Radovan Zak

Cell Proliferation During Cardiac Growth

Abstract During the embryonic development of the myocardium both undifferentiated cells and cells containing muscle-specific proteins divide. As the heart grows and approaches maturity, its muscle cells progressively lose their mitotic activity; myocardial cell enlargement then becomes the principal process by which the heart as a whole enlarges. Mitotic figures in nuclei of heart muscle cells are frequent in the neonatal rat but become very rare at about the third month of postnatal life. Both in the developing and adult animal the work load is one of the determinants of cardiac size. The cytologic features of cardiac enlargement depend on the stage of development of the heart at the time when the stimulus to growth occurs. A work load imposed on embryonic or early neonatal hearts results in enlargement characterized by an increase in both the number and size of myocardial cells. The adult heart enlarges only by enlargement of its component muscle cells. Division of ventricular muscle cells in mammals is not activated after cardiac injury. The inability of ventricular myocardial cells to regenerate stands in sharp contrast to skeletal muscle, which is capable of considerable tissue repair, involving both regeneration of individual fibers and reconstitution of the whole muscle.

Biochemical Correlates of Cardiac Hypertrophy I. Experimental Model; Changes in Heart Weight, RNA Content, and Nuclear RNA Polymerase Activity

Cardiac hypertrophy occurred in mature rats after producing supravalvular aortic stenosis with a specially designed silver clip. For 2 weeks following this procedure, heart weight, body weight, and RNA content of the myocardium were serially determined. Heart weight and RNA content increased within 24 hours of aortic banding, reaching a maximal level in 2 days and remaining elevated during the 2 weeks of observation. Nuclei were isolated and purified from heart muscle homogenates, and changes in RNA polymerase activity following aortic banding were determined. The nearest neighbor frequency of the bases of the RNA synthesized by the polymerase from nuclear preparations was identical in both the banded animals and the sham-operated controls. Both groups could thus be compared on the basis of the enzyme assay. RNA polymerase activity in nuclei from the hearts of banded rats rose rapidly when compared with the activity in sham-operated rats; peak values were reached on the second day, the earliest detectable change being around 12 hours. The increase in RNA polymerase activity represents one of the earliest biochemical events that take place in the myocardium following aortic banding.

Regression of myocardial hypertrophy. I. Experimental model, changes in heart weight, nucleic acids and collagen.

Left ventricular hypertrophy was induced by placing a constricting silver band around the ascending aorta in rats. These bands were then removed either 10 days (early debanding) or 28 days (late debanding) after banding. After early debanding the left ventricular mass decreased rapidly from 35% to 11% above the control values (P < 0.001) 3 days post-debanding. Left ventricular weights of the debanded and control groups were not statistically different 7 to 28 days after debanding except at 10 days. The left ventricular RNA of the debanded animals fell from 37% to 19% above the control group 3 days after surgery (P < 0.05), and remained at control levels for the rest of the study period. The left ventricular DNA of the debanded group, however, remained elevated; i.e., 32% above control levels (P < 0.001), 10 days post-debanding, and 12% at 28 days (P = n.s.). Left ventricular hydroxyproline levels of the debanded group at these times were 90% (P < 0.001) and 80% (P < 0.025), respectively, above those of controls. In the late debanding study, both left ventricular mass and RNA fell less than in the early study. The left ventricular mass remained significantly elevated, i.e., 12% above that of controls (P < 0.025), 21 days post-debanding, and left ventricular RNA was 34% above control values (P < 0.01) at 14 days. Both decreased to control values thereafter. Left ventricular DNA and hydroxyproline content did not decline, remaining 25% (P < 0.02) and 118% (P < 0.001), respectively, above the control values 28 days post debanding. The data show that while left ventricular mass and RNA content decrease after relief of a pressure overload, left ventricular DNA and hydroxyproline content does not.

Mitochondria and Cardiac Hypertrophy

• Normally, myocardial metabolism is almost exclusively aerobic. The substantial quantities of adenosine triphosphate (ATP) required for muscle contraction and the much smaller amounts necessary for the maintenance of functions such as ion transport, rhythmicity, and conduction and the synthesis of membrane and protein constituents of the myocardium are supplied almost exclusively by mitochondrial oxidation of fatty acid and carbohydrate substrates. Even under conditions of stress such as those that exist during severe exercise, when the cardiac ATP requirement may be increased considerably, the capacity of the mitochondria for oxidative phosphorylation appears to be adequate to meet the requirements. The high level of and the capacity for oxidative metabolism in the heart are reflected morphologically in the remarkable observation that mitochondria constitute more than 35% of the cardiac cell volume as measured by quantitative electron microscopic stereological procedures (1). Acute changes in energy requirements such as those that occur during strenuous exercise appear to be effectively met by increased mitochondrial synthesis of ATP, and the synthesis is finely adjusted to ATP requirements by the respiratory control mechanism. Increased utilization of ATP results in transient accumulation of adenosine diphosphate (ADP), which acts as a phosphate acceptor and stimulates the mitochondrial oxidative rate by decreasing the level of mitochondrial high-energy intermediates (2) or the proton-motive force across the inner mitochondrial membrane (3), according to the chemical or chemiosmotic theories of oxidative phosphorylation, respectively. Sustained levels of increased preload or afterload, however, activate another more slowly re

Long-term cell culture of adult mammalian cardiac myocytes: Electron microscopic and immunofluorescent analyses of myofibrillar structure

Adult rat heart was dissociated into a single-cell suspension by a retrograde perfusion technique with collagenase and hyaluronidase in Krebs-Ringer phosphate buffer. Long-term culture of these isolated single cardiac muscle cells was established for up to 45 days. Transmission electron microscopy and immunofluorescence analysis with monoclonal antibodies to cardiac myosin were used to examine sequentially the external and internal structural organization of the cardiac myocytes. Most of the cardiac myocytes exhibited prominent alterations in their external and internal structural organization during the first two weeks of culture. As they attached to the substrate and spread out, the myocytes assumed various shapes and sizes, with the exception of a few which maintained their original cylindrical shape. Electron microscopy of 2 to 4-day cultures revealed that most of the muscle cells contained disorganized myofibrils and surface blebs with enclosed mitochondria and myofilaments, which were eventually extruded from the cytoplasm. With progressive culture, the cardiac myocytes appeared to lose myofibrillar material; fewer myofilaments or sacromere fragments with interfibrillar mitochondria were observed in the sarcoplasm. Such cells resembled cultured embryonic or neonatal cardiac myocytes. However, some muscle cells retained closely packed, well organized myofibrils characteristic of freshly dissociated or in vivo cardiac myocytes. Immunofluorescence microscopy demonstrated that the cultured cardiac myocytes were strongly myosin positive throughout their morphological changes and subsequent maintenance in culture. Two patterns of fluorescence were observed in these cells in correlation with the fine structural evidence for myofibrillar distribution. One pattern exhibited bright fluorescence near the central region of the cell with a more weakly diffuse fluorescence throughout the cytoplasm; the other pattern was characterized by bright fluorescence throughout the sarcoplasm. Most of the myocytes retained their contractility throughout the culture period excepting the initial 24 to 48 h of cell attachment and flattening. These studies demonstrate the feasibility of maintaining contractile cardiac muscle cells from adult rats for at least 1 1/2 months in monolayer culture, although some variability in myofibrillar organization has been observed.

Myofibrillar Mass in Rat and Rabbit Heart Muscle: CORRELATION OF MICROCHEMICAL AND STEREOLOGICAL MEASUREMENTS IN NORMAL AND HYPERTROPHIC HEARTS

A sequestered fraction of myofibrillar magnesium presumably bound to thin filaments during polymerization of actin was used to determine myofibrillar mass in rat left ventricles and rabbit hearts. Myofibrillar volume was also determined by stereological measurements on electron micrographs of rat ventricles. Myofibrillar Mg (1.11 mmoles/kg dry ventricle) can be determined in glycerinated ventricle either by measuring 28Mg-inexchangeable Mg or Mg remaining after extraction with EDTA, KCl, and a nonionic detergent. These measurements, combined with the Mg content of myofibrillar suspensions similarly extracted (3.2 mmoles/kg protein), indicated that myofibrillar mass was ∼347 g myofibrillar protein/kg dry ventricle. In rabbit hearts, increases in myofibrillar Mg per unit dry weight were as follows: auricular appendage < right ventricle < left ventricle ≅ interventricular septum. After production of left ventricular hypertrophy in rats by constriction of the ascending aorta, both myofibrillar Mg and volume increased within 24 hours. After 10 or more days, myofibrillar Mg and myofibrillar volume increased proportionately more than tissue dry mass and cell volume, and the ratio of mitochondrial volume to cell volume decreased.

Evidence for expression of a common myosin heavy chain phenotype in future fast and slow skeletal muscle during initial stages of avian embryogenesis

We have utilized a key biochemical determinant of muscle fiber type, myosin isoform expression, to investigate the initial developmental program of future fast and slow skeletal muscle fibers. We examined myosin heavy chain (HC) phenotype from the onset of myogenesis in the limb bud muscle masses of the chick embryo through the differentiation of individual fast and slow muscle masses, as well as in newly formed myotubes generated in adult muscle by weight overload. Myosin HC isoform expression was analyzed by immunofluorescence localization with a battery of anti-myosin antibodies and by electrophoretic separation with SDS-PAGE. Results showed that the initial myosin phenotype in all skeletal muscle cells formed during the embryonic period (until at least 8 days in ovo) consisted of expression of a myosin HC which shares antigenic and electrophoretic migratory properties with ventricular myosin and a distinct myosin HC which shares antigenic and electrophoretic migratory properties with fast skeletal isomyosin. Similar results were observed in newly formed myotubes in adult muscle. Future fast and slow muscle fibers could only be discriminated from each other in developing limb bud muscles by the onset of expression of slow skeletal myosin HC at 6 days in ovo. Slow skeletal myosin HC was expressed only in myotubes which became slow fibers. These findings suggest that the initial commitment of skeletal muscle progenitor cells is to a common skeletal muscle lineage and that commitment to a fiber-specific lineage may not occur until after localization of myogenic cells in appropriate premuscle masses. Thus, the process of localization, or events which occur soon thereafter, may be involved in determining fiber type.

Biochemical Correlates of Cardiac Hypertrophy V. LABELING OF COLLAGEN, MYOSIN, AND NUCLEAR DNA DURING EXPERIMENTAL MYOCARDIAL HYPERTROPHY IN THE RAT

The incorporation of [2, 3-3H]proline into collagen hydroxyproline and into noncollagenous protein was measured during development of cardiac hypertrophy produced by surgical constriction of the ascending aorta in rats. Heart weight increased sharply during the first 4 days after aortic banding and then slowly rose to a plateau. Free intracellular proline concentration remained unaltered 2 days after banding and increased by 38% on the fifth postoperative day. Specific radioactivity of free proline 3 hours after injection of [3H]proline was elevated by 40% on the second postoperative day but was unchanged on the fifth day. Incorporation of [3H]proline into collagen hydroxyproline peaked sharply on the second day after aortic constriction (550% above control) in one experiment, and on the fourth day (330% above control) in another. The peak labeling of noncollagenous protein by [3H]proline (100% over control) was less than that of collagen. To further evaluate the differences in synthetic response of muscle cells and interstitial cells to increased work load, incorporation of radioactive precursors into myosin, collagen, nuclear DNA, and noncollagenous protein were measured simultaneously, after aortic constriction. Collagen labeling was maximal either on day 2 or 4, depending on the degree of hypertrophy. Labeling of myosin and noncollagenous protein reached a plateau on day 4. Incorporation of [3H]thymidine into nuclear DNA peaked on day 7. Thus both muscle cells and connective tissue cells respond independently during cardiac hypertrophy with an increased synthesis of specific proteins.

Transitions in cardiac isomyosin expression during differentiation of the embryonic chick heart.

The expression of different isoforms of the contractile protein myosin plays a major role in determining contractile characteristics in both cardiac and skeletal muscle in the adult. There is little evidence pertaining to putative changes in myosin phenotype during cardiac embryogenesis or if such changes could play a role in modulating the contractile characteristics of the developing heart. We examined isomyosin expression during cardiogenesis in the chick by indirect immunofluorescence microscopy with monoclonal antibodies to adult ventricular and atrial myosin heavy chains. Antibody specificity was characterized in the adult on the basis of immunofluorescence localization, ELISA, and protein blot immunoassay. Results show that the early embryonic chick heart has a different myosin phenotype than the later embryonic or adult heart. Both the embryonic ventricular and atrial myocardia initially expressed a myosin heavy chain that was recognized by antibody specific (in the adult) for ventricular myosin heavy chain. The ventricles remained reactive throughout life with the ventricular antibody, but reactivity of the atrial myocardium was confined to the initial 6 days of embryonic development. On the other hand, reactivity of the embryonic heart with multiple antibodies specific (in the adult) for atrial myosin was confined to the atrial myocardium throughout development. Thus, the distribution of myosin isoforms became similar to that of the adult myocardium by the time the embryonic heart achieved a 4-chambered configuration at 6 days in ovo.

Correlations between a nuclear and a mitochondrial mRNA of cytochrome c oxidase subunits, enzymatic activity and total mRNA content, in rat tissues

SummaryCytochrome c oxidase (COX), like other multi-subunit components of the respiratory chain, is controlled by both the nuclear and the mitochondrial genome. In order to find wether there is a close relationship between mRNAs encoded by the nucleus and by the mitochondrion, and between these mRNAs and enzyme activity, we compared six rat tissues (ventricle, liver, m. soleus, m. plantaris, and the white and red portions of m. gastrocnemius). We found a tenfold range for COX activity, a tenfold range for the contents of mRNA III (mitochondrial) and mRNA VIc (nuclear), a threefold range for total [poly(A)+] mRNA content and a sevenfold range for total RNA content in these tissues. The ratio of mRNA III to mRNA VIc was equal in each tissue, indicating the presence of a mechanism that coordinates the two genomes. There was a good correlation between mRNA content and COX activity (r = 0.78 for VIc, r = 0.77 for 111; p < 0.0001), demonstrating that the expression of this enzyme is mainly under pretranslational control.