**3. Discussion**

In this three-armed crossover study, we investigated the miRNA profiles in young, healthy athletes before and after various resistance training interventions (HI and LI). In addition, we employed peripheral blood flow restriction (LI-BFR) that was achieved by applying an external cuff during the resistance training at LI. We assumed that BFR can mimic the occlusion of a larger artery, leading to an increased collateral flow, and would therefore serve as an external stimulus of arteriogenesis.

The BFR application led to a decreased blood flow velocity in the popliteal artery, confirming our first hypothesis. The HI intervention showed the largest effects on lactate, and all interventions led to a comparable heart rate response. The LI intervention resulted in the smallest pre to post differences. Hypothesis 2 was thus partially verified (depending on the outcome). Our results further sugges<sup>t</sup> that miRNA profiles were acutely affected by HI training and LI-BFR training but not by LI training alone. In particular, miR-143-3p expression correlated with training intensity, which verifies Hypothesis 3.

The fact that BFR application led to a decrease in the blood flow velocity (Hypothesis 1) confirms the validity of BFR. The reduction of 7.6 cm/s is far beyond the standard error of measurement found in inter-rater reliability analyses [35] and can thus be considered as clinically relevant. The mean blood flow velocity in the popliteal artery in PAD patients was recently found to be 41 ± 17 (SD) cm/s [36], which is considerably lower than the velocity we found in the no-cuff condition and is comparable to the one we found in the blood flow-restricted condition. Consequently, one may consider our model valid in terms of blood flow restriction and velocity, although the study population does not fully mimic the PAD caused by atherosclerosis.

All interventions induced increases in lactate and heart rate; the largest effects on lactate occurred after the HI intervention, whereas all interventions led to a comparable heart rate response. The LI intervention resulted in the smallest differences in pre- and post-intervention values in objective and participant-reported outcomes. The finding of a BFR-induced increase in lactate concentration is in accordance with previous reports [23]. The metabolic response thus varies with varying training intensity. If BFR training effects comparable to those of 70% 1RM sessions without BFR are to be reached, the current literature recommends BFR training at 30% of the individual 1RM [18]. Though we followed this recommendation, BFR elicited lower values/effects in some of the outcomes than during/after HI training; this is in contrast to our second hypothesis, which was only partially verified. One possible reason may be due to our sample. The majority of current studies adopting BFR training or interventions at 30% of the 1RM refer to an elderly or untrained population [18]. For a trained study population, a minority of studies in the literature sugges<sup>t</sup> that 50% of the 1RM should be used to achieve a sufficient metabolic response [24].

We further analyzed the profiles of circulating miRNAs before and after resistance training. Our results of the quantification in each intervention group sugges<sup>t</sup> that the profile of circulating miRNAs is altered as an acute effect of resistance training. In particular, we identified six miRNAs (miR-139-5p, miR-143-3p, miR-195-5p, miR-197-3p, miR-30a-5p, and miR-10b-5p) that are up-regulated after HI training. Only miR-143 was found to be down-regulated after LI-BFR training. The LI training, in contrast, had no systematic effect of either miRNA. Other studies also demonstrated both acute effects of intensive stimuli (miRNA-21, miRNA-146a, miRNA-221, and miRNA-222) and training effects (miRNA-146a, miRNA-222, and miRNA-20a) [37]. Consequently, we conclude that increased training intensity leads to increased miR-143-3p. The correlation of the lactate difference (BFR-LI, LI, and HI) and miR-143-3p abundance further indicates a decisive role depending on training intensity. In detail, 3.5 mmol/L of pre-to-post lactate difference was determined as a potential threshold from miR-143-3p down-regulation to its up-regulation. Consequently, a major share, but not only the lactate concentration (or the intensity of the training), is decisive for miRNA expression—as is (to a minor share) the type of training. More concretely, BFR seems not only to lead to lower lactate increases but also tendentiously leads to a down-regulation of miRNA-143-3p, whereas LI seems to be able to increase lactate concentration but does not affect miRNA-143-3p expression. The HI, in contrast, seemed to be able to both up-regulate lactate and miRNA-143-3p. Whether the differences between the conditions are due to the lower intensity or the type of training may be finally delineated by using the intensity increase up to 50% during BFR, as described above.

We have previously shown an association of miR-195-5p and miR-143-3p with collateral growth [38]. Both of these species were found to be highly up-regulated in the vascular tissue itself, and miR-143-3p was identified as an essential factor for proper collateral formation following femoral artery ligation in mice. The acute and local blockade of miR-143-3p in these mice completely abrogated arteriogenesis. In blood vessels, miR-143 is one of the most-studied miRNAs expressed by vascular smooth muscle cells, and, together with miR-145, this miRNA is thought to play a pivotal role in smooth muscle cell differentiation and vascular disease [8,13,39]. Furthermore, circulating miR-143-3p has been associated with cardiovascular disease [40] and is considered to be a predictor of aging and the acute adaptive response to resistance exercise [41].

Small volumes of capillary blood are routinely used for lactate diagnostics. We aimed to establish this routinely used, less invasive method of fingertip blood drawing for obtaining cell-free, non-hemolytic plasma samples suitable for the isolation and quantification of circulating miRNAs. Indeed, this method was successful in yielding plasma samples reproducible in quality and volume. All miRNAs identified, with the exception of miR-10b-5p, were validated in terms of independency of hemolytic score. For miRNA profiling, we included plasma samples of four participants with an OD414 < 0.3 and an increased lactate concentration after training intervention, a maximal heart rate during training of at least 60% of maximal calculated heart rate, and a participant-reported "very hard" intensity on the Borg scale of at least 16 points (data not shown).

Our results are in line with the proposed epigenetic potential of lifestyle interventions that may alter gene expression [42,43]. Therefore, we postulate that in order to maximize the beneficial role of miR-143-3p in collateral growth, training intensity will have to be adjusted. For symptomatic PAD patients, controlled training is an efficient, conservative therapy that is a good alternative to invasive therapies. The formation of collaterals and compensatory blood flow is the goal of conservative treatment. However, since the success of training varies, responders and non-responders must be identified. Thus, future studies are needed to (1) confirm our findings in PAD patients, (2) delineate the mechanisms of how miRNA-143-3p may be decisive in response or non-response to resistance training, and (3) determine how a pre-intervention screening of miRNA-143-3p or other microRNAs can be used to stratify responders and non-responders for the individualization of intervention/training goals.

### **4. Materials and Methods**

### *4.1. Ethical Standard and Study Design*

The study had a randomized-balanced crossover design. Ethical approval was obtained from the local institutional review board (protocol number 2018-16, 17.06.2018, Ethics Committee Department 5 Psychology and Sports Sciences Goethe-University Frankfurt). The trial was conducted in accordance with the ethical standards set down by the declaration of Helsinki (World medical Association) Declaration of Helsinki–Ethical Principles for Medical Research Involving Human Subjects) with its recent modification of 2013 (Fortaleza). All participants gave written informed consent prior to study enrollment.
