Type II Muscle Fiber Capillarization Is an Important Determinant of Post-Exercise Microvascular Perfusion in Older Adults

Abstract

Introduction

Microvascular perfusion is essential for post-exercise skeletal muscle recovery to ensure adequate delivery of nutrients and growth factors. This study assessed the relationship between various indices of muscle fiber capillarization and microvascular perfusion assessed by contrast-enhanced ultrasound (CEUS) at rest and during recovery from a bout of resistance exercise in older adults.

Methods

Sixteen older adults (72 ± 6 y, 5/11 male/female) participated in an experimental test day during which a muscle biopsy was collected from the vastus lateralis and microvascular perfusion was determined by CEUS at rest and at 10 and 40 min following a bout of resistance exercise. Immunohistochemistry was performed on muscle tissue samples to determine various indices of both mixed and fiber-type-specific muscle fiber capillarization.

Results

Microvascular blood volume at t = 10 min was higher compared with rest and t = 40 min (27.2 ± 4.7 vs. 3.9 ± 4.0 and 7.0 ± 4.9 AU, respectively, both p < 0.001). Microvascular blood volume at t = 40 min was higher compared with rest (p < 0.001). No associations were observed between different indices of mixed muscle fiber capillarization and microvascular blood volume at rest and following exercise. A moderate (r = 0.59, p < 0.05) and strong (r = 0.81, p < 0.001) correlation was observed between type II muscle fiber capillary-to-fiber ratio and the microvascular blood volume increase from rest to t = 10 and t = 40 min, respectively. In addition, type II muscle fiber capillary contacts and capillary-to-fiber perimeter exchange index were strongly correlated with the microvascular blood volume increase from rest to t = 40 min (r = 0.66, p < 0.01 and r = 0.64, p < 0.01, respectively).

Conclusion

Resistance exercise strongly increases microvascular blood volume for at least 40 min after exercise cessation in older adults. This resistance exercise-induced increase in microvascular blood volume is strongly associated with type II muscle fiber capillarization in older adults.

Introduction

Tissue perfusion is essential for skeletal muscle homeostasis and function. When the metabolic rate in skeletal muscle fibers increases upon contraction, blood flow is rapidly and equally increased (up to a 100-fold) to match the required substrate delivery and metabolite removal [1]. Although this hyperemic response is complex, it has been suggested that the linear relationship between blood flow and metabolic demand is mainly regulated by the cellular interaction between the contracting muscle fibers and the local vascular cells [2]. This is further evidenced by the ability to direct blood flow specifically to active muscle fibers within the tissue [3, 4]. The ability to direct blood flow to active muscle tissue requires a close relationship between muscle fibers and vascular cells, as well as inter-communication along the microvascular network to coordinate the microvascular response [5].

The delivery of oxygen, substrates, metabolites, hormones, and other bioactive signaling molecules to the muscle fibers is ultimately limited by the surface area of the capillary bed [6]. Blood flow is generally quite low in capillaries within resting skeletal muscle tissue. However, upon muscle contraction, capillary flow via arteriole dilation is significantly enhanced to facilitate a dramatic increase in blood volume to support the increased oxygen and substrate demands [7]. Hence, muscle fiber perfusion is a multifaceted process that is regulated by both anatomical and functional factors. Previously, we [8–11] as well as others [12–14] have applied immunohistochemistry combined with fluorescence microscopy to assess the anatomy of the muscle fiber capillary network in muscle biopsy samples obtained from humans at rest and/or in response to prolonged physical (in)activity interventions. With this method, various indices of capillarization can be calculated that represent differences in muscle fiber perfusion characteristics based upon the anatomical positioning of the capillaries among the muscle fibers [15]. However, a muscle biopsy only provides information on the anatomical perfusion potential and does not reflect actual perfusion dynamics. In contrast, contrast-enhanced ultrasound (CEUS) has been developed to assess tissue perfusion dynamics and can be applied effectively to study skeletal muscle tissue perfusion in vivo in humans [16, 17]. This technique relies on acoustic signals by intravenously infused inert gas microbubbles and allows calculation of microvascular blood volume, velocity, and flow (i.e., the product of volume and velocity). Changes in CEUS-derived microvascular muscle tissue perfusion have been reported following food intake [18, 19], a single bout of exercise [20, 21], exercise training [21], as well as the administration of various pharmacological agents [22–24].

Both muscle fiber capillarization, assessed by fluorescent microscopy, and microvascular perfusion, assessed by CEUS, provide important information on the regulation of muscle fiber perfusion. Yet, the relationship between these two remains poorly defined in vivo in humans. Previously, several indices of capillarization have been shown to be moderately correlated with CEUS-derived microvascular perfusion at rest [25, 26]. However, whether the anatomy of the muscle capillary bed is more strongly related to microvascular perfusion in a situation of extensive muscle fiber capillary flow (i.e., following exercise) has not been assessed. In this study, we investigated the relationship between various indices of muscle fiber capillarization as assessed by microscopy and microvascular perfusion as assessed by CEUS at rest and during recovery from a single bout of resistance exercise in older adults.

Methods

Participants

A convenience sample of sixteen older adults aged 65–85 years with a body mass index between 18.5 and 30 kg⋅m⁻² were recruited to participate in the current study. Participants’ characteristics are presented in Table 1. During the initial screening, medical history was assessed to exclude known medical conditions such as cancer, cardiovascular disease, arthritic conditions, neuromuscular problems, renal disorders, and/or pulmonary diseases. Resting blood pressure was assessed (HEM-907, OMRON Healthcare Europe B.V., Hoofddorp, the Netherlands) in a seated position by taking the average of three consecutive readings to exclude individuals with hypertension (i.e., above 140/90 mm Hg systolic/diastolic). If participants were classified as hypertensive when assessed in our laboratory but not when assessed by their general practitioner, they were still included. An oral glucose tolerance test was performed to exclude type II diabetes mellitus patients [27]. Participants were informed of the nature and possible risks of the experimental procedures before their written informed consent was obtained. The study was approved by the Medical Research Ethics Committee Academic Hospital Maastricht/Maastricht University, the Netherlands (METC 18-060) and complied with the guidelines set out in the most recent version of the Declaration of Helsinki. This study was part of a larger project registered at the International Clinical Trials Registry Platform (https://trialsearch.who.int) as NTR7681 and was independently monitored by the Clinical Trial Center Maastricht.

Table 1.

Participants’ characteristics

Sex (male/female)	5/11
Age, years	72±6
Stature, m	1.67±0.06
Body mass, kg	71.6±8.9
BMI, kg·m−2	25.6±2.5
Whole body fat mass, %	33.8±5.1
Whole body lean mass, kg	45.0±7.8
Fasting plasma glucose concentration, mmol·L−1	5.5±0.7
2-h plasma glucose concentration, mmol·L−1	6.9±1.9
Systolic blood pressure, mm Hg	131±14
Diastolic blood pressure, mm Hg	70±8
1RM leg muscle strength, kg	210±50
Absolute VO2peak, mL·min−1	2,018±339
Relative VO2peak, mL·min−1·kg−1	28.4±4.2
Peak aerobic power, W	165±41
Type I muscle fiber size, μm2	4,899±1,140
Type II muscle fiber size, μm2	3,351±1,274
Muscle fiber-type distribution, % type I	45±13
Muscle fiber-type distribution, % CSA type I	55±16

Values are mean±SD or counts, n = 16. BMI, body mass index. CSA, cross-sectional area. 1RM, one-repetition maximum. VO_2peak, peak oxygen consumption.

Aerobic Capacity

Participants performed a VO_2peak test on a calibrated cycle ergometer (Lode Excalibur Sport, Groningen, the Netherlands), while O₂ consumption and CO₂ production were captured and measured continuously (Omnical, Maastricht University, the Netherlands). In addition, heart rate was measured during the test with a chest-strap heart rate monitor (Kalenji, France). Following a 1-min warm-up at 30 W, the load was increased by 1 W every 4 s. Participants were instructed to maintain a cadence between 70 and 90 rpm. The test was terminated when the cadence dropped below 60 rpm for more than 10 s, or when voluntary fatigue was reached. The VO_2peak was determined by taking the highest average of three consecutive VO₂ measurements (i.e., 15 s).

Leg Muscle Strength

Leg muscle strength was assessed by the 1 repetition maximum (1RM) strength test on both a leg press and leg extension machine (Technogym, Italy). During the initial visit, participants were familiarized with both exercises, and 1RM was estimated using the multiple repetition testing procedure [28]. This estimation was used during the subsequent 1RM tests, as described previously [29]. Leg muscle strength was calculated as the summation of the leg press and leg extension 1RM.

Experimental Test Day

Participants refrained from any strenuous physical activity for 3 days prior to the experimental test day and arrived at the university by car or public transportation. Due to limited availability of testing facilities during the COVID-19 pandemic (2019-2021), participants were tested either in the morning following an overnight fast (n = 7) or semi-fasted in the late afternoon (n = 9) following a standardized breakfast and lunch (4.0 ± 0.4 MJ, with 50 energy percent (%En) from carbohydrate, 35%En from fat, and 15%En from protein) consumed before 12:30 h. Time of testing (morning vs. late afternoon) did not influence any of the outcomes tested (see online suppl. Table 1; for all online suppl. material, see https://doi.org/10.1159/000535831). First, a muscle biopsy sample was obtained from the middle region of the m. vastus lateralis, approximately 15 cm above the patella and 3 cm below entry through the fascia, by using the percutaneous needle biopsy technique [30]. Muscle samples were dissected carefully and freed from any visible non-muscle material. The biopsy sample was embedded in Tissue-Tek (Sakura Finetek, Zoeterwoude, the Netherlands) and frozen in liquid nitrogen-cooled isopentane. All samples were stored at −80°C until further analysis. Next, following at least 30 min of bed rest in a supine position, resting microvascular perfusion in the m. vastus lateralis of the contralateral leg was assessed by CEUS. All CEUS measurements were performed using a high-end ultrasound machine (Affinity 70 G, Philips, the Netherlands) with a linear array probe (eL18-4, Philips, the Netherlands). The ultrasound probe was fixed in a custom-made holder to visualize a cross-sectional image of the m. vastus lateralis at 1/3rd distance between the superior patellar border and the anterior superior iliac spine. This position was marked on the leg by pen and a screenshot in ultrasound B-mode was taken to ensure repeated measurements at the exact same position. A 5-s clip was acquired in color Doppler mode to be used during subsequent analysis. Next, an infusion of gas-filled microbubbles (SonoVue, Bracco, concentration: 8 μL⋅mL⁻¹) was initiated via a catheter placed in an antecubital vein. For each CEUS measurement, a 10 mL suspension of microbubbles was infused for 6 min at 85 mL⋅h⁻¹. Following 3 min of infusion to achieve a steady state of circulating microbubbles, six 30 s recordings were acquired using contrast mode (8 Hz with a mechanical index [MI] of 0.07). At the start of each recording, a high MI flash (0.53 MI) was given to destroy all visible microbubbles, and the subsequent replenishment of microbubbles was recorded (see Fig. 1). Following the assessment of resting microvascular perfusion, participants performed a single bout of resistance exercise. This exercise bout consisted of 4 sets of 8 repetitions with 80% 1RM on the leg press and leg extension machines. Two min of rest between sets and 3 min of rest between exercises were allowed. Finally, CEUS measurements were repeated at t = 10 and 40 min following cessation of exercise to assess post-exercise microvascular perfusion.

Fig. 1.

Representation of contrast-enhanced ultrasound (CEUS) analyses of muscle microvascular perfusion in older adults. First, a high mechanical index (MI) flash (b) is given to destroy all microbubbles (c). Next, microbubbles are replenished until a plateau is reached (d). A region of interest (ROI) is set to exclude rapidly filling vessels and connective tissue. Video intensity within the ROI is analyzed for every frame, resulting in a replenishment curve (a). A curve (red line in figure) is fitted to the equation: y = A[1-e^-βt], where A is the plateau video intensity (i.e., microvascular blood volume) and β reflects the rate of rise of video intensity (i.e., microvascular blood velocity). Microvascular blood flow is calculated as the product of microvascular blood volume and velocity.

Microvascular Perfusion Analysis

All CEUS recordings were analyzed using ImageJ (version 2.1.0/1.53c). First, the Color Doppler clip was used to create a region of interest (ROI) that excludes larger blood vessels and connective tissue. This ROI was then used for the 30-s microbubble replenishment recording to determine video intensity for every frame. A background correction was applied by subtracting the average video intensity of the first 4 frames (i.e., 0.5 s) following the high MI flash from all data points. Video intensity data were plotted in GraphPad Prism (version 8.3), and a curve was fitted to the equation: y = A(1-e^-βt), where A is the plateau video intensity (i.e., microvascular blood volume) and β reflects the rate of rise of video intensity (i.e., microvascular blood velocity; see Fig. 1) [31]. Microvascular blood flow was calculated as the product of volume and velocity. For one CEUS measurement, the average of the six 30-s recordings was taken for microvascular blood volume, velocity, and flow. All microvascular perfusion analyses were completed in a blinded fashion.

Immunohistochemistry

Frozen muscle biopsies were cut into 5-μm-thick cryosections using a cryostat at −20°C and thaw-mounted onto uncoated, pre-cleaned glass slides. Care was taken to properly align the samples for cross-sectional orientation of the muscle fibers. Muscle cross-sections were stained for muscle fiber type, laminin, and capillaries. Following 5 min fixation in acetone and subsequent 15 min air drying at room temperature, the muscle cross-sections were incubated for 45 min with the first primary antibody CD31 (dilution 1/50; M0823; Dako, Glostrup, Denmark) in 0.05% Tween-phosphate-buffered saline (PBS). Slides were washed 3 × 5 min with PBS. Next, slides were incubated for 45 min in HAM Biotine (1:500, Vector Laboratories) in 0.05% Tween/PBS. Slides were again washed 3 × 5 min with PBS, followed by a 45-min incubation with Avidine Texas Red (A2006, dilution 1/400; Vector Laboratories) and antibodies against Myosin Heavy Chain (MHC)-I (BA-F8, dilution 1/10; DSHB) and laminin (polyclonal rabbit anti-laminin, dilution 1/50; Sigma) in 0.05% Tween/PBS. Following a 3 × 5 min washing step in PBS, samples were finally incubated for 30 min with the appropriate secondary antibodies goat anti-mouse (GAM) IgG2b AlexaFluor488 and goat anti-rabbit (GAR) IgG AlexaFluor647 (Molecular Probes). After the final washing (3 × 5 min PBS), slides were mounted with Mowiol (Calbiochem). The staining procedure resulted in images with laminin in white, MHC-I in green, and CD31 in red (see Fig. 2).

Fig. 2.

Representative images of the analyses for muscle fiber size and fiber-type-specific capillarization in older adults. a Laminin (white; cell borders), MHC1 (green; type I muscle fibers), CD31 (red; capillaries). b Laminin (white), CD31 (red). c MHC1 (green), CD31 (red). d CD31 (red) only. Green fibers indicate type I muscle fibers.

Slides were viewed and automatically captured using ×20 objective on a modified Olympus BX51 fluorescence microscope with a customized spinning disk unit (DSU, Olympus), computer-controlled excitation and emission filter wheels (Olympus), 3-axis high-accuracy computer-controlled stepping motor specimen stage (Grid Encoded Stage, Ludl Electronic Products, Hawthorne, NY, USA), ultra-high-sensitivity monochrome electron multiplier CCD camera (C9100-02, Hamamatsu Photonics, Hamamatsu City, Japan), and controlling software (StereoInvestigator; MBF BioScience, Williston, VT, USA). Quantitative analyses were performed using ImageJ version 2.1.0/1.53c. On average, 215 ± 70 muscle fibers were analyzed per participant to determine muscle fiber type distribution and size. The quantification of muscle fiber capillaries was performed on 30 type I and 30 type II muscle fibers based on the work of Hepple et al. [32]. Quantification was made of (i) capillary contacts (CC), (ii) the capillary-to-fiber ratio (C/Fi), (iii) capillary-to-fiber perimeter exchange (CFPE) index, and (iv) capillary density (CD). Mixed capillarization outcomes were calculated based on the % of total cross-sectional area (CSA) covered by type I or II muscle fibers. All immunofluorescence analyses were completed in a blinded fashion.

Statistical Analyses

All data are expressed as mean ± SD. Normal distribution of all parameters was verified by the Shapiro-Wilk test. Microvascular blood volume, velocity, and flow at rest, t = 10, and 40 min of post-exercise recovery were compared using a one-factor repeated-measures ANOVA with time as within-subjects factor or a Friedman and consecutive Wilcoxon matched-pair signed-rank tests when parameters were not normally distributed. Bonferroni correction was applied for multiple comparisons. Differences between type I and type II muscle fiber capillarization indices and differences between the microvascular perfusion increase from rest to t = 10 and 40 min of post-exercise recovery were analyzed using Students’ paired t tests. Effect sizes were calculated by means of Cohen’s d. Pearson (r) correlation analyses were performed when parameters were normally distributed, while Spearman rank (ρ) correlation analyses were used when parameters were not normally distributed. Correlation analyses were performed between different indices of muscle fiber capillarization and microvascular blood volume, velocity, and flow at rest, 10 min post-exercise, and 40 min post-exercise. A significant r- or ρ-value between 0 and 0.19 was regarded as “very weak,” between 0.2 and 0.39 as “weak,” between 0.40 and 0.59 as “moderate,” between 0.6 and 0.79 as “strong,” and between 0.8 and 1 as “very strong” correlation. Statistical significance was accepted as p < 0.05. All calculations were performed using SPSS (version 27.0, IBM Corp., USA).

Results

Participants’ Characteristics

The study population included 5 males and 11 females with an age ranging from 65 to 83 years old. BMI ranged from 21 to 30 kg·m⁻² with 63% being classified as overweight (>25 kg·m⁻²). Resting systolic and diastolic blood pressure ranged from 101 to 156 and 54–85 mm Hg, respectively. Based on fasting blood glucose values, 75% of all participants had a normal and 25% had impaired fasting glucose (<5.6 versus > 5.6 mmol⋅L⁻¹, respectively). Based on the glucose concentration at the 2-h time point during the OGTT, 69% of participants showed normal (<7.8 mmol⋅L⁻¹) and 31% had impaired glucose tolerance (>7.8 mmol⋅L⁻¹). One participant was categorized as diabetic based on fasting glucose (>7 mmol⋅L⁻¹), but none based on 2-h glucose concentrations (>11.1 mmol⋅L⁻¹). In the collected vastus lateralis muscle biopsy sample, we observed that type II muscle fiber size (range from 1,533 to 5,282 μm²) was significantly smaller when compared to type I muscle fiber size (range 2,940–6,625 μm², p < 0.001; d: 1.45). Fiber-type distribution ranged from 24 to 83% of CSA occupied by type I fibers.

Muscle Fiber Capillarization Anatomy

Based on immunofluorescent staining of the muscle fiber capillaries in the collected muscle biopsy samples, we observed that CC, C/Fi, and CFPE index were significantly higher in type I compared with type II muscle fibers in older adults (Table 2). In contrast, no significant differences were observed between type I and type II muscle fiber CD (Table 2). We observed that various indices of mixed muscle fiber capillarization were significantly associated with mixed muscle fiber size (CC: r = 0.79, p < 0.001; C/Fi: r = 0.81, p < 0.001; and CFPE index: r = 0.63, p < 0.01).

Table 2.

Muscle fiber capillarization

	Mixed	Type I	Type II	Type I versus II
				p value	Cohen’s D
CC	3.5±1.0	3.9±1.1	2.9±0.8	<0.001	1.49
C/Fi	1.6±0.4	1.8±0.5	1.4±0.4	<0.001	1.46
CFPE index, capillaries 1,000 μm−2	5.5±0.9	6.0±1.1	4.7±0.9	<0.001	1.75
CD, capillaries mm−2	396±64	398±64	399±73	=0.468	−0.20

Values are mean±SD, n = 16. CC, capillary contacts. C/Fi, capillary-to-fiber ratio. CFPE, capillary-to-fiber perimeter exchange. CD, capillary density.

Microvascular Perfusion Dynamics

Based on CEUS, microvascular perfusion dynamics were assessed at rest and at t = 10 and 40 min of post-exercise recovery. Microvascular blood volume was significantly higher at t = 10 (15.6 ± 5.6 AU, p < 0.001, d: 3.01) and 40 min (7.0 ± 4.9 AU, p < 0.001, d: 1.28) compared with rest (3.9 ± 4.0 AU; Fig. 3a). Microvascular blood velocity was significantly higher at t = 10 (0.16 ± 0.04 AU, p = 0.004, d: 0.86) but not at 40 min (0.08 ± 0.04 AU) compared with rest (Fig. 3b). Microvascular blood flow was significantly higher at t = 10 (2.5 ± 1.3 AU, p < 0.001, d: 2.13) and 40 min (0.60 ± 0.9 AU, p = 0.041, d: 0.56) compared with rest (0.27 ± 0.4 AU; Fig. 3c).

Fig. 3.

Microvascular blood volume (a), velocity (b), and flow (c) assessed with CEUS at rest and during recovery from a single bout of resistance exercise (t = 10 and t = 40 min) in older adults (n = 16). Bars represent mean ± SD. Circles represent females and triangles represent males. *Significantly higher compared with rest, p < 0.05. ^§Significantly higher compared with t = 40, p < 0.05. ^#Significantly higher compared with rest to t = 40, p < 0.05.

Mixed Muscle Fiber Capillarization versus Microvascular Perfusion

No significant correlations were observed between mixed muscle fiber CC, C/Fi, CFPE index, or CD and microvascular blood volume, velocity, or flow at rest, t = 10, or t = 40 min of post-exercise recovery. Furthermore, no significant correlations were observed between mixed muscle fiber CC, C/Fi, CFPE index, or CD and the microvascular blood volume, velocity, or flow increase from rest to t = 10 or t = 40 min of post-exercise recovery.

Muscle Fiber-Type-Specific Capillarization versus Microvascular Perfusion Response to Exercise

Whereas no significant correlations were observed between type I muscle fiber CC, C/Fi, CFPE index, or CD and the microvascular perfusion response to exercise, significant associations were observed for type II muscle fibers. Type II muscle fiber CC tended (p = 0.059) to be correlated with the microvascular blood volume increase from rest to t = 10 min of post-exercise recovery (r = 0.48, Fig. 4a). A significant moderate correlation was observed between type II muscle fiber C/Fi and the microvascular blood volume increase from rest to t = 10 min of post-exercise recovery (r = 0.59, p < 0.05, Fig. 4c). A strong correlation was observed between type II muscle fiber CC as well as CFPE index and the microvascular blood volume increase from rest to t = 40 min of post-exercise recovery (r = 0.66, p < 0.01 and r = 0.64, p < 0.01, respectively, Fig. 4b, f). A very strong correlation was observed between type II muscle fiber C/Fi and the microvascular blood volume increase from rest to t = 40 min of post-exercise recovery (r = 0.81, p < 0.001, Fig. 4d). No significant correlations were observed between type II muscle fiber CC, C/Fi, CFPE index, or CD and the microvascular blood velocity or flow response to exercise.

Fig. 4.

Associations between various indices of type II muscle fiber capillarization and the microvascular blood volume response to a single bout of resistance exercise in older adults (n = 16). Pearson correlation coefficient (r) between type II muscle fiber CC (a, b), C/Fi (c, d), CFPE index (e, f), and CD (g, h) and the microvascular blood volume increase from rest to t = 10 and t = 40 min of post-exercise recovery. Circles represent females and triangles represent males. Straight lines represent linear regression, and bands represent the 95% confidence interval. CC, capillary contacts. C/Fi, capillary-to-fiber ratio. CFPE, capillary-to-fiber exchange. CD, capillary density.

Discussion

The present study shows that resistance exercise strongly increases microvascular perfusion for at least 40 min after exercise cessation in older adults. No associations were observed between mixed or type I muscle fiber capillarization and microvascular perfusion at rest or following exercise. Interestingly, various indices of type II muscle fiber capillarization were strongly associated with the microvascular perfusion response to a single bout of resistance exercise in older adults.

Perfusion is essential for post-exercise skeletal muscle recovery to ensure adequate delivery of nutrients and growth factors and removal of accumulated waste products. Muscle microvascular perfusion is regulated by both anatomical and functional factors, such as density of the capillary network and dilation of terminal arterioles, respectively. Muscle fiber capillarization, assessed by fluorescent microscopy, provides information on the anatomical perfusion potential, while CEUS allows the assessment of muscle fiber capillary perfusion dynamics (Fig. 1, 2). In the present study, microvascular perfusion (i.e., microvascular blood volume and velocity) was assessed by CEUS at rest and after 10 and 40 min of recovery from a single bout of resistance exercise in older adults. We show that microvascular blood volume increased ∼ 6-fold from rest to 10 min of post-exercise recovery (Fig. 3a). Together with a ∼2.5-fold increase in microvascular blood velocity (Fig. 3b), this resulted in a near 20-fold increase in microvascular blood flow (i.e., the product of volume and velocity) at 10 min of post-exercise recovery (Fig. 3c). This increase in microvascular perfusion appears to be greater when compared with others showing a 2-fold increase in microvascular blood volume [21] or 11-fold increase in microvascular blood flow [33] immediately following resistance exercise. However, in these previous studies [21, 33], the exercise volume and intensity (i.e., 1 set of 6 knee extensions at 50% 1RM [21] or arm flexions for 2 min with an 8 kg bar [33]) were much lower compared with what was performed in the current study (i.e., 4 sets of 8 repetitions with 80% 1RM for both the leg press and leg extension machine). Importantly, the near 20-fold increase in microvascular perfusion observed in the present study implies that the capillary network itself is not restrictive to muscle tissue perfusion at rest in muscle tissue of older participants. In support, we observed no significant correlations between various indices of muscle fiber capillarization and microvascular blood volume, velocity, and flow at rest. This contradicts previous work by Weber et al. [25, 26] reporting significant associations between various indices of muscle fiber capillarization and microvascular blood volume at rest. A possible explanation for this discrepancy is the relative homogenous group of older (72 ± 6 years; range: 65–83 years) adults included in the present study, whereas a more heterogeneous group of middle-aged (median age 54 years; range: 39–65 years) adults was included by Weber et al. [25, 26]. In addition, any relationship between the capillary network and resting muscle perfusion observed in middle-aged adults [25, 26] may be disrupted in older adults by age-related changes in the microcirculation, such as capillary rarefaction [34] and increased capillary basement membrane thickness [35]. In response to exercise (i.e., muscle contraction), capillary flow is substantially enhanced to support the dramatic increases in oxygen and substrate demands. Interestingly, we show that microvascular blood volume remained elevated as microvascular blood velocity returned to baseline at 40 min after cessation of exercise. The sustained increase in microvascular blood volume supports the concept that nutrient delivery is largely determined by the increase in microvascular blood volume (i.e., enhanced microvascular/capillary flow) [36, 37]. Nevertheless, the exercise-induced increase in microvascular blood volume and/or flow in the present study, both at 10 and 40 min following the exercise bout, did not strongly depend on various indices of mixed muscle fiber capillarization. From these data, it would appear that, in older adults, muscle microvascular perfusion at rest and in response to exercise is more dependent on functional factors of the microvasculature (such as dilatory responsiveness of upstream arterioles and/or nitric oxide production) than the anatomical structural characteristics of the capillary network.

To further investigate the relationship between exercise-induced muscle perfusion and the capillary network, we determined muscle fiber capillarization for type I and II fibers specifically. Enhanced capillary blood flow can be very local and depending on the type of stimulation provided. Animal work has previously shown that stimulation of small fiber bundles (5–6 muscle fibers) specifically increases capillary blood flow towards those, and not the surrounding, fibers [4]. In the present study, a single bout of resistance exercise was performed at a non-damaging (80% of 1RM) exercise intensity, which has been shown to mainly activate type II muscle fibers [38]. To our knowledge, no other study has previously assessed whether muscle fiber-type-specific capillarization relates to microvascular perfusion dynamics in response to a single exercise session. Whereas no associations were observed with type I muscle fibers, we report moderate to very strong correlations between various indices of type II muscle fiber capillarization and microvascular perfusion parameters in response to the single bout of exercise (Fig. 4). Interestingly, the strongest correlations (r = 0.64-0.81) were observed between type II muscle fiber capillarization and the increased microvascular blood volume at t = 40 min after exercise cessation. Hence, up to as much as 65% of the variation in the increase in microvascular blood volume at t = 40 min could be explained by its linear relation with type II muscle fiber capillarization. Such a strong relation between the fiber-type-specific capillarization parameters and the perfusion data is remarkable, especially since no relations were observed when mixed-fiber-type capillarization parameters were used. In fact, this would argue in favor of the importance of the anatomical capillary network characteristics for microvascular perfusion responsiveness, be it in a much more subtle context, i.e., potentially representing exercise- and fiber-type-specific responsiveness. Furthermore, we speculate that the stronger correlations at the t = 40 min time point relate to the importance of a prolonged elevation of microvascular blood volume to ensure adequate delivery of nutrients and growth factors and removal of accumulated waste products during post-exercise recovery. These results are of particular relevance in older adults, as age-related capillary rarefaction predominantly occurs in type II muscle fibers [34]. In other words, increasing type II muscle fiber capillarization may represent a therapeutic target to enhance post-exercise microvascular perfusion to subsequently improve the delivery of nutrients and growth factors to support recovery and adaptation. Obviously, the lack of a young control group in the present study limits us from deciphering whether these associations between muscle fiber capillarization and microvascular perfusion during post-exercise recovery are somehow linked to an aging phenotype or whether it is equally present in young adults. Nevertheless, our findings support the hypothesis that type II muscle fiber capillarization may be a key determinant for microvascular perfusion in response to a single bout of resistance exercise in older adults.

Although a relative homogenous group of older adults was included, the data clearly show a high inter-individual variation in muscle fiber morphology (e.g., fiber-type distribution, fiber size, and capillarization) as well as muscle tissue perfusion response during post-exercise recovery. Whether these differences may be explained by habitual physical activity levels, previous periods of performed exercise training and/or genetic predisposition remains to be investigated. In addition, due to the limited sample size, we were not able to assess whether the observed outcomes were different for males compared with females. Nevertheless, our findings provide important insights into the potential relationship between type II muscle fiber capillarization and the muscle perfusion response to a bout of resistance exercise in older adults. It has previously been proposed that type II muscle fiber capillarization is a critical factor for the acute muscle stem cell response to a single bout of resistance exercise [8] and the muscle fiber hypertrophy response to prolonged resistance exercise training in older adults [9, 14]. Based on the results of the present study, we speculate that the muscle microvascular perfusion response to resistance exercise may be limited by type II muscle fiber capillarization in the muscle tissue of older adults, which may subsequently impair the muscle fiber hypertrophic response to resistance exercise training in older adults. Hence, we hypothesize that exercise preconditioning to increase type II muscle fiber capillarization will optimize the skeletal muscle hypertrophic response to subsequent resistance exercise training in the older population. However, prolonged exercise training intervention studies in older adults will be required to test this hypothesis.

In conclusion, resistance exercise greatly increases microvascular perfusion for up to 40 min after exercise cessation in older adults. Type II muscle fiber capillarization is strongly associated with the resistance exercise-induced increase in microvascular blood volume. Hence, the resistance exercise-induced increase in microvascular blood volume may be restricted by type II muscle fiber capillarization in the muscle tissue of older adults. This provides further support for the need to improve type II muscle fiber capillarization to allow optimal muscle reconditioning to resistance exercise training in older adults.

Statement of Ethics

Participants were informed of the nature and possible risks of the experimental procedures before their written informed consent was obtained. The study was approved by the Medical Research Ethics Committee Academic Hospital Maastricht/Maastricht University, the Netherlands (METC 18-060) and complied with the guidelines set out in the most recent version of the Declaration of Helsinki.

Conflict of Interest Statement

Luc J.C. van Loon has received research grants, consulting fees, speaking honoraria, or a combination of these for research on the impact of exercise and nutrition on muscle metabolism. A full overview of research funding is provided at https://www.maastrichtuniversity.nl/l.vanloon. Lex B. Verdijk has received research funding from FrieslandCampina BV (not related to the work presented here). Alfons J.H.M. Houben is part of a European consortium financed by the European Union: European Commission, European Research Executive Agency, Marie Skłodowska-Curie Actions and Support to Experts; MSCA Doctoral Networks NR. 954798. All other authors have no conflicts of interest to declare.

Funding Sources

This research was supported by funding from the Dutch Research Council (NWO) and ZonMw-veni (016.196.008). The funders had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Author Contributions

Milan W. Betz, Alfons J.H.M. Houben, Lex B. Verdijk, Luc J.C. van Loon, and Tim Snijders contributed to the design of the study. Milan W. Betz, Floris K. Hendriks, and Mathias D.G. van den Eynde contributed to data acquisition. Milan W. Betz and Mathias D.G. van den Eynde contributed to data analysis. Milan W. Betz, Floris K. Hendriks, Alfons J.H.M. Houben, Mathias D.G. van den Eynde, Lex B. Verdijk, Luc J.C. van Loon, and Tim Snijders contributed to the interpretation of the data. Milan W. Betz wrote the first draft of the manuscript. Floris K. Hendriks, Alfons J.H.M. Houben, Mathias D.G. van den Eynde, Lex B. Verdijk, Luc J.C. van Loon, and Tim Snijders reviewed the current work critically for important intellectual content.

Funding Statement

This research was supported by funding from the Dutch Research Council (NWO) and ZonMw-veni (016.196.008). The funders had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Data Availability Statement

All data generated or analyzed during this study are included in this article and its online supplementary material files. Further inquiries can be directed to the corresponding author.