0.3%) versus both control (2.5 +/- 0.2%) and needle-treated (2.5 +/- 0.3%) hearts (P<.05). In addition, muscle disarray was seen adjacent to channel-associated fibrosis. We observed injected pigment within fibrosis associated with the channels and in surrounding myocardium. We also found vessels that appeared to be connected to channels; however, there was no overall increase in capillary density. CONCLUSIONS We were able to protect the heart against coronary artery occlusion by making transmural channels 2 months before occlusion. Channels created by a needle provided greater protection than channels created by a laser, probably because they caused less initial injury. Our results are consistent with the concept that the channels were able to provide blood flow to the tissue directly from the ventricular cavity; however, we cannot rule out the possibility that other mechanisms of protection may be involved.

The effect of AT1 receptor antagonist on chronic cardiac response to coronary artery ligation in rats.

OBJECTIVE The aim was to study the effect of the AT1 receptor antagonist losartan on hemodynamic and morphometric changes following experimental infarction. METHODS Experimental infarction was produced in adult male rats by ligating the coronary artery. Treatment with losartan was compared to untreated controls, in rats with experimental infarction and sham-operated animals. RESULTS Infarcted hearts were characterized by significant decreases in left ventricular developed pressure, as well as positive and negative (dP/dt)max, whereas left ventricular end-diastolic pressure (LVEDP), relaxation constant tau and right ventricular systolic pressure (RVSP) significantly increased. Treatment with losartan decreased the LVEDP, the relaxation constant tau and RVSP in the infarcted hearts. Right ventricular weight significantly increased in rats with infarction; this was attenuated by losartan. Infarct size was not significantly influenced by losartan treatment. Morphometric data revealed decreased capillary supply in infarcted hearts, especially in regions close to infarction; the decrease was less pronounced after losartan treatment. Capillary density in near infarct region decreased from 2826/mm2 to 1471/mm2 in untreated animals but in the treated animals it decreased from 2982/mm2 to only 2037/mm2. Simultaneous significant decrease in myocyte-to-capillary ratio in treated animals compared to untreated rats (0.87 to 0.67) seems to indicate formation of new capillary channels after losartan treatment. LVEDP was dependent on the size of infarction in untreated but not in treated animals. A close correlation between LVEDP and capillary density was found. CONCLUSIONS Decreased ventricular contractility, prolonged relaxation and decreased coronary capillary density in rat experimental cardiac infarction confirm and amplify previous reports dealing with this experimental model. Moreover, we have found evidence of improved hemodynamics and coronary angiogenesis after losartan treatment.

Regional capillary supply in the normal and hypertrophied rat heart.

Abstract Capillary density and the distribution of tissue located in various distances from the nearest capillary were studied in normal and hypertrophic rat hearts. Measurements were made on midwall and subendocardial regions of the left ventricular wall. The number of capillaries and muscle fibers per square millimeter were similar in both regions of the normal hearts while in the hypertrophic hearts these numbers were significantly lower in the subendocardial region. The distribution of diffusion distances within the tissues was measured by the method of concentric circles and the results were compared with more conventional methods. The pattern of distribution was once again similar in both regions of the normal heart while in the hypertrophic hearts a significantly higher proportion of the tissue was located farther from the capillary in the subendocardial region than in the midwall region. If one compares the results in the normal and hypertrophic hearts, both regions exhibited less favorable capillary supply in the hypertrophic hearts which was more pronounced in the subendocardial region.

The influence of aging and growth on the postnatal development of cardiac muscle in rats.

We studied the effects of different rates of postnatal body growth on the growth of rat myocytes. Growth rates were altered by adjusting the number of newborn animals per litter to 4 (fast-growing), 8 (normal), or 16 (slow-growing). On the 21st postnatal day, average left ventricular weights (mg ± SEM) were 80 ± 5 in slow- growing 131 ± 8 in normal, and 183 ± 5 in fast-growing rats. Average myocyte diameters, obtained from direct microscopic measurements in longitudinal sections prepared from eight animals in each group, were 8.6 ± 0.3, 9.8 ± 0.2, and 10.2 ± 0.3 μm, respectively. The total number and total length of myocytes constituting the left ventricle were estimated, assuming a constant length-to-width ratio and cyclindrical model of myocytes. The estimated total number of myocytes constituting the respective left ventricles were 23.6 ± 2.0, 24.5 ± 2.2, and 31.1 ± 2.1 millions. All differences between experimental groups were significant by analysis of variance. When the estimated total numbers of myocytes and their estimated total lengths were related to the left ventricular weights, a significant linear correlation was obtained, which indicated their dependence on the organ growth and their relative independence of age. These indications were strengthened further by analysis of covariance, which showed that the differences in all morphometric data among the three groups of hearts may be explained entirely by different growth rates of the left ventricle. These results also suggest that the accelerated growth of the heart during the early postnatal period is associated with increased proliferation of myocytes.

Lognormal distribution of intercapillary distance in normal and hypertrophic rat heart as estimated by the method of concentric circles: its effect on tissue oxygenation

The inhomogeneity of the capillary net in the cardiac muscle was estimated using our morphometric measurements in normal and hypertrophic rats hearts. As entry data we used the distribution of tissue at different distances from the nearest capillary as measured by the method of concentric circles and the mean intercapillary distance independently calculated from the capillary density. The derived distribution of intercapillary distances was approximated by lognormal distribution in which the spread can be characterized by a single parameter, namely the log standard deviation.The effect of the log standard deviation on tissue oxygenation was evaluated in normal and hypertrophic hearts, at normoxia and at hypoxia. The mean tissuePO2 and the percentage of anoxic tissue at the venous end of the tissue cylinder were calculated using Kroghs model. Two boundary situations were considered: A) the end-capillaryPO2 was assumed to be equal in all capillaries due to compensatory adjustment in blood flow; B) the same flow in all capillaries was assumed resulting in varying end-capillaryPO2. The real situation is expected to be between situations A and B. Increased variability of intercapillary distance proved to impair considerably the tissue oxygenation, especially when the results were expressed as a percentage of anoxic tissue. The percentage of anoxic tissue turned out to be a better index of tissue oxygenation than the meanPO2 particularly at hypoxia. The results suggest the presence of at least a partial adjustment of blood flow with respect to the width of tissue cylinder. Without such adjustment, a large part of tissue would become anoxic already in normal hearts at normoxia and this would be further aggravated by hypertrophy and/or hypoxia.

Mast cells in the rat heart during normal growth and in cardiac hypertrophy.

Mast cells in rat hearts were studied quantitatively during normal postnatal growth and in two types of cardiac hypertrophy. Normally, cardiac mast cell density in 11-12-day-old animals is very low, but increases markedly in the following 2-3 weeks to its highest values, with a subsequent decline toward adult values. At the peak of mast cell density, the percentage of mast cells in close proximity to capillaries is also highest. In adult animals, mast cell counts are significantly higher in the right ventricle than in the left. This relation is preserved even when the right ventricle is hypertrophic, as in rats born at simulated high altitude. Chronic hypertension and swimming have little effect on the mast cell density in rat hearts. Conspicuous changes in the mast cell density at the time of capillary proliferation seem to indicate a special role played by these cells in the formation of new vessels.

Morphometry and ultrastructure of heart hypertrophy induced by chronic volume overload (aorto-caval fistula in the rat).

Abstract Heart hypertrophy due to chronic volume overload was produced in adult female rats by experimental aorto-caval fistula. Animals were sacrificed 5 days, 1 and 6 months after the operation. Morphology of cardiac myocytes from the left ventricle was studied in light and electron microscope. Cardiac weight of operated rats almost doubled the normal heart weight during the first month, while later the rate of cardiac growth was similar in both operated and control animals. Cell length increased to the same extent as the cell width, indicating harmonious growth. Increase in the cell volume was more pronounced in the subendocardial region than in the midwall layers. Similarly, degenerative changes were found more often in the subendocardium. Average increase in the cell volume was similar to the increase in cardiac weight, which seems to suggest hypertrophic growth. Cytological features of active cell growth were found not only in the early stages of pathological growth (5 days and 1 month) but also 6 months after the operation. This observation is consonant with the persistence of increased proteosynthesis not only in the period of the stable hypertrophy but also in the later stages, often associated with cardiac failure.

Geometry of capillary networks in hypertrophied rat heart

Capillary geometry was examined in normal and hypertrophic myocardium. Hypertrophy was induced by aortic constriction in neonatal rats. Morphometric data were obtained from tissue sections exposed to a staining technique that distinguished the arteriolar and venular portions of capillaries by color. In sham-operated controls, the theoretical tissue region supplied by a single capillary decreased from the arteriolar to venular side (499 +/- 3 microns 2 and 456 +/- 5 microns 2, P less than 0.05; mean +/- SE) of capillaries. In hypertrophy, only arteriolar capillary tissue regions increased in size, thus enlarging the difference between arteriolar and venular ends (547 +/- 6 microns 2 and 464 +/- 5 microns 2, P less than 0.01). Intercapillary distances, measured at various levels along the capillary path length, decreased in a stepwise manner in both normal and hypertrophic hearts. In hypertrophic hearts, mean capillary path length was significantly longer than in controls, but the total length of the individual capillary nets was reduced. In both groups, arteriolar capillary segment length was longer (P less than 0.01) than venular capillary segment length. Given that PO2 values are lower on the venular side of capillaries, this spatially distinctive geometry in normal myocardium: smaller domains, shorter intercapillary distances and segment lengths, would provide favorable geometric conditions for oxygen diffusion. In hypertrophy, average intercapillary distance increased, and the distinction between arteriolar and venular portions of capillaries was further exacerbated.

Effect of nifedipine on coronary capillary geometry in normotensive and hypertensive rats.

The aim of this study was to describe quantitatively changes in the coronary capillary network resulting from hypertrophy in spontaneously hypertensive rats (SHR) and a potential effect of long-term treatment of these animals with nifedipine. Age-matched male SHR and Wistar-Kyoto (WKY) rats were treated for 27 weeks. Four experimental groups were analyzed: (1) untreated SHR, (2) nifedipine-treated SHR, (3) untreated control WKY rats, and (4) nifedipine-treated WKY rats. Treatment significantly decreased systolic blood pressure in SHR, although normotensive pressures were not reached. SHR had significantly higher cardiac weight, which decreased in nifedipine-treated rats, but values remained above those in control animals. Morphometric evaluation revealed lower capillary density and larger capillary domain area in hearts from SHR, which were partially attenuated by treatment with nifedipine. Capillary domain area was also significantly larger at arteriolar portions compared with domains supplied at venular portions. Capillary segment length was consistently shorter on the venular than arteriolar portion of the capillary, whereas no differences were observed between hearts from WKY rats and SHR. Treatment with nifedipine resulted in a prolongation of segment length. Reconstruction of the three-dimensional capillary supply unit (capillary domain area times capillary segment length) revealed significant differences between the amount of tissue supplied by a capillary at its arteriolar portion than more distally, which was detectable in all experimental groups. In hypertrophic hearts from SHR this tissue volume is increased mainly because of longer intercapillary distances and larger domains, especially on arteriolar portions.(ABSTRACT TRUNCATED AT 250 WORDS)