Stimulatory Effects of (+)-Epicatechin on Mitochondrial Biogenesis and Function in Skeletal Muscle of Aged Rats: Underlying Mechanisms

Abstract

Mitochondrial dysfunction affects skeletal muscle (SkM) function and is critical in the etiology of age-related sarcopenia. The sirtuin 1-PGC1α pathway is a key regulator of mitochondrial mass, structure, and function. However, pathway activity decreases with aging. Cacao flavanols show promise in their ability to activate mitochondrial pathways. We evaluated the capacity of the flavanol (+)-epicatechin (+Epi) to stimulate such a pathway and favorably impact mitochondrial and oxidative stress (OS)-associated endpoints in aged SkM. Using 23-month-old male Sprague-Dawley rats, an 8-week oral administration of +Epi (1 mg/kg/day) was implemented, and results were compared versus vehicle-treated controls. Assessments included the nicotinamide adenine dinucleotide (NAD)/sirtuin 1/PGC1α pathway, acetylated proteins levels, mitochondrial function and biogenesis, as well as OS-related endpoints in SkM. +Epi increased the NAD/NADH ratio, activation of sirtuin 1, the deacetylation of nuclear protein content, including that of PGC1α. Also, +Epi induced increases of TFAM and NRF1 mRNA levels, deacetylation of mitochondrial complex I and V, increases in complex I activity, sirtuin 3, complexes I and V, mitofilin, and TFAM protein levels. SkM citrate synthase activity and ATP content increased with +Epi. OS markers in proteins and lipids were reduced, while buffering systems (superoxide dismutase 2 and catalase protein and activities) increased. In white blood cells, we documented serial reductions in mitochondrial DNA content and citrate synthase activity with aging, which were either fully or partially reversed with +Epi. Results demonstrate that +Epi treatment yields positive effects on mitochondrial biogenesis and function, leading to decreased OS and improved SkM bioenergetics in aged rats.

Keywords

atrophy epicatechin mitochondria

INTRODUCTION

Aging is associated with a loss of skeletal muscle (SkM) mass (sarcopenia), leading to a progressive reduction in functional capacity.¹ It is well established that with aging, reductions in mitochondrial mass and function, and thus ATP generation underpins in many organs losses in function as seen in SkM and kidney.² Aging-associated mitochondrial dysfunction is also linked with the production of excess free oxygen radicals (i.e., oxidative stress; OS) and inflammation.³ It is proposed that the sustained presence of such processes likely facilitates and/or promotes a progressive loss of SkM mass.¹ Conversely, to the extent that mitochondrial dysfunction can be prevented or reversed, a preservation of SkM mass and function is likely to occur. Currently, only exercise has been demonstrated to limit aging-associated loss of SkM, yielding significant reductions in functional decline.⁴ However, in older individuals a sustained exercise regimen can prove to be difficult to achieve and/or sustain as per the presence of diseases such as osteoarthritis. Thus, there is great interest in identifying safe and effective alternatives for the targeting of sarcopenia.

The dysregulation of protein acetylation/deacetylation rates in mitochondria appears as an important cause for organelle dysfunction.^5,6 With aging, protein acetylation levels increase, leading to perturbations in mitochondrial homeostasis.^5,6 In cells, protein deacetylation is in part executed by sirtuins, which are nicotinamide adenine dinucleotide (NAD)+ dependent protein deacetylase enzymes.⁶ While sirtuins can modulate mitochondrial function, they also influence the production of free radicals as per their ability to regulate buffering systems.⁷ The protein sirtuin 1, which is located in the nucleus, is strongly expressed in SkM and plays a pivotal role in transcription factor deacetylation and of other key proteins.⁷ Sirtuin-1 is involved in the modulation of multiple cellular processes, including metabolism, DNA repair, inflammation, and aging,⁷ and as such has been identified as an important therapeutic target. Mitochondrial biogenesis is also recognized as being partly regulated in the nucleus by the sirtuin 1-PGC1α axis, primarily because the sirtuin deacetylates PGC1α and increases its transcriptional activity⁸ leading to the stimulation of this process. Inside mitochondria, sirtuin 3 deacetylates enzymes and proteins in the citric acid cycle and electron transport chain, thereby promoting oxidative phosphorylation.⁹ Another key role of sirtuin 3 is to deacetylate superoxide dismutase 2 (SOD2), enhancing its enzymatic activity.¹⁰ Sirtuin enzymatic activity is dependent on adequate levels of its substrate NAD^+.6 It has been documented that the deacetylation capacity of sirtuins decreases with aging due in part, to a diminished cellular NAD⁺ pool.¹¹ Several experimental approaches have been employed to sustain cellular NAD⁺ levels, and therefore, boost sirtuin deacetylation in order to ameliorate mitochondrial dysfunction, including the pharmacological inhibition of CD38 (one of the most NAD⁺-utilizing enzymes), caloric restriction, fasting, exercise training, and resveratrol treatment.¹² However, there is a limited amount of knowledge on the impact of targeted treatments on NAD⁺, sirtuins, and relevant protein activity status in aged SkM mitochondria-related endpoints.

We previously reported on the stimulatory effects of the flavanol (-)-epicatechin (Epi) on SkM mitochondrial biogenesis, function, and OS buffering systems in conditions such as aging and obesity or in the setting of diseases such as muscular dystrophy or Gulf War illness.^13–16 We also recently reported on the capacity of the epimer +Epi to reverse SkM atrophy and capillary rarefaction in 25 month old male rats.^17,18 Using samples from the same cohort of aged rats, we wished to evaluate the capacity of +Epi to improve mitochondrial indicators of organelle density and function as well as of OS status and associated buffer system-related endpoints. We also explored for underlying mechanisms associated with mitochondrial stimulatory effects with an emphasis on the regulation of the sirtuin 1-PGC1α axis, while blood samples were used to explore select relevant endpoints and the effects of treatment.

MATERIALS AND METHODS

Please see the Supplementary Data for detailed materials and methods.

RESULTS

Effects on NAD/NADH and deacetylation activity

Figure 1 denotes the levels of NAD⁺ (A), NADH (B), as well as the NAD⁺/NADH ratio (C) in SkM. Animals treated with +Epi demonstrated an ∼80% increase in NAD⁺, no difference in NADH, and therefore, an elevated (>100%) NAD⁺/NADH ratio vs. controls. Assessment of nuclear NAD⁺-dependent deacetylase sirtuin 1 activity (phosphorylation) was evaluated by Westerns after nuclear protein isolation from SkM (D). +Epi-treated animals demonstrated ∼60% higher levels of phosphorylation vs. controls. Nuclear protein acetylation levels were evaluated by Westerns using an anti-acetylated lysin antibody (E). Control animals demonstrated increased levels of total nuclear protein acetylation vs. +Epi-treated rats, which were reduced by ∼50%.

FIG. 1.

Effects of +Epi on aged gastrocnemius NAD⁺, nuclear SIRT1 activation and nuclear protein acetylation levels. (A, B, and C) report on gastrocnemius content of NAD⁺ (A), NADH, (B) and NAD⁺/NADH ratio (C) in control and +Epi groups. Panel (D) illustrates SIRT1 activation (phosphorylation) and (E) reports on nuclear protein acetylation by Westerns. Data are mean ± SEM, n = 6/subgroup. *P < .05 by t-test.

Effects on regulators of mitochondrial biogenesis

Acetylated PGC1α was determined after nuclear protein isolation and lysin immunoprecipitation (Fig. 2A) and was quantified to report relative amounts using nuclear PGC1α and tata-binding protein (TBP) as nuclear and protein loading controls (B). Results demonstrate ∼70% decreased level of acetylated PGC1α in +Epi-treated animals vs. controls. Figures 2C and D report on the relative amount of TFAM and NRF1 mRNA levels. +Epi-treated animals demonstrated ∼50% increased amounts for both mRNAs. mtDNA copy number (E) shows a trend for increased levels in +Epi-treated animals.

FIG. 2.

Effects of +Epi on aged on gastrocnemius acetylated PGC1α, TFAM, NRF1 mRNAs and mtDNA levels. (A and B) illustrate and report on gastrocnemius nuclear acetylation status of PGC1α. (C and D) denotes mRNA levels of TFAM and NRF1. (E) illustrates mtDNA levels in control and +Epi groups. Data are mean ± SEM, n = 6/subgroup. *P < .05 by t-test.

Effects on mitochondrial structure and function

The NAD⁺-dependent deacetylase sirtuin 3 was evaluated in SkM by Westerns (Fig. 3). +Epi-treated animals demonstrated ∼35% increased amount of sirtuin 3 versus controls (A). Acetylated mitochondrial complexs I and V were determined after lysin immunoprecipitation (B). +Epi-treated animals demonstrated ∼50% decreased amounts of acetylated complex I and acetylated complex V vs. controls. Mitochondrial complex I activity was evaluated (C) and +Epi increased it by ∼100%. Figure 4 reports on complex I and V, mitofilin, and TFAM relative protein levels as evaluated by Westerns. Results demonstrate ∼20–30% increased content for such proteins in SkM of +Epi treated animals vs. controls (A). Mitochondrial function surrogates citrate synthase activity (B) and ATP content (C) denoted increases of > 200% and ∼90%, respectively, with +Epi treatment.

FIG. 3.

Effects of +Epi on aged gastrocnemius SITR3, acetylated mitochondrial complex I and V and complex I levels and/or activity. (A) report on gastrocnemius SIRT3 levels. (B) reports on complex I and V acetylation status. (C) illustrates the complex I activity in control and +Epi groups. Data are mean ± SEM, n = 6/subgroup. *P < .05 by t-test.

FIG. 4.

Effects of +Epi on aged gastrocnemius mitochondrial proteins and function. (A) illustrates gastrocnemius complex I, V, mitofilin, and TFAM relative protein levels by Westerns. (B) reports on citrate synthase activity and (C) on ATP content in control and +Epi groups. Data are mean ± SEM, n = 6/subgroup. *P < .05 by t-test.

Effects on OS and buffering systems

Figure 5 denotes the assessment of OS-related endpoints in plasma and gastrocnemius of aged rats. Panel A illustrates a time course analysis of protein carbonylation in plasma of rats at 6, 12, 18, 23, and 25 months of age. From the 6 to 25-month time point, plasma carbonyl protein content was consistently increased to more than double over time. However, 8 weeks of +Epi treatment in rats (from 23 to 25 month) yielded significant decreases of ∼40% for this endpoint at the end of treatment. Panel B illustrates protein carbonyl content, while panel C denotes malondialdehyde levels in gastrocnemius. +Epi-treated animals demonstrated ∼60% decreased content of both OS markers in gastrocnemius vs. controls. Panel D reports on total levels of oxidized proteins analyzed by Westerns. +Epi-treated animals demonstrated ∼50% decreased oxidized protein content in SkM versus controls. Figure 6 denotes antioxidant enzyme assessment in gastrocnemius of aged rats. Panel A illustrates SOD2 and catalase content evaluated by Westerns. +Epi-treated animals demonstrated ∼100% increased content of both antioxidant enzymes in gastrocnemius vs. controls. Panels B and C illustrate SOD2 and catalase enzyme activity in gastrocnemius. +Epi-treated animals demonstrated increased activity of ∼60% and ∼100%, respectively, versus controls.

FIG. 5.

Effects of +Epi on oxidative stress markers in plasma and gastrocnemius of aged rats. (A) illustrates protein carbonylation content in a time course (6–25 months) assessment in plasma. (B) reports on protein carbonylation and panel (C) reports on malondialdehyde levels in gastrocnemius. (D) reports on relative protein oxidation levels in gastrocnemius. Data are mean ± SEM, n = 6/subgroup. *P < .05 by t-test.

FIG. 6.

Effects of +Epi on antioxidant buffering systems of aged muscle. (A) illustrates on superoxide dismutase 2 and catalase protein levels by Westerns (left panel) and their relative amounts (right panel). (B) reports on superoxide dismutase 2 and panel C reports on catalase activities in muscle of control and +Epi groups. Data are mean ± SEM, n = 6/subgroup. *P < .05 by t-test.

Effects on mitochondria white blood cell endpoints

Figure 7 denotes the serial assessment of mitochondrial DNA copy number (mtDNA) and citrate synthase activity in white blood cells collected at 6, 12, 18, 23, and 25 months of age. From the 6 to 12-month time point, mtDNA content was unchanged (A). However, from 12 to 25 months of age, mtDNA decreased over time (A). The 8 weeks of +Epi intervention in rats at the 23-month time point yielded a trend for increases in mtDNA until the end of treatment (25 months of age). Panel B illustrates a time course analysis of citrate synthase in white blood cells of rats at 6, 12, 18, 23, and 25 months of age. From the 6 to 25-month time point, citrate synthase activity decreased by ∼60% over time. However, 8 weeks of +Epi treatment yielded a significant ∼100% increase in activity by 25 months of age.

FIG. 7.

Effects of +Epi on white blood cell mitochondrial DNA and citrate synthase activity of aged rats. (A) illustrates the content of mitochondrial DNA copy number in white blood cells in a time course assessment as determined by by qPCR. (B) reports on citrate synthase activity in white blood cells in control and +Epi groups. Data are mean ± SEM, n = 6/subgroup. *P < .05 by t-test.

DISCUSSION

Unique results from this study demonstrate that in a rat model of aging-induced sarcopenia, +Epi increases the SkM NAD/NADH ratio, sirtuin 1 activity, and consequently, the deacetylation of nuclear proteins, including PGC1α. Effects are accompanied by increases in TFAM and NRF1 mRNA levels, increases in sirtuin 3, complex I and V, mitofilin and TFAM protein levels, deacetylation of mitochondrial complexes I and V, and increased complex I activity. SkM citrate synthase activity and ATP content increase. OS was reduced while buffering system protein levels and activities increased. In white blood cells, we also documented serial reductions in mtDNA content and citrate synthase activity with aging, which were partially or fully reversed with +Epi. Altogether, the concerted activity of such biological actions suggests that part of the beneficial effects of +Epi on sarcopenia relates to the favorable activation of mitochondria-related axis.

Aging-associated conditions and/or diseases have become a great burden to health care systems, and for many of these entities, therapeutic options remain limited. Sarcopenia in particular, has gained high levels of attention as per its close relationship with frailty, falls, and consequently, higher mortality.¹⁹ While exercise represents a useful tool to counter sarcopenia, its widespread use is limited by conditions such as osteoarthritis and obesity or access to suitable training facilities.²⁰ Thus, the high level of interest in identifying candidate agents that target sarcopenia. In aging populations, agents that can be provided orally and carry exceptional safety profiles would be preferred. Flavonoids are polyphenolic compounds found ubiquitously in a wide range of plants, fruits, and vegetables.²¹ Many of the positive health effects derived from the consumption of such products are attributed to the presence of flavonoids. Evidence is derived from epidemiological studies that associate that regular consumption of products rich in flavonoids with decreased cardiometabolic and other chronic disease risk.^22–24 Thus, the interest in identifying and isolating such agents so as to explore their pharmacological properties, therapeutic potential, and mechanisms of action.²¹

Over the course of several years, a large body of related literature has been generated whereby specific flavonoid isolates have been examined for their potential to exert positive effects on various systems, conditions, and/or diseases.²¹ We recently published a review and meta-analysis on preclinical and clinical studies reporting on the beneficial effects of specific polyphenolic compounds or flavonoids on SkM health.²⁵ It is readily apparent that compounds such as quercetin and green tea catechins have the capacity to favorably impact SkM health, including mass and function. However, there is a lack of a systematic analysis for specific flavonoids, and there can be large discrepancies on key issues such as the doses used, types of outcomes reported, and clinical applicability.

Cacao contains high amounts of the flavonoid Epi, and the health-promoting effects of the moderate consumption of cocoa products are linked to the actions of this compound. Kuna Indians who regularly consume a cacao-based beverage are protected against age-dependent reductions in vascular and renal function²⁶ and demonstrate extremely low mortality indices for chronic diseases.²⁷ In support of these observations, clinical studies have demonstrated reductions in blood and organ inflammation and OS levels with cocoa/dark chocolate consumption.²⁸ We have previously explored the potential of +Epi to exert stimulatory effects under controlled conditions on cultured SkM cells with promising results.²⁹ Furthermore, in humans and animal models, we examined the pharmacokinetic and partial pharmacodynamic properties of +Epi, with recent results suggesting a higher efficacy profile vs. the epimer -Epi.³⁰ Using SkM samples obtained from the same-aged animals used in this study, we previously reported that treatment with +Epi led to significant increases in front paw grip strength, treadmill time, and muscle mass vs. controls.¹⁷ Treatment also led to the activation of key intracellular pathways associated with the regulation of muscle mass. +Epi also significantly reversed adverse changes in plasma and/or SkM TNF-α, IGF-1, atrophy and protein synthesis related endpoints vs. controls. In a separate recent publication, using the same samples we also reported on the angiogenic effect of +Epi on SkM, whereby increases in capillary density were reported vs. controls an action, that was linked to the activation of the VEGF signaling pathway.¹⁸ As a whole, stimulatory effects on angiogenesis and atrophy-related pathways appear to provide a coherent and integrative framework for how +Epi can effectively translate its effects into enhanced structure and function in the setting of aging-induced sarcopenia.

However, strong biological links have been reported between mitochondrial status and changes in muscle mass. In animal models, causal relationships have been reported whereby increased mitochondrial density via the forced expression of the organelles leads to the stimulation of myofiber formation.³¹ In a study by Always et al., after the induction of SkM cryoinjury in mice, the transplantation of mitochondria led to an enhanced restoration of muscle mass and function.³² Conversely, in conditions whereby a loss of mitochondria density, structure, and/or function takes place, muscle atrophy can ensue.^33,34 In humans, studies have linked impaired mitochondrial structure, function, or density with sarcopenia, while positive effects on muscle mass have been linked to the beneficial effects of exercise on this organelle.^4,35 Thus, mitochondria appear to play critical roles in determining the preservation of muscle mass and function as per it’s capacity to produce ATP and ameliorate OS levels. Despite age-associated mitochondrial dysfunction being extensively investigated, the precise mechanisms that drive such changes remain to be determined. Candidate mechanisms to drive such detrimental changes may include decreased mitochondrial biogenesis, function, and/or diminished mitochondrial DNA levels.³⁵

Accumulating evidence suggests that sirtuin 1 and PGC-1α play a key role as upstream activators of mitochondrial biogenesis.⁸ Sirtuin 1-mediated deacetylation activates PGC-1α through a functional protein-protein interaction, and once PGC-1α is active, it works in concert with several transcription factors to stimulate the expression of a broad set of mitochondrial genes.^36,37 These transcriptions factors include NRF-1 and -2 whose activation increases the expression of multiple mitochondrial proteins, including oxidative phosphorylation complexes and TFAM.³⁸ Reduced levels of PGC-1α have been found in aged animals, and loss of PGC-1α has been suggested to be an important contributor to mitochondrial dysfunction in aging-associated diseases.³⁹ As per the results reported in this study, +Epi activates the sirtuin 1 and PGC-1α mitochondrial biogenesis-related pathway, leading to enhanced levels of key mitochondrial proteins, which ultimately translate into improved bioenergetics given the noted increases in citrate synthase activity and SkM ATP levels.

Using aged mice, we previously reported that 2 weeks of -Epi treatment of aged (26-month-old) mice reduces SkM OS levels as determined by protein-free carbonyl content while restoring the buffering system levels toward those noted for young animals.¹⁴ Using a high-fat diet mouse model of obesity and insulin resistance, we also previously reported increases in SkM protein carbonylation and nitrotyrosilation, as well as a decrease in SOD2 and catalase activity.⁴⁰ -Epi treatment reversed such changes toward those of normal mouse muscle. Thus, Epi has the capacity to effectively modulate ROS buffering systems in aged or diseased SkM that translate into reduced levels of OS. The deacetylase sirtuin 3 which is primarily located in mitochondria, is known to play an important role on the regulation of ROS buffering systems. A major target of sirtuin 3 deacetylation is SOD2.¹⁰ In this study, +Epi treatment increased sirtuin 3, SOD2, and catalase protein levels in aged SkM, documenting the manner by which +Epi led to reduced OS levels.

With the intent of documenting broader effects of +Epi beyond those noted for SkM, we also evaluated changes in PBMC mtDNA copy number and citrate synthase activity. The analysis of samples collected at 6, 12, and 23 months indicates an age-dependent decrease in such endpoints, which were apparently either fully restored (mtDNA) or partially reversed (citrate synthase activity) with 1 month of treatment with +Epi. Thus, as we have documented in our previous reports, Epi has the capacity to positively impact mitochondria-related system in a broad manner and not act in a tissue-selective manner. This property is seen as advantageous as per the adverse impact that aging has on SkM and other systems such as the vasculature.

CONCLUSIONS

In conclusion, +Epi has the capacity to positively impact on pathways that activate key regulatory systems in mitochondria and ROS buffering systems. Such effects translate into improved SkM bioenergetics and reduced levels of OS. As per the well-documented safety profile of Epi and favorable pharmacokinetics, clinical trials are warranted to explore its potential to be used as an agent that favorably impacts either in a preventive or therapeutic manner, sarcopenia.

AUTHORS’ CONTRIBUTIONS

I.R.-S.: Investigation, data curation, writing—original draft preparation, reviewing, editing. R.O.-R.: Investigation, data curation, writing—original draft preparation. V.N.: Investigation, data curation, writing—original draft preparation. G.C.: Supervision, data curation, formal analysis, original draft preparation, reviewing, editing. F.V.: Conceptualization, funding acquisition, project administration, supervision, data curation, writing—original draft preparation, reviewing, editing.

Footnotes

AUTHOR DISCLOSURE STATEMENT

F.V. (co-founder) and G.C. are stockholders of Epirium Bio Inc.

FUNDING INFORMATION

This study was partly supported by DoD GW180044 and VA I01BX3230, I01BX5894 to Dr. Villarreal and IPN 20195988 and CONACYT 283938 to Dr. Ramirez-Sanchez.

References

Larsson

, Degens

, Li

, et al. Sarcopenia: Aging-related loss of muscle mass and function. Physiol Rev, 2019; 99(1):427–511; doi: 10.1152/physrev.00061.2017

Chistiakov

, Sobenin

, Revin

, et al. Mitochondrial aging and age-related dysfunction of mitochondria. Biomed Res Int, 2014; 2014:238463; doi: 10.1155/2014/238463

Conte

, Martucci

, Chiariello

, et al. Mitochondria, immunosenescence and inflammaging: A role for mitokines? Semin Immunopathol, 2020; 42(5):607–617; doi: 10.1007/s00281-020-00813-0

Alizadeh Pahlavani

, Laher

, Knechtle

, et al. Exercise and mitochondrial mechanisms in patients with sarcopenia. Front Physiol, 2022; 13:1040381; doi: 10.3389/fphys.2022.1040381

Wagner

, Payne

. Widespread and enzyme-independent Nepsilon-acetylation and Nepsilon-succinylation of proteins in the chemical conditions of the mitochondrial matrix. J Biol Chem, 2013; 288(40):29036–29045; doi: 10.1074/jbc.M113.486753

Sack

, Finkel

. Mitochondrial metabolism, sirtuins, and aging. Cold Spring Harb Perspect Biol, 2012; 4(12):a013102; doi: 10.1101/cshperspect.a013102

Michan

, Sinclair

. Sirtuins in mammals: Insights into their biological function. Biochem J, 2007; 404(1):1–13; doi: 10.1042/BJ20070140

Feige

, Auwerx

. Transcriptional coregulators in the control of energy homeostasis. Trends Cell Biol, 2007; 17(6):292–301; doi: 10.1016/j.tcb.2007.04.001

Lombard

, Alt

, Cheng

H-L

, et al. Mammalian Sir2 homolog SIRT3 regulates global mitochondrial lysine acetylation. Mol Cell Biol, 2007; 27(24):8807–8814; doi: 10.1128/MCB.01636-07

10.

Tao

, Coleman

, Pennington

, et al. Sirt3-mediated deacetylation of evolutionarily conserved lysine 122 regulates MnSOD activity in response to stress. Mol Cell, 2010; 40(6):893–904; doi: 10.1016/j.molcel.2010.12.013

11.

Yeo

, Kang

, Ji

. Aging alters acetylation status in skeletal and cardiac muscles. Geroscience, 2020; 42(3):963–976; doi: 10.1007/s11357-020-00171-7

12.

Mouchiroud

, Houtkooper

, Auwerx

. NAD(+) metabolism: A therapeutic target for age-related metabolic disease. Crit Rev Biochem Mol Biol, 2013; 48(4):397–408; doi: 10.3109/10409238.2013.789479

13.

Nogueira

, Ramirez-Sanchez

, Perkins

, et al. (-)-Epicatechin enhances fatigue resistance and oxidative capacity in mouse muscle. J Physiol, 2011; 589(Pt 18):4615–4631; doi: 10.1113/jphysiol.2011.209924

14.

Moreno-Ulloa

, Nogueira

, Rodriguez

, et al. Recovery of indicators of mitochondrial biogenesis, oxidative stress, and aging with (-)-epicatechin in senile mice. J Gerontol A Biol Sci Med Sci, 2015; 70(11):1370–1378; doi: 10.1093/gerona/glu131

15.

Varela

, Rodriguez

, Romero-Valdovinos

, et al. Browning effects of (-)-epicatechin on adipocytes and white adipose tissue. Eur J Pharmacol, 2017; 811:48–59; doi: 10.1016/j.ejphar.2017.05.051

16.

Ramirez-Sanchez

, Navarrete-Yañez

, Garate-Carrillo

, et al. Restorative potential of (-)-epicatechin in a rat model of Gulf War illness muscle atrophy and fatigue. Sci Rep, 2021; 11(1):21861; doi: 10.1038/s41598-021-01093-w

17.

Ramirez-Sanchez

, Navarrete-Yañez

, Ramirez

, et al. Restorative effects of (+)-epicatechin in a rodent model of aging induced muscle atrophy: Underlying mechanisms. Food Funct, 2024; 15(7):3669–3679; doi: 10.1039/d3fo04004f

18.

Ramirez-Sanchez

, Ramirez

, Navarrete-Yañez

, et al. Stimulation of skeletal muscle angiogenesis in aged rats by (+)-epicatechin: Identification of underlying mechanisms. Food Biosci, 2024; 61:104989; doi: 10.1016/j.fbio.2024.104989

19.

Yeung

SSY

, Reijnierse

, Pham

, et al. Sarcopenia and its association with falls and fractures in older adults: A systematic review and meta-analysis. J Cachexia Sarcopenia Muscle, 2019; 10(3):485–500; doi: 10.1002/jcsm.12411

20.

Shen

, Shi

, Nong

, et al. Exercise for sarcopenia in older people: A systematic review and network meta-analysis. J Cachexia Sarcopenia Muscle, 2023; 14(3):1199–1211; doi: 10.1002/jcsm.13225

21.

Ullah

, Munir

, Badshah

, et al. Important flavonoids and their role as a therapeutic agent. Molecules, 2020; 25(22):5243; doi: 10.3390/molecules25225243

22.

Barreca

, Alessandro

, Corrado

. Effects of flavonoids on cancer, cardiovascular and neurodegenerative diseases: Role of NF-kappaB signaling pathway. Int J Mol Sci, 2023; 24(11):9236; doi: 10.3390/ijms24119236

23.

Fang

, Zhao

, Zhang

, et al. Beneficial effects of flavonoids on cardiovascular diseases by influencing NLRP3 inflammasome. Inflammopharmacology, 2023; 31(4):1715–1729; doi: 10.1007/s10787-023-01249-2

24.

Ciumarnean

, Milaciu

, Runcan

, et al. The effects of flavonoids in cardiovascular diseases. Molecules, 2020; 25(18).

25.

Munguía

, Ortiz

, González

, et al. Beneficial effects of flavonoids on skeletal muscle health: A systematic review and meta-analysis. J Med Food, 2022; 25(5):465–486; doi: 10.1089/jmf.2021.0054

26.

McCullough

, Chevaux

, Jackson

, et al. Hypertension, the Kuna, and the epidemiology of flavanols. J Cardiovasc Pharmacol, 2006; 47(Suppl 2):S103–S109; doi: 10.1097/00005344-200606001-00003

27.

Hollenberg

, Fisher

, McCullough

. Flavanols, the Kuna, cocoa consumption, and nitric oxide. J Am Soc Hypertens, 2009; 3(2):105–112; doi: 10.1016/j.jash.2008.11.001

28.

Hooper

, Kay

, Abdelhamid

, et al. Effects of chocolate, cocoa, and flavan-3-ols on cardiovascular health: A systematic review and meta-analysis of randomized trials. Am J Clin Nutr, 2012; 95(3):740–751; doi: 10.3945/ajcn.111.023457

29.

Dugar

, Villarreal

, Hollinger

, et al. 11-beta-hydroxysterols as possible endogenous stimulators of mitochondrial biogenesis as inferred from epicatechin molecular mimicry. Pharmacol Res, 2020; 151:104540; doi: 10.1016/j.phrs.2019.104540

30.

Moreno-Ulloa

, Nájera-García

, Hernández

, et al. A pilot study on clinical pharmacokinetics and preclinical pharmacodynamics of (+)-epicatechin on cardiometabolic endpoints. Food Funct, 2018; 9(1):307–319; doi: 10.1039/c7fo01028a

31.

Lin

, Wu

, Tarr

, et al. Transcriptional co-activator PGC-1 alpha drives the formation of slow-twitch muscle fibres. Nature, 2002; 418(6899):797–801; doi: 10.1038/nature00904

32.

Alway

, Paez

, Pitzer

, et al. Mitochondria transplant therapy improves regeneration and restoration of injured skeletal muscle. J Cachexia Sarcopenia Muscle, 2023; 14(1):493–507; doi: 10.1002/jcsm.13153

33.

Moon

, Dilthey

, Guan

, et al. Genetic deletion of skeletal muscle iPLA(2)gamma results in mitochondrial dysfunction, muscle atrophy and alterations in whole-body energy metabolism. iScience, 2023; 26(6):106895; doi: 10.1016/j.isci.2023.106895

34.

Chen

, Ji

, Liu

, et al. Mitochondrial dysfunction: Roles in skeletal muscle atrophy. J Transl Med, 2023; 21(1):503; doi: 10.1186/s12967-023-04369-z

35.

Bellanti

, Lo Buglio

, Vendemiale

. Mitochondrial Impairment in Sarcopenia. Biology (Basel), 2021; 10(1):31; doi: 10.3390/biology10010031

36.

Nemoto

, Fergusson

, Finkel

. SIRT1 functionally interacts with the metabolic regulator and transcriptional coactivator PGC-1alpha. J Biol Chem, 2005; 280(16):16456–16460; doi: 10.1074/jbc.M501485200

37.

Aquilano

, Vigilanza

, Baldelli

, et al. Peroxisome proliferator-activated receptor gamma co-activator 1alpha (PGC-1alpha) and sirtuin 1 (SIRT1) reside in mitochondria: Possible direct function in mitochondrial biogenesis. J Biol Chem, 2010; 285(28):21590–21599; doi: 10.1074/jbc.M109.070169

38.

Hock

, Kralli

. Transcriptional control of mitochondrial biogenesis and function. Annu Rev Physiol, 2009; 71:177–203; doi: 10.1146/annurev.physiol.010908.163119

39.

Dillon

, Rebelo

, Moraes

. The role of PGC-1 coactivators in aging skeletal muscle and heart. IUBMB Life, 2012; 64(3):231–241; doi: 10.1002/iub.608

40.

Ramirez-Sanchez

, Taub

, Ciaraldi

, et al. (-)-Epicatechin rich cocoa mediated modulation of oxidative stress regulators in skeletal muscle of heart failure and type 2 diabetes patients. Int J Cardiol, 2013; 168(4):3982–3990; doi: 10.1016/j.ijcard.2013.06.089