The extract of Ginkgo biloba EGb 761 reactivates a juvenile profile in the skeletal muscle of sarcopenic rats by transcriptional reprogramming

Abstract

Background: Sarcopenia is a major public health problem in industrialized nations, placing an increasing burden on public healthcare systems because the loss of skeletal muscle mass and strength that characterizes this affection increases the dependence and the risk of injury caused by sudden falls in elderly people. Albeit exercise and caloric restriction improve sarcopenia-associated decline of the muscular performances, a more suitable and focused pharmacological treatment is still lacking.

Methodology/principal findings: In order to evaluate such a possible treatment, we investigated the effects of EGb 761, a Ginkgo biloba extract used in chronic age-dependent neurological disorders, on the function of the soleus muscle in aged rats. EGb 761 induced a gain in muscular mass that was associated with an improvement of the muscular performances as assessed by biochemical and electrophysiological tests. DNA microarray analysis shows that these modifications are accompanied by the transcriptional reprogramming of genes related to myogenesis through the TGFbeta signaling pathway and to energy production via fatty acids and glucose oxidation. EGb 761 restored a more juvenile gene expression pattern by regenerating the aged muscle and reversing the age-related metabolic shift from lipids to glucose utilization.

Conclusions/significance: Thus, EGb 761 may represent a novel treatment for sarcopenia both more manageable and less cumbersome than exercise and caloric restriction.

Figure 1. Contractility studies.

Representative examples of isometric contractile force in isolated soleus muscle from aged controls (a, c), and EGb 761-treated rats (b, d). In a and b the recordings were obtained by direct muscle stimulation and in c and d by nerve stimulation. In each recording (a to d) are shown evoked twitches (0.2 Hz stimulation) and tetanic contractions (40 Hz, 600 ms stimulation). The force is expressed in Newton (N). Histograms represent mean values±S.E.M. of the force (expressed in N/g) for the single twitch (e) and for tetanic stimulation (f). Mean ratio values between treated and controls are indicated in respective boxes with number of values (n) used for calculations. * Significantly different from controls p<0.05.

Figure 2. Force-frequency relationship.

Peak force during isometric tetanic responses in isolated soleus muscles from aged controls (○) and EGb 761-treated rats (•) obtained by direct muscle stimulation (A) and by nerve stimulation (B) at frequencies indicated (abscissa) during trains of 600 ms duration. An interval of 30 s was used between trains. Each circle represent the mean±S.E.M. (n = 3). * Significant different from controls p<0.05.

Figure 3. Microarray data analysis and clustering.

Comparison of gene expression profiles of soleus muscle in young rats (Y), aged control rats (AC) and aged treated rats (AT). Genes are clustered using Pearson's correlation. Down-regulated genes are represented in green, up-regulated genes, in red, number of genes is reported in parentheses.

Figure 4. Graphic representation of gene expression variations.

Comparison between variations of gene expression in the different groups of rats according either to DNA microarray analysis (blue lines) or to qPCR (red lines). Values of the AC (aged control) group are standardized to 1 for each gene.

Figure 5. Schematic representation of the interactions between the genes regulated by EGb 761 associated to myogenesis/regeneration in the soleus muscle of aged rats.

Regulated genes are included in frames. indicates up-regulation of gene expression by EGb 761. indicates down-regulation of gene expression by EGb 761. → = activation. -- = inhibition. Activins and BMPs may be diverted into alternative pathways through interaction with soluble and membrane-bound binding proteins Activin, myostatin and BMPs signal via type II activin receptor. The access of these TGF-β2 superfamily members to their type II receptors is blocked by extracellular binding proteins (follistatine, Fstl1 and α2-macroglubulin) and membrane-bound pseudoreceptors (BAMBI for activin signaling).

Figure 6. Schematic representation of the interactions between the genes regulated by EGb 761 associated with regulation of energy metabolism in mitochondria in the soleus muscle of aged rats.

Regulated genes are included in frames. indicates up-regulation of gene expression by EGb 761. indicates down-regulation of gene expression by EGb 761. → = activation. ---- = inhibition. FA (Free Fatty Acids) are taken up by the fatty acid transporter protein Cd36 and activated to fatty acyl-coenzyme A (FA-CoA). FA-CoA oxidation would increase the ratios of acetyl-CoA/CoA and of NADH/NAD+, which inhibit the pyruvate dehydogenase (PDH) complex by activating the pyruvate dehydrogenase kinase 4 (Pdk4). Increase FA oxidation products as citrate should further inhibit phosphofructokinase (Pfk) and hexokinase. These changes would slow down oxidation of glucose and pyruvate and glycogen stock is maintained. In the same time, fatty acid synthase (Fasn) and mitochondrial 3-oxoacyl-Coenzyme A thiolase (Acaa2) are inhibited resulting in the lipid synthesis inhibition and a preferential use of these lipids for ATP synthesis.