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.

Isometric force production and ATPase activity were determined simultaneously in single human skeletal muscle fibers (n = 97) from five healthy volunteers and nine patients with chronic heart failure (CHF) at 20 degrees C. The fibers were permeabilized by means of Triton X-100 (1% vol/vol). ATPase activity was determined by enzymatic coupling of ATP resynthesis to the oxidation of NADH. Calcium-activated actomyosin (AM) ATPase activity was obtained by subtracting the activity measured in relaxing (pCa = 9) solutions from that obtained in maximally activating (pCa = 4.4) solutions. Fiber type was determined on the basis of myosin heavy chain isoform composition by polyacrylamide SDS gel electrophoresis. AM ATPase activity per liter cell volume (+/-SE) in the control and patient group, respectively, amounted to 134 +/- 24 and 77 +/- 9 microM/s in type I fibers (n = 11 and 16), 248 +/- 17 and 188 +/- 13 microM/s in type IIA fibers (n = 14 and 32), 291 +/- 29 and 126 +/- 21 microM/s in type IIA/X fibers (n = 3 and 5), and 325 +/- 32 and 205 +/- 21 microM/s in type IIX fibers (n = 7 and 9). The maximal isometric force per cross-sectional area amounted to 64 +/- 7 and 43 +/- 5 kN/m(2) in type I fibers, 86 +/- 11 and 58 +/- 4 kN/m(2) in type IIA fibers, 85 +/- 6 and 42 +/- 9 kN/m(2) in type IIA/X fibers, and 90 +/- 5 and 59 +/- 5 kN/m(2) in type IIX fibers in the control and patient group, respectively. These results indicate that, in CHF patients, significant reductions occur in isometric force and AM ATPase activity but that tension cost for each fiber type remains the same. This suggests that, in skeletal muscle from CHF patients, a decline in density of contractile proteins takes place and/or a reduction in the rate of cross-bridge attachment of approximately 30%, which exacerbates skeletal muscle weakness due to muscle atrophy.

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.

Myoglobin plays various roles in oxygen supply to muscle mitochondria. It is difficult, and in some cases impossible, to study the relationship between the myoglobin concentration and the oxidative capacity of individual muscle cells because myoglobin has to be fixed in situ whereas determination of oxidative capacity, for example, succinate dehydrogenase activity, requires unfixed cryostat sections. We have investigated whether a vapour-fixation technique allows the use of serial sections to study the relationship between myoglobin and succinate dehydrogenase activity. The technique is used to study a rat soleus muscle, two human skeletal muscle biopsies and biopsies of two patients with chronic heart failure, and in a control and hypertrophied rat heart. Staining intensities were quantified by microdensitometry. The absorbance values were calibrated using sections cut from gelatine blocks containing known amounts of myoglobin. The results show that it is possible to use serial sections for the determination of the myoglobin concentration and succinate dehydrogenase activity, and indicate that myoglobin can lead to a substantial reduction (18-60%) of the extracellular oxygen tension required to prevent an anoxic core in muscle cells.

Previous studies indicate that the low maximum rate of oxygen consumption (VO2max) of chronic heart failure (CHF) patients is not because of impaired pump function of the heart. We hypothesize that VO2 during maximum exercise is determined by the total oxidative capacity of skeletal muscle. VO2max of six controls and 14 CHF patients, New York Heart Association class I-III, was determined using an incremental bicycle ergometer test. Cryostat sections of a biopsy from the quadriceps femoris muscle were incubated for succinate dehydrogenase (SDH) using quantitative histochemistry. VO2max (range: 29 ml O2 kg muscle(-1) min(-1) in a class III patient to 118 ml O2 kg muscle(-1) min(-1) in a control subject) correlates with the mean SDH activity of skeletal muscle fibres (r=0.79 or r=0.81, including or excluding oxygen uptake at rest, respectively; P<0.001). The relationship between VO2max and SDH activity is similar to that determined previously using isolated single muscle fibres and myocardial trabeculae under hyperoxic conditions. From the product of SDH activity and the cross-sectional area of the fibre (i.e. spatially integrated SDH activity), it is possible to calculate the maximum oxygen uptake rate per unit muscle fibre length. This uptake rate is linearly related to the number of capillaries per fibre (r=0.76, P<0.001) in all subjects, suggesting that oxidative capacity of skeletal muscle fibres in CHF patients decreases in proportion to the oxygen supply capacity of the microcirculation.