Rationale: Recently it was suggested that patients with pulmonary hypertension (PH) suffer from inspiratory muscle dysfunction. However, the nature of inspiratory muscle weakness in PH remains unclear.
Objectives: To assess whether alterations in contractile performance and in morphology of the diaphragm underlie inspiratory muscle weakness in PH.
Methods: PH was induced in Wistar rats by a single injection of monocrotaline (60 mg/kg). Diaphragm (PH n = 8; controls n = 7) and extensor digitorum longus (PH n = 5; controls n = 7) muscles were excised for determination of in vitro contractile properties and cross-sectional area (CSA) of the muscle fibers. In addition, important determinants of protein synthesis and degradation were determined. Finally, muscle fiber CSA was determined in diaphragm and quadriceps of patients with PH, and the contractile performance of single fibers of the diaphragm.
Measurements and Main Results: In rats with PH, twitch and maximal tetanic force generation of diaphragm strips were significantly lower, and the force-frequency relation was shifted to the right (i.e., impaired relative force generation) compared with control subjects. Diaphragm fiber CSA was significantly smaller in rats with PH compared with controls, and was associated with increased expression of E3-ligases MAFbx and MuRF-1. No significant differences in contractility and morphology of extensor digitorum longus muscle fibers were found between rats with PH and controls. In line with the rat data, studies on patients with PH revealed significantly reduced CSA and impaired contractility of diaphragm muscle fibers compared with control subjects, with no changes in quadriceps muscle.
Conclusions: PH induces selective diaphragm muscle fiber weakness and atrophy.
Recent studies suggest that in pulmonary hypertension the sensation of dyspnea, a symptom observed in most patients, is a consequence of inspiratory muscle weakness. However, the nature of inspiratory muscle weakness in pulmonary hypertension is unclear.
This study illustrates profound diaphragm muscle fiber weakness and atrophy in a rat model of pulmonary hypertension, with no changes in peripheral skeletal muscle. Importantly, similar findings were obtained in patients with pulmonary hypertension. We reason that patients with pulmonary hypertension suffer from specific diaphragm muscle weakness, which likely contributes to the sensation of dyspnea.
Recently, it was speculated that patients with PH suffer from inspiratory muscle dysfunction (3). This speculation was based on the notion that in PH the inspiratory muscles are weakened and subjected to increased activity. The notion that in PH the inspiratory muscles are more active comes from the observation that patients with PH hyperventilate during exercise, but also at rest, and even during sleep (3–5). This continuous hyperventilation with increased respiratory frequency increases the demand on the inspiratory muscles. Inspiratory muscle weakness in PH was suggested by recent studies showing that volitionally assessed maximal inspiratory mouth pressures (3), and nonvolitionally assessed transdiaphragmatic pressures during bilateral anterior magnetic phrenic nerve stimulation (6), were markedly lower in patients with PH compared with control subjects. Thus, as postulated previously by Naeije (7), patients with PH need to breathe more with weaker inspiratory muscles.
This imbalance between the demand placed on the inspiratory muscle and the impaired capacity to generate force is likely to contribute to the reduced exercise capacity and quality of life of patients with PH (6). In addition, inspiratory muscle weakness is an important determinant of dyspnea, a symptom that affects most patients with PH (8).
The nature of inspiratory muscle weakness in PH remains to be elucidated. The capacity to generate negative intrathoracic or transdiaphragmatic pressure is an indirect measure of inspiratory muscle strength: the outcome depends not only on muscle fiber function, but also on central drive, nerve function, and neuromuscular transmission.
In the present study, we tested the hypothesis that alterations in contractile performance and in morphology of the diaphragm, the main muscle of inspiration, underlie inspiratory muscle weakness in PH. Some of the results of these studies have been previously reported in the form of an abstract (9).
This study was approved by the Institutional Animal Care and Use Committee at the VU University and the ethics committee of the VU University Medical Center, Amsterdam, The Netherlands, and CPP IDF VII, CHU Bicêtre, Le Kremlin-Bicêtre, France.
Male Wistar rats were used (150–175 g; Harlan, Horst, The Netherlands). PH was induced by a single subcutaneous injection of 60 mg/kg monocrotaline (MCT; Sigma-Aldrich, Zwijndrecht, The Netherlands) dissolved in sterile saline; the control group was injected only with saline. Description of the sugen hypoxia (SuHx) and pulmonary artery banding (PAB) model can be found in the online supplement. When right heart failure developed (defined as >5% loss of body mass per day, or respiratory distress, cyanosis, and lethargy) (10), rats were hemodynamically evaluated by echocardiography and invasive right ventricular (RV) pressure measurements (10). In a subset of 10 rats (five control and five PH), breathing frequencies were determined by use of telemetry (see online supplement) (11). Subsequently, diaphragm and extensor digitorum longus (EDL) muscle were excised for further analyses.
For intact muscle experiments, diaphragm (seven control and eight PH) and EDL (seven control and five PH) muscles were quickly dissected from rats with PH and controls, and mounted vertically in a tissue bath between a dual-mode lever arm and a fixed hook (1200A Intact Muscle Test System; Aurora Scientific, Aurora, ON, Canada), as described previously (see online supplement) (12).
Antibody clones brd-5 and sc-71 were used to assess cross-sectional area (CSA) of slow and fast muscle fibers, respectively (13), and capillary density and oxidative enzyme activity were assessed as described previously (14). Furthermore, protein kinase B (Akt)-activation, myosin heavy chain expression (MyHC), E3-ligase (MAFbx and MuRF-1) mRNA expression, proteasome-activity, and proteasome content were determined (see online supplement) (15).
Diaphragm and quadriceps biopsies were obtained from six patients with PH and six control subjects. Tissue slides were stained for hematoxylin and eosin to assess CSA of the muscle fibers. In two patients with PH and two control subjects, contractile force measurements were performed on single diaphragm muscle fibers isolated from biopsies (see online supplement) (16, 17).
All data are presented as mean ± SEM and were verified for normal distribution. A P value less than 0.05 was considered significant. Differences between control and PH in hemodynamics, diaphragm contractile measurements, muscle fiber CSA, oxidative enzyme activity, and molecular alterations were analyzed by independent t tests. Two-way repeated measure analysis of variance was used to analyze the differences in respiratory rate and force-frequency between control and PH, with Bonferroni post hoc tests.
Compared with controls, rats with PH had clear signs of pulmonary vascular remodeling (increased RV systolic pressure and pulmonary vascular resistance), RV dysfunction (decreased cardiac output, tricuspid annular plane systolic excursion), and increased RV remodeling (increased RV wall thickness, RV end-diastolic diameter) (Table 1). To investigate whether breathing frequency is increased in rats with PH, similar to clinical PH, we assessed breathing frequencies at baseline and when rats had developed overt right heart failure (Figure 1A). As shown in Figure 1B, breathing frequencies in rats with PH were significantly (P < 0.001) increased by 86%.
|RVSP, mm Hg||28.1 ± 1.2||70.6 ± 4.7||<0.001|
|RVEDP, mm Hg||3.6 ± 0.4||8.8 ± 1.1||<0.01|
|RVWT, mm||0.87 ± 0.02||1.34 ± 0.02||<0.001|
|RVEDD, mm||3.68 ± 0.05||7.31 ± 0.17||<0.001|
|CO, ml/min||102 ± 9||32 ± 5||<0.001|
|TAPSE, mm||3.8 ± 0.1||1.7 ± 0.2||<0.001|
| PVR, mm Hg/ml/min||0.33 ± 0.05||2.05 ± 0.27||<0.001|
To compare the findings of the MCT rat model with two other models for PH, we also investigated the SuHX and PAB model. The hemodynamic characteristics of both models were comparable with the MCT model. The SuHx model had elevated RV systolic pressure (78.6 ± 4.8 mm Hg) and RV hypertrophy (RV/LV+S 0.74 ± 0.12); the PAB model demonstrated increased RV systolic pressure (66.7 ± 15.7 mm Hg) and moderate RV hypertrophy (RV/LV+S 0.48 ± 0.09). Additional hemodynamic characteristics and comparisons of both models have been described previously (18).
We investigated whether the morphology of diaphragm muscle fibers was altered in rats with PH. As shown in Figure 2B, the CSA of both slow and fast diaphragm fibers are significantly smaller in rats with PH than in controls. To confirm the findings obtained in the MCT model, we also assessed diaphragm muscle fiber CSA in a second model for PH, the SuHx model. As shown in Figure 2B, the SuHx model showed a comparable magnitude of diaphragm muscle fiber atrophy. In contrast, no atrophy was observed in a model for PAB (Figure 2B).
To assess whether the observed muscle fiber atrophy was diaphragm specific, we also assessed muscle fiber CSA of a peripheral skeletal muscle, the EDL muscle. No changes in the CSA of EDL muscle fibers were observed between rats with PH and controls (slow fibers, 1,334 ± 70 vs. 1,366 ± 73 μm2; fast fibers, 3,079 ± 164 vs. 2,857 ± 134 μm2; control vs. PH, respectively). These findings indicate specific atrophy of diaphragm muscle fibers in PH.
Because the diaphragm muscle fiber atrophy could be an adaptation mechanism to the reduced cardiac output, we investigated oxygen handling of the diaphragm muscle fibers. However, capillary density and fiber oxidative enzyme activity were similar in controls and rats with PH (Figure 3).
Muscle fiber atrophy is caused by an imbalance between the rate of protein synthesis and proteolysis (19). Akt activation and MyHC expression were comparable between rats with PH and controls (Figure 4), suggesting no major alterations in protein synthesis. On the contrary, we observed increased expression of the E3-ligases MAFbx (Figure 5A) and MuRF-1 (Figure 5B), with no alterations in proteasome content and activity (Figures 5C and 5D).
We investigated whether diaphragm muscle function is impaired in rats with PH by measuring the contractile performance of intact diaphragm muscle strips. Both twitch force (P = 0.01) and maximal tetanic force (P = 0.01) were significantly reduced in PH, compared with control (Figures 6C and 6D).
In vivo, the diaphragm mainly undergoes submaximal, rather than maximal activation. Therefore, the force-generating capacity of the diaphragm at various submaximal stimulation frequencies was determined. The absolute force-frequency relation clearly demonstrates reduced force-generating capacity in rats with PH, at all stimulation frequencies, compared with controls (P < 0.001; see Figure 6E). The normalized force-frequency relation was shifted to the right in rats with PH (P < 0.05; Figure 6F), indicating impaired relative force generation.
To determine whether the changes in muscle function in PH are specific for the diaphragm, as opposed to a generalized muscle weakness, we also studied the contractile performance of the EDL (12). No differences were observed in the force-generating capacity of the EDL muscle in rats with PH compared with controls (see Figure E1).
The general characteristics of the subjects are described in Table E1. To determine whether the findings in rats with PH extrapolate to clinical PH, we first compared the CSA of diaphragm fibers of patients with PH (n = 6) with those of control subjects (n = 6). A marked reduction in the CSA of diaphragm muscle fibers from patients with PH was observed (Figure 7A), indicating severe diaphragm fiber atrophy in patients with PH. To evaluate whether this fiber atrophy was specific for the diaphragm, we also analyzed the CSA of quadriceps muscle fibers. In contrast, CSA of quadriceps muscle in patients with PH was on average increased compared with control subjects; however, this did not reach statistical significance (Figure 7B).
Second, the contractile performance was determined of demembranated (i.e., skinned) muscle fibers isolated from diaphragm biopsies of three patients with PH and three control subjects. These measurements cannot be performed on autopsy-derived biopsies, and were therefore only feasible on the two biopsies that were obtained during surgery. The maximal force-generating capacity of diaphragm muscle fibers was severely impaired (>50% reduction) (Figure 8) in patients with PH compared with control subjects.
Thus, the findings obtained from diaphragm specimens of patients with PH are in line with the results from our rat model, and indicate specific diaphragm muscle dysfunction in patients with PH.
To the best of our knowledge, this is the first study that investigated diaphragm muscle fiber function in rats and patients with PH. Using a multidisciplinary approach to assess muscle fiber contractility and structure, we have demonstrated that in PH (1) manifest diaphragm muscle fiber atrophy occurs, most likely caused by elevated proteolysis, and (2) diaphragm force-generating capacity is severely impaired. These results suggest an important role for diaphragm muscle weakness in the pathogenesis of dyspnea in patients with PH and warrant further investigation.
We observed marked atrophy of diaphragm muscle fibers in rats with PH. Muscle fiber CSA depends on the balance between the rates of protein synthesis and degradation (19). The diaphragm fiber atrophy in rats with PH seems not to be caused by changes in protein synthesis (Figure 4), but rather by elevated degradation. We found a marked increase in the expression of the E3 ligases MAFbx and MuRF-1 (Figure 5), which are considered key markers of proteolytic activity in muscle (20, 21). The bulk of sarcomeric protein degradation occurs via the ubiquitin–proteasome pathway (20, 22). In this pathway, muscle-specific E3-ligases, such as MAFbx and MuRF-1, ubiquitinate proteins that are subsequently degraded by the proteasome. Despite the increase in E3-ligase activity in PH diaphragm, we observed no increase in proteasome activity. However, it should be noted that the baseline activity of proteasomes is very high because it is responsible for most normal protein turnover in muscle (22). Therefore, we speculate that the threshold for detecting a relatively small increase in proteasome activity might have been too high for the activity assay used in the present study.
In addition to diaphragm muscle fiber atrophy, we found an approximately 30% reduction in the maximal force-generating capacity of diaphragm muscle strips from rats with PH, whereas in these rats EDL muscle function was preserved. This suggests that diaphragm muscle weakness in PH results from a local process, and is not part of a generalized muscle weakness.
Because force was normalized to the CSA of the muscle strip, and because we observed no changes in extracellular area fraction (data not shown), our findings suggest that in PH the intrinsic capacity of diaphragm fibers to generate force is impaired. The nature of this intrinsic diaphragm fiber weakness remains to be established, but could involve sarcomeric injury, or loss of myosin, the main contractile protein. Both have been reported to occur in animal models for congestive heart failure (13, 23–25) and chronic obstructive pulmonary disease, and in diaphragm fibers of patients with chronic obstructive pulmonary disease (16, 26). Because typically the force-generating capacity of a muscle fiber is proportional to its CSA, these findings suggest that, on top of the intrinsic diaphragm fiber weakness, diaphragm strength is further impaired by fiber atrophy.
The observed diaphragm muscle atrophy might be regarded as an adaptation to the low cardiac output that accompanies severe PH (Table 1) (27), by reducing oxygen diffusion distance to prevent hypoxia. Although no alterations in oxidative enzyme activity were observed, the preserved capillary density in PH diaphragm (Figure 3) reduces the oxygen diffusion distance, thereby potentially improving muscle fiber oxygenation (14). However, the observed magnitude of atrophy, combined with the intrinsic muscle fiber weakness, is expected to result in an approximately 60% loss of maximum force- and power-generating capacity of the diaphragm in rats with PH. Therefore, it is unlikely that the observed atrophy is merely an adaptive mechanism, without compromising inspiratory muscle function.
An important finding of the present study was that we also observed severe diaphragm fiber atrophy in patients with PH (Figure 7). These findings likely provide a cellular basis for the impaired in vivo inspiratory muscle function in patients with PH, which has been described previously (3, 6). The magnitude of diaphragm fiber atrophy was even much more pronounced in the patients with PH compared with the magnitude of atrophy in rats with PH (diaphragm fiber CSA reduced by ∼ 75% in patients vs. ∼ 40% in rats). The reason for this discrepancy is unclear but might involve the difference in duration to PH exposure. Muscle fiber remodeling, such as atrophy, requires time and clearly patients with PH had more time for diaphragm remodeling to occur than rats with PH, who had only approximately 25 days between induction of PH and death.
In addition to severe atrophy, we also observed impaired force-generating capacity of diaphragm muscle fibers in patients with PH. In these patients, intact muscle fiber function cannot be assessed because the biopsies are obtained from the middle of the diaphragm. Hence, the fiber ends are not sealed by their tendons, which disrupts normal excitation–contraction coupling. Therefore, we evaluated diaphragm muscle fiber function in demembranated (or skinned) muscle fibers. In skinned muscle fibers, the membranous structures, such as the sarcolemma and sarcoplasmic reticulum, are made highly permeable, while leaving the contractile machinery intact. By exposing these skinned fibers to exogenous calcium, their contractile performance was evaluated. These experiments revealed a greater than 50% reduction of the force-generating capacity of diaphragm muscle fibers from patients with PH, even when force was normalized to fiber CSA (Figure 8).
Thus, similar to our observations in rats with PH, diaphragm function in patients with PH is impaired by both muscle fiber atrophy and intrinsic muscle fiber weakness.
In line with the rat model for PH, the diaphragm atrophy in patients with PH was not part of a generalized process, because quadriceps muscle fibers from patients with PH showed no signs of atrophy (Figure 7). Data describing skeletal muscle function and structure in patients with PH are scarce (3, 28, 29). In line with our findings, Mainguy and coworkers (29) reported no significant changes in quadriceps muscle fiber size in patients with PH compared with control subjects. Nevertheless, they did observe a mild decrease in in vivo quadriceps strength in patients with PH (29). This might have been caused by extramuscular phenomena, such as altered nerve function or neuromuscular transmission. These extramuscular phenomena do not play a role in our contractile force measurements on EDL muscle strips, in which muscle fibers are directly activated through electrical field stimulation, thereby bypassing the neuromuscular junction.
It has been reported that patients with PH, and rats with MCT-induced PH, have elevated levels of circulating proinflammatory cytokines, such as IL-6 and tumor necrosis factor-α (30–33). Animal studies have shown that cytokines initiate striated muscle injury, impair contractile protein function (34), and stimulate proteolysis (35). Thus, systemic inflammation might be involved in the development of diaphragm weakness in PH. However, if such a systemic etiology is at play, alterations in the diaphragm and peripheral skeletal muscles are expected to share a high degree of similarity and, to a certain extent, develop simultaneously. In support of this, previous work has shown that the effects of tumor necrosis factor-α and IL-6 on diaphragm and peripheral muscle function are comparable (34, 36). Thus, because our findings suggest that the diaphragm and peripheral muscles are affected differentially in PH, a major role for circulating cytokines seems unlikely.
Instead, we propose that the increased activity of the inspiratory muscles in PH plays a role (see Figure 9 for a schematic overview). Patients with PH are subjected to continuous hyperventilation. This is indicated by increased ventilatory equivalents, low end-tidal carbon dioxide pressures (PetCO2), and reduced PaCO2 (4, 37, 38). The cause of this hyperventilation is not completely understood. It might be a compensatory mechanism for increased dead-space ventilation, as has been described in left heart failure. However, the observation that both PetCO2 and PaCO2 are reduced in patients with PH suggests a role for increased sympathetic overdrive (5). However, as a consequence of hyperventilation, inspiratory muscle activity is substantially increased. We propose that in PH, the inspiratory muscles are not able to adapt to this increase in demand, eventually leading to marked diaphragm weakness (Figure 9).
To investigate diaphragm and peripheral muscle function in rats with PH, we have used the MCT rat model. A limitation of this model is that induction of PH with MCT induces systemic inflammation (39). Systemic inflammation can affect muscle function (34, 36); therefore, the diaphragm weakness in rats with PH might have been a consequence of MCT-induced systemic inflammation rather than of PH. However, our findings in the MCT rat model were confirmed in another widely used animal model for PH, the SuHx model. In addition, because systemic inflammation is expected to cause a generalized muscle weakness, it is unlikely that MCT-induced systemic inflammation is a major contributor to diaphragm dysfunction in this model. Furthermore, we observed a high degree of similarity between the observations in the MCT PH rat model and in the patients with PH.
The present study demonstrates an association, but not a causal relation, between increased breathing frequencies and diaphragm muscle weakness in PH. Clearly, a role for other factors cannot be ruled out. For instance, hypoperfusion of the diaphragm muscle, caused by reduced cardiac output in PH, might be involved. However, hypoperfusion is expected to also affect skeletal muscle function, as has previously been described for left heart failure (40–42). In the present study we did not observe alterations in skeletal muscles from rats and from patients with PH.
This is the first study revealing diaphragm muscle fiber weakness in PH. We propose that this weakness is not part of a generalized muscle weakness but is specific for the diaphragm, and we speculate that it is at least partly caused by the chronic increase of diaphragm activity in PH. The identification of the pathogenesis of diaphragm muscle weakness may provide new therapeutic targets to reduce the sensation of dyspnea and eventually improve the quality of life of patients with PH.
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