Synopsis

Keywords: Muscle, Aging, Multi-contrast, Metabolism, Non-proton, Spectroscopy

The age-related loss of muscle mass, strength and quality is a multifactorial process whose mechanisms are incompletely understood. NMR allows investigating numerous physiological and biochemical variables in vivo dynamically. Here, 51 physically active subjects performed a normalized plantar flexion exercise to evaluate vascular response (blood flow, T₂*, water compartmentalization), tissue oxygenation and oxidative energy metabolism (pH, mitochondrial oxidative capacity, work to [ADP] ratio). Results showed that the vascular and metabolic function was preserved in old subjects relative to young and middle-aged participants. We conclude that vascular and oxidative metabolism impairments are not a necessary condition of old age.

Purpose

The age-related loss of muscle mass, strength and quality is a multifactorial process whose mechanisms are incompletely understood. NMR allows investigating numerous physiological and biochemical variables in vivo during a dynamic paradigm. For instance, tissue blood flow and blood oxygen level dependent contrast effects can be mapped dynamically, and tissue oxygenation and high-energy phosphates turnover can be monitored using ¹H and ³¹P MRS, respectively. Mitochondrial ATP production rate and ADP-control can also be monitored by quantifying the work performed. Here, we applied a multi-nuclear interleaved measurements to evaluate age-related changes of these functional parameters in the lower leg and performed multi-compartment T₂ measurements to evaluate changes in water compartments in response to an exercise

Methods

Experimental Setup
Experiments were done at 3T on 51 healthy volunteers (28 women, aged from 20 to 81 y.o). The RF coil used (RAPID Biomedical) combined a ¹H birdcage transmitter, a 18Rx phased-array receiver and a ³¹P 1Tx/3Rx semi-cylindrical transceiver.
Exercise paradigm
Plantar flexions were performed every 2.5 s for 13 min, increasing the load every minute from 10% to 25% of the maximum voluntary torque. An ergometer interfaced with LabView (v2014, National Instruments) was employed and work values were recorded. Dynamic NMR lasted 25 min, starting 2 min before the exercise onset.
Interleaved NMR
An interleaved 1H/31P pulse sequence¹ (fig.2A) consecutively acquired, every 2.5 s, a non-localized ³¹P spectrum, a ¹H spectrum for desaturated myoglobin (dMb) detection, followed by colocalized radially-encoded Golden-Angle perfusion-weighted² and T₂*-weighted images.
Multi-compartment T₂
A fat-suppressed ISIS-CPMG acquisition measured the water T₂ decay³ (150 echoes, 2.5 ms inter-echo spacing, 35x35x35 mm³ voxel, TR=9 s, 2m42s acquisition time) before the exercise and at the 10th minute of recovery in the gastrocnemius medialis.
Data Analysis
Interleaved NMR
Data analysis was performed in Matlab as described elsewhere¹. No motion correction was applied. T₂* values were calculated using an exponential decay of the multi-echo GRE data. The rephosphorylation rate of phosphocreatine (PCr), τPCr, was estimated with an exponential function during exercise recovery. Adenosine diphosphate (ADP) was calculated assuming creatine kinase equilibrium⁴ (K_CK):
[ADP]=[ATP]*([TCr]/[PCr]-1)/([H⁺]K_CK))
where total creatine (TCr) and ATP were assumed 42.5 and 8.2 mM, respectively and K_CK=1.66x10⁹ M^-1.
A normalized work output per minute was calculated by dividing the measured work to the gastrocnemius muscle surface at its thickest section.
Multi-compartment T₂
Water T₂ times (T_2-H2O) and their relative fractions were estimated using a non-negative least squares algorithm3 (480 ms upper limit). A short (<50 ms) and a long (>50 ms) T_2-H2O component are reported.
Statistical analysis
Subjects were divided into groups: Young (G_Y, 20-35 y.o., n=13), Middle-aged (G_M, 35-60 y.o., n=24) and Old (G_O, above 60 y.o., n=14). Wilcoxon tests were used for pairwise group mean comparisons.

Results

Fig.1 shows the participants’ demographics. Fig.2 shows the time courses of the gastrocnemius and soleus blood perfusion and T₂*; intracellular pH; PCr and Pi; and dMb signals. dMb was detected in 43±3% of the subjects in each group, consistent with a mild-to-moderate aerobic exercise. Few significant differences were found, notably the maximum pH reached during exercise was higher in G_Y than in G_M; and resting T₂* in soleus was higher in G_Y than in G_O (fig.3A,B). No differences in τPCr were found (fig.3C). The end-of-exercise PCr depletion was borderline(p=0.057) lower in G_O than in G_M. For blood perfusion and T₂* in the gastrocnemius, no age differences were found for peak recovery or end-of-exercise values. To evaluate the coupling between work and mitochondrial stimulation, the ratio between normalized work and [ADP] was estimated (fig.4). The Work/[ADP] ratio during the first minute was systematically higher, as expected from the initial PCr breakdown as energy buffer and early glycolytic pathway activation during exercise. For the remainder of the exercise, G_M and G_O showed stable values, while G_Y showed a trend of continuous decline until the 10th minute of exercise (fig.4). No significant Work/[ADP] differences were observed among the groups, suggesting no impaired control of oxidative activity with age⁴. The relative fractions of the long T_2-H2O, the short T_2-H2O and the long T_2-H2O, showed no significant differences before and after exercise (fig.5A,B,C, respectively). Only the long T_2-H2O fractions were larger in G_O than in G_Y, both at rest and after exercise. By ignoring the extreme long T_2-H2O that reached the model’s upper limit, then a significant increase of long T_2-H2O in G_O is found (fig.5D). This result should nevertheless be considered with caution.

Discussion and Conclusion

Our results show that despite a tendency for a higher PCr depletion during exercise in G_O relative to G_Y, no significant differences in vascular compartments, blood perfusion or oxydative energy metabolism were found between age groups during an aerobic, moderate and normalized exercise paradigm. In particular, no mitochondrial functional impairments were found, as shown by the similar τPCr and work/[ADP] ratios. This is in agreement with a previous study evaluating blood flow, tissue oxygenation and energy metabolism⁵ but in disagreement with a study evaluating energy metabolism and carnosine concentration⁷. Unlike the cited study⁶, our participants were physically active and no BMI differences were found, which correlates to aerobic capacity. Our results point that blood flow and oxidative energy metabolism impairments are not a necessary condition of ageing.

Acknowledgements

No acknowledgement found.