Search

The performance of the heart depends on the concentrations of free calcium ions in the cytoplasm of the myocytes. However, direct evidence for changes in free Ca concentration in physiological events during response to drugs and in pathogenesis has been difficult to obtain because of technical problems in measuring free Ca at 10(-7) M in cells with a volume of only a few picolitres. Here we describe measurements made with the Ca-sensitive photoprotein aequorin in single ventricular myocytes isolated from rat heart. We have detected signals from resting and contracting cells, and from cells exposed to media of altered ionic composition (raised K, lowered Na), ouabain and metabolic inhibitors. We report that free Ca in metabolically-poisoned myocytes is remarkably stable and that severe injury to the cell occurs before the free Ca concentration rises above 1-3 X 10(-7) M, hence cell damage seems to be a cause, not a consequence, of a rise in free Ca. The technique used here should help to resolve many uncertainties regarding free Ca in heart function, and should be particularly valuable for investigating the role of free Ca in ischaemic pathogenesis.

Electron microscopic autoradiographic investigations to study the localization of Ca in muscle were performed using heart muscle. The distribution of autoradiographic grains, originating from Ca-45 were compared above myofibrils and interfibrillar spaces of the ventricle, auricle and sinus node of the heart. Our aim was to get new data on the role of Ca in the mechanical activity of muscle with special consideration of the Ca content of interfibrillar spaces. The different distribution of grains in the muscle fibrils and interfibrillar spaces of various parts of the heart can be in connection with the different activities of these parts. The more Ca content in myofibrils of ventricle cells may be in connection with its greater mechanical activity and its work.

Atomic absorption spectrometry was used to measure magnesium, calcium, and sodium, and emission spectrometry to measure potassium, in myocardium (left and right ventricles) of 26 control subjects who died of acute trauma. Results were expressed in mumol/g of proteins. Mg/Ca and K/Na ratios were also determined. The same measurements were made in 24 patients who died from acute myocardial infarction. Samples were also taken from the necrotic area. Mg/Ca and K/Na ratios were significantly higher in the left ventricle of both populations, thus providing evidence of anatomical and physiological differences between the two ventricles. As a result of cytolysis and anoxia, the Mg/Ca ratio was very significantly inverted, and the K/Na ratio very significantly smaller, for samples from the necrotic area. In these clinical conditions arrhythmias could certainly be considered likely, and there is reason to believe that magnesium depletion may be a cause of arrhythmias.

The concentration of osmium has been measured by destructive chemical analysis in glutaraldehyde fixed heart tissue postfixed with osmium tetroxide and embedded in epoxy resin. After such treatment, the mean atomic number of the specimen (Z) is close to 10, which permits a quantitative analysis of calcium (Ca) by the continuum method, using Z2/A as a correcting factor (A: atomic weight). Wavelength-dispersive X-ray microanalysis has been used to determine the Ca concentration of frog cardiac tissue fixed in glutaraldehyde and embedded in resin. These measurements have been repeated on tissue postfixed in osmium tetroxide; contrary to expectations, the apparent Ca concentration is much higher in osmium treated than in nontreated tissue. However, this result is observed with OsO4 solutions prepared in glass, not with solutions prepared in plastic. It is shown by energy dispersive X-ray analysis of droplets that OsO4 solutions prepared in glass contain large amounts of calcium, potassium and silicon. Care must be taken in preparing OsO4 fixatives when the fixed tissues are to be subjected to X-ray microanalysis of such elements as Ca or Si.

A non-enzymatic method is presented for isolating mitochondria from small-sized human cardiac samples, including ventricular needle biopsies of 15-25 mg of wet weight. Electron microscopy demonstrates that these fractions are rich in structurally well preserved mitochondria. Calcium and magnesium levels of fractions are determined by atomic absorption flame spectroscopy. Comparative analyses are made in similar fractions of the mouse ventricle. Calcium concentrations of mitocondria isolated in the presence of ruthenium red do not differ significantly between the human auricle and ventricle, averaging 61 nmol Ca/mg protein and 68 nmol Ca/mg protein, respectively. Mitochondrial calcium level is lower in the mouse ventricular fractions, averaging 7 nmol Ca/mg protein. Mitochondrial magnesium amounts to slightly less than 60% of the calcium levels in the human heart, while it exceeds the calcium level by more than 100 per cent in the mouse heart. There is no significant difference of mitochondrial calcium between normal auricles, and, auricles of patients with increased right atrial mean pressure and/or volume overload.

The calcium content and inulin space of the atrial and ventricular muscle were determined in isolated perfused heart of the toad, bullfrog, guinea pig, rat, and cat. In all species studied, the total and "cellular" calcium content of the atrium were higher than those of the ventricle. A high calcium content of the atrial muscle was also observed in fresh unperfused hearts. The total calcium content of atrial and ventricular muscles increased when extracellular phosphate concentration was increased. When severe ventricular arrhythmia such as fibrillation occurred during perfusion, the total calcium content of the ventricle increased. The decay of tissue calcium content with calcium washout was examined in the toad, guinea pig, and rat heart. It was revealed that about 80 percent of total calcium existed as "exchangeable" calcium in both atrial and ventricular muscles. When the extracellular calcium concentration ([Ca]o) was altered in the toad, guinea pig, and rat heart preparation, the cellular calcium content of atrial muscle varied in proportion to [Ca]o, whereas that of ventricular muscle remained fairly constant at a higher [Ca]o value. The development of contractile tension in the atrial and ventricular muscles at various [Ca]o values corresponded well to these changes in the cellular calcium content except for the rat ventricle in which the contractile tension was almost proportional to [Ca]o. The relationship between the development of contractile tension and the level of cellular calcium content or [Ca]o was discussed.

We tested if interaction between the thick and thin filaments, as revealed by the rate of cross-bridge cycling or the pattern of calcium activation, influences the relation between tension and free calcium concentration (pCa) of cardiac muscle. Bundles of rabbit ventricular cells which contained either fast-cycling RV1 myosin or slowly-cycling RV3 myosin and bundles of human atrial cells were chemically skinned by exposure to saponin. Skinned bundles were rapidly activated with calcium using the method of Moisescu and Thieleczek (1978). Our results suggest that the relation between pCa and tension is not different for skinned bundles of rabbit ventricular cells that contain either fast or slowly cycling myosin cross-bridges. The pattern of calcium activation, i.e. maximal to submaximal or submaximal to maximal, was also found not to influence the relation between tension and pCa for either rabbit ventricular or human atrial muscle. The tension-pCa relation of cardiac muscle was not altered by conditions that have been shown to alter the tension-pCa relation of skeletal muscle. These results suggest that the mechanisms responsible for regulating force development at the level of the thick and thin filaments that have been reported for skeletal muscle, are absent in cardiac muscle.

Microelectrodes filled with neutral carrier selective to Ca2+ were used to measure the free intracellular Ca2+ concentration [( Ca2+]i) in sheep cardiac tissue and frog skeletal muscle. Calibration of the electrodes was performed in the presence of a solution resembling the cationic composition of the cytoplasm. [Ca2+]i at rest in normal physiological saline (20-22 degrees C) was 240 nM in Purkinje fibres, 270 nM in ventricular muscle, and 52 nM in skeletal muscle. In Purkinje fibres, elevation of [Ca2+]o from 1.8 mM to 5.4 mM produced a 1.7-fold increase in [Ca2+]i. Elevation of [Ca2+]o from 1.8 mM to 18 mM induced a 2.6-fold increase in [Ca2+]i. Exposure to Na+-free solution (Li+-substituted) gave rise to elevation of [Ca2+]i by factors of 5.8 and 14 in ventricular muscle and Purkinje fibres, respectively. These latter changes in [Ca2+]i were associated with the development of contractures which reached 34% and 172% of the corresponding twitch tension.

We have measured intracellular free Ca2+ concentration in rat and guinea pig ventricular myocytes using the fluorescent Ca2+-indicator quin2. Our results indicate a resting concentration in heart cells that is considerably lower than previous estimates. The mean value of 137 nM for rat ventricle that we have recorded is consistent with the hypothesis that resting intracellular [Ca2+] is controlled by a voltage-dependent, sarcolemmal exchange mechanism. Furthermore, we show that activation of plasma membrane Ca2+-channels is involved when intracellular free Ca2+ increases in response to K+-depolarization.