W.J. van der Laarse

Opposite Effects of Training in Rats With Stable and Progressive Pulmonary Hypertension

Background— Exercise training in pulmonary arterial hypertension (PH) is a promising adjunct to medical treatment. However, it is still unclear whether training is beneficial for all PH patients. We hypothesized that right ventricular adaptation plays a pivotal role in the response to training. Methods and Results— Two different dosages of monocrotaline were used in rats to model stable PH with preserved cardiac output and progressive PH developing right heart failure. Two weeks after injection, PH was confirmed by echocardiography, and treadmill training was initiated. Rats were trained for 4 weeks unless manifest right heart failure developed earlier. At the end of the study protocol, all rats were functionally assessed by endurance testing, echocardiography, and invasive pressure measurements. Lungs and hearts were further analyzed in quantitative histomorphologic analyses. In stable PH, exercise training was well tolerated and markedly increased exercise endurance (from 25±3.9 to 62±3.9 minutes; P<0.001). Moreover, capillary density increased significantly (from 1.21±0.12 to 1.51±0.07 capillaries per cardiomyocyte; P<0.05). However, in progressive PH, exercise training worsened survival (hazard ratio, 2.7; 95% confidence interval, 1.1 to 14.2) and increased pulmonary vascular remodeling. In addition, training induced widespread leukocyte infiltration into the right ventricle (from 135±14 to 276±18 leukocytes per 1 mm2; P<0.001). Conclusions— In our rat model, exercise training was found to be beneficial in stable PH but detrimental in progressive PH. Future studies are necessary to address the clinical implications of our findings.

The muscle fiber type-fiber size paradox: hypertrophy or oxidative metabolism?

An inverse relationship exists between striated muscle fiber size and its oxidative capacity. This relationship implies that muscle fibers, which are triggered to simultaneously increase their mass/strength (hypertrophy) and fatigue resistance (oxidative capacity), increase these properties (strength or fatigue resistance) to a lesser extent compared to fibers increasing either of these alone. Muscle fiber size and oxidative capacity are determined by the balance between myofibrillar protein synthesis, mitochondrial biosynthesis and degradation. New experimental data and an inventory of critical stimuli and state of activation of the signaling pathways involved in regulating contractile and metabolic protein turnover reveal: (1) higher capacity for protein synthesis in high compared to low oxidative fibers; (2) competition between signaling pathways for synthesis of myofibrillar proteins and proteins associated with oxidative metabolism; i.e., increased mitochondrial biogenesis via AMP-activated protein kinase attenuates the rate of protein synthesis; (3) relatively higher expression levels of E3-ligases and proteasome-mediated protein degradation in high oxidative fibers. These observations could explain the fiber type–fiber size paradox that despite the high capacity for protein synthesis in high oxidative fibers, these fibers remain relatively small. However, it remains challenging to understand the mechanisms by which contractile activity, mechanical loading, cellular energy status and cellular oxygen tension affect regulation of fiber size. Therefore, one needs to know the relative contribution of the signaling pathways to protein turnover in high and low oxidative fibers. The outcome and ideas presented are relevant to optimizing treatment and training in the fields of sports, cardiology, oncology, pulmonology and rehabilitation medicine.

Iron deficiency is common in idiopathic pulmonary arterial hypertension

The aims of this study were to assess the prevalence of iron deficiency in idiopathic pulmonary arterial hypertension (IPAH) and investigate whether oral iron supplementation has effects in iron-deficient patients. Iron parameters were measure for all IPAH patients attending our centre (VU University Medical Center, Amsterdam, the Netherlands) between May 2009 and February 2010. Iron data were related to clinical parameters, including 6-min walking distance (6MWD), and haemodynamic parameters measured during right heart catheterisation. In a subset of iron-deficient patients, the uptake of iron from the bowel was studied after administering oral iron for 4 weeks. Iron deficiency was found in 30 (43%) out of 70 patients. 6MWD was reduced in iron-deficient patients compared with iron-sufficient patients (mean±sd 390±138 versus 460±143 m; p<0.05) irrespective of the existence of anaemia. In a subset of 18 patients that received oral iron, ferritin levels were significantly increased, although eight patients only slightly increased their iron storage. This study shows that iron deficiency is frequently present in IPAH and is associated with a lower exercise capacity. The small response to oral iron in 44% of the treated patients suggests impaired iron absorption in these patients.

Maximum rate of oxygen consumption and quantitative histochemistry of succinate dehydrogenase in single muscle fibres of Xenopus laevis

SummaryThree different types of single living muscle fibre were dissected from the iliofibularis muscle ofXenopus laevis. The fibres were mounted in a glass chamber and their rate of oxygen consumption was determined as a function of twitch frequency at 20‡ C. The rate of oxygen consumption increased with twitch frequency until it levelled off and reached a maximum. The maximum rate of oxygen consumption varied between fibres (0.019 to 0.161 nmol O2 s−1 mm−3) and was reached at different twitch frequencies (<0.2 to 5.7 stimuli s−1). After the determination of the maximum rate of oxygen consumption, the succinate dehydrogenase activity in cross sections of the fibre was determined by means of a quantitative histochemical method. A proportional relationship between the maximum rate of oxygen consumption and the succinate dehydrogenase activity was found. The maximum rate of oxygen consumption and the succinate dehydrogenase activity are also proportional to the volume density of mitochrondria in the three fibre types reported by Smith and Ovalle (1973;J. Anat., Lond.116, 1–24). It is concluded that quantitative histochemistry of succinate dehydrogenase reliably predicts the maximum rate of oxygen consumption of muscle fibres inXenopus laevis and that the maximum rate of oxygen consumption of single muscle fibres is determined by the volume density of mitochondria.

Fasudil reduces monocrotaline-induced pulmonary arterial hypertension: comparison with bosentan and sildenafil

Pulmonary arterial hypertension (PAH) still cannot be cured, warranting the search for novel treatments. Fasudil (a Rho kinase inhibitor) was compared with bosentan (an endothelin receptor blocker) and sildenafil (a phosphodiesterase 5 inhibitor), with emphasis on right ventricular (RV) function, in a reversal rat model of monocrotaline (MCT)-induced PAH. In addition, the effects of combining bosentan or sildenafil with fasudil were studied. MCT (40 mg·kg body weight−1) induced clear PAH in male Wistar rats (n = 9). After 28 days, echocardiography, RV catheterisation and histochemistry showed that cardiac frequency, stroke volume and RV contractility had deteriorated, accompanied by RV dilatation and hypertrophy, and marked pulmonary arterial wall thickening. Mean pulmonary arterial pressure and pulmonary vascular resistance increased significantly compared to healthy rats (n = 9). After 14 days, MCT-treated rats received a 14-day oral treatment with bosentan, sildenafil, fasudil or a combination of fasudil with either bosentan or sildenafil (all n = 9). All treatments preserved cardiac frequency, stroke volume and RV contractility, and reduced pulmonary vascular resistance and RV dilatation. Fasudil lowered RV systolic pressure and mean pulmonary arterial pressure significantly, by reducing pulmonary arterial remodelling, which reduced RV hypertrophy. Combining bosentan or sildenafil with fasudil had no synergistic effect. Fasudil significantly improved PAH, to a greater degree than did bosentan and sildenafil.

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 (PO2,crit) required by cardiomyocytes to prevent hypoxia during hypertrophic growth, and determinants of PO2,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 PO2,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 1α can be demonstrated by immunohistochemistry in cardiomyocytes with high PO2,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. Microsc. Res. Tech. 58:412–420, 2002.

Myocardial force development and structural changes associated with monocrotaline induced cardiac hypertrophy and heart failure

In this study alterations are characterized which occur, in myocardial force development morphological appearance and protein composition, during the development of cardiac hypertrophy and heart failure in monocrotaline (MCT) treated rats. The transition from cardiac hypertrophy to heart failure was studied by comparing the results from control (CON) and two MCT groups (40 and 44 mg/kg body weight). The three experimental groups consisted of at least five animals each. Parameters studied were: body weight (measured daily), lung/body weight ratio, right ventricular wall volume and thickness, and force development in thin right ventricular trabeculae at 27°C, using different extracellular calcium concentrations and pacing frequencies. MCT injection resulted in marked right ventricular hypertrophy and heart failure as evidenced by an up to 2-fold increase in lung/body weight ratio and a 1.7-fold increase in wall volume. The MCT groups showed a negative force–frequency relation and maximum force was up to 2-fold less than in the CON group. Protein analysis by means of one- and two-dimensional gel electrophoresis revealed a marked (7-fold) up-regulation of the slow myosin heavy chain isoform as well as a 4.5-fold increase in the content of the cytoskeletal protein desmin, whereas the mitochondrial protein ATP-synthase content was reduced. Hence MCT-induced cardiac hypertrophy and heart failure result in altered cellular calcium handling, depression of maximum force output, an increase in the economy of myocardial contraction and changes in cytoskeletal structure and energy supply.

Calibration of Quantitative Histochemical Methods: Estimation of Glycogen Content of Muscle Fibers Using the PAS Reaction

A fairly simple method for calibrating microdensitometric histochemical assays is described. The method is based on paired biochemical and histochemical assays on single freeze-dried skeletal muscle fibers which differ widely in their properties. As an example, the method is applied to investigate the validity of the periodic acid-Schiff (PAS) reaction for the microdensitometric estimation of glycogen content. Some problems that may interfere with the calibration are discussed.

SIZE PRINCIPLE OF STRIATED MUSCLE CELLS

We have investigated the relationship between cross-sectional area (CSA) and maximum rate of oxygen consumption (VO2max, in nmol .mm-3 .s-1) of heart and skeletal muscle cells from different species. VO2max and CSA were determined for single muscle fibres of Xenopus laevis at 20°C and for cardiomyocytes in thin trabeculae dissected from the right ventricle of rats at 38°C. Succinate dehydrogenase activity was determined using a quantitative histochemical method to estimate VO2max in mammalian skeletal muscle fibres. Literature values of volume density of mitochondria were used to estimate VO2max in some mammalian cardiomyocytes. We found that an inverse relationship exists between VO2max (range 1.5 to 0.024 nmol . mm-3 . s-1) and cross-sectional area (0.0002 to 0.018 mm2, respectively): VO2max = constant/CSA, where the value of the constant equals 0.39±0.18 pmol . mm-1 .s-1 (mean ± S.D., n = 14). Such a relationship is predicted by a simple Hill-type model for oxygen diffusion in cilindrical cells, if it is assumed that muscle cells are evolved so that anoxic cores in muscle cells are prevented. This indicates that the product of endurance of muscle cells (which is proportional to VO2max) and force production (which is proportional to cross-sectional area) is limited by oxygen diffusion, and that adaptation of heart and skeletal muscle cells to increased workload is limited by the interstitial oxygen tension.

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

Abstract 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) μm, 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) μm, 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.