Giorgio Olivetti

Apoptosis in the Failing Human Heart

BACKGROUND Loss of myocytes is an important mechanism in the development of cardiac failure of either ischemic or nonischemic origin. However, whether programmed cell death (apoptosis) is implicated in the terminal stages of heart failure is not known. We therefore studied the magnitude of myocyte apoptosis in patients with intractable congestive heart failure. METHODS Myocardial samples were obtained from the hearts of 36 patients who underwent cardiac transplantation and from the hearts of 3 patients who died soon after myocardial infarction. Samples from 11 normal hearts were used as controls. Apoptosis was evaluated histochemically, biochemically, and by a combination of histochemical analysis and confocal microscopy. The expression of two proto-oncogenes that influence apoptosis, BCL2 and BAX, was also determined. RESULTS Heart failure was characterized morphologically by a 232-fold increase in myocyte apoptosis and biochemically by DNA laddering (an indicator of apoptosis). The histochemical demonstration of DNA-strand breaks in myocyte nuclei was coupled with the documentation of chromatin condensation and fragmentation by confocal microscopy. All these findings reflect apoptosis of myocytes. The percentage of myocytes labeled with BCL2 (which protects cells against apoptosis) was 1.8 times as high in the hearts of patients with cardiac failure as in the normal hearts, whereas labeling with BAX (which promotes apoptosis) remained constant. The near doubling of the expression of BCL2 in the cardiac tissue of patients with heart failure was confirmed by Western blotting. CONCLUSIONS Programmed death of myocytes occurs in the decompensated human heart in spite of the enhanced expression of BCL2; this phenomenon may contribute to the progression of cardiac dysfunction.

Gender differences and aging: Effects on the human heart

OBJECTIVES This study investigated the changes in myocyte size and number in the left and right ventricles that occur with aging in the female and male heart. BACKGROUND Differences in life span between women and men may be related to a better preservation of myocardial structure in the female heart with aging. On this basis, the hypothesis was advanced that the aging process has a different impact on the integrity of the myocardium in the two genders. METHODS Morphometric methodologies were applied to analyze the changes in number and size of ventricular myocytes in the hearts of 53 women and 53 men. The changes in mononucleated and binucleated myocytes with age were determined in enzymatically dissociated cells. The age interval examined varied from 17 to 95 years. RESULTS Aging was associated with a preservation of ventricular myocardial mass, aggregate number of mononucleated and binucleated myocytes, average cell diameter and volume in the female heart. In contrast, nearly 1 g/year of myocardium was lost in the male heart, and this phenomenon accounted for the loss of approximately 64 million cells. This detrimental effect involved the left and right sides of the heart. In the remaining cells, myocyte cell volume increased at a rate of 158 microns3/year in the left and 167 microns3/year in the right ventricle. CONCLUSIONS Aging does not lead to myocyte cell loss and myocyte cellular reactive hypertrophy in women, indicating that gender differences may play a significant role in the detrimental effects of the aging process on the heart.

PTX3, A Prototypical Long Pentraxin, Is an Early Indicator of Acute Myocardial Infarction in Humans

BACKGROUND Inflammation is an important component of ischemic heart disease. PTX3 is a long pentraxin whose expression is induced by cytokines in endothelial cells, mononuclear phagocytes, and myocardium. The possibility that PTX3 is altered in patients with acute myocardial infarction (AMI) has not yet been tested. METHODS AND RESULTS Blood samples were collected from 37 patients admitted to the coronary care unit (CCU) with symptoms of AMI. PTX3 plasma concentrations, as measured by ELISA, higher than the mean+2 SD of age-matched controls (2.01 ng/mL) were found in 27 patients within the first 24 hours of CCU admission. PTX3 peaked at 7.5 hours after CCU admission, and mean peak concentration was 6.94+/-11.26 ng/mL. Plasma concentrations of PTX3 returned to normal in all but 3 patients at hospital discharge and were unrelated to AMI site or extent, Killip class at entry, hours from symptom onset, and thrombolysis. C-reactive protein peaked in plasma at 24 hours after CCU admission, much later than PTX3 (P<0.001). Patients >64 years old and women had significantly higher PTX3 concentrations at 24 hours (P<0.05). PTX3 was detected by immunohistochemistry in normal but not in necrotic myocytes. CONCLUSIONS PTX3 is present in the intact myocardium, increases in the blood of patients with AMI, and disappears from damaged myocytes. We suggest that PTX3 is an early indicator of myocyte irreversible injury in ischemic cardiomyopathy.

Quantitative Structural Analysis of the Myocardium During Physiologic Growth and Induced Cardiac Hypertrophy: A Review

The quantitative structural properties of the ventricular myocardium during postnatal physiologic growth are compared with those accompanying an increased load in the adult rat heart to determine whether induced cardiac hypertrophy is a pathologic condition or simply a form of well compensated accelerated growth. The expansion of the ventricular myocardium during maturation shows a remarkable degree of well balanced compensatory response, because the capillary microvasculature, parenchymal cells and subcellular components of myocytes all grow in proportion to the increase in cardiac mass. In contrast, the increases in myocyte diameter and length caused by pressure hypertrophy, volume hypertrophy and infarction-induced hypertrophy are consistent with concentric, eccentric and a combination of concentric and eccentric hypertrophic growth of the whole ventricle, respectively. These cellular shape changes may represent a compensatory response of the myocardium at the cellular level of organization that tends to minimize the effects of an increased pressure or volume load, or both, on the heart. Cardiac hypertrophy, however, may also show alterations affecting capillary luminal volume and surface and the mitochondrial to myofibril volume ratio, which indicate an inadequate growth adaptation of the component structures responsible for tissue oxygenation and energy production. Thus, hypertrophy of the adult heart differs from that during physiologic growth, and the hypertrophied myocardium may exhibit structural abnormalities that can be expected to increase its vulnerability to ischemia.

The cellular basis of dilated cardiomyopathy in humans

The present investigation was designed to evaluate whether end-stage cardiac failure in patients affected by dilated cardiomyopathy (DC) was dependent upon extensive myocyte cell death with reduction in muscle mass or was the consequence of collagen accumulation in the myocardium independently from myocyte cell loss. In addition, the mechanisms of ventricular dilation were analysed in order to determine whether the changes in cardiac anatomy were important variables in the development of intractable congestive heart failure. DC is characterized by chamber dilation, myocardial scarring and myocyte hypertrophy in the absence of significant coronary atherosclerosis. However, the relative contribution of each of these factors to the remodeling of the ventricle is currently unknown. Moreover, no information is available concerning the potential etiology of collagen deposition in the myocardium and the changes in number and size of ventricular myocytes with this disease. Morphometric methodologies were applied to the analysis of 10 DC hearts obtained from patients undergoing cardiac transplantation. An identical number of control hearts was collected from individuals who died from causes other than cardiovascular diseases. DC produced a 2.2-fold and 4.2-fold increase in left ventricular weight and chamber volume resulting in a 48% reduction in mass-to-volume ratio. In the right ventricle, tissue weight and chamber size were both nearly doubled. Left ventricular dilation was the result of a 59% lengthening of myocytes and a 20% increase in the transverse circumference due to slippage of myocytes within the wall. Myocardial scarring represented by segmental, replacement and interstitial fibrosis occupied approximately 20% of each ventricle, and was indicative of extensive myocyte cell loss. However, myocyte number was not reduced and average cell volume increased 2-fold in both ventricles. In conclusion, reactive growth processes in myocytes and architectural rearrangement of the muscle compartment of the myocardium appear to be the major determinants of ventricular remodeling and the occurrence of cardiac failure in DC.

Myocyte cell loss and myocyte hypertrophy in the aging rat heart

To determine the effects of age on the myocardium, the functional and structural characteristics of the heart were studied in rats at 3, 10 to 12 and 19 to 21 months of age. Systemic arterial pressure, left ventricular pressure and its first derivative (dP/dt) and heart rate were comparable in the three animal groups. In the interval between 3 and 10 to 12 months, mean myocyte cell volume per nucleus increased 53 and 26% in the left and the right ventricle, respectively. The total number of myocyte nuclei remained constant in either ventricle. In the following period, between 10 to 12 and 19 to 21 months, a 39% further cellular hypertrophy on the left side of the heart was found in association with an 18% loss of cells in the ventricle. Cell loss was accompanied by discrete areas of interstitial and replacement fibrosis in the subendocardium. In contrast, no myocardial damage was observed in the right ventricle, and the measured 35% additional enlargement of myocytes occurred without a change in cell number. Thus, the aging left ventricle is composed of a smaller number of hypertrophied cells. Cellular hypertrophy may explain the unaltered cardiac function of the aged myocardium.

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

The Cellular Basis of Pacing-Induced Dilated Cardiomyopathy Myocyte Cell Loss and Myocyte Cellular Reactive Hypertrophy

BACKGROUND Rapid ventricular pacing leads to a cardiac myopathy consisting of an increase in chamber dimension, mural thinning, elevation in ventricular wall stress, and congestive heart failure, mimicking dilated cardiomyopathy in humans. However, contrasting results have been obtained concerning the mechanisms of ventricular dilation and the existence of myocardial hypertrophy. Moreover, questions have been raised regarding the occurrence of myocardial damage and cell loss in the development of the experimental myopathy. METHODS AND RESULTS The functional and structural characteristics of the heart were studied in conscious dogs subjected to left ventricular pacing at 210 beats per minute for 3 weeks and 240 beats per minute for an additional week. At the time the animals were killed, measurements of myocardial structural integrity and myocyte shape, size, and number were determined by morphometric analysis of the myocardium in situ and enzymatically dissociated cells. The experimental protocol used was associated with overt cardiac failure documented by an increase in left ventricular end-diastolic pressure and a decrease in left ventricular systolic pressure and +dP/dt in combination with tachycardia, ascites, and pulmonary congestion. Although cardiac weights were not altered, cavitary diameter was increased and wall thickness was decreased from the base to the apex of the heart. Multiple foci of replacement fibrosis, comprising 6% of the myocardium, were detected across the left ventricular wall. Measurements of myocyte size and number documented a 39% loss of cells in the entire ventricle and a 61% increase in volume of the remaining viable myocytes. Myocyte hypertrophy was characterized by a 33% increase in cell length and a 23% increase in transverse area, resulting in a 23% increase in the cell length-to-cell diameter ratio. Pacing did not alter the relative proportion of mononucleated, binucleated, and multinucleated myocytes in the myocardium. CONCLUSIONS Myocyte cell loss and myocyte reactive hypertrophy are the major components of ventricular remodeling in pacing-induced dilated cardiomyopathy.

Cellular basis of ventricular remodeling after myocardial infarction

To determine whether acute left ventricular failure associated with myocardial infarction leads to architectural changes in the spared nonischemic portion of the ventricular wall, large infarcts were produced in rats, and the animals were sacrificed 2 days after surgery. Left ventricular end-diastolic pressure was increased, whereas left ventricular dP/dt and systolic pressure were decreased, indicating the presence of severe ventricular dysfunction. Absolute infarct size, determined by measuring the fraction of myocyte nuclei lost from the left ventricular free wall, averaged 63%. Transverse midchamber diameter increased by 20%, and wall thickness diminished by 33%. The number of mural myocytes in this spared region of the left ventricular free wall decreased by 36% and the capillary profiles by 40%. Thus, side-to-side slippage of myocytes in the myocardium occurs acutely in association with ventricular dilation after a large myocardial infarction. In order to analyze the chronic consequences of myocardial infarction on ventricular remodeling, a second group of experiments was performed in which the left coronary artery was ligated and the functional and structural properties of the heart were examined 1 month later. In infarcts affecting an average 38% of the free wall of the left ventricle (small infarcts), reactive hypertrophy in the spared myocardium resulted in a complete reconstitution of functioning tissue. However, left ventricular end-diastolic pressure was increased, left ventricular dP/dt was decreased, and diastolic wall stress was increased 2.4-fold. After infarctions resulting in a 60% loss of mass (large infarcts), a 10% deficit was present in the recovery of viable myocardium. Functionally, ventricular performance was markedly depressed, and diastolic wall stress was increased 9-fold. The alterations in loading of the spared myocardium were due to an increase in chamber volume and a decrease in the myocardial mass/chamber volume ratio that affected both infarct groups. Thus, decompensated eccentric ventricular hypertrophy develops chronically after infarction and growth processes in myocytes are inadequate for normalization of wall stress when myocyte loss involves nearly 40% or more of the cells of the left ventricular free wall. The persistence of elevated myocardial and cellular loads may sustain the progression of the disease state toward end-stage congestive heart failure.

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.