Why was \(\dot{\mathrm{V}}\)O2peak maintained after reducing Hbmass to pre-training values?
Despite normalising Hb
mass and BV, the ET-induced improvements in
\(\dot{V}\)O
2peak and
\(\dot{Q}\)peak were maintained during upright cycling. This contradicts studies using a similar experimental design, which suggest that
\(\dot{V}\)O
2peak and
\(\dot{Q}\)peak return to pre-ET levels after removing the ET-induced elevations in BV (Bonne et al.
2014) and RBCV (Montero et al.
2015a). We speculate that the discrepancies between studies originate from the magnitude of the ET-induced BV expansion. For Bonne et al. (
2014) and Montero et al. (
2015a), the ET-induced increases in BV were 382 ml (7%) and 310 ml (6%), respectively, compared to 181 ml (4%) in the present study. Yet, improvements in
\(\dot{V}\)O
2peak (9–11%) and
\(\dot{Q}\)peak (7–10%) during upright cycling were similar in the three studies. Thus, ET-induced increases in
\(\dot{V}\)O
2peak and
\(\dot{Q}\)peak of this magnitude does not depend on BV expansion alone, and
\(\dot{Q}\)peak improved partly due to different mechanisms in the three studies. This is supported by increased LV mass in the present study, as opposed to no change in the study by Bonne et al. (
2014), and suggests that multifactorial mechanisms explain the ET-induced increases in
\(\dot{Q}\)peak and
\(\dot{V}\)O
2peak as suggested in classical studies (Saltin et al.
1968).
Because of the small BV withdrawal, little hypovolemia-induced impairment on venous return was likely elicited in our subjects. Supported by no reductions in \(\dot{Q}\)peak, \(\dot{V}\)O2peak and submaximal SV during upright cycling after phlebotomy. Consequently, we were unable to test one of our main hypotheses that when \(\dot{V}\)O2peak and \(\dot{Q}\)peak during upright cycling were reversed to pre-ET levels after phlebotomy, the improvements would be preserved during supine cycling owing to the beneficial gravitational effects on venous return.
Apparently, it may be that the cardiovascular system can maintain venous return to the heart, despite small BV reductions by redistributing venous volumes through increased vasomotor activity acting on capacitance vessels. This is supported by the unchanged LV EDV, SV and peak mitral inflow velocity during early diastole from before to just after the phlebotomy procedure. There is, however, some uncertainty in extrapolating these responses measured during supine rest to upright peak exercise, where LV filling times are shorter and exert a major challenge on cardiac preload (Gledhill et al.
1994). Similarly, indications of maintained venous return despite small reductions in BV have been observed in studies examining fluid loss after heat stress and prolonged exercise (Saltin
1964; Saltin and Stenberg
1964). In those studies, a small-to-moderate reduction in PV was not sufficient to decrease
\(\dot{Q}\)peak, assessed in normothermic conditions. Thus, it was argued that an effective contribution from the muscle pump and increased vasomotor activity enabled a normal SV despite reduced BV. In contrast to small BV losses, phlebotomy of one unit of blood (450 ml) or more reduces
\(\dot{V}\)O
2peak by lowering venous return (Kanstrup and Ekblom
1984; Krip et al.
1997). Therefore, a certain threshold might exist, at which small reductions in BV are not detrimental to venous return, acting as a mechanism for the circulation to cope with small BV losses and PV reductions induced by, e.g., dehydration.
Although Hb
mass and BV were restored to pre-ET levels, transcapillary fluid shifts may occur during and after the phlebotomy procedure. The first exercise trial started 45 min after phlebotomy as compared with ~ 15 min in the studies by Bonne et al. (
2014) and Montero et al. (
2015a). To obtain an indication of fluid shifts, we measured [Hb] repeatedly during the phlebotomy day in six of the subjects. From before (15.6 ± 1.0 g dl
−1) to just after the phlebotomy procedure, the venous [Hb] was slightly decreased (15.2 ± 1.0 g dl
−1;
P = 0.19), with no further change until initiation of the first (15.2 ± 1.2 g dl
−1) and second (15.2 ± 1.1 g dl
−1) cycling exercise trials. Therefore, at the end of, or gradually during the phlebotomy procedure, a gross movement of fluid from the interstitium to the plasma appears to have counteracted the blood withdrawal. Hence, the PV (and BV) may have been slightly higher during the phlebotomy exercise trials than reported in the present study, which may have contributed to the maintained
\(\dot{Q}\)peak. However, since the reduction in [Hb] exclusively occurred from before to just after the phlebotomy, this mechanism has likely also affected the subjects in the Bonne et al. (
2014) and Montero et al. (
2015a) studies. Also, the Hb
mass was re-established to pre-ET levels independent of potential PV shifts.
We have focused on the role of BV for
\(\dot{Q}\)peak, i.e. one of the main determinants of venous return and cardiac preload. However, an ET-induced reduction in afterload through a lowering of total peripheral resistance may also increase
\(\dot{Q}\)peak. For instance, after ET of both legs separately, Klausen et al. (
1982) found a reduction in mean arterial pressure (MAP) and total peripheral resistance that likely contributed to the increased
\(\dot{Q}\)peak after ET. This mechanism can also have facilitated the increased
\(\dot{Q}\)peak in the present study.
The magnitude of haematological adaptations
The changes in BV reported after 6–8 weeks ET (3–4 sessions week
−1) typically range from 140 to 550 ml (Bonne et al.
2014; Helgerud et al.
2007; Montero et al.
2017,
2015a; Montero and Lundby
2017b), whereas a meta-analysis reported a mean increase of 267 ml after ~ 15 weeks of ET (range 1–51 weeks) (Montero and Lundby
2017a). A complex interplay of mechanisms causes the BV expansion in ET that may be affected by the training program, the training status of the subjects and their nutritional status (Montero and Lundby
2018; Sawka et al.
2000). Although our BV expansion was within the expected range, the smaller increase compared to that by Bonne et al. (
2014) and Montero et al. (
2015a) is unlikely caused by iron deficiency, as indicated by the normal and maintained ferritin levels. Lean body mass was maintained, indicating sufficient protein and caloric intake during the ET period. Over 10 weeks, the subjects performed 27–30 ET sessions as compared to only 18–20 sessions over 6 weeks in the studies by Bonne et al. (
2014) and Montero et al. (
2015a), using a similar training intensity. Hence, the training intensity and the total volume and length of the training intervention cannot explain the different findings.
Central vs peripheral limitations to \(\dot{\mathrm{V}}\)O2peak
There were no statistically significant changes in estimated a-
\(\bar{\text{v}}\)O
2diff from pre- to post-ET, potentially indicating no substantial contribution of peripheral adaptations to the improvements in
\(\dot{V}\)O
2peak. Based on the average of all maximal exercise tests conducted pre- (
n = 2; upright and supine cycling) and post-ET (
n = 4; before and after phlebotomy),
\(\dot{V}\)O
2peak increased by 318 ± 147 ml min
−1. Of this increase, the increase in
\(\dot{Q}\)peak account for 221 ± 202 ml min
−1. Hence, on average, a-
\(\bar{\text{v}}\)O
2diff was elevated by 5 ± 12 ml l
−1 (154 ± 22 vs 159 ± 25 ml l
−1) and account for 30% of the increase in
\(\dot{V}\)O
2peak. Therefore, our data support that
\(\dot{V}\)O
2peak is mainly limited by convective O
2 delivery (Montero et al.
2015b; Mortensen et al.
2005), but also supports calculations indicating that 70–75% of the limitations lie within the central circulation and that 25–30% are determined by peripheral factors (di Prampero
2003; di Prampero and Ferretti
1990).
O
2 extraction and blood flow are interdependent. For example, by decreasing
\(\dot{\mathrm{Q}}\)peak and leg blood flow using β-adrenergic blockade, systemic and leg a-
\(\bar{\text{v}}\)O
2diff increases during submaximal and maximal exercise, facilitated by increased erythrocyte capillary mean transit time (MTT) (Ekblom et al.
1972; Pawelczyk et al.
1985). In the present study, muscle fibre hypertrophy was accompanied by only a minor increase in the capillary-to-fibre ratio, causing no change in capillary density. If we calculate the capillary volume within the leg muscle mass engaged during cycling (Boushel et al.
2014) and subtract a non-leg blood flow of 6.5 l min
−1 from the total
\(\dot{Q}\)peak (Calbet et al.
1985,
2006; Lundby et al.
1985; Mortensen et al.
2005), there would be a trend towards shorter erythrocyte MTT after ET during upright peak exercise (508 ± 138 vs 452 ± 132 ms before and after ET, respectively). Therefore, due to reduced time for O
2 unloading, peripheral adaptations such as increased muscle oxidative capacity (CS and COX-IV) may have been crucial in maintaining the pre-ET level of a-
\(\bar{\text{v}}\)O
2diff. This is substantiated by a correlation between the percent change in a-
\(\bar{\text{v}}\)O
2diff during upright cycling and the percent change in CS content from before to after ET (
r = 0.73;
n = 10;
P = 0.017). Further improvements in a-
\(\bar{\text{v}}\)O
2diff, at least large enough to evoke statistical significance, may likely only be detected if peripheral adaptations largely surpass the changes in
\(\dot{Q}\)peak and peripheral blood flow.
After years of training, elite endurance athletes have a higher leg O
2 extraction than untrained individuals (> 90% vs ~ 70%, respectively) (Calbet et al.
2005; Roca et al.
1985). A similar situation can be evoked by relative short periods of one-legged ET inducing robust peripheral adaptations without stimulating the central circulation, and improve leg a-
\(\bar{\text{v}}\)O
2diff by 5–10 ml l
−1 (Klausen et al.
1982; Rud et al.
2012). Thus, ET improves the muscles’ ability to extract O
2 but may be masked by improvements in
\(\dot{Q}\)peak and peripheral blood flow after short periods of whole-body ET (Montero et al.
2015b). Furthermore, the improvements seen after 7–8 weeks of one-legged ET by Rud et al. (
2012) and Klausen et al. (
1982) were in the range of 5–10 ml l
−1 as assessed by arterial and femoral venous blood sampling. Accordingly, small potential improvements in systemic a-
\(\bar{\text{v}}\)O
2diff, as indicated in the present study (5 ml l
−1), may be difficult to detect when calculated from
\(\dot{V}\)O
2peak and non-invasively determined
\(\dot{Q}\)peak.
Cardiac remodelling
LV mass increased without any change in EDV. This contradicts the classic model of athletic cardiac remodelling that predicts increased EDV following ET due to the haemodynamic stimulus of volume loading on the ventricles (Morganroth et al.
1975). However, longitudinal studies demonstrate concentric remodelling at the commencement of ET, with the adaptations gradually switching into eccentric remodelling. For instance, after 3 months of ET, Arbab-Zadeh et al. (
2014) found a 10% increase in LV mass-to-volume ratio before it returned to pre-ET levels after 9–12 months of ET. Therefore, with an increase in LV mass-to-volume ratio of 14% after 10 weeks of ET, our data support that the initial ET-induced cardiac remodelling is concentric (Arbab-Zadeh et al.
2014; Bjerring et al.
2019; Weiner et al.
2015).
Despite unchanged LV EDV and diastolic and systolic functional parameters at rest, the submaximal exercise SVs and SV
peak were increased after ET. This indicates an increased capacity of the heart to utilise the Frank–Starling mechanism to increase SV from rest to peak exercise after ET (Crawford et al.
1985; Rerych et al.
1980). This could be due to increased chamber compliance (Arbab-Zadeh et al.
2014; Levine et al.
1991), increased filling rates (Ferguson et al.
2001; Gledhill et al.
1994), a lower rise in MAP during exercise due to reduced total peripheral resistance (Klausen et al.
1982) or a combination. Since the SV
peak was elevated even after phlebotomy, increased filling rates through expanded BV are unlikely to have made any major contribution. However, with the present experimental design, we cannot determine whether increased LV mass, enhanced venous return through other mechanisms than elevated BV, reduced total peripheral resistance, or improved qualitative properties of the heart (e.g. contractility, compliance, faster ventricular relaxation) were facilitating the elevated SV
peak and
\(\dot{Q}\)peak.
Study considerations
Although Hb
mass and BV were restored to pre-ET levels by phlebotomy, transcapillary fluid shifts may have occurred, and it is uncertain whether BV normalisation was preserved during exercise. Some of the subjects were unfamiliar with cycling exercise before the study, and no one had tried supine cycling. Therefore, we cannot exclude the possibility that some subjects became better able to maintain venous return and cardiac filling at peak exercise after ET due to familiarisation. The findings were obtained from a small sample size. But the main finding was that the ET-induced increase in
\(\dot{V}\)O
2peak was preserved despite removing the increase in Hb
mass, and this conclusion is based on methods with low typical error (
\(\dot{V}\)O
2peak 2.9% and Hb
mass 1.1%). Impedance cardiography is associated with a larger measurement error than the invasive gold-standard methods (Del Torto et al.
2019; Richard et al.
2001). Besides, when calculating the a-
\(\bar{\text{v}}\)O
2diff from
\(\dot{V}\)O
2 and
\(\dot{Q}\) inherent of its measurements errors, a larger measurement error is expected as compared with deriving a-
\(\bar{\text{v}}\)O
2diff using arterial and venous blood sampling.