Myoglobin concentration in skeletal muscle fibers of chronic heart failure patients.

The purpose of this study was to determine the myoglobin concentration in skeletal muscle fibers of chronic heart failure (CHF) patients and to calculate the effect of myoglobin on oxygen buffering and facilitated diffusion. Myoglobin concentration, succinate dehydrogenase (SDH) activity, and cross-sectional area of individual muscle fibers from the vastus lateralis of five control and nine CHF patients were determined using calibrated histochemistry. CHF patients compared with control subjects were similar with respect to myoglobin concentration: type I fibers 0.69 +/- 0.11 mM (mean +/- SD), type II fibers 0.52 +/- 0.07 mM in CHF vs. type I fibers 0.70 +/- 0.09 mM, type II fibers 0.49 +/- 0.07 mM in control, whereas SDH activity was significantly lower in CHF in both fiber types (P < 0.01). The myoglobin concentration in type I fibers was higher than in type II fibers (P < 0.01). Consequently, the oxygen buffering capacity, calculated from myoglobin concentration/SDH activity was increased in CHF: type I fibers 11.4 +/- 2.1 s, type II fibers 13.6 +/- 3.9 s in CHF vs. type I fibers 7.8 +/- 0.9 s, type II fibers 7.5 +/- 1.0 s in control, all P < 0.01). The calculated extracellular oxygen tension required to prevent core anoxia (Po2(crit)) in muscle fibers was similar when controls were compared with patients in type I fibers 10.3 +/- 0.9 Torr in CHF and 11.5 +/- 3.3 Torr in control, but was lower in type II fibers of patients 6.1 +/- 2.8 Torr in CHF and 14.7 +/- 6.2 Torr in control, P < 0.01. The lower Po2(crit) of type II fibers may facilitate oxygen extraction from capillaries. Reduced exercise tolerance in CHF is not due to myoglobin deficiency.

Sarcoplasmic reticulum ATPase activity in type I and II skeletal muscle fibres of chronic heart failure patients.

BACKGROUND: Reduced exercise tolerance and muscle weakness are present in patients with CHF. Altered metabolism, histology and function in skeletal muscle of patients with CHF have been reported. The sarcoplasmic reticulum (SR) has control of Ca(++) release and uptake required for contraction and relaxation, respectively, and uses a considerable amount of energy. Little is known about SR alterations in CHF. We determined sarcoplasmic reticulum adenosine triphosphatase (SR ATPase) activity in different types of skeletal muscle fibres of CHF patients. METHODS: SR ATPase activity, succinate dehydrogenase (SDH) activity and myofibrillar adenosine triphosphatase (M ATPase) activity in single fibres of the vastus lateralis muscle in 16 CHF patients and 5 controls was determined using quantitative enzyme histochemistry. RESULTS: SR ATPase activity of type II skeletal muscle fibers was significantly higher compared to type I fibres. SR ATPase activity in type II skeletal muscle fibres of CHF patients was higher than in control subjects. CONCLUSION: Increased skeletal muscle SR ATPase activity contributes to reduced exercise tolerance in CHF patients.

Cardioprotection via activation of protein kinase C-delta depends on modulation of the reverse mode of the Na+/Ca2+ exchanger.

BACKGROUND: Pretreatment with the volatile anesthetic sevoflurane protects cardiomyocytes against subsequent ischemic episodes caused by a protein kinase C (PKC)-delta mediated preconditioning effect. Sevoflurane directly modulates cardiac Ca2+ handling, and because Ca2+ also serves as a mediator in other cardioprotective signaling pathways, possible involvement of the Na+/Ca2+ exchanger (NCX) in relation with PKC-delta in sevoflurane-induced cardioprotection was investigated. METHODS AND RESULTS: Isolated right ventricular rat trabeculae were subjected to simulated ischemia and reperfusion (SI/R), consisting of superfusion with hypoxic glucose-free buffer for 40 minutes after rigor development, followed by reperfusion with normoxic glucose containing buffer. Preconditioning with sevoflurane before SI/R improved isometric force development during contractile recovery at 60 minutes after the end of hypoxic superfusion (83+/-7% [sevo] versus 57+/-2% [SI/R];n=8; P<0.01). Inhibition of the reverse mode of the NCX by KB-R7943 (10 micromol/L) or SEA0400 (1 micromol/L) during preconditioning attenuated the protective effect of sevoflurane. KB-R7943 and SEA0400 did not have intrinsic effects on the contractile recovery. Furthermore, inhibition of the NCX in trabeculae exposed to sevoflurane reduced sevoflurane-induced PKC-delta translocation toward the sarcolemma, as demonstrated by digital imaging fluorescent microscopy. The degree of PKC-delta phosphorylation at serine643 as determined by western blot analysis was not affected by sevoflurane. CONCLUSIONS: Sevoflurane-induced cardioprotection depends on the NCX preceding PKC-delta translocation presumably via increased NCX-mediated Ca2+ influx. This may suggest that increased myocardial Ca2+ load triggers the cardioprotective signaling cascade elicited by volatile anesthetic agents similar to other modes of preconditioning.

Krogh's diffusion coefficient for oxygen in isolated Xenopus skeletal muscle fibers and rat myocardial trabeculae at maximum rates of oxygen consumption.

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.

Immunohistochemical determination of cytosolic cytochrome C concentration in cardiomyocytes.

Cytochrome c release from the intermembrane space of mitochondria is one of the triggers of apoptosis. There is no histochemical method available to demonstrate cytochrome c in cryostat sections, possibly because small cytosolic proteins diffuse readily into aqueous fixation media. This report shows that it is possible to demonstrate cytochrome c release in cardiomyocytes in failing myocardium using vapor fixation of cryostat sections and immunohistochemistry. The method is calibrated using sections from gelatin blocks containing known concentrations of cytochrome c. The method is applied to the hypertrophied right ventricular wall of rats in which pulmonary hypertension was induced by monocrotaline. Cytochrome c release is found in a fraction of the cardiomyocytes, leading to a mosaic-staining pattern. Cytochrome c release was found in myocytes over the full range of cross-sectional area (from 1 to 3.9 times control) in the hypertrophied myocardium. Cytosolic cytochrome c concentrations up to 0.4-0.5 mM occur frequently.

Reactive oxygen species precede protein kinase C-delta activation independent of adenosine triphosphate-sensitive mitochondrial channel opening in sevoflurane-induced cardioprotection.

BACKGROUND: In the current study, the authors investigated the distinct role and relative order of protein kinase C (PKC)-delta, adenosine triphosphate-sensitive mitochondrial K+ (mito K+(ATP)) channels, and reactive oxygen species (ROS) in the signal transduction of sevoflurane-induced cardioprotection and specifically addressed their mechanistic link. METHODS: Isolated rat trabeculae were preconditioned with 3.8% sevoflurane and subsequently subjected to an ischemic protocol by superfusion of trabeculae with hypoxic, glucose-free buffer (40 min) followed by 60 min of reperfusion. In addition, the acute affect of sevoflurane on PKC-delta and PKC-epsilon translocation and nitrotyrosine formation was established with use of immunofluorescent analysis. The inhibitors chelerythrine (6 microM), rottlerin (1 microM), 5-hydroxydecanoic acid sodium (100 microM), and n-(2-mercaptopropionyl)-glycine (300 microM) were used to study the particular role of PKC, PKC-delta, mito K+(ATP), and ROS in sevoflurane-related intracellular signaling. RESULTS: Preconditioning of trabeculae with sevoflurane preserved contractile function after ischemia. This contractile preservation was dependent on PKC-delta activation, mito K+(ATP) channel opening, and ROS production. In addition, on acute stimulation by sevoflurane, PKC-delta but not PKC-epsilon translocated to the sarcolemmal membrane. This translocation was inhibited by PKC inhibitors and ROS scavenging but not by inhibition of mito K+(ATP) channels. Furthermore, sevoflurane directly induced nitrosylation of sarcolemmal proteins, suggesting the formation of peroxynitrite. CONCLUSIONS: In sevoflurane-induced cardioprotection, ROS release but not mito K+(ATP) channel opening precedes PKC-delta activation. Sevoflurane induces sarcolemmal nitrotyrosine formation, which might be involved in the recruitment of PKC-delta to the cell membrane.

Determination of myoglobin concentration and oxidative capacity in cryostat sections of human and rat skeletal muscle fibres and rat cardiomyocytes.

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.

Maximum rate of oxygen consumption related to succinate dehydrogenase activity in skeletal muscle fibres of chronic heart failure patients and controls.

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.