Reinnervation of Vastus lateralis is increased significantly in seniors (70-years old) with a lifelong history of high-level exercise (2013, revisited here in 2022)

Abstract

In 2013 we presented results showing that at the histological level lifelong increased physical activity promotes reinnervation of muscle fibers in aging muscles. Indeed, in muscle biopsies from 70-year old men with a lifelong history of high-level physical activity, we observed a considerable increase in fiber-type groupings (F-TG), almost exclusively of the slow type. Slow-type transformation by denervation-reinnervation in senior sportsmen seems to fluctuate from those with scarce fiber-type transformation and groupings to almost fully transformed muscle, going through a process in which isolated fibers co-expressing fast and slow Myosin Heavy Chains (MHCs) seems to fill the gaps. Taken together, our results suggest that, beyond the direct effects of aging on the muscle fibers, changes occurring in skeletal muscle tissue appear to be largely, although not solely, a result of sparse denervation-reinnervation. The lifelong exercise allows the body to adapt to the consequences of the age-related denervation and to preserve muscle structure and function by saving otherwise lost muscle fibers through recruitment to different, mainly slow, motor units. These beneficial effects of high-level life-long exercise on motoneurons, specifically on the slow type motoneurones that are those with higher daily activity, and on muscle fibers, serve to maintain size, structure and function of muscles, delaying the functional decline and loss of independence that are commonly seen in late aging. Several studies of independent reserchers with independent analyses confirmed and cited our 2013 results. Thus, the results we presented in our paper in 2013 seem to have held up rather well. Trial Registration: ClinicalTrials.gov: NCT01679977

Ethical Publication Statement

We confirm that we have read the Journal’s position on issues involved in ethical publication and affirm that this report is consistent with those guidelines.

It has long been accepted that histological changes seen in aging muscle suggest that denervation significantly contributes to tissue atrophy.^1,2 Corroborating evidence of a progressive loss of α - motoneurons has been described with aging.³ Electrophysiological studies have confirmed a decrease in the number of motor units with some increase in their size, suggesting reinnervation events.⁴ Further evidence supporting rounds of denervation and reinnervation is based on the observation that in young humans, fiber types appear randomly distributed across the muscle but become increasingly clustered or grouped together with age.⁵ Therefore, it has been proposed that apoptosis of motoneurons in the spinal cord (with subsequent incomplete reinnervation of fibers by surviving motoneurons) contributes to the loss of muscle strength and mass that occurs with age.⁶ All of these processes are accompanied by a progressive increase in slow muscle fibers, although the literature provides some contradictions (see a recent review).⁷ Some of this discrepancy has been dispelled by comparisons of muscle from active and immobile patients: the immobile elderly have a shift toward fast isoform expression, as is common in “unloaded” muscle (e.g., during spaceflight or limb immobilization), whereas muscle wasting is accompanied by a shift toward a fast twitch phenotype.⁸ Thus the actual expression pattern of myosin isoforms in the elderly is modulated by complex factors because it depends upon the conflicting influences of both aging and reduced activity tending to shift toward slow and fast isoforms, respectively.⁹ To further complicate the situation, conflicting results regarding fast to slow myosin transition arise in endurance training studies using animal models and in clinical trials of humans involving either voluntary exercise or electrical stimulation (directly to muscle or indirectly through nerve stimulation).^8,10-15 Whether these shifts are under neural control or the direct effect of use/disuse on muscle fibers remains to be clarified. In the presents study, we analyzed muscle biopsies harvested from the Vastus lateralis of senior (65 to 79 years) amateur sportsmen (i.e., subjects who routinely practice sport activities usually more than three times a week, up to the time of biopsy). In agreements with some previous studies of master athletes,^16-18 we show that lifelong high-level physical activity considerably increases the percentage of slow-type myofibers and the number of muscle fiber-type groupings (F-TG). Slow-type transformation by reinnervation in senior sportsmen appears to be a clinically relevant mechanism because, despite the facts that the biopsies from our subjects vary in the degree to which they have undergone slow-type transformation and that numerous factors can affect fiber type transition, the analyses of our data demonstrate that the senior sportsmen have a significantly greater level of slow type fiber groupings, demonstrating that their muscle has undergone significant reinnervation. Indeed, in recent meetings, we have reported that muscle properties of these senior amateur sportsmen are more similar to those of active young men than to those of sedentary seniors.^19,20 Thus our studies support the concept that lifelong high-level exercise has a beneficial effect on the motoneurons and, through them, on the muscle fibers, resulting in maintainance of muscle size, structure and function, thereby delaying the functional decline and loss of independence that are commonly seen in aging adults.

Materials and Methods

All subjects recruited for the study were volunteers who received detailed information and all signed an informed consent. Approval from the national committee for medical ethics was obtained before study onset (EK08-102-0608). Groups of young men (n=16), seniors with normal life style (sedentary, n=16) and seniors with a lifelong history of high-level recreational sport activities (n=16) were enrolled. All subjects were healthy and declared not to have any specific mobility impairment or disease. Upon enrollment in the study, needle muscle biopsies were harvested through a small skin incision (6 mm) from the right and left Vastus lateralis muscles of each patient and then frozen for light microscopy as described.¹²

Light microscopy and quantitative histological analyses

Serial cryosections (8 μm) from frozen muscle biopsies were mounted on polysine™ glass slides, air-dried and stained either with Hematoxylin and Eosin (H&E) or using conventional techniques for myofibrillar ATPases to evaluate muscle fiber types.²¹ In the latter method, slow-type muscle fibers are dark while the fast-type fibers are lightly stained following preincubation at pH 4.35. The reverse is true after preincubation at pH 9.4. Morphometric analyses of the fiber diameter and of the fiber type distribution were performed on cryosections using Scion Image for Windows version Beta 4.0.2 (2000 Scion Corporation) as previously describe^{.12, 19, 21-24}

Statistical analysis

ANOVA tests were performed with statistics algorithms of Origin™, OriginLab Corporation, USA. The level of statistical significance was set at p<0.05.

Results

From our previous studies on skeletal muscle biopsies of paraplegic patients we know that muscle disuse resulting from decades of years of denervation (after upper motor neuron lesion) induces at most a 50% decrease in size (i.e., from a myofiber diameter of approximately 70 μm to 35 μm),²⁴ while lower motor neuron denervated skeletal muscle (one year. after denervation) shows muscle fibers with a diameter less than 30 μm.^12,22,23,25 Based upon these findings, we are confident in defining those muscle fibers having a diameter smaller than 30 μm as denervated. This interpretation is strengthened by the fact that several small myofibers have angular aspects.²⁰ In the biopsies analyzed here, small angular muscle fibers have the size and the morphology of denervated muscle fibers and they are more frequent in sedentary septuagenarians than in young men and septuagenarians with a lifelong history of high-level exercise (Table 1). Muscle fibers with a diameter less than 30 μm are seldom observed (< 0.5 %) in the muscle biopsies of young men, while biopsies harvested from the sedentary seniors contain the highest percentage (6.9 %) of denervated muscle fibers among the three groups (Table 1). When muscle fibers with diameters less than 25 μm are counted the percentages decrease by approximately 50% for each group, however, the sedentary seniors still maintain the highst values. ANOVA tests on these data confirm that the higher percentages of small angular fibers in sedentary seniors relative to both young subjects and senior sportsmen are statistically significant. This is not the case when young subjects and senior sportsmen are compared.

Table 1.

Small angular muscle fibers in young men and in septuagenarians either sedentary or sportsmen

		Myofiber diameter
		< 30 µm		<25 µm
Subjects	(size)	%	ANOVA	%	ANOVA
Young men	(16)	0.5 +/- 0.7	YES	0.3+/-0.5	YES (versus. Sedentary)
Seniors
Sedentary	(16)	6.9 +/- 3.6	YES	3.0+/- 2.0	YES (vs. Sportsmen)
Sportmen	(16)	1.0 +/- 1.7	NO	0.2 +/- 0.3	NO (vs.Youngs)

YES or NO, significance of ANOVA test.

Analyses of F-TG demonstrate that, although not statistically significant, the percentage of fast fiber types is markedly higher in the sedentary seniors than in either the senior sportsmen or the young men. The percentage of slow type fibers, however, is significantly higher in the senior men (both sedentary and sportsmen) than in the younger men. Most interestingly, the percentage of slow-type fibers in the senior sportsmen is signficantly higher than in the sedentary seniors (Table 2).

Fiber-type grouping is identified on the basis that one myofiber is completely surrounded by fibers of the same phenotype. Because two or more slow type fibers were not always easily distinguished one from another in alkaline-resistant ATPase specimens, we confirmed our fiber border delineations with the less ambiguous method of acid resistant ATPase staining of specimens following preincubation at pH 4.35. In figure 1 some examples of ATPase staining of muscle biopsies harvested from high-level recreational sportsmen are shown, beginning with one which contains a one-to- one proportion of slow-to-fast fibers (as in normal adult muscles) and escalating to a sample in which almost all the muscle biopsy is covered by very large slow F-TG.

Table 2 shows that some fast F-TG were present in the biopsies harvested from sedentary seniors: the central fibers characterizing fast F-TG were 3.6% of the total muscle fibers, while those of slow-type were around 0.5%. Even more evident is the fact that, in the biopsies harvested from senior sportsmen, the slow type fibers are grouped in larger areas (mean 8.4 %, see Table 2), almost reaching the 92% in the extreme cases (Table 3).

It has long been recognized that the histological changes seen in aging muscle suggest that denervation significantly contributes to muscle decay,^1,26,27 and that immobility accelerates the deterioration process,⁹ while running activity sustained for decades (as that performed by master athletes) protects against the age-related loss of motor units,^28-30 and, thereby, protects lean muscle mass.³¹ However, the degree to which denervation causes muscle fiber transformation and loss of myofibers is an open issue in humans, since reinnervation events may compensate long-term for motor neuron loss in spinal cord and/or axonal abnormalities in peripheral nerves.^4,5,32,33

Table 2.

Fiber-type groupings in young men and in septuagenarians either sedentary or sportsmen

		Fiber-type groupings (as % of central fibers in clustered areas vs. total fibers)
Subjects	(size)	Fast (% )	ANOVA	Slow (%)	ANOVA
Young men	(8)	01.1 +/- 2.2	NO	< 0.1+/-0.1	NO (vs. Sedentary)
Seniors
Sedentary	(8)	3.6 +/- 5.1	NO	0.5+/- 0.6	YES (vs. Sportsmen)
Sportmen	(8)	0.1 +/- 0.1	NO	8.4 +/- 7.9	YES (vs.Youngs)

YES or NO, significance of ANOVA test.

Fig 1.

Fiber type distribution by ATPase staining (pH 4.35) in 70-year sportsmen shows a high occurrence of slow type fibers (dark stained myofibers). Biopsies are ordered from panel A to panel L according to their increasing percentage of slow fibers. The majority has around 70% of slow type, ranging from 51% (panel A), to 92% (panel L). See also Table 3. All panels are at the same magnification, bar = 1 mm.

Table 3.

Slow fibers (%) and type groupings (#) in Vastus lateralis of 70-year sportsmen

Panel	Slow	Slow
	(%)	(#)
A	51	2
B	68	6
C	69	3
D	70	19
E	71	6
F	75	4
G	76	18
H	81	23
I	85	>23
L	92	>23

Discussion

In the present study we used histochemical ATPase methods to analyze muscle biopsies harvested from septuagenarian sportsmen and compared their relative amount of: i) small angular myofibers (denervated muscle fibers), ii) fast and slow muscle fibers (muscle plasticity), and iii) central muscle fibers of fiber-type clusters (reinnervated muscle fibers) with those in muscle biopsies of sedentary septuagenarians and young men. The main results are: i) biopsies from young men seldom contain denervated and reinnervated muscle fibers or transforming myofibers; ii) biopsies from sedentary seniors contain both denervated and a few reinnervated clustered myofibers of the fast type; and iii) senior sportsmen present with a larger percentage of slow myofibers, up to 90%, which appear clustered in slow F-TG.

Our data suggest that slow-type transformation by reinnervation in senior sportsmen is a clinically relevant mechanism despite the facts: i) that subject biopsies vary from those with scarce fiber-type transformation and groupings to those with almost fully transformed muscles in which isolated fibers co-expressing fast and slow MHCs fill in the gaps (Mosole et al.);^34,35 and ii) there are potential confounding factors such as the sampling of a heterogeneous muscle, individual genetic backgrounds, difference in kind and extent of the high level activities.

Indeed, in recent meetings we reported that the muscle properties of this group of senior sportsmen are more similar to that of active young men than those of sedentary seniors.

Specifically, the results indicate that relative to their sedentary cohorts, senior sportsmen have greater muscle maximal isometric force and function and better preserved muscle morphology and ultrastructure.^19,20 Taken together our results suggest that, beyond the direct effects of aging on the muscle fibers, changes occurring in skeletal muscle tissue appear to be largely, although not solely, a result of sparse incremental denervation.

In senior sportsmen the increase in slow clustered fiber percentage is conceivably the result of the positive effect of lifelong physical activity on the motoneuron pool, which has spared the slow motoneurons from age related lesion/death, increasing the chance that peripheral reinnervation occurs due to sprouting of slow axons. Lifelong exercise seems to allow the body to adapt to the consequences of age- related denervation and to preserve muscle structure and function by saving otherwise lost muscle fibers through recruitment to different, mainly slow, motor units. Regular physical activity is a good strategy to attenuate muscle functional decline and ultrastructural abnormalities associated with aging. Certainly other mechanisms contribute to lifelong muscle health, however, our present data support the concept that lifelong high-level exercise has a beneficial effect on the motoneurons, specifically on the slow type motoneurones that are those with higher daily activity, and, through them, on muscle fibers.

In conclusion, the beneficial effects of lifelong high-level exercise on motor neurons and, of course, muscle fibers serve to maintain muscle fiber size, structure and function, delaying functional decline and loss of independence commonly observed in progressive aging. Several confirmatory studies by independent researchers with independent analyzes have been published to date citing our 2013 results.³⁶ Those results, here presented, seem to have held up quite well..

List of acronyms

F-TG

fiber-type groupings

MHC

myosin heavy chains

Funding Statement

Funding: The authors would like to acknowledge the support of: (1) the European Regional Development Fund for funding the Cross Border Cooperation Programme Slovakia – Austria 2007 – 2013 (Interreg-IVa), project Mobilität im Alter, MOBIL, N_00033 (partners: Ludwig Boltzmann Institute of Electrical Stimulation and Physical Rehabilitation, Austria, Center for Medical Physics and Biomedical Engineering, Medical University of Vienna, Austria and Faculty of Physical Education and Sports, Comenius University in Bratislava, Slovakia), (2) the Austrian national co- financing of the Austrian Federal Ministry of Science and Research, and (3) MIUR Italy, Funds ex-60% to UC, Laboratory of Translation Myology, Department of Biomedical Sciences, University of Padua, Italy. (4) U.C. thanks the A&C M-C Foundation for Translational Myology, Padua, Italy for sponsoring this publication