Calibrated histochemistry applied to oxygen supply and demand in hypertrophied rat myocardium.

Oxygen supply and demand of individual cardiomyocytes during the development of myocardial hypertrophy is studied using calibrated histochemical methods. An oxygen diffusion model is used to calculate the critical extracellular oxygen tension (PO(2,crit)) required by cardiomyocytes to prevent hypoxia during hypertrophic growth, and determinants of PO(2,crit) are estimated using calibrated histochemical methods for succinate dehydrogenase activity, cardiomyocyte cross-sectional area, and myoglobin concentration. The model calculation demonstrates that it is essential to calibrate the histochemical methods, so that absolute values for the relevant parameters are obtained. The succinate dehydrogenase activity, which is proportional to the maximum rate of oxygen consumption, and the myoglobin concentration hardly change while the cardiomyocytes grow. The cross-sectional area of the cardiomyocytes, which increases up to threefold in the right ventricular wall due to pulmonary hypertension in monocrotaline-treated rats, is the most important determinant of PO(2,crit) in this model of myocardial hypertrophy. The relationship between oxygen supply and demand at the level of the cardiomyocyte can be investigated using paired determinations of spatially integrated succinate dehydrogenase activity and capillary density. Hypoxia-inducible factor 1alpha can be demonstrated by immunohistochemistry in cardiomyocytes with high PO(2,crit) and increased spatially integrated succinate dehydrogenase activity, indicating that limited oxygen supply affects gene expression in these cells. We conclude that a mismatch of oxygen supply and demand may develop during hypertrophic growth, which can play a role in the transition from myocardial hypertrophy to heart failure.

Twitch and tetanic tension during culture of mature Xenopus laevis single muscle fibres.

Investigation of the mechanisms of muscle adaptation requires independent control of the regulating factors. The aim of the present study was to develop a serum-free medium to culture mature single muscle fibres of Xenopus laevis. As an example, we used the culture system to study adaptation of twitch and tetanic force characteristics, number of sarcomeres in series and fibre cross-section. Fibres dissected from m. iliofibularis (n = 10) were kept in culture at a fibre mean sarcomere length of 2.3 microm in a culture medium without serum. Twitch and tetanic tension were determined daily. Before and after culture the number of sarcomeres was determined by laser diffraction and fibre cross-sectional area (CSA) was determined by microscopy. For five fibres twitch tension increased during culture and tetanic tension was stable for periods varying from 8 to 14 days ('stable fibres'), after which fibres were removed from culture for analysis. Fibre CSA and the number of sarcomeres in series remained constant during culture. Five other fibres showed a substantial reduction in twitch and tetanic tension within the first five days of culture ('unstable fibres'). After 7-9 days of culture, three of these fibres died. For two of the unstable fibres, after the substantial force reduction, twitch and tetanic tension increased again. Finally at day 14 and 18 of culture, respectively, the tensions attained values higher than their original values. For stable fibres, twitch contraction time, twitch half-relaxation time and tetanus 10%-relaxation time increased during culture. For unstable fibres these parameters fluctuated. For all fibres the stimulus threshold fluctuated during the first two days, and then remained constant, even for the fibres that were cultured for at least two weeks. It is concluded that the present culture system for mature muscle fibres allows long-term studies within a well-defined medium. Unfortunately, initial tetanic and twitch force are poor predictors of the long-term behaviour of the fibres.

Calibrated histochemistry of myoglobin concentration in cardiomyocytes.

This article describes the calibration of a histochemical method to determine the myoglobin concentration in individual cardiomyocytes. Calibration is based on paired microdensitometric determinations in sections stained for myoglobin and on biochemical myoglobin determinations in tissue samples from different hearts. In addition, the staining intensity of sections from gelatin blocks containing known amounts of myoglobin is determined. To construct a calibration line, sections stained for myoglobin must be corrected for the degree of shrinkage caused by glutaraldehyde fixation and biochemical myoglobin determinations must be corrected for interstitial space. As an example, the method is used to determine the myoglobin concentration in individual skeletal muscle fibers and in control and hypertrophied rat cardiomyocytes. The amount of myoglobin per cardiomyocyte nucleus is increased two- to threefold in hypertrophied cardiomyocytes, whereas changes in myoglobin concentration depend on the model of hypertrophy used.

Effects of in vivo-like activation frequency on the length-dependent force generation of skeletal muscle fibre bundles.

It is known that a range of firing frequencies can be observed during in vivo muscle activity, yet information is lacking as to how different in vivo-like frequencies may affect force generation of skeletal muscle. This study examined the effects of constant (CSF, constant within one contraction) and decreasing stimulation frequencies (DSF) on mean sarcomere length-force characteristics of rat gastrocnemius medialis fibre bundles. The CSF resulted in an optimal mean sarcomere length (lso) of 2.30 (SEM 0.02), 2.46 (SEM 0.03), 2.76 (SEM 0.03) and more than 2.99 (SEM 0.07) lm, for 100, 50, 30 and 15 Hz, respectively. Compared to 100-Hz stimulation, both lso and the ascending limb of the relationship significantly shifted to higher lengths with lower frequencies. No shift was encountered for the initial part of the descending limb. The DSF reduced the frequency-induced shift to higher mean lengths [lso 2.33 (SEM 0.02), 2.52 (SEM 0.08) and more than 2.92 (SEM 0.10) microm, respectively, for 50, 30 and 15 Hz]. No effect of activation time on length-force characteristics was observed. It was concluded from these studies that the frequency and history of stimulation is a major determinant of the length-force characteristics of muscle fibre bundles, and should be taken into account when analysing animal and human locomotion. The previously observed frequency-induced shift in whole muscle length-force relationship resides mainly at the level of fibre bundles.

Twitch characteristics and energy metabolites of mature muscle fibres of Xenopus laevis in culture.

Mature, high-oxidative, skeletal muscle fibres of Xenopus laevis were kept in culture in L-(Leibovitz's)-15 medium supplemented with creatine and antibiotics and some other additions. Single fibres were mounted at a fixed sarcomere length in a flow-through culture chamber which accommodates stimulus electrodes and a force transducer. Twitch characteristics were determined daily. Depending on culture conditions, fibres remained excitable electrically for up to two weeks at 20 degrees C when foetal calf serum and/or phosphate were added to the culture medium. During the second week, fibres lost phosphocreatine and ATP, but relatively small changes (if any) in total creatine, glycogen and protein contents, fibre volume and dry weight occurred. Succinate dehydrogenase activity decreased after 9 days-when ATP was reduced already. Fibres which were inexcitable electrically contracted normally when exposed to caffeine, indicating that excitation-contraction coupling failed and that the contractile apparatus was still functional.

Histochemistry of sarcoplasmic reticulum Ca-ATPase using dysprosium as capturing reagent.

This report describes the development of a histochemical method for the demonstration of sarcoplasmic reticulum Ca-ATPase activity in cross-sections of skeletal muscle. The demonstration of sarcoplasmic reticulum Ca-ATPase activity is complicated by the fact that capturing reagents for phosphate inhibit the enzyme. We present a minimal model for heavy-metal-phosphate precipitation reactions which gives a theoretical description of the effect of enzyme inhibition on the rate of phosphate precipitation in the section. The model indicates that the choice of capturing reagent is crucial: whether or not ATPase activity can be demonstrated depends mainly on the inhibition constant and the solubility product of the phosphate salt of the capturing reagent (but also on a fairly large number of other factors). All lanthanides tested can be used to demonstrate sarcoplasmic reticulum Ca-ATPase activity, but dysprosium results in the highest staining intensity. This suggests that dysprosium inhibits sarcoplasmic reticulum Ca-ATPase to a lesser degree than the other lanthanides and/or the solubility product of its phosphate salt is smaller. As an example, the method is used to investigate the effect of thyroid hormone on sarcoplasmic reticulum Ca-ATPase activity in individual fibres of the rat soleus muscle.

Mean sarcomere length-force relationship of rat muscle fibre bundles.

To study how sarcomere length inhomogeneities and the duration of activation affect sarcomere length-force characteristics of muscle, the mean sarcomere length-force relationship was determined for twitches and at 100 and 300 ms during tetanic activation for rat extensor digitorum longus and gastrocnemius medialis muscle fibre bundles. Mean sarcomere length is the mean length of all sarcomeres within the fibre, calculated by dividing fibre length by the number of sarcomeres in series in the fibre. The twitch mean sarcomere length-force relationship is shifted to larger sarcomere lengths (optimum mean sarcomere length = 2.69 microns) compared to the relationships determined at 100 or 300 ms of tetanic activation (optimum mean sarcomere length = 2.38 microns), which were the same. It is shown that the normalized Gordon et al. rationale results in a large overestimate of force (at most 68% of force at a sarcomere length of 1.60 microns) for mean sarcomere lengths between 1.4 and 2.0 microns, and in an underestimate of force between 2.3 and 3.0 microns. It is concluded that modelling skeletal mammalian muscle length-force relationships can be improved by using mean sarcomere length-force relations of mammalian fibres instead of the normalized rationale of Gordon et al. derived from a selected homogeneous part of frog fibre.