The purine nucleotide cycle catalyzes the net reaction: aspartate + GTP + H2O----fumarate + NH3 + GDP + Pi. The cycle leads to regeneration of AMP. In skeletal muscle the cycle's rate of operation increases severalfold in response to a corresponding increase in work load. This results in a net increase in citric-acid-cycle intermediates and in release of ammonia. The same may be expected in heart muscle, which, like skeletal muscle, possesses the enzymes of the purine nucleotide cycle. Isolated and working rat hearts were therefore perfused for 45 min at low or high work load (0.16 vs. 0.42 kg X m/min per g dry wt.) with glucose (5 mM) as substrate. Release of ammonia into the perfusate as well as the content of citric-acid-cycle intermediates (citrate, isocitrate, 2-oxoglutarate, malate, and oxaloacetate), related amino acids (aspartate, glutamate, and glutamine), adenine nucleotides, and creatine phosphate were measured in freeze-clamped tissue. There was no significant change between low and high work load in the sum of the citric-acid-cycle intermediates (1.295 vs. 1.313 mumole/g dry wt.), in aspartate (13.21 vs. 14.32 mumole/g dry wt.), in glutamate (15.58 vs. 15.67 mumole/g dry wt.), ATP (19.06 vs. 19.17 mumole/g dry wt.), ADP (5.00 vs. 4.11 mumole/g dry wt.), AMP (1.45 vs. 1.00 mumole/g dry wt.) or in creatine phosphate (22.58 vs. 25.80 mumole/g dry wt.). Ammonia release was 26 and 22 mumole/hr per g dry wt. at low and high work load, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)
Catecholamine-induced myocardial hypertrophy and necrosis in rats have been measured and compared following treatment with different catecholamines. Significant degrees of both hypertrophy (whether measured as biventricular weight or biventricular/body weight ratio) and necrosis (measured by enzyme histochemical techniques on a standardized series of cryostat sections through the apex of each heart) occurred following 10 days' treatment with daily isoproterenol (0.5 or 5 mg/kg s.c.) or dobutamine (5 mg/kg s.c.) (N = 6-12). These agents given to conscious restrained animals lowered blood pressure and increased heart rate for 3, 6, or 1 hr, respectively. Neither hypertrophy nor necrosis occurred after norepinephrine (1 mg/kg) or dopamine (5 mg/kg); both these agents acutely increased blood pressure for about 30 min. Hemodynamic factors may therefore contribute to catecholamine-induced necrosis, which may in turn contribute to the associated hypertrophy.
The ultrastructure and electrophysiological properties of ventricle cells from newborn rats were studied before and after explantation. The cultured cells were dissociated either with trypsin or with collagenase, the latter enzyme being used with and without stirring with a magnetic bar. The explanted cells were studied 10 hr and 48 hr or more after explantation. At 10 hr after explantation, the cells exhibited fast-rising action potentials, but their myofibrils were disorganized, except for stirred collagenase-dispersed cells, which were also depolarized and inexcitable. At 2 days and later after explantation, all preparations had well-defined sarcomeres and myofibrils oriented in parallel similar to the ventricle before explantation, but the cells showed slow-response action potentials together with spontaneous activity. These findings suggest that the disorganization of myofibrils does not reflect damage to the surface membrane. Moreover, collagenase seems more damaging to the cells than trypsin under similar conditions (comparable periods of mechanical stirring), especially 10 hr after explantation.
Fine structural changes in the sarcoplasmic reticulum (SR) in myocardial ischemia, induced by occlusion of the anterior descending branch of the left coronary artery in the canine heart, were studied by the freeze-fracture technique in situ and in vitro and compared to the alterations in Ca2+-stimulated ATPase activity and sodium dodecyl sulfate gel electrophoresis of the isolated SR. Both SR in situ and the isolated SR exhibited the typical intramembranous particles of 70-90 A in freeze-fracture replicas, and the numbers of particles were more numerous in the concave face (PF) than in the convex face (EF). The numbers of particles in the PF were 2748/micron 2 on the average. In ischemia for 1-2 hr, a significant decrease in the numbers of the particles was found in SR in situ, and corresponding changes were noted in the isolated SR. Decreases in Ca2+-stimulated ATPase activity and in the major protein band of ATPase were recognized simultaneously. The close correlation of the changing patterns between the reduction in Ca2+-ATPase and that in the intramembranous particle density during ischemia supports the suggestion that a large part of the intramembranous particles represents ATPase protein itself. The decrease in the particles of SR membrane indicates the degradation of ATPase in the process of ischemic myocardial necrosis.
Ischemia and hypoxia both cause a rapid loss of potassium from myocardial cells. We have investigated the relationship between the accumulation of potassium in the extracellular fluid and the early loss of contractility. Experiments were performed on the isolated rabbit heart perfused with physiological saline at 36 degrees C, paced at 3 Hz. Tension was recorded from the apex. Extracellular potassium concentration [( K+]o) was recorded with small ion-selective electrodes. After the onset of global ischemia, [K+]o rose within 15 sec and reached 9.5 +/- 1.1 mmoles/liter after 5 min. Developed tension (T) fell to 9 +/- 2% of control over the same period. During substrate-free hypoxia, T declined at a similar rate, and [K+]o rose slowly to 5.5 +/- 0.1 mmoles/liter after 5 min. The relationship between [K]o and T during normal perfusion and oxygenation was investigated by incrementally increasing the perfusate [K+]. T dropped to 78.6 +/- 4.5% of control at a [K+]o of 9 mmoles/liter. Comparison of the relationship between [K+]o and T during high-potassium perfusion, ischemia, and hypoxia shows that extracellular potassium accumulation per se makes almost no contribution to the decline of contractile function in ischemia or hypoxia. (Values are means +/- S.E., N = 5.)
Experiments were conducted on 18 dogs using an in situ blood-perfused canine heart model. Intracoronary infusion of AMP resulted in increased ATP and total adenine nucleotide levels. On reperfusion following a 15-min period of ischemia, ATP and total adenine nucleotide levels were significantly higher than control. Most important, contractile function recovered more rapidly in the AMP-treated dogs. It is therefore concluded that the delayed functional recovery noted after periods of ischemia is likely to be a direct result of delayed ATP resynthesis.
Previously, we reported that amrinone increases isometric twitch force but relaxes K+-induced contracture in muscles from normal cat right ventricle. This study evaluated its effects on diseased cardiac tissue. Right-ventricular papillary muscles were obtained from cats with subacute right-ventricular failure (3-14 days after partial pulmonary-artery ligation) and studied in vitro during stimulation (0.5 Hz) and exposure to high-K+ Tyrode solution. Active isometric twitch force and rate of force development (dP/dt) were significantly lower in muscles from hearts with right-ventricular failure compared to control muscles. In addition, while time to peak force was not different, duration of the twitch was significantly longer. In contrast to its positive inotropic actions in control muscles, amrinone (5.3 X 10(-4) M) had no significant effects on twitch force and dP/dt in muscles from failed ventricles. Time to peak force was not changed by amrinone in either group, but unlike its action in control muscle, duration of the twitch was reduced in failed muscle. Amrinone reduced K+-contracture force similarly in both control and failed muscles. Isoproterenol (10(-6) M) significantly increased twitch force and dP/dt and reduced K+-contracture force in both muscle groups. Since amrinone appears to be a phosphodiesterase inhibitor, our data indicate that cyclic AMP (cAMP)-related relaxation processes, but not cAMP-related contractile processes, can be enhanced by phosphodiesterase inhibitors in experimental heart failure. Furthermore, amrinone's reduced positive inotropic effect in failed myocardium suggests that its improvement of ventricular function in patients reflects, in part, enhancement of relaxation.
Cyanide-resistant respiration in heart homogenates supplemented with 1 mM NADH was greater in hypertrophied homogenates (60 days banding) with respect to control homogenates, particularly when the homogenates were incubated in 100% oxygen. The intermyofibrillar mitochondria from hypertrophied hearts produced more superoxide radicals than sub-sarcolemmal mitochondria, and both values were greater than in the unbanded group. H2O2 formation was more evident in the intact mitochondria prepared from hypertrophied hearts than in those of the control hearts. Moreover, the perfusion of isolated hearts in anoxic and reoxygenated conditions caused a greater lipoperoxidative and functional damage at the mitochondrial level in hypertrophied hearts than in the control hearts. These results, correlate with the reduction in mitochondrial function found in the overloaded hearts, suggest an involvement of the reactive species of oxygen in the formation of cardiac damage induced by prolonged aortic banding.
Isolated papillary-muscle preparations from the cat and rabbit were used to study the phenomenon of reoxygenation contracture and whether is is amenable to intervention independently of the preceding hypoxic insult. Reduction of extracellular Ca2+ to "0" mM abolished reoxygenation contracture, but subsequent replacement of Ca2+ resulted in severe contracture and death due to the "calcium paradox." Lowering of Ca2+ to 0.125 mM and its stepwise replacement to 2.5 mM resulted in no contracture with good mechanical recovery. Gradual reoxygenation, Mg2+ (30 mM), Mn2+ (8 mM), or H+ (pH 6.5) ions, or diltiazem (10(-4) M) delayed but did not prevent the development of contracture and contractile failure. Unlike diltiazem, verapamil (10(-4) M) and lidoflazine (2 X 10(-5) M) did not significantly affect the contracture.
A mathematical model of simple oxygen diffusion into a homogeneous cylindrical muscle is developed. The model incorporates a variable sigmoidal relationship between oxygen consumption and oxygen concentration. For any given consumption-concentration relationship, the simulated mean basal metabolic rate (averaged over the radial extent of the muscle) is computed. This calculation is repeated for a range of muscle diameters, yielding the basal metabolic rate-muscle size relationship. This theoretical relationship, which is specific for the underlying oxygen consumption-concentration relationship, is then compared to observed resting heat production-muscle size data reported in the literature. Simulated results fail to explain observed data unless the underlying oxygen consumption-concentration relationship is of a highly improbable form. It is suggested that agreement between theoretical results, based on realistic oxygen consumption-concentration relationships, and experimental observations might be achieved if the mathematical model were extended to include a contribution by myoglobin-facilitated oxygen diffusion to the total oxygen flux.
The ultrastructure of calcium-tolerant isolated myocytes has been compared with that of intact heart myocytes using morphometric techniques. Isolated myocytes were prepared by the method of Powell et al. [1] from four adult rat hearts, and multiple left ventricular tissue samples were obtained from a further four rat hearts after fixation by Langendorff perfusion. The subcellular-component volumes of myofibrils, mitochondria, nuclei, transverse tubules, sarcoplasmic reticulum, lipid droplets, and cytoplasmic space have been estimated using a point-counting method on thin sections of intact myocardium and isolated myocytes. No significant differences between the two sample types were found. With the use of freeze-fracture, the numerical density and distribution of sarcolemmal intramembrane particles were analyzed by the method of Jones et al. [2]. The intramembrane particle density of isolated myocyte E-faces was very similar to that of myocytes of intact myocardium. However, isolated myocytes showed a slight reduction in P-face intramembrane particle density (less than 10%), although intramembrane particle distribution remained unaltered on both fracture faces. Our findings show that the ultrastructural features of the isolated myocytes closely resemble those of their counterparts in the intact myocardium.
This chapter describes the basic properties of integral membrane channels from both cardiac sarcoplasmic reticulum and sarcolemma. Channels are studied, under voltage-clamp conditions, following their incorporation into planar phospholipid bilayers by fusion of isolated native membrane vesicles with preformed membranes. The rate of fusion of vesicles may be influenced by a number of factors including divalent cations and negatively charged phospholipids in the preformed bilayer. Mammalian cardiac sarcoplasmic reticulum contains a monovalent cation selective channel with a single-channel conductance of approximately 150 pico-Siemens in the presence of symmetrical solutions of 500 mM K+ at holding potentials ranging from -60 to +60 mV. The probability of the channel being in the open state is high at positive holding potentials and low at negative holding potentials. Mammalian cardiac sarcolemma contains at least three K+-selective channels and one Cl(-)-selective channel.
The extracellular matrix of heart muscle contains a considerable variety of structures. We have systematically studied the morphology of these structures using several methods of fixation and microscopy. Endomysial connections between cells are comprised of struts of collagen [1] as well as combinations of elastin fibers, collagen fibers, and microfibrils. The rest of the extracellular matrix is filled with a polyanionic lattice of unit collagen fibrils, microthreads, and granules. In the course of these investigations, we have observed regions of structural continuity across the sarcolemma, from endomysial collagen struts to Z-bands. We have also correlated the mechanical resistance to stretch with orientation of epimysial collagen fibers and sarcomere lengths in living as well as fixed rat papillary muscles. Our observations suggest that the extracellular skeletal framework plays an important role in normal cardiac function.
The mechanisms and regulatory factors involved in cardiac proteolysis are incompletely understood. Agents that interfere with lysosomal function (e.g., chloroquine, leupeptin, methyladenine) cause a 25-30% reduction in the overall rate of protein degradation. In the same hearts, however, the rate of myosin breakdown remains unchanged. Disaggregation of micro-tubules with colchicine is accompanied by a 15% reduction in the rate of degradation of total protein and of myosin. In the same hearts, the degradation of "organellar" protein, including mitochondrial cytochromes, is reduced by over 30%. Thus, it appears that the degradation of different classes of cardiac proteins may be accomplished and regulated by different processes. Lysosomes are important in overall proteolysis, but appear not to be involved in the regulation of myosin breakdown. Microtubules are also involved in the proteolytic process, and appear to be especially important for the breakdown of proteins from mitochondria and perhaps other organelles.
Several independent studies have demonstrated that there is a degradation of membrane phospholipids during myocardial ischemia. At present, most of the data support the initial activation of a phospholipase A pathway of phospholipid degradation. The extent of total phospholipid degradation is in the nanomole per gram wet weight quantity, as opposed to ischemic liver, in which the extent of phospholipid depletion approaches the micromole per gram wet weight level. However, in vitro studies suggest that calcium permeability properties and other myocardial cell membrane functions are sensitive to nanomole levels of phospholipid degradation. Clearly, further work is necessary in intact cell and heart preparations to correlate the degradation of phospholipid with the development of irreversible membrane injury during ATP depletion and hypoxia.
Cardiac muscle cells are equipped with three distinct types of intercellular junction--gap junctions, "spot" desmosomes, and "sheet" desmosomes (or fasciae adherentes)--located in a specialized portion of the plasma membrane, the intercalated disk. Gap junctions are responsible for electrical coupling and the transfer of small molecules between cells, whereas the desmosomelike junctions (also known as adherens junctions) provide strong intercellular adhesion. The adhesion sites formed by the "spot" desmosome anchor the intermediate-filament cytoskeleton of the cell; those formed by the fascia adherens anchor the contractile apparatus. An understanding of the ultrastructure of these junctions helps explain how they carry out their functions, and new observations in this field have been made through the application of ultrarapid freezing techniques in conjunction with freeze-fracture electron microscopy. With recent findings from biochemical and immunocytochemical studies, this understanding is now being extended to the molecular level.
The localization in cardiac muscle and the biochemical properties of fibronectin, filamin, and vinculin were studied. Fibronectin was localized between cardiomyocytes. Filamin was identified in the Z-line region of sarcomers and in the intercalated disks of heart muscle. Vinculin was found to be present in intercalated disks and near the plasma membrane at the cell periphery between external myofibrils and sarcolemma. It was suggested that fibronectin, filamin, and vinculin play an important role in intercellular and intracellular linkages in cardiac muscle.
A cell is described that has enabled isolated Langendorff-perfused ferret hearts to be studied in a Bruker WM200 widebore superconducting nuclear magnetic resonance (NMR) spectrometer. Left ventricular pressure was monitored with a latex balloon catheter, and the hearts were paced with a stimulator triggered from the spectrometer's central computer, enabling gated studies to be performed. Suitable radiofrequency filtering for the pacing leads is described. Phosphorus (31P) NMR was used to determine internal pH and the concentration of phosphorylated metabolites under resting conditions. The perfusion rate is shown to affect the phosphocreatine/ATP ratio at low flow rates, but the removal of phosphate from the perfusate is shown not to affect metabolite levels or the internal pH. The time resolution of the method is assessed and its potential for monitoring transient effects illustrated by studies of the effects of acetylcholine and cyanide-induced anoxia. The cardiac gated 31P NMR experiment is discussed and four spectra, corresponding to mid- and end systole and mid- and end diastolic are presented. No effects of cycling of high-energy phosphates are evident in these results.
This chapter presents a brief review of measurements of rapid inward sodium current in single cardiac cells. It is shown that the simplified morphology of the individual cells, with a lack of restricted extracellular space, has been exploited to provide improved spatial and temporal voltage control, resulting in the first recordings of both the activation and inactivation phases of rapid inward sodium current. It is to be expected that future research will produce much interesting data on this component of membrane current, which will have direct relevance to many processes concerned with cardiac function at the cellular level.
