Complex Network Model Reveals the Impact of Inspiratory Muscle Pre-Activation on Interactions among Physiological Responses and Muscle Oxygenation during Running and Passive Recovery

ough several studies have focused on the adaptations provided by inspiratory mus- cle (IM) training on physical demands, the warm-up or pre-activation (PA) of these muscles alone appears to generate positive effects on physiological responses and performance. This study aimed to understand the effects of inspiratory muscle pre-activation (IM PA) on high-intensity running and passive recovery, as applied to active subjects. In an original and innovative investigation of the impacts of IM PAon high-intensity running, we proposed the identi cation of the interactions among physical characteristics, physiological responses and muscle oxygenation in more and less active muscle to a running exercise using a complex network model. For this, fteen male subjects were submitted to all-out 30 s tethered running efforts preceded or not preceded by IM PA, composed of 2 15 repetitions (1 min interval between them) at 40% of the maximum individual inspiratory pressure using a respiratory exercise device. During running and recovery, we monitored the physio- logical responses (heart rate, blood

complex network model. For this, fteen male subjects were submitted to all-out 30 s tethered running efforts preceded or not preceded by IM PA, composed of 2 15 repetitions (1 min interval between them) at 40% of the maximum individual inspiratory pressure using a respiratory exercise device. During running and recovery, we monitored the physio- logical responses (heart rate, blood lactate, oxygen saturation) and muscle oxygenation (in vastus Biology2022,11, 963.

Biology2022,11, 963 2 of 23 lateralis and biceps brachii) by wearable near-infrared spectroscopy (NIRS). Thus, we investigated four scenarios: two in the tethered running exercise (with or without IM PA) and two built into the recovery process (after the all-out 30 s), under the same conditions. Undirected weighted graphs were constructed, and four centrality metrics were analyzed (Degree, Betweenness, Eigenvector, and Pagerank). The IM PA(40% of the maximum inspiratory pressure) was effective in increasing the peak and mean relative running power, and the analysis of the complex networks advanced the interpretation of the effects of physiological adjustments related to the IM PAon exercise and recovery. Centrality metrics highlighted the nodes related to muscle oxygenation responses (in more and less active muscles) as signi cant to all scenarios, and systemic physiological responses mediated this impact, especially after IM PAapplication. Our results suggest that this respiratory strategy enhances exercise, recovery and the multidimensional approach to understanding the effects of physiological adjustments on these conditions. Keywords: acute inspiratory loading; performance; near-infrared spectroscopy (NIRS); muscle oxygenation; tethered running; computational modelling 1. Introduction Inspiratory muscles (IM) are essential for performing physical effort at different in- tensities [1], as well as for maintaining the quality of post-exercise recovery [2,3]. Aiming to improve respiratory muscle ef ciency, speci c respiratory muscle training has been the target of many investigations in sports performance [4–8] and has been used as a proce- dure to boost the physical capacity and physiological state of active individuals [9,10] and patients [11–13]. With long-term training, IM showed positive adaptations, such as the reduction of the respiratory metabore ex mechanism, which contributes to the redirection of blood ow to more active muscles during physical effort [14,15]. Although several studies have focused on the adaptations provided by IM train- ing, such as a signi cant resource to improve performance [6,14], the warm-up or pre- activation (PA) of these muscles already seems to generate positive effects on physiological responses [16,17] and performance [18,19]. So, this individualized and licit strategy has been highlighted as ergogenic for elite swimmers [19], badminton players [20], judo ath- letes [21] and

provided by IM train- ing, such as a signi cant resource to improve performance [6,14], the warm-up or pre- activation (PA) of these muscles already seems to generate positive effects on physiological responses [16,17] and performance [18,19]. So, this individualized and licit strategy has been highlighted as ergogenic for elite swimmers [19], badminton players [20], judo ath- letes [21] and runners [18]. On the other hand, the literature is scarce about the effects of inspiratory muscle pre-activation (IMPA) on non-athlete participants, revealing some positive effects on pulmonary function [16] and breathlessness sensations and an improve- ment in exercise tolerance [22] with inspiratory exercise at 40% of the maximal inspiratory pressure (MIP) before the main exercise. Based on pre-activation concepts, it is probable that the IM warm-up using individual load ow restriction by a mechanical device (i.e., external mechanical loading) [23] acts as an important pre-activator of the organism, preparing it for high-intensity demands and recovery processes and boosting performance. In addition to studies that investigate electroencephalographic (EEG) activity prior to motor activation [24] and diaphragm motor-evoked potentials [25], the monitoring of physiological responses (e.g., HR, VO2 and SpO2) during effort and recovery seems to contribute to the analysis of the impact of IMPAon the organism. Metabolically, lactate can develop a signi cant role during different processes, including exercise and recovery. Currently, it is considered a major energy source for mitochondrial respiration, the major gluconeogenic precursor and a signaling molecule [26]. This metabolite, produced during high-intensity exercise [27,28], can be used as an important fuel by different tissues, including less-active muscles after effort. Regarding IM warm-up, Lin et al. [20] observed a reduction in blood lactate accumulation during an incremental eld test using this strategy. In this way, we strongly believe that the effects of IMPAon high-intensity running effort and recovery can be related to blood lactate shuttle.

Biology2022,11, 963 3 of 23 Furthermore, the analysis of muscle oxygenation by wearable near-infrared spec- troscopy (NIRS) [29,30] offers the possibility of exploring O2balance in skeletal muscle continuously, both during and after exercise. The NIRS technique has been applied suc- cessfully to measure the muscle oxygenation changes in a single muscle [31,32] and in two or more muscles [33–37]. Regarding the use of the NIRS analysis associated with the acute loading of IM on exercise, there are few investigations in the literature. In 2015, Ohya et al. [38] investigated the effects of IM warm-up on locomotor muscle oxygenation (vastus lateralis—VL) in healthy males submitted to cycling exercise. They did not observe signi cant performance enhancement or muscle oxygenation during high-intensity inter- mittent sprint cycling in untrained participants. Recently, Richard & Billaut [39] conducted an interesting study applying the IM warm-up to elite speed skaters before 3000 m time trials. Corroborating the results of Ohya et al. [38], no differences in speed performance nor in the tissue saturation index (TSI) and total hemoglobin (tHb) after an IM warm-up strategy were observed, at least in the VL muscle. However, investigations using portable NIRS in less and more active muscle concomitantly are scarce, but they can contribute strongly to the comprehension of the physiological effects promoted by IMPA. The IMPAseems to provoke positive effects on performance and physiological adjust- ments, but this is not the consensus in the literature. However, to the best of our knowledge, only one recent study conducted by our group involving this strategy is based on complex analysis—rather than only conventional statistics—to interpret these data [21]. In this case, by the complex network model, the positive impact of the IMPAat 40% of the MIP was con rmed. So, it is possible that important physiological changes occur in athletes or non-athletes submitted to IMPAprior to different exercise types but are not always detectable by statistical analyses based on “cause-effect” approaches. In previous studies, we have worked with more integrated models to investigate the physical exercise [40,41] and sports-related context [21,42,43], applying the concepts of complex networks [44,45] for biological data

is possible that important physiological changes occur in athletes or non-athletes submitted to IMPAprior to different exercise types but are not always detectable by statistical analyses based on “cause-effect” approaches. In previous studies, we have worked with more integrated models to investigate the physical exercise [40,41] and sports-related context [21,42,43], applying the concepts of complex networks [44,45] for biological data interpretation [46,47]. This line of investigation has contributed strongly to understanding biological responses that do not depend only on an isolated factor [46–51], which occurs with physiological adjustments in effort and recovery. In this way, we believe that this computational model can improve the knowledge about IMPA, especially by inte- grating analyses conducted with many responses during exercise and recovery. Basically, in complex network models, the centrality metrics that are returned from the graphs built for different scenarios are able to highlight, within a dataset, those which are the main players in an integrative context. Centrality measures are capable of quantifying the capacity of a node to in uence or be in uenced by other parameters in a connection topology [52]. Among the many available centrality metrics, we highlight here the analysis of the Degree, Betweenness, Eigenvector and Pagerank, each with a purpose in studies with complex networks [21,42,43,52–56]. Here, we investigated the impact of one mechanical IMPAprotocol (load at 40% of the MIP) on tethered running power on a non-motorized treadmill. Thus, by applying the concept of complex networks for integrative analysis among physiological responses, we aimed to understand the effects of this speci c IMPAon important physiological measures for effort and recovery, such as blood lactate, HR and muscle oxygenation in the biceps brachii (BB) and the vastus lateralis (VL), which are, respectively, less and more active in high-intensity running. We investigated four scenarios: two in the tethered running exercise (with or without IMPA) and two others built to understand the recovery process after the all-out 30 s (AO30) running effort under the same conditions. We hypothesize that the IMPApresents changes in physiological responses in both exercise and recovery, and the complex network model is sensitive enough to

high-intensity running. We investigated four scenarios: two in the tethered running exercise (with or without IMPA) and two others built to understand the recovery process after the all-out 30 s (AO30) running effort under the same conditions. We hypothesize that the IMPApresents changes in physiological responses in both exercise and recovery, and the complex network model is sensitive enough to detect the effects of this pre-activation strategy on physiological connections.

Biology2022,11, 963 4 of 23 2. Methods 2.1. Subjects Fifteen physically active young men participated in the study (23 1 years, 73.2 2.0 kg, 1.77 0.02 m, 6.5 0.5% body fat, 144.3 10.2 cm H2O of MIP, mean and peak global strength index of IMs (S-Index) equal to 123.7 3.7 and 139.1 3.4 cm H2O, respectively). Firstly, the subjects answered the International Physical Activity Questionnaire (IPAQ), in which the minimum score required to classify them as “physically active” was used as the inclusion criterion [57]. All of the subjects reported no cardiovascular, respiratory, metabolic or orthopaedic disease and no use of drugs, ergogenic supplements or medications. This study was conducted in agreement within the ethical recommendations of the Declaration of Helsinki, and all experiments were approved by the Research Ethics Committee of The School of Medical Sciences (protocol number 99783318.4.0000.5404). The participants were only evaluated after having received information about the experimental procedures and risks and signing an informed consent form. 2.2. Experimental Design The experimental design consisted of four laboratory visits, separated by 24 72 h (Figure, Panel A). Firstly, the subjects received information about the experimental design and signed a consent form. They answered the IPAQ and a questionnaire for health characterization. Next, the participants were submitted to anthropometric and body composition measurements, including body mass (balance portable digital scale, with a maximum capacity of 150 kg and an accuracy of 100 g, Toledo ® model 2098, S¢o Bernardo do Campo, Brazil) and height (by a stadiometer (Cescorf ® , Porto Alegre, Brazil). To estimate the percentage of body fat (%BF), seven skinfold thicknesses of the right side of the body were evaluated (triceps, chest, subscapular, midaxillary, suprailiac, abdominal and thigh by the Lange ® skinfold caliper, Beta Technology, Santa Cruz, USA), and the equation proposed by Jackson & Pollock, following by Siri et al., was applied [58,59]. On the second day, the maximal inspiratory pressure (MIP) and S-Index (SI) were obtained (with a 1 h interval between them), and the tethered running familiarization was conducted on a non-motorized treadmill (NMT). The third and fourth days were randomly

Technology, Santa Cruz, USA), and the equation proposed by Jackson & Pollock, following by Siri et al., was applied [58,59]. On the second day, the maximal inspiratory pressure (MIP) and S-Index (SI) were obtained (with a 1 h interval between them), and the tethered running familiarization was conducted on a non-motorized treadmill (NMT). The third and fourth days were randomly dedicated to AO30 in NMT, preceded or not preceded by the IMPA, aiming to determine the power in tethered running and physiological measurements in two conditions. In these sections, the subjects were equipped with two NIRS devices (one in BB and the other in VL) to acquire muscle oxygenation signals and with the HR monitor. After the running test, the participants remained at rest (18 min in the dorsal decubitus position) for the continuous monitoring of HR, SpO2and muscle oxygenation responses. Additionally, blood samples and Borg scale scores [60] were collected at rest immediately after the effort and after each 2 min of passive recovery in order to assess blood lactate and perceived exertion (RPE). All of the trials were completed under controlled laboratory conditions at a similar temperature, relative humidity and luminosity. 2.3. Maximal Inspiratory Pressure and S-Index Determination The maximal inspiratory pressure (MIP) measurement was obtained from residual volume in accordance with Hartz et al. [6]. The evaluation was conducted by a trained researcher who demonstrated the correct performance of the respiratory maneuver. The volunteers remained seated in a chair, wearing a nose clip and a plastic mouthpiece that was connected to an analogical manovacuometer ( 300 cmH2O; GER-AR, S¢o Paulo, SP, Brazil) used to measure maximal pressures. A small hole (2 mm) was introduced in the rigid mouthpiece in order to prevent glottic closure. The volunteers were instructed to complete three to ve acceptable and reproducible maximal maneuvers (i.e., differences of 10% or less between values). Each inspiratory effort was sustained for at least 1 s, and the highest value reached was registered for further analysis. An interval of about 1 min was allowed between efforts [61].

instructed to complete three to ve acceptable and reproducible maximal maneuvers (i.e., differences of 10% or less between values). Each inspiratory effort was sustained for at least 1 s, and the highest value reached was registered for further analysis. An interval of about 1 min was allowed between efforts [61].

Biology2022,11, 963 5 of 23Biology 2022, 11, x FOR PEER REVIEW 5 of 25 Figure 1. Panel A. Experimental design. First and second visits were conducted for sample characterization and familiarization.Additionally, the maximal inspiratory pressure (MIP) and global strength index of

Investigates the impact of inspiratory muscle pre-activation on physiological responses during running.