Paola Valdivieso

Early Changes in Costameric and Mitochondrial Protein Expression with Unloading Are Muscle Specific

We hypothesised that load-sensitive expression of costameric proteins, which hold the sarcomere in place and position the mitochondria, contributes to the early adaptations of antigravity muscle to unloading and would depend on muscle fibre composition and chymotrypsin activity of the proteasome. Biopsies were obtained from vastus lateralis (VL) and soleus (SOL) muscles of eight men before and after 3 days of unilateral lower limb suspension (ULLS) and subjected to fibre typing and measures for costameric (FAK and FRNK), mitochondrial (NDUFA9, SDHA, UQCRC1, UCP3, and ATP5A1), and MHCI protein and RNA content. Mean cross-sectional area (MCSA) of types I and II muscle fibres in VL and type I fibres in SOL demonstrated a trend for a reduction after ULLS (0.05 ≤ P < 0.10). FAK phosphorylation at tyrosine 397 showed a 20% reduction in VL muscle (P = 0.029). SOL muscle demonstrated a specific reduction in UCP3 content (−23%; P = 0.012). Muscle-specific effects of ULLS were identified for linear relationships between measured proteins, chymotrypsin activity and fibre MCSA. The molecular modifications in costamere turnover and energy homoeostasis identify that aspects of atrophy and fibre transformation are detectable at the protein level in weight-bearing muscles within 3 days of unloading.

CaMKII content affects contractile, but not mitochondrial, characteristics in regenerating skeletal muscle

BackgroundThe multi-meric calcium/calmodulin-dependent protein kinase II (CaMKII) is the main CaMK in skeletal muscle and its expression increases with endurance training. CaMK family members are implicated in contraction-induced regulation of calcium handling, fast myosin type IIA expression and mitochondrial biogenesis. The objective of this study was to investigate the role of an increased CaMKII content for the expression of the contractile and mitochondrial phenotype in vivo. Towards this end we attempted to co-express alpha- and beta-CaMKII isoforms in skeletal muscle and characterised the effect on the contractile and mitochondrial phenotype.ResultsFast-twitch muscle m. gastrocnemius (GM) and slow-twitch muscle m. soleus (SOL) of the right leg of 3-month old rats were transfected via electro-transfer of injected expression plasmids for native α/β CaMKII. Effects were identified from the comparison to control-transfected muscles of the contralateral leg and non-transfected muscles. α/β CaMKII content in muscle fibres was 4-5-fold increased 7 days after transfection. The transfection rate was more pronounced in SOL than GM muscle (i.e. 12.6 vs. 3.5%). The overexpressed α/β CaMKII was functional as shown through increased threonine 287 phosphorylation of β-CaMKII after isometric exercise and down-regulated transcripts COXI, COXIV, SDHB after high-intensity exercise in situ. α/β CaMKII overexpression under normal cage activity accelerated excitation-contraction coupling and relaxation in SOL muscle in association with increased SERCA2, ANXV and fast myosin type IIA/X content but did not affect mitochondrial protein content. These effects were observed on a background of regenerating muscle fibres.ConclusionElevated CaMKII content promotes a slow-to-fast type fibre shift in regenerating muscle but is not sufficient to stimulate mitochondrial biogenesis in the absence of an endurance stimulus.

T/T homozygosity of the tenascin-C gene polymorphism rs2104772 negatively influences exercise-induced angiogenesis

Background Mechanical stress, including blood pressure related factors, up-regulate expression of the pro-angiogenic extracellular matrix protein tenascin-C in skeletal muscle. We hypothesized that increased capillarization of skeletal muscle with the repeated augmentation in perfusion during endurance training is associated with blood vessel-related expression of tenascin-C and would be affected by the single-nucleotide polymorphism (SNP) rs2104772, which characterizes the non-synonymous exchange of thymidine (T)-to-adenosine (A) in the amino acid codon 1677 of tenascin-C. Methods Sixty-one healthy, untrained, male white participants of Swiss descent performed thirty 30-min bouts of endurance exercise on consecutive weekdays using a cycling ergometer. Genotype and training interactions were called significant at Bonferroni-corrected p-value of 5% (repeated measures ANOVA). Results Endurance training increased capillary-to-fiber-ratio (+11%), capillary density (+7%), and mitochondrial volume density (+30%) in m. vastus lateralis. Tenascin-C protein expression in this muscle was confined to arterioles and venules (80% of cases) and increased after training in A-allele carriers. Prior to training, volume densities of subsarcolemmal and myofibrillar mitochondria in m. vastus lateralis muscle were 49% and 18%, respectively, higher in A/A homozygotes relative to T-nucleotide carriers (A/T and T/T). Training specifically increased capillary-to-fiber ratio in A-nucleotide carriers but not in T/T homozygotes. Genotype specific regulation of angiogenesis was reflected by the expression response of 8 angiogenesis-associated transcripts after exercise, and confirmed by training-induced alterations of the shear stress related factors, vimentin and VEGF A. Conclusion Our findings provide evidence for a negative influence of T/T homozygosity in rs2104772 on capillary remodeling with endurance exercise.

ACE inhibition modifies exercise-induced pro-angiogenic and mitochondrial gene transcript expression

Skeletal muscle responds to endurance exercise with an improvement of biochemical pathways that support substrate supply and oxygen‐dependent metabolism. This is reflected by enhanced expression of associated factors after exercise and is specifically modulated by tissue perfusion and oxygenation. We hypothesized that transcript expression of pro‐angiogenic factors (VEGF, tenascin‐C, Angpt1, Angpt1R) and oxygen metabolism (COX4I1, COX4I2, HIF‐1α) in human muscle after an endurance stimulus depends on vasoconstriction, and would be modulated through angiotensin‐converting enzyme inhibition by intake of lisinopril. Fourteen non‐specifically trained, male Caucasians subjects, carried out a single bout of standardized one‐legged bicycle exercise. Seven of the participants consumed lisinopril in the 3 days before exercise. Biopsies were collected pre‐ and 3 h post‐exercise from the m. vastus lateralis. COX4I1 (P = 0.03), COX4I2 (P = 0.04) mRNA and HIF‐1α (P = 0.05) mRNA and protein levels (P = 0.01) showed an exercise‐induced increase in the group not consuming the ACE inhibitor. Conversely, there was a specific exercise‐induced increase in VEGF transcript (P = 0.04) and protein levels (P = 0.03) and a trend for increased tenascin‐c transcript levels (P = 0.09) for subjects consuming lisinopril. The observations indicate that exercise‐induced expression of transcripts involved in angiogenesis and mitochondrial energy metabolism are to some extent regulated via a hypoxia‐related ACE‐dependent mechanism.

Genomic and lipidomic actions of nandrolone on detached rotator cuff muscle in sheep

Reversal of fatty infiltration of pennate rotator cuff muscle after tendon release is hitherto impossible. The administration of nandrolone starting at the time of tendon release prevents the increase in fat content, but does not revert established fatty infiltration. We hypothesised that tendon release and myotendinous retraction cause alterations in lipid related gene expression leading to fatty muscle infiltration, which can be suppressed by nandrolone through its genomic actions if applied immediately after tendon release. The effects of infraspinatus tendon release and subsequent tendon repair at 16 weeks were studied in six Swiss Alpine sheep. In the interventional groups, 150mg nandrolone was administered weekly after tendon release until sacrifice (N22W, n=6) or starting at the time of repair (N6W, n=6). Infraspinatus volume, composition, expressed transcripts, lipids, and selected proteins were analyzed at baseline, 16 and 22 weeks. Tendon release reduced infraspinatus volume by 22% and increased fat content from 11% to 38%. These changes were not affected by repair. Fatty infiltration was associated with up-regulation of 227 lipid species, and increased levels of the adipocyte differentiation marker PPARG2 (peroxisome proliferator-activated receptor gamma 2). Nandrolone abrogated lipid accumulation, halved the loss in fiber area percentage, and up-regulated androgen receptor levels and transcript expression in the N22W but not the N6W group. The results document that nandrolone mitigates muscle-to-fat transformation after tendon release via a general down-regulation of lipid accumulation concomitantly with up-regulated expression of its nuclear receptor and downstream transcripts in skeletal muscle. Reduced responsiveness of retracted muscle to nandrolone as observed in the N6W group is reflected by a down-regulated transcript response.

Muscle Degeneration Associated With Rotator Cuff Tendon Release and/or Denervation in Sheep

Background: The effect of an additional neurological injury (suprascapular nerve traction injury) to a chronically retracted rotator cuff muscle is incompletely understood and warrants clarification. Purpose: To investigate the microscopic and macroscopic muscle degeneration patterns caused by tendon release and/or muscle denervation in a sheep rotator cuff model. Study Design: Controlled laboratory study. Methods: Infraspinatus muscle biopsy specimens (for histological analysis) were obtained from 18 Swiss alpine sheep before and 16 weeks after release of the infraspinatus tendon (tenotomy [T] group; n = 6), transection of the suprascapular nerve (neurectomy [N] group; n = 6), or tendon release plus nerve transection (tenotomy + neurectomy [T&N] group; n = 6). Magnetic resonance imaging (MRI) and computed tomography (CT) were used to assess retraction (CT), muscle density (CT), volume (MRI T2), and fat fraction (MRI Dixon). Stiffness of the infraspinatus was measured with a spring scale. Results: At 16 weeks postoperatively, the mean infraspinatus muscle volume had decreased significantly more after neurectomy (to 47% ± 7% of the original volume; P = .001) and tenotomy plus neurectomy (48% ± 13%; P = .005) than after tenotomy alone (78% ± 11%). Conversely, the mean amount of intramuscular fat (CT/MRI Dixon) was not significantly different in the 3 groups (T group: 50% ± 9%; N group: 40% ± 11%; T&N group: 46% ± 10%) after 16 weeks. The mean myotendinous retraction (CT) was not significantly different in the T and T&N groups (5.8 ± 1.0 cm and 6.4 ± 0.4 cm, respectively; P = .26). Stiffness was, however, most increased after additional neurectomy. In contrast to muscle changes after tendon release, denervation of the muscle led to a decrease in the pennation angle of lengthened muscle fibers, with a reduced mean cross-sectional area of pooled muscle fibers, a slow- to fast-type transformation, and an increase in the area percentage of hybrid fibers, leading to overall significantly greater atrophy of the corresponding muscle. Conclusion: Although it is unclear which experimental group (T or T&N) most accurately reflects the clinical scenario in a given case, these findings provide baseline information for clinical differentiation between muscle changes caused by denervation or rotator cuff tendon lesions. Clinical Relevance: The findings of this study help to understand how and to which extent a neurological lesion of the supplying suprascapular nerve could influence the pattern of anatomic-physiological muscular changes after rotator cuff tendon tears.

Gene-Pharmacologial Effects on Exercise-Induced Muscle Gene Expression in Healthy Men

Angiotensin 2 is a major vasoconstrictor and subject to complex regulation. It is produced by the angiotensinconverting enzyme (ACE), which is itself regulated by blood flow and represents a target for pharmacological (lisinopril) and genetical (ACE-I/D polymorphism) influences. 32 healthy Caucasian men of British descent, including six subjects that were treated with lisinopril, completed standardized one-legged cycling-type endurance exercise to assess ACE-I/D and lisinopril dependent levels alterations of ACE and hypoxia-modulated transcripts in knee extensor muscle. Lisinopril treatment revealed significantly decreased transcript expression at baseline (COX4I1-88%; COX4I2 -67%; HIF-1α -93%) and 3 hours into recovery (HIF-1α -78%; ACE -62%). Furthermore, during recovery from cycling-type endurance exercise, COX4I2 mRNA expression was increased by 281% in ACE-II genotypes (p <0.05) but not in subjects carrying the ACE D-allele. Additionally, ACE-DD genotypes showed a trend for superior expression of ACE mRNA (p =0.07), whereas for carriers of the ACE I-allele a decrease (ACE-ID-35%; ACE-II -67%) was observed. Changes in COX4I2 and ACE transcript expression were correlated to HIF-1α protein levels prior to exercise, which was highest in ACE-DD genotypes. The interpretative hypothesis is suggested that exercise-induced COX4I2 transcript expression is sensitized in dependence of the I-allele, which silences ACE expression. The findings implicate a pronounced influence of anti-hypertensive treatment on muscle gene expression, which is modified by the ACE-I/D genotype. The observed effects of lisinopril and the ACE I-allele suggests a possible role of shear stress and tissue oxygenation on the expression of oxygen-associated transcripts during recovery from the sympatholytic challenge of endurance exercise.

The Metabolic Response of Skeletal Muscle to Endurance Exercise Is Modified by the ACE-I/D Gene Polymorphism and Training State

The insertion/deletion polymorphism in the gene for the regulator of vascular tone, angiotensin-converting enzyme (ACE), is the prototype of a genetic influence on physical fitness and this involves an influence on capillary supply lines and dependent aerobic metabolism in skeletal muscle. The respective interaction of ACE-I/D genotype and training status on local metabolic and angiogenic reactions in exercised muscle is not known. Toward this end we characterized the metabolomic and angiogenic response in knee extensor muscle, m. vastus lateralis, in 18 untrained and 34 endurance-trained (physically active, V˙O2max > 50 mL min−1 kg−1) white British men to an exhaustive bout of one-legged cycling exercise. We hypothesized that training status and ACE-I/D genotype affect supply-related muscle characteristics of exercise performance in correspondence to ACE expression and angiotensin 2 levels. ACE-I/D genotype and training status developed an interaction effect on the cross-sectional area (CSA) of m. vastus lateralis and mean CSA of slow type fibers, which correlated with peak power output (r ≥ 0.44). Genotype × training interactions in muscle also resolved for exercise-induced alterations of 22 metabolites, 8 lipids, glycogen concentration (p = 0.016), ACE transcript levels (p = 0.037), and by trend for the pro-angiogenic factor tenascin-C post exercise (p = 0.064). Capillary density (p = 0.001), capillary-to-fiber ratio (p = 0.010), systolic blood pressure (p = 0.014), and exercise-induced alterations in the pro-angiogenic protein VEGF (p = 0.043) depended on the ACE-I/D genotype alone. Our observations indicate that variability in aerobic performance in the studied subjects was in part reflected by an ACE-I/D-genotype-modulated metabolic phenotype of a major locomotor muscle. Repeated endurance exercise appeared to override this genetic influence in skeletal muscle by altering the ACE-related metabolic response and molecular aspects of the angiogenic response to endurance exercise.

Neurectomy preserves fast fibers when combined with tenotomy of infraspinatus muscle via upregulation of myogenesis: Protein expression with denervation and tenotomy

Introduction: We evaluated the contribution of denervation‐related molecular processes to rotator cuff muscle degeneration after tendon release. Methods: We assessed the levels of myogenic (myogenin and myogenic differentiation factor [myoD]) and proadipogenic (peroxisome proliferator‐activated receptor γ) transcription factors; the denervation‐associated proteins tenascin‐C, laminin‐2, and calcium/calmodulin‐dependent kinase II (CaMKII); and cellular alterations in sheep after infraspinatus tenotomy (TEN), suprascapular neurectomy (NEU), or both (TEN‐NEU). Results: Extracellular ground substance increased at the expense of contractile tissue 16 weeks after surgery, correlating with CaMKII isoform levels. Sheep undergoing NEU and TEN‐NEU had exaggerated infraspinatus atrophy and increased fast fibers compared with TEN sheep. The βMCaMKII isoform levels increased with TEN, and myoD levels tripled after denervation and were associated with slow fibers. Discussion: In sheep, denervation did not affect muscle‐to‐fat conversion after TEN of the infraspinatus. Furthermore, concurrent NEU mitigated the loss of fast fibers after TEN by inducing a fast‐contractile phenotype. Muscle Nerve 59:100–107, 2019

Does a Better Perfusion of Deconditioned Muscle Tissue Release Chronic Low Back Pain

Non-specific chronic low back pain (nsCLBP) is a multifactorial condition of unknown etiology and pathogenesis. Physical and genetic factors may influence the predisposition of individuals to CLBP, which in many instances share a musculoskeletal origin. A reduced pain level in low back pain patients that participate in exercise therapy highlights that disuse-related muscle deconditioning may predispose individuals to nsCLBP. In this context, musculoskeletal pain may be the consequence of capillary rarefaction in inactive muscle as this would lower local tissue drainage and washing out of toxic waste. Muscle activity is translated into an angio-adaptative process, which implicates angiogenic-gene expression and individual response differences due to heritable modifications of such genes (gene polymorphisms). The pathophysiologic mechanism underlying nsCLBP is still largely unaddressed. We hypothesize that capillary rarefaction due to a deconditioning of dorsal muscle groups exacerbates nsCLBP by increasing noxious sensation, reducing muscle strength and fatigue resistance by initiating a downward spiral of local deconditioning of back muscles which diminishes their load-bearing capacity. We address the idea that specific factors such as angiotensin-converting enzyme and Tenascin-C might play an important role in altering susceptibility to nsCLBP via their effects on microvascular perfusion and vascular remodeling of skeletal muscle, inflammation, and pain sensation. The genetic profile may help to explain the individual predisposition to nsCLBP, thus identifying subgroups of patients, which could benefit from ad hoc treatment types. Future therapeutic approaches aimed at relieving the pain associated with nsCLBP should be based on the verification of mechanistic processes of activity-induced angio-adaptation and muscle-perfusion.