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.

The value of the diffusion coefficient for oxygen in muscle is uncertain. The diffusion coefficient is important because it is a determinant of the extracellular oxygen tension at which the core of muscle fibers becomes anoxic (Po(2crit)). Anoxic cores in muscle fibers impair muscular function and may limit adaptation of muscle cells to increased load and/or activity. We used Hill's diffusion equations to determine Krogh's diffusion coefficient (Dalpha) for oxygen in single skeletal muscle fibers from Xenopus laevis at 20 degrees C (n = 6) and in myocardial trabeculae from the rat at 37 degrees C (n = 9). The trabeculae were dissected from the right ventricular myocardium of control (n = 4) and monocrotaline-treated, pulmonary hypertensive rats (n = 5). The cross-sectional area of the preparations, the maximum rate of oxygen consumption (Vo(2 max)), and Po(2crit) were determined. Dalpha increased in the following order: Xenopus muscle fibers Dalpha = 1.23 nM.mm(2).mmHg(-1).s(-1) (SD 0.12), control rat trabeculae Dalpha = 2.29 nM.mm(2).mmHg(-1).s(-1) (SD 0.24) (P = 0.0012 vs. Xenopus), and hypertrophied rat trabeculae Dalpha = 6.0 nM.mm(2).mmHg(-1).s(-1) (SD 2.8) (P = 0.039 vs. control rat trabeculae). Dalpha increased with extracellular space in the preparation (Spearman's rank correlation coefficient = 0.92, P < 0.001). The values for Dalpha indicate that Xenopus muscle fibers cannot reach Vo(2 max) in vivo because Po(2crit) can be higher than arterial Po(2) and that hypertrophied rat cardiomyocytes can become hypoxic at the maximum heart rate.

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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.

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 mm 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.