Protein degradation pathways
Although MAFbx was not significantly different between groups, we detected an elevated expression of the key atrophic marker MuRF1 in sedentary HF, but not in AET mice, as compared with sham (Fig. 4A and B). We subsequently assessed key pathways of protein degradation, the ubiquitin proteasome and calpain systems, and found their activity to be reduced in HF + AET compared with HF alone (Fig. 4C and D).
Our findings show, for the first time, that regular AET prevented diaphragm contractile dysfunction in HF, and when compared with sedentary HF mice, this was associated with significant reductions in both the oxidative modifications of key contractile proteins (i.e., actin and creatine kinase) and the activity of proteolytic pathways associated with muscle atrophy (i.e., ubiquitin proteasome and calpain systems). Interestingly, however, although we did find some evidence that certain markers of oxidative stress and proteolysis were higher in the diaphragm of sedentary HF mice compared with shams, as demonstrated for example by increased XO activity and MuRF1 expression, these measures were not significantly different compared with HF + AET mice, with our data also showing that additional indices of oxidative stress (i.e., carbonylated proteins) and proteolysis (i.e., proteasome and calpain systems) were not consistently elevated in sedentary HF mice versus shams. Overall, therefore, it remains unclear whether the key mechanism(s) involved in AET protecting the diaphragm from contractile dysfunction in HF is related to alterations in oxidative stress and/or protein degradation.
Exercise training and respiratory muscle function
The close link between respiratory muscle weakness, symptoms, and prognosis in HF suggests the development of therapies focused on improving the main muscle of respiration, the diaphragm, is likely critical (20). In the present study, we investigated the therapeutic intervention of AET on the diaphragm in HF to assess whether this could benefit contractile function as well as modulate putative underlying mechanisms related to oxidative stress and proteolysis. To date, numerous patient studies in HF have demonstrated that exercise training (whole body or respiratory muscle) can improve inspiratory muscle strength, exercise capacity, and also quality of life (1,8,11,22,25,38). Nevertheless, up until now, it remained unknown whether diaphragm contractile function per se improves after exercise training in HF, as patient studies had previously assessed inspiratory muscle strength noninvasively, which provides an indirect measure of diaphragm function fraught with limitations. In addition, none of the patient studies provided any underlying molecular and cellular mechanisms explaining the benefits observed after training.
The current data, therefore, are the first to directly show AET in HF prevents contractile dysfunction in diaphragm fiber bundles, while providing novel evidence on potential underlying mechanisms. Further, our data support the contention that around 10 wk of AET seems sufficient to induce benefits to the diaphragm, which is in accordance with a patient study where 8 wk of AET improved inspiratory muscle strength (38). Interestingly, we did not find diaphragm fibers to be more fatigable in sedentary HF mice compared with AET + HF and sham mice, with twitch kinetics also not affected. One explanation may be related to calcium function not being altered in our HF mice, as such impairments are known to have a greater influence on force production at low frequencies, on twitch properties, and during fatiguing contractions (24). By contrast, however, it may also be related to the “matched-stimulus” frequency fatigue protocol we used rather than a “matched-initial specific force” fatigue protocol, with the latter suggested to provide a similar metabolic challenge that is likely a more appropriate assessment of fiber fatigue (14,20).
Mechanisms preventing diaphragm dysfunction in HF after exercise training
It has been suggested that the key mechanisms underpinning diaphragm dysfunction in HF include increased protein degradation (leading to loss of muscle mass) (27,36,37) as well as elevated oxidant levels (leading to contractile dysfunction) (4,9). Interestingly, research directed toward limb skeletal muscle in HF has previously revealed that the severity of muscle wasting and contractile dysfunction can be attenuated after AET (6,19), which is further associated with a reduced expression of atrogenes, lower proteolytic activity, increased antioxidant enzyme activity, improved mitochondrial function, and reduced inflammatory cytokine levels (6,19). Indeed, although the present study provided direct evidence that diaphragm contractile dysfunction induced by HF can be prevented by AET, this was not consistently accompanied by a significant reduction in all markers of oxidative stress and proteolysis between the mice that did or did not perform exercise training (e.g., XO activity was not significantly different, nor were MuRF1 and MAFbx levels). Furthermore, markers of oxidative stress and protein degradation were also not consistently elevated between HF and sham mice. For example, although XO activity and MuRF1 levels in the diaphragm were increased in HF mice, oxidized proteins along with proteasome and calpain activity were not significantly different compared with sham mice. The reason for this discrepancy compared with previous studies remains unclear (2,4,9,35); however, in the present study, it may be related to a low statistical power because of the small sample size of groups (a typical feature of using this animal model) combined with the addition of multiple group comparisons, as statistical significance has usually been achieved in the past with comparison of only two groups (i.e., sham vs HF) (2,4,9,35). Indeed, when we used a t-test to compare sham and sedentary HF mice, we then found significant differences in terms of carbonylated actin and creatine kinase, proteasome activity, MAFbx protein expression, and also glutathione peroxidase activity. However, our study was designed to detect a statistical difference in our primary variable of interest, that is, diaphragm function, as based on previous studies in rodents (2,4,9,35). As such, an increased sample size of groups would likely be required to tease out the dominant molecular mechanisms responsible for protecting diaphragm function in HF after AET.
However, our data do provide some initial evidence that exercise was able to modulate oxidative stress and proteolysis that may have influenced diaphragm contractile function. Specifically, we found both the oxidative modifications of key proteins (i.e., actin and creatine kinase) and the activity of proteolytic pathways associated with muscle atrophy (i.e., ubiquitin proteasome and calpain systems) were significantly lower in the diaphragm of HF + AET mice compared with HF mice alone. These findings likely represent a complex interplay where ROS mediate protein degradation on multiple levels: one by acting as direct signaling molecules to increase rates of proteolysis (e.g., via targeting specific transcription factors such as FOXO and nuclear factor-kB) (3), whereas another by targeting proteins for oxidative modifications, which then leads to increased proteolytic activity to dispose of these damaged proteins (16). In contrast to previous studies in healthy mice, however, antioxidative enzyme activities were not increased after AET (26,33), which seemingly excludes a key role for antioxidants in maintaining diaphragm function in HF after exercise training. As such, the present findings suggest that AET in HF targets more upstream mechanisms related to ROS production rather than increasing antioxidant capacity in the diaphragm, and this subsequently influences downstream factors such as oxidative modifications of contractile proteins and upregulation of catabolic factors.
Although further studies are required in HF to elucidate how AET modulates upstream ROS production in the diaphragm, current evidence indicates that inflammatory cytokines likely play a key role, with our laboratory showing that AET can prevent tumor necrosis factor-α–induced diaphragm dysfunction concomitant with lower oxidative stress and proteolysis (26). As exercise also reduces inflammatory cytokine levels in HF patients (15), we propose that in the present study, exercise may have reduced systemic and/or local inflammation that subsequently lowered ROS and proteolytic activity, thus helping maintain normal diaphragm function. This is also supported where 4 wk of exercise training attenuated respiratory muscle weakness in HF patients in combination with reduced plasma concentrations of inflammatory cytokines (8).
We cannot confirm categorically whether AET reversed diaphragm dysfunction or merely maintained function in HF. However, data from our laboratory recently demonstrated that 3-d after myocardial infarction diaphragm function is impaired by ~20%, which was associated with increased oxidative stress but not an upregulation in markers of proteolysis (4). Collectively, therefore, data from our laboratory, although speculative, suggest that the following events may occur in the diaphragm after infarction: 1) early response—where at 3 d, muscle function is rapidly impaired because of increased oxidation of contractile proteins; 2) late response—where at 10 wk after HF development, muscle function is still impaired consequent to elevated proteolysis in combination with increased protein oxidation; 3) AET modulated response—where at 10 wk, muscle function is normalized after AET, by potentially limiting in part the initial protein oxidation and the subsequent secondary increase in proteolysis. Nevertheless, to confirm such as a notion, a temporal study measuring diaphragm function after infarction is required.
In addition, we are unable to provide the precise exercise intensity that our mice trained at but it was likely that of moderate (i.e., mice ran ~40% of their peak treadmill speed). We selected the current exercise training regime based on evidence from our laboratory where we have shown these treadmill speeds are sufficient to induce beneficial circulatory and muscular adaptations in mice (26,28). Nevertheless, the addition of standard measurements of training adaptations and exercise tolerance (e.g., maximal oxygen uptake, ventilatory variables, and blood lactate) would have significantly strengthened the present study to better translate our findings to other species and also the clinical setting. As such, future studies will be required to confirm the optimal training intensity and duration required for preventing diaphragm dysfunction in HF. Moreover, we are also unable to confirm whether the molecular alterations associated with exercise are specific to the HF syndrome alone as we did not have a sham group that performed exercise training, while we are also unable to rule out the contribution of other key factors not determined in the present study, which may have, in part, also contributed to the exercise-related benefits, such as improved calcium handling (7) and reduced ROS production from NADPH oxidase (2) and the mitochondria (21).
Further, although not statistically significant, heart dysfunction was ~25% more severe in the sedentary HF mice compared with those that performed AET. The reason for this discrepancy remains unclear, as cardiac dysfunction assessed by echocardiography before the exercise intervention at 1 wk after myocardial infarction was near-identical between the sedentary and the trained HF mice. As such, it remains a possibility that AET conferred some cardiac protection during the training period that attenuated LV infarct size and pump dysfunction (12,32), which in turn may have contributed to the normalized diaphragm forces we observed in HF + AET mice. Indeed, additional measures of LV dysfunction and the HF syndrome, such as invasive LV filling pressures and those of exercise capacity (30), may have therefore provided greater insight into this question.
Regular AET in mice prevented diaphragm contractile dysfunction in HF, but this was not consistently associated with lower oxidative stress and proteolysis when compared with sedentary HF mice. As such, our findings suggest that although AET protects against diaphragm muscle weakness induced by HF, it remains unclear whether the predominant mechanism underpinning this benefit is mediated by reduced levels of oxidative stress and/or protein degradation.
T. S. B. is a recipient of a postdoctoral research fellowship from the Alexander von Humboldt Foundation.
The authors declare no professional relationships.
The results of the present study do not constitute endorsement by the American College of Sports Medicine and are presented clearly, honestly, and without fabrication, falsification, or inappropriate data manipulation.
Norman Mangner and T. Scott Bowen contributed equally.
1. Adamopoulos S, Schmid JP, Dendale P, et al. Combined aerobic/inspiratory muscle training vs. aerobic training in patients with chronic heart failure: the Vent-HeFT trial: a European prospective multicentre randomized trial. Eur J Heart Fail
2. Ahn B, Beharry AW, Frye GS, Judge AR, Ferreira LF. NAD(P)H oxidase subunit p47phox is elevated, and p47phox knockout prevents diaphragm contractile dysfunction in heart failure. Am J Physiol Lung Cell Mol Physiol
3. Betters JL, Criswell DS, Shanely RA, et al. Trolox attenuates mechanical ventilation-induced diaphragmatic dysfunction and proteolysis. Am J Respir Crit Care Med
4. Bowen TS, Mangner N, Werner S, et al. Diaphragm muscle weakness in mice is early-onset post-myocardial infarction
and associated with elevated protein oxidation. J Appl Physiol (1985)
5. Bowen TS, Rolim NP, Fischer T, et al. Heart failure with preserved ejection fraction induces molecular, mitochondrial, histological, and functional alterations in rat respiratory and limb skeletal muscle
. Eur J Heart Fail
6. Bowen TS, Schuler G, Adams V. Skeletal muscle
wasting in cachexia and sarcopenia: molecular pathophysiology and impact of exercise training. J Cachexia Sarcopenia Muscle
7. Bueno CR Jr, Ferreira JC, Pereira MG, Bacurau AV, Brum PC. Aerobic exercise training improves skeletal muscle
function and Ca2+
handling-related protein expression in sympathetic hyperactivity-induced heart failure. J Appl Physiol (1985)
8. Chiappa GR, Roseguini BT, Vieira PJ, et al. Inspiratory muscle training improves blood flow to resting and exercising limbs in patients with chronic heart failure. J Am Coll Cardiol
9. Coirault C, Guellich A, Barbry T, Samuel JL, Riou B, Lecarpentier Y. Oxidative stress
of myosin contributes to skeletal muscle
dysfunction in rats with chronic heart failure. Am J Physiol Heart Circ Physiol
10. Cunha TF, Bacurau AV, Moreira JB, et al. Exercise training prevents oxidative stress
and ubiquitin-proteasome system overactivity and reverse skeletal muscle atrophy
in heart failure. PLoS One
11. Dall’Ago P, Chiappa GR, Guths H, Stein R, Ribeiro JP. Inspiratory muscle training in patients with heart failure and inspiratory muscle weakness: a randomized trial. J Am Coll Cardiol
12. de Waard MC, van der Velden J, Bito V, et al. Early exercise training normalizes myofilament function and attenuates left ventricular pump dysfunction in mice with a large myocardial infarction
. Circ Res
13. Esposito F, Reese V, Shabetai R, Wagner PD, Richardson RS. Isolated quadriceps training increases maximal exercise capacity in chronic heart failure: the role of skeletal muscle
convective and diffusive oxygen transport. J Am Coll Cardiol
14. Ferreira LF, Moylan JS, Gilliam LA, Smith JD, Nikolova-Karakashian M, Reid MB. Sphingomyelinase stimulates oxidant signaling to weaken skeletal muscle
and promote fatigue. Am J Physiol Cell Physiol
15. Gielen S, Adams V, Mobius-Winkler S, et al. Anti-inflammatory effects of exercise training in the skeletal muscle
of patients with chronic heart failure. J Am Coll Cardiol
16. Grune T, Merker K, Sandig G, Davies KJ. Selective degradation of oxidatively modified protein substrates by the proteasome. Biochem Biophys Res Commun
17. Hammond MD, Bauer KA, Sharp JT, Rocha RD. Respiratory muscle strength in congestive heart failure. Chest
18. Hirai DM, Copp SW, Holdsworth CT, et al. Skeletal muscle
microvascular oxygenation dynamics in heart failure: exercise training and nitric oxide-mediated function. Am J Physiol Heart Circ Physiol
19. Hirai DM, Musch TI, Poole DC. Exercise training in chronic heart failure: improving skeletal muscle
transport and utilization. Am J Physiol Heart Circ Physiol
20. Kelley RC, Ferreira LF. Diaphragm abnormalities in heart failure and aging: mechanisms and integration of cardiovascular and respiratory pathophysiology. Heart Fail Rev
. 2016 In press.
21. Laitano O, Ahn BS, Patel N, et al. Pharmacological targeting of mitochondrial reactive oxygen species counteracts diaphragm weakness in chronic heart failure. J Appl Physiol (1985)
22. Laoutaris I, Dritsas A, Brown MD, Manginas A, Alivizatos PA, Cokkinos DV. Inspiratory muscle training using an incremental endurance test alleviates dyspnea and improves functional status in patients with chronic heart failure. Eur J Cardiovasc Prev Rehabil
23. Linke A, Adams V, Schulze PC, et al. Antioxidative effects of exercise training in patients with chronic heart failure: increase in radical scavenger enzyme activity in skeletal muscle
24. Lunde PK, Sejersted OM, Thorud HM, et al. Effects of congestive heart failure on Ca2+
handling in skeletal muscle
during fatigue. Circ Res
25. Mancini DM, Henson D, La Manca J, Donchez L, Levine S. Benefit of selective respiratory muscle training on exercise capacity in patients with chronic congestive heart failure. Circulation
26. Mangner N, Linke A, Oberbach A, et al. Exercise training prevents TNF-α induced loss of force in the diaphragm of mice. PLoS One
27. Mangner N, Weikert B, Bowen TS, et al. Skeletal muscle
alterations in chronic heart failure: differential effects on quadriceps and diaphragm. J Cachexia Sarcopenia Muscle
28. Matsumoto Y, Adams V, Jacob S, Mangner N, Schuler G, Linke A. Regular exercise training prevents aortic valve disease in low-density lipoprotein-receptor-deficient mice. Circulation
29. McParland C, Krishnan B, Wang Y, Gallagher CG. Inspiratory muscle weakness and dyspnea in chronic heart failure. Am Rev Respir Dis
30. Musch TI, Moore RL, Leathers DJ, Bruno A, Zelis R. Endurance training in rats with chronic heart failure induced by myocardial infarction
31. Musch TI, Nguyen CT, Pham HV, Moore RL. Training effects on the regional blood flow response to exercise in myocardial infarcted rats. Am J Physiol
. 1992;262(6 pt 2):H1846–52.
32. Oh BH, Ono S, Rockman HA, Ross J Jr. Myocardial hypertrophy in the ischemic zone induced by exercise in rats after coronary reperfusion. Circulation
33. Powers SK, Criswell D, Lawler J, et al. Regional training-induced alterations in diaphragmatic oxidative and antioxidant enzymes. Respir Physiol
34. Supinski G, DiMarco A, Dibner-Dunlap M. Alterations in diaphragm strength and fatiguability in congestive heart failure. J Appl Physiol (1985)
35. Supinski GS, Callahan LA. Diaphragmatic free radical generation increases in an animal model of heart failure. J Appl Physiol
36. van Hees HW, Li YP, Ottenheijm CA, et al. Proteasome inhibition improves diaphragm function in congestive heart failure rats. Am J Physiol Lung Cell Mol Physiol
37. van Hees HW, van der Heijden HF, Ottenheijm CA, et al. Diaphragm single-fiber weakness and loss of myosin in congestive heart failure rats. Am J Physiol Heart Circ Physiol
38. Vibarel N, Hayot M, Ledermann B, Messner Pellenc P, Ramonatxo M, Prefaut C. Effect of aerobic exercise training on inspiratory muscle performance and dyspnoea in patients with chronic heart failure. Eur J Heart Fail
Keywords:© 2016 American College of Sports Medicine
MYOCARDIAL INFARCTION; SKELETAL MUSCLE; OXIDATIVE STRESS; MOUSE; CHF; ATROPHY