Abstract
Peripheral muscle weakness in PAH patients is at least partly caused by sarcomeric dysfunction http://ow.ly/J8LuE
To the Editor:
Despite improvements in disease targeted therapies, pulmonary arterial hypertension (PAH) is a progressive disease and PAH patients remain symptomatic [1]. Exercise intolerance is one of the main symptoms, which limit PAH patients in their daily life activities. Reduced exercise capacity is generally attributed to right ventricular dysfunction [1]. However, as with other cardiac and pulmonary diseases, PAH patients develop respiratory [2] and peripheral muscle [3, 4] weakness, which might also contribute to exercise intolerance. Indeed, exercise training improves exercise capacity in PAH patients and maximal oxygen consumption of PAH patients correlates with the functional decline of peripheral muscle strength [5, 6]. The underlying cause of the reduction in muscle strength is unclear. Some studies have reported muscle fibre atrophy and a shift towards more fast-twitch fatigable fibres in skeletal muscles of PAH patients [3, 7]; however, these are not consistent findings [4, 8].
Recently, we have shown that weakness of the respiratory muscles in PAH patients [8] and in pulmonary hypertension rats [9] is, at least partly, caused by impaired contractility of the sarcomeres, the smallest contractile units in muscle. Whether sarcomere contractility is also affected in peripheral muscles of PAH patients is yet unknown. Physical activity declines in PAH patients and muscle disuse is known to affect sarcomere function [10]. Therefore, in the present study, we hypothesised that sarcomere contractility is impaired in peripheral muscles of PAH patients.
To test this hypothesis, we measured sarcomeric function in permeabilised individual muscle fibres of idiopathic PAH patients. In permeabilised muscle fibres, sarcomeric contractility can be studied without the confounding effects of processes upstream in the excitation–contraction coupling cascade. The fibres were isolated from quadriceps muscle (vastus lateralis) biopsies of female PAH patients (N=11) and were compared to those from healthy, age- and sex-matched control subjects (N=8). For patients' characteristics, see figure 1a. All subjects gave written informed consent before inclusion. Ethical approval was given by the institutional review board at VU University Medical Center, Amsterdam, the Netherlands. This report focuses on results obtained on fast-twitch fibres because of the low number of slow-twitch muscle fibres present in the biopsies (PAH: number of patients (Npatients)=4, number of fibres (nfibres)=5; control: Npatients=5, nfibres=13).
Individual fibres were mounted between a force transducer and servomotor, and exposed to activating calcium solutions, as described previously [9]. Maximal tension (i.e. maximal force normalised to muscle fibre cross-sectional area (CSA)) was significantly lower in fast-twitch muscle fibres of PAH patients (Npatients=11, nfibres=112) than in control subjects (Npatients=8, nfibres=62) (mean±sem 192±5 versus 226±8 mN·mm−2, p<0.005; fig. 1b). Based on our permeabilised muscle fibre measurements, we observed no significant difference in fast-twitch muscle fibre CSA between groups (PAH versus control: 3387±280 versus 3307±242 µm2; p=0.84).
In muscle fibres, force is generated by the cyclic interaction between myosin-based cross-bridges and actin. Thus, to determine the underlying cause of the reduction in maximal tension, we studied cross-bridge cycling kinetics. In permeabilised muscle fibres, active force generation is determined by: 1) the fraction of strongly bound cross-bridges; 2) the number of available cross-bridges; and 3) the force generated per cross-bridge. First, to estimate the fraction of strongly bound cross-bridges we measured the rate constant of force redevelopment (ktr) during maximal activation. No significant difference in ktr was observed in fast-twitch muscle fibres (fig. 1c), suggesting that the fraction of strongly bound cross-bridges was unaltered. Secondly, to estimate the number of available cross-bridges, we measured active stiffness by imposing fast (<1 ms), small length changes on the fibre during maximal activation. Active stiffness reflects the number of attached cross-bridges during activation, which is determined by both the fraction of strongly bound cross-bridges (ktr) as well as the number of available cross-bridges. We found a significant decrease in active stiffness in fast-twitch muscle fibres of PAH patients (fig. 1d). Since ktr was unaltered, this suggests that the number of available cross-bridges is reduced in quadriceps muscle fibres of PAH patients. Finally, we estimated the force generated per cross-bridge by calculating the tension/stiffness ratio. No significant difference was observed between groups (fig. 1e), indicating that the force generated per cross-bridge is unaltered.
During daily life activities, quadriceps muscles are generally not maximally activated but activated at submaximal motor neuron firing rates, which yield submaximal calcium concentrations ([Ca2+]). Therefore, we measured force at submaximal [Ca2+]. The tension–[Ca2+] relationship shows that at high [Ca2+], tension was significantly lower (fig. 1f). The [Ca2+] at which 50% of the maximal force is reached was unaffected in fast-twitch muscle fibres of PAH patients. Thus, changes in the Ca2+ sensitivity of force do not contribute to contractile muscle fibre weakness in PAH patients.
For optimal active force generation, structural integrity of the sarcomere is indispensable. This structural integrity is regulated by the passive elastic properties of the giant sarcomeric protein titin. Therefore, we measured passive tension in muscle fibres by stretching the fibre from slack length (∼1.9 µm) to a sarcomere length of ∼3.2 µm (velocity, 10% length change per second) while in relaxing solution ([Ca2+] 1 nM). A significant upward shift in passive tension–sarcomere length relationship was observed in fast-twitch muscle fibres (fig. 1g), indicating that the passive tension of quadriceps muscle fibres is increased in PAH patients.
This is the first study to show sarcomeric contractile weakness in the quadriceps muscle of PAH patients. This weakness is caused by a reduction in the number of available cross-bridges, which might result from a loss of the major contractile protein myosin. Notably, the reduction in maximal tension (∼15%) was less than the reduction in muscle strength measured in vivo (20–30%) [3], suggesting that extrasarcomeric changes, for example, in the process of excitation–contraction coupling, and/or muscle atrophy also contribute to peripheral muscle weakness.
A limitation of the present study is that our findings are restricted to fast-twitch muscle fibres, whereas the vastus lateralis in humans, including PAH patients, contains 30–40% slow-twitch fibres [7]. The low number of slow-twitch muscle fibres found in the biopsies might be a result of sampling error. Therefore, caution is warranted when extrapolating the current findings to the whole muscle. Measurements were performed shortly after the biopsy was taken to avoid deterioration of muscle fibres over time. The researcher involved in the experiments was also involved in obtaining the biopsies. Therefore, the experiments were not performed blind, which is another limitation of this study.
In addition, we observed that passive tension of quadriceps muscle fibres was increased in PAH patients. This was an unexpected finding, as muscle disuse and loss of myosin are associated with a reduction in passive tension [11]. Passive tension was already increased by 39% at a sarcomere length of 2.5 µm, with a more pronounced increase at higher sarcomere lengths. As in vivo, muscle contraction is estimated to occur at a sarcomere length ranging from 2.5 to 3.2 µm [12], the increase in passive tension is likely to affect in vivo muscle function. Increased titin-based passive tension is associated with an increase in the Ca2+ sensitivity of force generation, possibly by reducing myofilament lattice spacing [13]. Although speculative, the increase in passive tension in PAH patients might be a compensatory mechanism to maintain Ca2+ sensitivity and, thus, submaximal force generation.
As muscle dysfunction affects exercise capacity and quality of life of PAH patients, therapeutic options that specifically improve skeletal muscle function are warranted. While it is recognised that exercise training can improve muscle function and exercise capacity in PAH patients [5], its tolerability is limited in severely haemodynamically impaired PAH patients [6]. Calcium sensitisers, which specifically target sarcomeric function by improving submaximal force generation and reduced [Ca2+] pumping by the sarco/endoplasmic reticulum Ca2+ ATPase [14, 15], might be a pharmacological therapeutic option to improve muscle function in this patient population.
To conclude, peripheral muscle weakness in PAH patients is at least partly caused by sarcomeric dysfunction. As muscle weakness might contribute to the reduction in exercise capacity, therapeutic options to specifically improve muscle function should be studied.
Footnotes
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Conflict of interest: Disclosures can be found alongside the online version of this article at erj.ersjournals.com
- Received November 5, 2014.
- Accepted January 30, 2015.
- Copyright ©ERS 2015