Absolute morphometric study of myocardial hypertrophy in experimental hypertension. II. Ultrastructure of myocytes and interstitium.

The left ventricular myocardium of normal and hypertensive rats has been characterized morphometrically in the endocardial and epicardial zones. Compared to the epicardial regions, the normal endocardial regions contain 30 per cent more myocytes, 27 per cent less interstitial space, 48 per cent less capillary volume, 17 per cent less capillary surface, and the same capillary length per unit 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 is nearly identical. After 14 weeks of hypertension, induced by constriction of the left renal artery, left ventricular weight is increased by 30 per cent, wall thickness by 42 per cent. The number of myocytes and the total length of capillaries remain constant. The epicardial region enlarged 37 per cent with proportional increases of myocyte and interstitial volumes. In contrast, the endocardial enlargement was only 26 per cent, comprised of 21 per cent hypertrophy of myocytes and a 55 per cent increase in interstitial components. Expansion of capillary lumina accounted for much of the interstitial enlargement throughout the myocardium. Hypertrophy of myocytes is 76 per cent greater in the epicardial region and is accompanied by a reduced mitochondria to myofibril ratio and disproportionately large increases (2- to 3-fold) in both smooth endoplasmic reticulum and T-system volume and surface area. On a cellular basis the absolute 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.     

Myocardial response to infarction in the rat. Morphometric measurement of infarct size and myocyte cellular hypertrophy.

For determination of the effects of myocardial infarction on the recovery potential of muscle mass in the surviving tissue, ligation of the left coronary artery was performed in 3-month-old rats, and the infarcted ventricles were analyzed morphometrically a month after surgery. Comparisons were made with 4-month-old control rats that underwent sham operations and with 3-month-old control rats that were not operated upon for evaluation of the magnitude of infarct size and discrimination of the relative contribution of tissue growth that occurred in the surviving myocardium solely as a result of the change in age, from 3 to 4 months (postoperative tissue growth, or POTG), from the additional growth induced by infarction (hypertrophic growth, or HG). Coronary occlusion induced a 276-cu mm loss of ventricular tissue volume that corresponded to 43% of the total left ventricular mass, 648 cu mm. Over a 30-day period the remaining 372 cu mm of viable tissue expanded by 90% with an overall volume gain of 334 cu mm. This tissue augmentation consisted of 20% POTG, 67 cu mm, and 80% HG, 267 cu mm. Total myocyte volume increased 89%, from 302 cu mm to 571 cu mm, and average myocyte cell volume per nucleus increased 92%, from 16,500 cu mu to 31,600 cu mu. The expansion of the myocyte mass was the result of a 21% POTG and a 79% HG. Corresponding values for the myocyte population were 19% and 81%.     

A morphometric analysis of the hypertrophy of experimental liver cirrhosis

Virchows Archiv - The objective of this study was to better elucidate the composition of the hypertrophic cirrhotic liver. We induced cirrhosis with hypertrophy in rats by simultaneous treatment...     

Association of myocardial cell necrosis with experimental cardiac hypertrophy.

Cardiac hypertrophy was induced in rabbits by injecting thyroxine or isoprenaline, or by surgically constricting the abdominal aorta. An increase in heart weight was associated with a change in the ratios of bound to free forms of five lysosomal enzymes, a change in serum creatine phosphokinase and lactate dehydrogenase, and a change in the morphology of the myocardial cells. Isoprenaline treatment for 5 days induced a maximal change in heart weight, in the ratio of lysosomal enzymes, and in the serum enzymes. Thyroxine treatment was required for 15 days before maximal changes in heart weight, ratio, and serum enzymes were observed. In contrast, coarctation of the aorta caused a progressive change in heart weight, in the ratio of lysosomal enzymes, and in serum enzymes. These results suggest that necrosis of the myocardial cells does indeed accompany cardiac hypertrophy. It was further observed that autophagosomes, degenerating mitochondria in the myocardial cells during the induction of cardiac hypertrophy, and myofibril lysis were found, all of which confirms the suggestion of myocardial cell necrosis in the experimentally enlarged heart.     

肾上腺素在心肌细胞肥大中的作用

目的：探讨肾上腺素在心肌细胞肥大中的作用。方法：在培养新生大鼠心肌细胞上,通过测量心肌细胞[³H]-Leu的掺入量来判断心肌细胞肥大。结果：肾上腺素能明显增加心肌细胞[³H]-Leu的掺入量,酚妥拉明、心得安、百日咳毒素（PTX）和蛋白激酶C（PKC）抑制剂（calphostin C）均可抑制肾上腺素诱导的心肌细胞[³H]-Leu掺入量增加。结论：肾上腺素诱导的心肌细胞肥大反应与激动肾上腺能受体（α受体和β受体）有关,并通过G蛋白和PKC起作用。     

Pathologic changes in the long-term transplanted heart: a morphometric study of myocardial hypertrophy, vascularity, and fibrosis

Myocyte hypertrophy and myocardial fibrosis have been observed in transplanted human hearts, and both could potentially have an adverse effect on long-term cardiac function. There has been some concern that distant donor heart procurement and cyclosporine treatment increase the risk of these changes, but their incidence and severity have not been documented quantitatively in large numbers of cardiac transplant recipients. We used light microscopic morphometric methods to estimate myocardial collagen volume fraction and myocyte width in right ventricular endomyocardial biopsies from 95 recipients at 3 years posttransplantation, and electron microscopic stereology to estimate myocardial vascularity and myocyte myofibril content in 40 recipients, also at 3 years posttransplantation. We compared those with locally and distantly procured donor hearts (mean ischemic time 160 minutes) and cyclosporine versus noncyclosporine immunosuppression. Controls were pretransplant right ventricular biopsies from 20 donor hearts which were free of heart disease. We found no significant differences in myocardial collagen volume fractions. Myocyte hypertrophy was typical of all the transplant biopsies (mean myocyte width 20.2 microns, SD 3.0 in all transplants versus 11.8 microns, SD 2.2 in controls, P less than 0.001), but distant donor procurement and cyclosporine had no significant effect. There were significant reductions of myofibril volume fraction in the transplants, which raises the possibility of gradual decompensation in some patients. There were no significant differences in myocardial vascularity, although a few patients were well below the control range. We conclude that distant donor heart procurement, with ischemic times averaging less than 3 hours, and cyclosporine treatment are not responsible for significant hypertrophy or fibrosis in most transplants. Hypertrophy is typical of the transplanted heart, and it is possible that associated abnormalities might have an effect on cardiac function in some long-term survivors.     

Myocardial tissue troponins T and I. An immunohistochemical study in experimental models of myocardial ischemia.

BACKGROUND: Cardiac troponins T (cTnT) and I (cTnI) are proven diagnostic and risk stratification biomarkers in patients with acute coronary syndromes. To date, no immunohistochemical studies have been performed which allow visualization of the time course and pattern of myocardial troponin egress from the myocardium during the early evolution of ischemic injury in experimental systems. METHODS: We studied archival formalin-fixed, paraffin-embedded myocardium from 50 experimental animals (dogs, pigs and rats) that had undergone permanent coronary occlusion (n = 34) for 0.5-6 h or occlusion of 0.75-6 h followed by reperfusion (n = 16). Histologic sections that included ischemic and nonischemic myocardium were studied by immunohistochemistry with three different antibodies to human cTnI and one to cTnT, using a standard avidin-biotin-peroxidase system. RESULTS: All antibodies detected cTnT or cTnI in normal myocardium and its loss from necrotic myocardium, in some cases as early as 30 min after coronary occlusion, before histologic evidence of necrosis was present. Loss was nonuniform, being greater at the periphery of the infarcts then at their central regions. Usually, loss of cTnT appeared greater than loss of cTnI. With reperfusion, findings were similar to those after permanent occlusion, except that there was a greater contrast between loss at the periphery compared to the loss in the central region. Considerable residual staining persisted for hours after occlusion, indicating delayed release over time, concordant with sustained serum elevations in patients with acute myocardial infarction. No loss of staining was observed in nonnecrotic myocardium. CONCLUSIONS: Immunohistochemical staining using antibodies to human cTnT and cTnI can be used to visualize cardiac troponins and document their loss in histologic sections of myocardium in different animal species. Loss of cTnT and cTnI occurs very early following ischemic injury and may precede histologic evidence of necrosis, but does not occur in myocardium that is not necrotic. Immunohistochemical staining of hearts for cTnT and cTnI can assist in the often difficult recognition of myocardial necrosis at autopsy, in patients suspected of dying from acute myocardial ischemia.     

Myocyte loss in early left ventricular hypertrophy of experimental renovascular hypertension

Left ventricular hypertrophy (LVH) develops very early in experimental renovascular hypertension after clipping of one renal artery and is accompanied by a remodeling of cardiac structure which has not yet been investigated in detail. It was the aim of the present study to analyze changes in cardiomyocyte number and volume in LVH after 2 weeks of renovascular hypertension. Sprague-Dawley rats were subjected to clipping of the left renal artery (2K1C) or sham operation (sham). One group of 2K1C rats received antihypertensive treatment with dihydralazine. The experiment was terminated after 2 weeks. Hearts were investigated using stereological methods, electron microscopy, immunohistology for the proliferation marker proliferating cell nuclear antigen, the pro- and anti-apoptotic proteins Bax and Bcl-2 as well as the TUNEL technique. After 2 weeks, systolic blood pressure and relative left ventricular weight were significantly higher in untreated 2K1C animals than in sham and dihydralazine-treated 2K1C rats. Volume fraction of interstitial tissue and capillary length density were not different, whereas wall thickness of intramyocardial arteries was significantly higher in untreated 2K1C (5.12+/-0.7 micro m) than in sham (3.92+/-0.6 micro m) and in dihydralazine-treated 2K1C (3.91+/-0.7 micro m) rats. Cardiomyocyte diameter and volume were significantly higher in untreated 2K1C than in sham animals. The number of cardiomyocytes per left ventricle was significantly lower in untreated 2K1C rats (5.5+/-1.6 vs 3.9+/-6.9 x10(7)). Using immunohistochemistry, no direct evidence of apoptosis was found, but a relative higher expression of the anti-apoptotic protein bcl-2 expression was seen in untreated 2K1C than in sham animals. This may reflect a protective mechanism as a consequence of earlier occurring apoptosis. These observations document that experimental renovascular hypertension induces a rapidly developing LVH characterized by marked cardiac remodeling and substantial loss of cadiomyocytes.     

Immunohistochemical study of fibronectin in experimental myocardial infarction.

Light microscopic immunohistochemical studies were performed to evaluate the distribution of fibronectin in paraffin sections of p-formaldehyde-fixed normal rat hearts and the hearts of rats that had undergone ligation of the left coronary artery. A peroxidase-labeled antibody technique was used, together with appropriate immunohistochemical control procedures, for the localization of fibronectin in normal hearts and in the hearts of sham-operated animals. Fibronectin was localized in the interstitial space between myocytes, and beneath arterial, venous, and capillary endothelium. At 4 hours after coronary ligation, fibronectin was localized in a patchy fashion in the cytoplasm and interstitial space of some of the myocytes in the area supplied by the ligated vessel. At 24 hours, there was more intense, homogeneous staining in necrotic myocytes in the infarcted area and in the capillary endothelium in the border zone. At 48 hours, the intensity of staining for fibronectin was maximal in and between the necrotic myocytes in the center of the infarct and in proliferating and migrating capillaries and fibroblasts in the border zone. Similar patterns of localization were observed at 3 and 7 days after coronary ligation, but with progressive decreases in the intensity of staining. Two sources of fibronectin appeared to have contributed to these changes: plasma fibronectin diffusing through damaged blood vessels would account for the early staining observed in necrotic myocytes in the center of the infarct, whereas de novo synthesis of fibronectin by connective tissue cells and endothelial cells in sprouting capillaries would be responsible for the subsequent staining observed in viable capillaries in the border zone of the infarct. Known properties of fibronectin in vitro, combined with these in vivo observations, indicate that fibronectin may influence the thrombotic, inflammatory, angiogenic, and fibrotic processes involved in infarct healing.     

The development and progression of myocyte injury at the margins of experimental myocardial infarcts

Distinct differences in the extent and progression of the lateral and epicardial boundaries of evolving regional infarcts were demonstrated in isolated rabbit hearts. Ischemia was produced by interrupting (0-240 minutes) flow in the ventral interventricular branch of the left coronary artery, whilst the remainder of the heart was continuously perfused with oxygenated Krebs-Henseleit bicarbonate buffer. Perfusion fixed blocks were freeze-fractured then examined using back-scattered electron imaging in a scanning electron microscope. Control myocytes showed relatively smooth, continuous internal fracture faces. After 30 min of ischemia myocytes showed evidence of mild, probably reversible, injury in the form of prominence of pits and channels. Severe injury, characterized by separation of organelles and prominent intracellular spaces, developed after 60 or more min of ischemia, first in the subendocardial two thirds, and after 120 min across the full thickness of the ventricular wall. At the lateral margins of infarcts there was a distinct cell-to-cell boundary between control and severely injured myocytes, with only a few scattered mildly injured cells within 30 mu of the infarct. Although transmural progression of necrosis provides the potential for recovery of the external aspect of the myocardium in the ischemic zone by reperfusion, corresponding regions of salvageable myocytes at lateral infarct margins are very narrow.