Cinnamtannin A2, (−)-epicatechin tetramer, attenuates skeletal muscle wasting in disuse atrophy model mice induced by hindlimb suspension

Abstract

The impact of repeated administration of cinntamtannin A2 (A2, 25 μg/kg) on skeletal muscle disuse atrophy model mice induced by hindlimb suspension for 14 days was examined. In soleus, weight loss and a reduction in the average myofibre size with shifting to the smaller side of the peak were observed in the suspension-vehicle group, but A2 reduced these changes. Average myofibre size significantly increased in ground-A2 compared to ground-vehicle. A marked increase in the dephosphorylation of forkhead box O (FoxO) 3a by the suspension was reduced by A2. The phosphorylation of protein kinase B (Akt) and eukaryotic translation initiation factor 4E-binding protein (4EBP)-1 were significantly increased by the treatment of A2. In addition, a single dose of A2 increased dramatically in the 24-h excretion of catecholamines in urine. These results suggest that A2 administration results in sympathetic nerve activation and promotes hypertrophy while inhibiting the progress of disuse muscle atrophy.

Introduction

It has been well known that prolonged unloading of skeletal muscle results in massive metabolic and structural changes in slow-twitch fibers.^(1,2) Skeletal muscle is regulated by a balance between the rate of muscle protein synthesis and degradation.⁽³⁾ To prevent disuse muscle atrophy, a mechanistic understanding of the cellular signaling pathways that regulate both proteosynthesis and proteolysis in muscle is needed.⁽⁴⁾ Hindlimb suspension (HLS) leads to significant skeletal muscle atrophy. The rodent disuse atrophy model induced by HLS provides information on the morphological and molecular changes responsible for the mechanisms of disuse-induced muscle loss.^(5,6)

The B-type procyanidin cinnamtannin A2 (A2, Fig. 1A) is an (−)-epicatechin tetramer linked by C4–C8 bonds. A2 is present as an astringent substance in cocoa, red wine, immature apples, pine bark, and some other foods with (−)-epicatechin.^(7–9) The epicatechin tetramer cinnamtannin A2 (A2) has been described as a powerful bioactive compound promoting thermogenesis,⁽¹⁰⁾ glucagon-like peptide-1,⁽¹¹⁾ and a marked increase in skeletal muscle blood flow.⁽¹²⁾

Fig. 1.

Chemical structure of cinnamtannin A2 (A2), experimental procedures to examine the effect of repeated administration of 25 μg/kg cinnamtannin A2 for 14 days on the mouse model of disuse atrophy, and weights of skeletal muscles. (A) Chemical structure of A2, (B) experimental procedures, (C) weight of the soleus, (D) weight of the extensor digitorum longus. The values represent the mean ± SD (each group, n = 6). *p<0.05, **p<0.01 vs ground-vehicle, ^#p<0.05 vs suspension-vehicle (two-way ANOVA, followed by the post hoc Dunnett’s test).

An interventional trial reported that dark chocolate, a procyanidin-rich food, exhibits beneficial metabolic changes in skeletal muscle with improved mitochondrial efficiency.⁽¹³⁾ Our previous animal study also revealed that oral doses of cocoa procyanidins enhance energy expenditure and promote mitochondrial biogenesis in skeletal muscle.⁽¹⁴⁾ Furthermore, we found that oral administration of a single oral dose of A2 transiently increased plasma catecholamine levels in the blood.⁽¹⁰⁾

Catecholamines (CAs) are secreted from the end of the sympathetic nerve or adrenal medulla to the organ or blood following activation of the sympathetic nerve system (SNS),⁽¹⁵⁾ resulting in the activation of adrenergic receptors expressed by skeletal muscle. Plasma CAs comprising 60–70% sulfated conjugated metabolites are excreted into the urine. The change in SNS activity can be estimated by the CA level in urine in mice.⁽¹⁵⁾

In skeletal muscle, dominantly expressed β2 adrenaline receptors bind with plasma CA partially regulating protein synthesis and/or protein degradation,⁽¹⁶⁾ which is an independent pathway of insulin-like growth factor (IGF)-I.⁽¹⁷⁾ Clenbuterol, a β-adrenergic agonist, has been reported to induce skeletal muscle hypertrophy or anti-atrophy through the β2 adrenaline receptor.^(18,19)

In the present study, we examined the impact of repeated oral administration of A2 on a mouse model of disuse muscle atrophy induced by HLS in the soleus used by the method of histochemistry. Besides, we also measured the protein expressions or phosphorylation involved in proteosynthesis (IGF-1 receptor, protein kinase B (Akt) or eukaryotic translation initiation factor 4E-binding protein 1; 4EBP1) and proteolysis (forkhead box O3a; FoxO3a and ubiquitin). In addition, we also determined the changes in urinary CAs, which may have a role in this impact following a single oral administration of A2.

Materials and Methods

Materials

A2 was obtained from Phytolab GmbH & Co., KG (Vestenbergsgreuth, Germany). The other chemicals were purchased from FUJIFILM Wako Pure Chemicals Corporation (Tokyo, Japan). The chemical structure of A2 is shown in Fig. 1A.

Animals and diets

The study was approved by the Animal Care and Use Committee of the Shibaura Institute of Technology (Permit Number: AEA19016). All mice received humane care under the National Institutes of Health guide for the care and use of laboratory animals in this institution. All surgery was performed under anesthesia, and all efforts were made to minimize suffering. Male C57BL/6J mice aged 17 to 19 weeks were obtained from Charles River Laboratories Japan, Inc. (Tokyo, Japan). The mice were kept in a room with controlled lighting (12/12 h light/dark cycles) at a regulated temperature of 23–25°C. A certified rodent diet (MF^®) was obtained from Oriental Yeast Co., Ltd., Tokyo, Japan).

Impact of repeated oral dose of A2 on disuse atrophy induced by hindlimb suspension in mice

Twenty-four mice were fed a basal diet for 7 days and divided randomly into four groups: ground-vehicle, ground-A2, suspension-vehicle, and suspension-A2. Mice in the vehicle treatment groups were orally administered 20% glycerol in saline, whereas animals in A2 treatment groups were dosed with A2 dissolved in the vehicle via gavage administration (25 μg/kg body weight/day) for 14 days (Fig. 1B). Hindlimb unloading was performed using a tail suspension clip (MSR2015, Yamashita Giken Co.Ltd., Tokushima, Japan) equipped with a block that was movable in a vertical direction.⁽²⁰⁾ At the end of this treatment period, the animals were sacrificed under anesthesia with a mixture of the following agents (10 ml/kg, ip); medetomidine (0.75 mg/10 ml in saline, Nippon Zenyaku Kogyo Co., Ltd., Fukushima, Japan), midazolam (4 mg/10 ml in saline, Astellas Pharma Inc., Tokyo, Japan), and butorphanol (5 mg/10 ml in saline, Meiji Seika Pharma Co., Ltd., Tokyo, Japan).⁽²¹⁾ Major organs were removed from the mice and weighed. The soleus was stored at −80°C. For histological analysis, the soleus was blocked with FSC 22 Blue (LEICA, Tokyo, Japan) and frozen with isopentane on dry ice, and stored at −80°C until sectioning.

Histological analyses

All samples blocked with FSC were cut into 8-μm-thick sections using the LEICA CM1950 (LEICA, Wetzlar, Germany). Three tissue cross-sections were taken and stained with hematoxylin-eosin (HE). Microscopic observation was carried out with an Olympus CX41 light microscope (Olympus Co., Tokyo, Japan). The muscle cross-sectional area was quantified using ImageJ software (http://rsb.info.nih.gov/ij/index.html, 21 April 2021). The sections were analyzed by three investigators who were blinded to the experimental group or condition. The value of the cross-sectional area was averaged from the results analyzed by the three investigators.

Western blotting

The soleus was homogenized in a microtube with lysis buffer (CelLytic^TM MT cell lysis reagent; Sigma Aldrich, Milwaukee, WI) containing protease inhibitor (Sigma Aldrich, Japan) and (±)-dithiothreitol (Wako, Osaka, Japan) using a Qsonic Model XL-2000 Series Sonicator (Arc Scientific, San Diego, LA). Protein concentrations were determined by Coomassie Blue staining (Coomassie Plus^TM Protein Assay Reagent, Thermo Fisher Scientific, Waltham, MA). Proteins (50 μg) were separated by SDS-PAGE using a 5–12% and 10–20% Bis-Tris gel and transferred onto a 0.45-μm and 0.2-μm pore size polyvinylidene difluoride membrane (Life Technologies, Logan, UT). The membrane was blocked with membrane-blocking reagent (GE Healthcare, Buckinghamshire, UK) for 1 h. After being blocked, the membrane was incubated with a rabbit polyclonal primary antibody against IGF-1 receptor (1:1,000, #3027; Cell Signalling Technology, Inc., Danvers, MA), α-tubulin (1:1,000, ab4074; Abcam), or phospho-Akt (1:1,000, ab81283; Abcam, Cambridge, UK), Akt (1:2,000, ab28422; Abcam), phospho-4EBP1 (1:1,000, #2855; Cell Signalling Technology, Inc., MA), 4EBP1 (1:1,000, #9452; Cell Signalling Technology, Inc.), FoxO3a (1:1,000, #2497; Cell Signalling Technology, Inc.), phospho-FoxO3a (1:1,000, #13129; Cell Signalling Technology, Inc.), or ubiquitin (1:1,000, #3933; Cell Signalling Technology, Inc.) at 4°C overnight. After the primary antibody reaction, the membrane was incubated with appropriate horseradish peroxidase-conjugated secondary antibodies (1:100,000, Anti-Rabbit IgG, HRP-Linked Whole Ab Sheep, NA931, GE Healthcare, Buckinghamshire, UK) for 1 h. Immunoreactivity was detected by chemiluminescence using the ECL Select^TM Western Blotting Reagent (GE Healthcare, Chicago, IL). Chemiluminescent bands were detected and analyzed by the C-DiGit Blot Scanner (LI-COR Technology, Lincoln, NE).

Effect of a single dose of A2 on the excretion of catecholamines in urine

After being fed a basal diet for 14 days, sixteen mice were randomly divided into two treatment groups: vehicle (20% glycerol in saline or 25 μg/kg A2. Animals were placed in individual metabolic cages (Fig. 4A) and allowed free access to food and water. Following a 48-h habituation period, 24-h urine was collected post-oral administration of vehicle or A2 using a tube containing 20 μl of 2.5 mol/L HCl (Fig. 4B). The oral administration of vehicle or A2 was carried out between 10:00 and 11:00 am.

Fig. 4.

Scheme for the experimental procedure to determine urinary catecholamines (CAs) following a single dose of 25 μg/kg A2 in mice. (A) Scheme for the experimental procedure, (B) Photo of metabolic cage, (C) amount of 24-h urinary CAs. The values represent the mean ± SD (each group, n = 8). *p<0.05, **p<0.01, ***p<0.001 (Student’s t test).

Analysis of catecholamines in urine

We determined the concentration of urinary CAs (noradrenaline and adrenaline) and their metabolites by treatment with sulfatase from Helix pomatia Type H-2 (Sigma Aldrich, St. Louis, MO) according to a previous report.⁽²⁰⁾ Briefly, the urine was heated for 10 min following incubation with 500 U/ml of the enzyme at 37°C for 1 h. After the addition of isoprenaline (Sigma Aldrich, Milwaukee, WI) as the internal standard, CAs were purified using Monospin PBA^® (GL Sciences, Tokyo Japan). The HPLC system (Prominance HPLC System Shimazu Corporation, Kyoto Japan) consisted of a quaternary pump with a vacuum degasser, thermostatted column compartment, and an autosampler equipped with an electrochemical detector (ECD 700 S; Eicom Corporation, Kyoto, Japan). A reverse-phase column (Inertsil ODS-4, 250 × 3.0 mm ID, 5 μm; GL Sciences) was used and the column temperature was maintained at 35°C. The HPLC mobile phase was 24 mM acetate-citrate buffer (pH 3.5, -CH₃CN, 100/14.1 v/v). The mobile phase flow rate was 0.3 ml/min and the injection volume 20 μl. The eluents were detected and analyzed at 500 mV. Excretion of CA was expressed as a ratio with the urinary creatinine concentration measured using Laboassay creatinine (FUJIFILM Wako Pure Chemical Corporation).

Statistical analysis

All data were reported as mean ± SD with individual data. Statistical analyses were performed by Student’s t test, two-way ANOVA followed by post hoc comparisons between experimental groups using Dunnett’s test or non-parametric Wilcoxon and Mann–Whitney U tests using statistical software R ver. 4.1.3. A probability of p<0.05 was considered significant.

Results

Impact of repeated oral dose of A2 on disuse atrophy induced by hindlimb suspension in mice. The weight of the soleus (Fig. 1C) in the suspension-vehicle group was significantly decreased compared to the ground-vehicle group but this change was not observed in the extensor digitorum longus (Fig. 1D). Repeated oral administration of A2 reduced this change. The other weight of tissues was shown in Supplemental Table 1*.

We observed significant muscle atrophy in the soleus with 40% weight reduction compared to the ground-vehicle group after 14 days of HLS. In contrast, this change was markedly decreased by repeated oral doses of A2 (−25% vs ground-vehicle). In addition, the average myofibre size was markedly decreased in the suspension-vehicle group compared to the ground-vehicle group (−31%) along with a shifting peak to the smaller side. But these changes were reduced by treatment with A2 (−14% vs ground-vehicle in average myofibre size). Interestingly, the average myofibre size increased by 34.0% in the A2-vehicle group compared ground-vehicle group with a shift in the peak to a larger side.

We determined the level of IGF-1 receptor and ubiquitin, the ratio of p-Akt/Akt, p-4EBP1/4EBP1, and FoxO3a/p-FoxO3a in the soleus by western blotting (Fig. 3). There were no significant changes in the level of IGF-1 receptor and ubiquitin. The phosphorylation of Akt was increased slightly in the suspension-vehicle group, and significantly in groups by the treatment of A2 both with or without suspension. 4EBP1 phosphorylation was significantly increased in the ground-A2 group compared to the ground-vehicle group. The ratio of FoxO3a/p-FoxO3a was significantly increased in the suspension-vehicle group compared to the ground-vehicle group and this change was significantly reduced by A2 treatment.

Fig. 3.

Impact of repeated oral administration of 25 μg/kg cinnamtannin A2 (A2) on selected protein levels involving proteosynthesis and proteolysis in the soleus in the mouse model of disuse atrophy. IGF-1R, insulin-like growth factor-1 receptor; 4EBP1, eukaryotic translation initiation factor 4E-binding protein 1; FOXO, forkhead box-containing protein O. The values represent the mean ± SD (each group, n = 6). *p<0.05, **p<0.01, ***p<0.001 vs ground-vehicle, ^#p<0.05, ^##p<0.01 vs suspension-vehicle (two-way ANOVA, followed by the post hoc Dunnett’s test).

Urinary catecholamine excretion after a single oral dose of A2. The amount of excreted CAs in 24-h urine following a single dose of vehicle or 25 μg/kg A2 is shown in Fig. 4C. The marked increase in noradrenaline, adrenaline and total CAs was observed in 24-h urine following a single oral dose of A2.

Discussion

HLS is known to cause significant skeletal muscle atrophy, especially in slow-twitch muscle in mammals.⁽⁵⁾ We observed significant muscle atrophy with a 40% weight reduction in the soleus compared to the ground-vehicle group after 14 days of HLS. In contrast, this change was markedly decreased by repeated oral doses of A2 with this weight loss (−25% vs ground-vehicle). In addition, the average myofibre size was significantly reduced in the suspension-vehicle group compared to the ground-vehicle group (−31%), along with a shifting peak to the smaller side. But these changes were reduced by treatment with A2 (−14% vs ground-vehicle in average myofibre size). Interestingly, the average myofibre size increased by 34.0% in the A2-vehicle group compared ground-vehicle group, with a shift in the peak to a larger side.

In addition, A2 significantly increased Akt phosphorylation with or without suspension, accompanied by the phosphorylation of 4EBP1 was raised in the ground-A2 group compared to the ground-vehicle group (Fig. 3). Skeletal muscles adapt to changes in their workload by regulating fiber size via Akt/mechanistic target of rapamycin (mTOR), an anabolic signal, and the FoxO pathway, a catabolic signal.⁽²²⁾ The Akt/mTOR pathway is a key regulator in the translation initiation step of protein synthesis in skeletal muscle. Under normal conditions, IGF-1 and its receptor activate phosphatidylinositol-3-kinase (PI3K), generating phosphoinositide-3,4,5-triphosphate (PIP3), which acts in the subsequent phosphorylation of Akt. Phosphorylated Akt further activates mTORC1, which phosphorylates both 4EBP1 and ribosomal protein S6 kinase β-1, consequently leading to proteosynthesis.⁽²³⁾

Besides, FoxO3 was identified as the main transcription factor regulating ubiquitin ligase expression and autophagy coordinating the proteasomal-dependent removal of proteins.⁽²⁴⁾ In addition, FoxO3 transcription was reported to be inhibited by the activation of Akt. In the present results, FoxO3a/p-FoxO3a was markedly increased following HLS, and this change was reduced by the administration of A2. Taken together, our findings suggest that repeated supplementation with A2 inhibits activation of the catabolic FoxO pathway through accelerated Akt phosphorylation. On the other hand, adrenergic signals via β2-adrenergic receptors were reported to be a pathway to regulate skeletal muscle mass independent of IGF-1 and its receptor.⁽¹⁷⁾

In previous studies, A2 exhibited potent bioactivity showing increased peripheral blood flow and thermogenic alteration for brown adipose.^(10,12) We also found that these changes were reduced by co-treatment with adrenaline blockers. While the activation of SNS induced by exercise, cold exposure, or ingestion of spice is well known to alter hemodynamics and metabolism through CAs secreted for organs or blood.⁽²⁵⁾ Therefore, we measured the concentration of urinary CAs, which surrogated CAs in the blood and confirmed a significant increase in CAs excretion in 24-h urine following oral administration of A2 in this study (Fig. 4). To summarize the results of this study with these previous studies suggested that repeated oral doses with A2 induced the anabolic Akt/mTOR pathway in the soleus by activating the β2-adrenergic receptor via CAs secreted from the adrenal medulla to blood.

Recently, it has been reported that the consumption of various polyphenols induces alterations in skeletal muscle metabolism.⁽²⁶⁾ (−)-Epicatechin,⁽²⁷⁾ epigallocatechin gallate⁽²⁸⁾ or theaflavin,⁽²⁹⁾ which are classified as flavanols as well as A2, was reported to reduce the oxidative myolysis induced by HLS and may delay the progression of disuse muscle atrophy. However, further studies are needed to determine whether these flavanols modify skeletal muscle metabolism by a similar mechanism as A2 through SNS.

There are three major limitations in this study. First, this study focused on biochemical and morphometric alteration, but it is also necessary to examine skeletal muscle functions. Second, the soleus muscle of mice is known to contain both myosin heavy chain (MHC) I, the slow muscle component, and MHC II, the fast muscle component.⁽³⁰⁾ To clarify whether A2 affects both or either of them needs to be verified using immunostaining techniques. Third, to elucidate the SNS-mediated muscle hypertrophic activity of A2, it is necessary to observe downstream signals of adrenergic receptor activation in skeletal muscle, i.e., activate protein kinase A (PKA) and cAMP response element binding protein (CREB).⁽³¹⁾ These possibilities will be the subject of further studies.

In conclusion, we found that repeated doses of A2 reduced the disuse of muscle atrophy induced by HLS, additionally, A2 has the possibility to induce hypertrophy in the soleus. Repeated oral doses of A2 were suggested to induce both inhibitions of proteolysis and enhancement of proteosynthesis through the PIK3/Akt/FoxO pathway. We also found that a single oral dose of A2 increased urinary CAs excretion which reflected SNS activation. These results suggested that SNS hyperactivity was involved with the inhibition of skeletal muscle atrophy, and inducing hypertrophy, following the administration of A2 as summarized in Fig. 5. Further experiments are needed to elucidate the mechanisms of A2, including activation of the SNS.

Fig. 5.

Possible mechanism of cinnamtannin A2 (A2) on skeletal muscle. The repeated oral dose of A2 induced hyperactivity in the sympathetic nervous system, subsequently increasing catecholamine in blood activates the β2 adrenaline receptor expressed in the soleus. As a result, the activation of the Akt/mTORC1 pathway downstream of the β2 adrenaline receptor and the inactivation of FoxO3a is induced, resulting in an increase in skeletal muscle protein, resulting in inhibition of muscle atrophy or inducing hypertrophy.

Author Contributions

Conceptualization, RA and NO; Data curation, SO and OM; Formal analysis, TF and YF; Investigation, SO, KShimizu, KSuzuki, MO, SK and OM; Methodology, SO, OM, and YF; Project administration, RA and NO; Supervision, NO.; Validation, TF; Visualization preparation, SO and OM; Writing, NO.

Funding

This work was supported by JSPS KAKENHI (Grant Number: JP19H04036).

Fig. 2.

Histochemical observations following repeated oral administration of 25 μg/kg cinnamtannin A2 (A2) for 14 days on the soleus in the mouse model of disuse atrophy. (A) Histochemical observation of soleus, (B) average size of myofibres, (C) distribution of myofibre size. The values represent the mean ± SD (each group, n = 6). ***p<0.001 vs ground-vehicle, ^#p<0.05 vs suspension-vehicle (Wilcoxon and Mann–Whitney U tests).

Abbreviations

A2

cinntamtannin A2

CA

catecholamine

4EBP1

eukaryotic translation initiation factor 4E-binding protein 1

FoxO

forkhead box O

HE

hematoxylin-eosin

HLS

hindlimb suspension

IGF

insulin-like growth factor

MHC

myosin heavy chain

mTOR

mechanistic target of rapamycin

PI3K

phosphatidylinositol-3-kinase

PIP3

phosphoinositide-3,4,5-triphosphate

SNS

sympathetic nerve system

Conflict of Interest

No potential conflicts of interest were disclosed.