Fig 1.
Fig 1a) Study protocol with time schedule for exercise, Sonovue infusion, CEUS recordings, as well as femoral artery Doppler and brachial blood pressure measurements at rest, during isometric exercise and during post-exercise hyperemia. The time points of high-MI US-destruction of the Sonovue microbubbles are indicated by arrows, each of which was followed by a low-MI recording of Sonovue replenishment curves covering 25 s. Fig 1b) Example of a torque recording during isometric knee extension as controlled by the subject through visual feed-back.
Fig 2.
‘Ultrasound and MRT imaging of CEUS and Biopsy muscle site’.
Fig 2 a) US B-mode image of a typical combined vastus lateralis (VLat) and intermedius (VInt) CEUS scan with the scanner position chosen parallel to the VLat muscle fiber orientation (i.e. from proximal/lateral to distal/medial). The intramuscular septum separating both muscles is indicated, the mean depth was similar between middle aged (MA) and young (YG) subjects and not significantly different between the conditions before, during, and post-exercise (see also the ‘Methods‘ section on Contrast-enhances US (CEUS). d) Thigh MRT-imaging transversal section at the exact site of VLat muscle biopsy (middle) and CEUS recording as well as 1 cm proximal (left) and distal (right). Note that this MRT was obtained 3 h after a muscle biopsy to visualize the exact biopsy site (local fluid /blood accumulation) indicated by the arrow.
Fig 3.
‘Time courses of the mean contrast-agent CEUS signals’.
Time course of the mean (±SEM) Sonovue microbubble signal in the vastus lateralis (VLat; upper panel) and the vastus intermedius (VInt; lower panel) muscle region of interests (ROI) in middle-aged (MA, n = 15) and young (YG, n = 11) males in the experimental intervals: equilibration at rest (left; initial 180 s of Sonovue infusion), isometric exercise (middle; first 60 s of knee extension at 15% of PT), and post-exercise (right; initial 15 s after cessation of). Note the different time scales on the x-axis with these three conditions. The time intervals for repetitive Sonovue replenishment curve (RC) recording following high-MI US destruction of Sonovue microbubbles (See Fig 1) are presented separately in Fig 4A (mean RC curves) and Fig 4B (means of the individual regression lines obtained from individual RC curves). * for p<0.05 MA vs YG by unpaired Student’s t-test.
Fig 4.
‘Means of measured replenishment curves and individual regression lines’.
Fig 4a) Mean replenishment curves (RC) in the vastus lateralis (VLat; upper panel) and the vastus intemedius (VInt; lower panel) muscle ROI of middle-aged (MA, n = 15) compared to young (YG, n = 11) males during rest (left), after 70 s isometric exercise (middle), and 15 s post-exercise. Note differences in initial slope or in the plateau reached during or post-exercise. Fig 4b) Mean regression lines, corresponding to mean RC presented above in a) i.e. for the VLat (upper panel) and the VInt (lower panel) muscle ROI of middle-aged (MA, n = 15) compared to young (YG, n = 11) males during rest (left), after 70 s isometric exercise (middle), and 15 s post-exercise. Note the differences in initial slope or reached plateau during or post exercise. Furthermore, note that regression lines represent the mean of individual regression lines calculated for individual RCs (not the regression line calculated for mean RCs, presented above in a). For statistical differences between MA and YG regarding the RC-derived parameters of microvascular blood volume (MBV), flow velocity (MFV), and blood flow (MBF) please see Fig 5.
Table 1.
Anthropometry, cardiovascular risk factors as well as leg muscle mass, function and vascularisation.
Table 2.
Histomorphometry of vastus lateralis muscle capillarisation and fiber composition.
Fig 5.
‘Microvascular blood volume (MBV), flow velocity (MFV) and blood flow (MBF)’.
Mean (±SEM) microvascular blood volume (MBV, left), flow velocity (MFV, middle), and blood flow (MBF, right) in the vastus lateralis (VLat; upper panel) and the vastus intemedius (VInt; lower panel) muscle ROI of middle-aged (MA, n = 15) compared to young (YG, n = 11) at rest (two measurements), after 70 and 95 s of isometric exercise and 15, 30 60 and 90 s post-exercise. Note that these data were individually calculated from individual RC curve regression before averaging them for MA or YG (for mean RCs and regression lines per group see Fig 4A). # for p<0.05 by unpaired Student’s t-test middle-aged MA vs. YG. * for p<0.05, ** for p<0.01, and *** for p<0.001 by paired Student’s t-test for changes relative to rest (basline) within the group of MA or YG.
Fig 6.
‘Total leg blood flow and conductance’.
Mean (±SEM) total leg blood flow (femoral artery Duplex-Doppler flow), calculated total leg vascular conductance (leg blood flow per mean arterial pressure), and systolic as well as diastolic brachial arterial blood pressure at rest in middle-aged (MA, n = 15) compared to young (YG, n = 11) males after ~90 s of isometric exercise and ~60 s post-exercise. # for p<0.05 by unpaired Student’s t-test middle-aged MA vs. YG. * for p<0.05, ** for p<0.01, and *** for p<0.001 by paired Student’s t-test for changes relative to rest (baseline) within the group of MA or YG.