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Article

Hormone Replacement Therapy Protects Skeletal Muscle by Stabilizing the Neuromuscular Junction in Post-Menopausal Women

by
Maria Abutair
1,
Sara Elkhatib
1,
Rawan Flayyih
1,
Dalal Alarabid
1,
Mohamed Nacer
1,
Rizwan Qaisar
1,2,3,
Shah Hussain
4 and
Areesha Ashraf
1,*
1
Basic Medical Sciences, College of Medicine, University of Sharjah, Sharjah 27272, United Arab Emirates
2
Space Medicine Research Group, Research Institute of Medical and Health Sciences, University of Sharjah, Sharjah 27272, United Arab Emirates
3
Cardiovascular Research Group, Research Institute of Medical and Health Sciences, University of Sharjah, Sharjah 27272, United Arab Emirates
4
Medical Oncology Department, Hayatabad Medical Complex, Peshawar 25120, Pakistan
*
Author to whom correspondence should be addressed.
J. Oman Med. Assoc. 2024, 1(1), 69-80; https://doi.org/10.3390/joma1010008
Submission received: 11 August 2024 / Revised: 16 September 2024 / Accepted: 22 October 2024 / Published: 30 October 2024
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Abstract

:
Introduction: Hormone replacement therapy (HRT) prevents muscle loss associated with menopause; however, the relative role of the neuromuscular junction (NMJ) in post-menopausal women taking HRT is poorly known. We investigate the effects of HRT on plasma C-terminal agrin-fragment-22 (CAF22) in post-menopausal women taking HRT. Methods: We recruited three groups of women, including pre-menopausal (age = 45.3 ± 3.1 years, n = 48) post-menopausal HRT-users (age = 56.7 ± 4.1 years, n = 42) and non-users (age = 55.4 ± 3.9 years, n = 47) for measurements of handgrip strength (HGS), skeletal muscle mass index (SMI), short physical performance battery (SPPB; marker of physical capacity), and plasma CAF22 levels. Results: Post-menopausal non-users of HRT had lower HGS, SMI, gait speed, and SPPB scores and higher plasma CAF22 levels than pre-menopausal women (all p < 0.05). Conversely, HRT users had higher HGS and gait speed and lower plasma CAF22 than non-users among post-menopausal women. HRT users also exhibited SPPB scores similar to those of pre-menopausal women. We observed significant correlations of plasma CAF22 with HGS, gait speed, and total SPPB scores in pre-menopausal and post-menopausal women with HRT (ALL p < 0.05). Lastly, HRT users had lower markers of inflammation and oxidative stress than non-users among post-menopausal women (both p < 0.05). Conclusion: Altogether, menopause was associated with elevated markers of NMJ degradation along with reduced muscle strength and physical capacity. HRT partly reduced NMJ degradation and restored muscle strength and physical capacity in post-menopausal women.

1. Introduction

Menopause is a naturally inevitable process in women that denotes the end of menstrual cycles. The definition of menopause is the absence of menstruation for 12 consecutive months in women aged 44 to 55 years old; however, this age range is subjective [1]. Menopause and loss of estrogen are associated with a natural decline in bone mineral density (BMD), skeletal muscle mass, and strength [2]. The atrophy of skeletal muscle and weakness provoke the loss of physical capacity and functional independence and are associated with several diseases. As estrogen exhibits anabolic properties in skeletal muscles, hormone replacement therapy (HRT) is given to reduce these side effects by compensating for the loss of estrogen [3]. HRT works on multiple receptors in a tissue-specific manner. However, the mechanisms and subcellular effects of HRT on skeletal muscles are not well-known [4].
Sarcopenia is the age-related loss of the strength of the muscle and its mass that adversely impacts quality of life and physical activity and raises the risks of functional dependence, morbidity, and mortality due to multiple diseases [5]. The short physical performance battery (SPPB) is an effective tool for evaluating physical ability in clinical contexts. The measurement is accomplished by assessing three components, including maintaining postural balance, the five-times sit-to-stand test (5-STS), and the 4 m walking test (4-MWT) [6].
Several mechanisms of sarcopenia are reported, including mitochondrial dysfunction and the ongoing loss of motor neurons, which contribute to the progressive reduction of muscle fiber number and size [5]. Sarcopenia can also involve various cellular and subcellular abnormalities, such as sarcolemma damage, endoplasmic reticulum stress, heightened inflammation, and poor energy utilization. The neuromuscular junction (NMJ) is the synapse between motor neurons and the sarcolemma to transmit nerve impulses for generating muscle action potential. NMJ integration is crucial for the adequate functionality of skeletal muscle. Conversely, a compromise of NMJ structure and/or function may lead to muscle pathology [7]. Thus, conditions associated with the breakdown of NMJ result in accelerated muscle atrophy and weakness [7]. Agrin is a protein found in the post-synaptic component of NMJ. The breakdown of NMJ is associated with the fragmentation of agrin into multiple fragments that are released into circulation. Among them, c-terminal agrin fragment-22 (CAF22) can be detected in the bloodstream and has surfaced as a potential biomarker of NMJ disintegration [8]. We have previously reported a negative correlation between plasma CAF22 levels and muscle strength [8]. Similarly, plasma CAF22 levels also exhibit inverse associations with SPPB scores [9]. Together, these reports show that CAF22 levels can be used to predict muscle strength and physical capacity.
As previously noted, the risk of muscle atrophy is more significant in women after menopause. The loss of the anabolic estrogen leads to muscle weakness and atrophy, which cause physical disability, functional dependence, and diminished overall quality of life [3]. These effects may partly be due to the loss of several protective attributes of estrogen following menopause [10]. For example, estrogen enhances mitochondrial function and improves cellular bioenergetics. Similarly, estrogen is known to improve exercise endurance capacity and muscle strength. Accordingly, the loss of estrogen following menopause reduces mitochondrial ATP production, increases oxidative stress, and causes enhanced muscle apoptosis [10,11]. Estrogen deficiency also negatively affects the muscle contractile apparatus, which leads to muscle weakness independent of muscle mass [11]. Further, direct protective effects of estrogen on NMJ are suggested [12]. For example, post-menopausal women taking estradiol exhibit improved NMJ transmission and motor control than the age-matched controls without estradiol [12]. In conjunction with these findings, these women also exhibit reduced inflammation and muscle proteolysis and improved motor control [12]. Together, these reports suggest that loss of estrogen and its protective effects increases the risk of muscle decline, sarcopenia, and frailty in older women [10].
However, the effects of HRT on CAF22 levels in relation to muscle weakness and atrophy are not known. In addition, it is not known how HRT affects physical capacity with relevance to plasma CAF22 levels in post-menopausal women. The current study seeks to address this gap by exploring the effects of HRT on plasma CAF22 levels, skeletal muscle mass and strength, and physical capacity in post-menopausal women. We recruited three groups of women, consisting of pre-menopausal, post-menopausal with HRT, and post-menopausal non-users of HRT. We hypothesized that (1) menopause is associated with an increase in plasma CAF22 levels and a decline in muscle mass, strength, and physical capacity, and (2) HRT reduces plasma CAF22 levels and improves muscle mass, strength, and SPPB scores when compared to post-menopausal non-users of HRT.

2. Material and Methods

Design and participants: In this cross-sectional study, we recruited participants at the [Redacted] Medical Complex in Peshawar, Pakistan, after obtaining approval from the Clinical Research Ethics Committee of the hospital. (IRB number: 1186-A/HMC/B&PSC/2023). We recruited three groups of women, including pre-menopausal women (age = 45.3 ± 3.1 years, n = 48) and two groups of post-menopausal women, including HRT users (age = 56.7 ± 4.1 years, n = 42) and non-users (age = 55.4 ± 3.9 years, n = 47). The participants in the HRT group were receiving combinations of synthetic estrogen and progestins as tablet Prempro (one of the two formulations, including 0.45/1.5 mg or 0.625/2.5 mg tablet per day) or FemHT (one of the two formulations, including 0.5 mg or 1 mg tablet per day) for at least six months, as described elsewhere [13]. The HRT-users were taking HRT to manage the symptoms of bone pain, muscle weakness, and early fatigue. The non-users were not taking HRT due to multiple reasons, including financial issues, significant distance from the health-care facility, and management symptoms with other medications, such as non-steroidal anti-inflammatory drugs. The demographic data about age, treatment, and comorbidities were obtained from their clinical records. The information about daily step count was collected with a pedometer. All patients aged 40 and above who agreed to participate by providing written informed consent were included in this study. Briefly, participants with muscle and joint diseases, reduced ambulatory capacity, chronic illnesses, and prolonged bed rest were excluded. Any participant with muscle and bone disorders, recent major surgeries, and acute or chronic impairments of vital organs, such as lungs, kidneys, and heart, were also excluded [14]. This is because these conditions can affect skeletal muscle and physical capacity due to their direct myotoxic effects or indirectly by reducing the ambulatory capacity [15,16]. Further, participants with antibiotics or probiotics use in the past six weeks were also excluded from this study. This is partly because antibiotics alter the pharmacodynamics and biological functions of estrogen [17]. Similarly, probiotics may enhance skeletal muscle mass and/or strength [18], which may mask the protective effects of estrogen on skeletal muscle and physical capacity. The recruitment and evaluation of participants was performed from 23 February to 5 April 2024. We used the European Working Group on Sarcopenia in Old People diagnostic criteria, which included muscle strength (Handgrip strength; HGS < 16 kg), muscle atrophy (appendicular skeletal muscle mass index; ASMI < 5.5 kg/m2), and poor physical performance (gait speed < 0.8 m/s) [19,20]. Probable sarcopenia was defined when the first criteria were only met. Definite sarcopenia was defined when both the first and second criteria were met. Severe sarcopenia was defined when all three criteria were met. This study was conducted under the declaration of Helsinki [21].
HGS measurement: To measure the HGS of the participants, a digital handgrip dynamometer was used as described previously [22]. After the procedure was explained to the participants, they sat comfortably on a chair with a maintained elbow at a 90-degree angle. The participants held the dynamometer in their dominant hand and squeezed it with maximum strength for five seconds while maintaining posture and not performing jerking movements. This procedure was repeated three times, and the maximum reading that was recorded was used for the analysis [22].
Body composition analysis: Analysis of body composition was done using dual-energy X-ray absorptiometry (DEXA scan and GE Healthcare GmbH, Vienna, Austria) as mentioned previously [23]. The participants were asked to wear light clothes and remove metallic equipment, such as zippers, jewelry, buttons, etc. The information regarding lean body mass, appendicular skeletal muscle mass (ASM), lean body mass, body fats, and BMD were collected. ASM was corrected for height in meters to obtain SMI, as described previously [23].
SPPB measurement: SPPB was used to evaluate the physical capabilities of the participants. In this procedure, we evaluated the ability to maintain a balanced posture, a 4 m walk test, 4MWT, and five-times chair sit-to-stand test, 5-STS. A timer was set to measure the time needed to complete each task, and each participant was assigned a score ranging from zero to four, with zero being the worst performance and four being the best performance. The total score of three tests was calculated for data analysis as described elsewhere [19].
Measurement of circulating biomarkers: We measured biomarkers circulating in blood samples that were collected from cubital veins between 8 a.m. and 10 a.m. after an overnight fast. The samples were centrifuged at 3000 revolutions per minute for 10 min to separate the blood components from plasma, which was aliquoted and stored at −80 degrees centigrade. We used ELISA kits to measure plasma levels of CAF22 (NTCAF, ELISA, Neurotune, Schlieren-Zurich, Switzerland) and assess the levels according to the manufacturer’s instructions in [9]. Finally, we used the ELISA kits to evaluate 8-isoprostanes (a marker of systemic oxidative stress) (Cayman Chemical, Ann Arbor, MI, USA) and c-reactive proteins (CRP) (a marker of systemic inflammation) (R&D systems, Minneapolis, MN, USA) levels in the plasma. These markers were measured due to the well-recognized detrimental effects of oxidative stress and inflammation on skeletal muscle [24,25] and the antioxidant and anti-inflammatory properties of HRT [26]. The kit used for the measurements of 8-isoprostanes primarily measures 8-iso Prostaglandin F3a ethanolamide with 20% cross-reactivity to 8-iso Prostaglandin F3a. We also evaluated plasma creatine kinase levels as a marker of muscle damage.
Statistical analysis: The data were analyzed using the one-way analysis of variance test to compare sarcopenia parameters, plasma biomarkers, and SPPB scores between the three groups. We used Pearson correlation to investigate the strength of the association of plasma CAFF22 with sarcopenia markers, SPPB, and plasma biomarkers. Multiple linear regression analysis with least squares was used to investigate the associations of CAF22 with age, step count, and SMI. We conducted mediation analysis to investigate the mediating effects of age, SMI, CRP, and 8-isoprostanes on the relationship between CAF22 and HGS among HRT-user post-menopausal women. The analysis involved the effects of CAF22 on the mediator (path X) and HGS (path Z) and the effects of the mediator on HGS (path Y). The indirect and direct effects of CAF22 and HGS were used to calculate the percent mediation, as described elsewhere [27]. GraphPad Prism 8 (Graphstats technologies private limited, Bangalore 560035, India) was used to analyze the data. All the data were presented as mean and standard deviation, and a p-value <0.05 was considered statistically significant.

3. Results

Characteristics of the participants: At baseline, both groups of post-menopausal women, including HRT-users and non-users, were significantly older than pre-menopausal women (p < 0.05). In terms of body composition, we found no difference in body mass index (BMI), lean body mass, fat content, or BMD among the groups (Table 1). In terms of plasma biomarkers, we observed no significant differences in creatine kinase, albumin, white blood cell (WBC) count, HbA1c, total cholesterol, HDL-cholesterol, triglycerides, and haemoglobin levels across the groups. Notably, total protein levels were significantly lower in both post-menopausal groups compared to pre-menopausal women (p < 0.05), and post-menopausal non-users of HRT had significantly higher total protein levels compared to HRT-users (p < 0.05).
Sarcopenia parameters: We evaluated the effects of menopause and HRT on muscle function and sarcopenia (Figure 1). Pre-menopausal women demonstrated significantly higher HGS, SMI, and gait speed compared to post-menopausal women, regardless of HRT status (Figure 1A, Figure 1B, and Figure 1C, respectively) (all p < 0.05). Furthermore, pre-menopausal women had a lower prevalence of sarcopenia compared to post-menopausal non-users of HRT (Figure 1D).
Plasma biomarkers: We investigated the plasma levels of CAF22, CRP, and 8-isoprostanes in pre-menopausal and post-menopausal women, with and without HRT (Figure 2). Pre-menopausal women exhibited significantly lower CAF22, CRP, and 8-isoprostanes, levels compared to post-menopausal women, regardless of HRT status (Figure 2A, Figure 2B, and Figure 2C, respectively) (all p < 0.05). Lastly, post-menopausal women with HRT exhibited lower levels of plasma CAF22 and 8-isoprostanes than non-users.
SPPB and physical capacity: We investigated SPPB scores, including balance, 4MWT, and 5-STS, in pre-menopausal and post-menopausal women, including HRT-users and non-users (Figure 3). There were no significant differences in SPPB balance, 4MWT, and 5-STS scores among the groups (Figure 3A, Figure 3B, and Figure 3C, respectively). However, post-menopausal non-users of HRT had significantly lower cumulative SPPB scores compared to pre-menopausal women (p < 0.05) (Figure 3D). We found similar SPPB scores between the two groups of post-menopausal women.
Associations of plasma CAF22 with sarcopenia, SPPB, and plasma biomarkers: We examined the correlation between plasma CAF22 and various markers of sarcopenia, physical capacity, and plasma biomarkers in pre-menopausal and post-menopausal women, including HRT-users and non-users (Table 2). In pre-menopausal women, significant correlations of CAF22 were found with HGS, gait speed, total SPPB scores, CRP, and 8-isoprostanes (all p < 0.05). In post-menopausal non-users of HRT, significant correlations were observed between CAF22 and HGS, gait speed, and 8-isoprostanes (all p < 0.05). Lastly, in post-menopausal HRT-users, significant correlations were found between CAF22 and HGS, gait speed, total SPPB scores, and 8-isoprostanes levels (all p < 0.05) (Table 2).
Associations of plasma CAF22 with age, step count, and SMI: We conducted multivariate regression analysis to investigate the associations of plasma CAF22 with age, step count, and SMI in the study groups. CAF22 was not associated with the three parameters in the pre-menopausal group (Table 3). Conversely, in post-menopausal non-users, CAF22 was significantly associated with step count (coefficient = −3.257, p = 0.002). CAF22 also exhibited significant associations with step count (coefficient = −3.45, p = 0.001) and SMI (coefficient = −2.366, p = 0.023) in the post-menopausal HRT-users (Table 3).
Mediation analysis of the relation between CAF22 and HGS among HRT-user post-menopausal women: Lastly, we evaluated the mediating effects of age, SMI, CRP, and 8-isoprostanes on the relationship between CAF22 and HGS among HRT-user post-menopausal women (Figure 4A–D). We found significant mediating effects of SMI (Percent mediation = 29.2%) (Figure 4B) and 8-isoprostanes (Percent mediation = 13.37%) (Figure 4D) on the relationship between CAF22 and HGS. Conversely, age (Figure 4A) and CRP (Figure 4C) did not exhibit significant mediating effects on the relationship between CAF22 and HGS.

4. Discussion

Our findings show lower CAF22 and higher HGS and gait speed in the post-menopausal HRT-users than in the non-users. Moreover, both groups of post-menopausal women displayed higher CAF22 and lower HGS, SMI, and gait speed than the pre-menopausal group. This denotes the association of menopause and ageing with the degradation of the NMJ, leading to reduced muscle mass and strength, and indicates the role of HRT in partly preventing these deleterious changes.
We report lower HGS, SMI, and gait speeds, the parameters for diagnosing sarcopenia, in post-menopausal women, suggesting a higher risk of acquiring sarcopenia. Previous reports support these observations found on the negative effects of menopause on skeletal muscle [28,29]. In support of this observation, we found higher proportions of women with definite and severe sarcopenia in non-HRT post-menopausal women. Conversely, the post-menopausal women taking HRT had higher HGS and gait speed and lower prevalence of sarcopenia than the non-HRT post-menopausal women. These observations suggest that the replacement of anabolic estrogen after menopause boosts skeletal muscle mass and strength.
The correlation analysis between plasma CAF22 levels and various markers of sarcopenia, physical capacity, and plasma biomarkers revealed significant associations in both pre-menopausal and post-menopausal women, with or without HRT. These observations show that NMJ degradation is directly associated with muscle weakness, as demonstrated by the significant correlation with HGS. Since muscle weakness primarily drives physical capacity, we also observed notable correlations of plasma CAF22 with gait speed and SPPB. We have previously shown, using a transgenic mouse model, that the dysfunction of the NMJ directly leads to weakness and the atrophy of skeletal muscle fibers [7]. Our current findings of the correlation between CAF22 and HGS directly align with previous observations. An adequate amount of muscle mass and strength are essential for maintaining optimal physical capacity. Significant associations of CAF22 with total SPPB scores were found, which confirms and extends the above statement. Between the non-HRT and HRT groups, there were notable variations in the correlation analysis; the HRT group exhibited a weaker correlation of CAF22 with HGS and gait speed, which further affirms the efficacy of estrogen as a mitigator of the negative repercussions of menopause.
Furthermore, CRP and 8-isoprostanes levels were notably higher in the post-menopausal non-users of HRT, suggesting the presence of increased inflammation and oxidative stress. This remark in post-menopausal women may be due to the decrease in levels of estrogen, which exhibits antioxidant and anti-inflammatory properties [26]. In addition, due to their weak muscles and poor physical capacity, post-menopausal women are more likely to have a sedentary lifestyle, which may further exacerbate the elevated inflammation and oxidative stress in the body [30,31].
Additionally, pre-menopausal women exhibited higher bone mineral density, BMI, and SMI compared to post-menopausal women. Consistent with previous studies, our results show that lean body mass was lower across the menopausal transition irrespective of HRT use [32]. In contrast, the body fat percentage increased, with the highest proportions observed in the HRT group. This observation may be explained by the prevalence of a more sedentary lifestyle in the post-menopausal years, leading to reduced fat oxidation and energy expenditure [33]. Thus, this lack of physical activity may be a significant contributor to the development of sarcopenia, as suggested elsewhere [34].
The lower SMI in the post- compared to pre-menopausal women suggests skeletal muscle wasting following menopause. This is a common finding in several studies investigating the effects of menopause on skeletal muscle [12,35]. The SMI is considered one of the three standard diagnostic indices of sarcopenia as suggested by the EWGSOP2 [20]. We did not observe a higher SMI among HRT-users than in non-users. Conversely, the HRT users exhibited significantly higher HGS and gait speed than the non-users. Thus, it appears that the protective effects of HRT are primarily related to muscle strength and physical capacity rather than muscle mass.
The mediation analysis revealed that the protective effects of HRT on HGS are partly mediated through SMI and reduced oxidative stress. Muscle strength is partly determined by muscle mass, as larger muscles generate higher force irrespective of the intrinsic force-generating properties of skeletal muscle. Thus, it appears that HRT improves HGS partly by boosting SMI. Estrogen is also recognized for its antioxidant properties [26]. In addition, oxidative stress is a well-recognized trigger of skeletal muscle weakness and atrophy in older adults [25]. The mediation analysis shows that HRT improves HGS partly by reducing oxidative stress. These findings provide a mechanistic insight into the protective effects of HRT on skeletal muscle.
We found higher levels of total cholesterols in post-menopausal than in pre-menopausal women. Dyslipidemia is considered a critical associated factor of sarcopenia in both genders [36]. Specifically, the loss of estrogen in post-menopausal women causes a higher production of triglycerides in the liver [36]. Our observation of higher triglycerides and total cholesterols in post-menopausal women is consistent with these reports. Hyperlipidemia is a significant risk factor for sarcopenia and may have contributed to muscle decline in our cohort of post-menopausal women. In addition, the loss of anti-inflammatory estrogen increases systemic inflammation in these women [10]. We found higher CRP levels in post-menopausal non-users of HRT, which is consistent with this observation. Interestingly, we did not observe a difference between CRP levels among pre-menopausal women and HRT-user post-menopausal women. Thus, it appears that HRT reduces systemic inflammation in post-menopausal women. The critical role of inflammation in sarcopenia phenotype is generally well-recognized [24]. Thus, the HRT-mediated reduction in inflammation may be a contributory factor to improved HGS and physical capacity in post-menopausal women.
We found a trend towards higher creatine kinase levels among post-menopausal than in pre-menopausal women. Creatine kinase is considered a marker of muscle damage, and its plasma levels are elevated in various muscle-atrophying conditions [37,38]. The higher creatine kinase levels in these women are consistent with lower SMI, suggesting accelerated muscle atrophy.
Our study has several strengths. The patients were recruited from a single hospital, which reduces the risk of heterogeneity in the clinical and experimental procedures. All participating women were recruited from a small geographical region with comparable racial, ethnic, genetic, and lifestyle profiles. The BMI and age ranges between the groups of women were closely matched, reducing any age-related bias, and controlling for BMI minimizes confounding factors that could affect muscle mass and physical performance.
The inclusion of CAF22 measurement uniquely enhanced the research by evaluating a biomarker related to physical activity and muscle function. This is particularly relevant for women who may be in a coma or non-ambulant, where their immobilized state impacts muscle mass and function. Favorably, the measurement of CAF22 levels only requires an ELISA test, which is easily available at most medical facilities. Lastly, the use of SPPB is a significant strength, as it is a well-validated standardized tool for assessing physical capacity in older adults; by including timed measurements of balance, gait speed, and chair stand tests, the study offers a comprehensive evaluation of physical performance, accurately identifying limitations associated with sarcopenia and ensuring reliable findings.
While our study provides valuable insights into the impact of menopause and HRT on skeletal muscle and physical capacity in women, it is important to acknowledge and address certain limitations that may have impacted the interpretation and generalizability of our findings. (1) The cross-sectional study design adapted in the study precludes establishing the temporal relationships of HRT with the NMJ and skeletal muscle. (2) Additionally, the varying durations of HRT use among the postmenopausal women introduces inhomogeneity within the group. (3) The study sample comprised 137 participants from a single hospital, limiting the generalizability of the findings. (4) The unequal distribution of participants among the three groups may have affected the accuracy and statistical power of the analyses. (5) The study did not take into consideration potential confounding factors, such as physical activity and diet, which may have influenced the outcomes of interest.
In conclusion, menopausal transition is significantly associated with changes in muscle function, sarcopenia parameters, and plasma biomarkers in women. The protective effects of HRT against loss of muscle mass and decline in physical capacity is evident from our findings and may be attributed to its role in the preservation of the NMJ and relative maintenance of CAF22 levels. The protective effects of HRT on skeletal muscle and the NMJ may partly involve a reduction in systemic inflammation and oxidative stress due to the well-recognized antioxidant and anti-inflammatory properties of estrogen. Our findings have translational potential and may suggest the NMJ as a therapeutic target for muscle loss in post-menopausal women. The serial measurements of CAF22 can be performed in most clinical settings in a cost-effective manner, offering a direct monitoring of NMJ structure in patients with diseases and therapies. We suggest long-term investigations to optimize the HRT dosage for protecting skeletal muscle and investigate the diagnostic potential of plasma CAF22 in muscle decline and sarcopenia in post-menopausal women.

Author Contributions

Conceptualization, M.A., S.E., R.F., D.A., M.N., R.Q., S.H. and A.A.; methodology, S.H. and R.Q.; software, R.Q.; validation, M.A., S.E., R.F., D.A., M.N., R.Q., S.H. and A.A.; formal analysis, R.Q.; investigation, M.A., S.E., R.F., D.A., M.N., R.Q., S.H. and A.A.; resources, S.H. and R.Q.; data curation, M.A., S.E., R.F., D.A., M.N., R.Q., S.H. and A.A.; writing—original draft preparation, M.A., S.E., R.F., D.A., M.N., R.Q., S.H. and A.A.; writing—review and editing, M.A., S.E., R.F., D.A., M.N., R.Q., S.H. and A.A.; visualization, A.A.; supervision, S.H.; project administration, S.H. and R.Q.; funding acquisition, R.Q. All authors have read and agreed to the published version of the manuscript.

Funding

The work described in this study is supported by a competitive grant (number: 22010901121) from the University of Sharjah to Rizwan Qaisar.

Institutional Review Board Statement

The study was conducted in accordance with the Declaration of Helsinki and approved by the Human Research Ethics Committee of Hayatabad Medical Complex, Peshawar Pakistan (protocol code: 1186-A/HMC/B&PSC/2023, date of approval: 25 March 2023).

Informed Consent Statement

All participants of this study provided a written informed consent.

Data Availability Statement

The data can be obtained from the corresponding author upon reasonable request.

Acknowledgments

No generative AI tools were used in the writing of this work.

Conflicts of Interest

The authors declare that they have no conflicts of interest.

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Joma 01 00008 g001
Figure 1. HGS (A), SMI (B), gait speed (C), and relative proportions of participants with probable, definite, and severe sarcopenia (D) in pre-menopausal (n = 48) and post-menopausal HRT-users (n = 42) or non-users (n = 47), * p < 0.05, ** p < 0.01, **** p < 0.0001. (HGS, handgrip strength; SMI, skeletal muscle mass index).
Figure 1. HGS (A), SMI (B), gait speed (C), and relative proportions of participants with probable, definite, and severe sarcopenia (D) in pre-menopausal (n = 48) and post-menopausal HRT-users (n = 42) or non-users (n = 47), * p < 0.05, ** p < 0.01, **** p < 0.0001. (HGS, handgrip strength; SMI, skeletal muscle mass index).
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Figure 2. Plasma levels of CAF22 (A), CRP (B), and 8-isoprostanes (C) in pre-menopausal (n = 48) and post-menopausal HRT-users (n = 42) or non-users (n = 47), * p < 0.05, ** p < 0.01, **** p < 0.0001. (CAF22, c-terminal agrin-fragment-22; CRP, C-reactive protein).
Figure 2. Plasma levels of CAF22 (A), CRP (B), and 8-isoprostanes (C) in pre-menopausal (n = 48) and post-menopausal HRT-users (n = 42) or non-users (n = 47), * p < 0.05, ** p < 0.01, **** p < 0.0001. (CAF22, c-terminal agrin-fragment-22; CRP, C-reactive protein).
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Figure 3. Violin plots showing the SPPB scores for balance (A), 4MWT (B), 5-STS (C), and cumulative SPPB scores (D) in pre-menopausal (n = 48) and post-menopausal HRT-users (n = 42) or non-users (n = 47) HRT treatment, * p < 0.05. (SPPB, short physical performance battery; 4MWT, 4-m walk test; 5-STS, five-times chair sit-to-stand test).
Figure 3. Violin plots showing the SPPB scores for balance (A), 4MWT (B), 5-STS (C), and cumulative SPPB scores (D) in pre-menopausal (n = 48) and post-menopausal HRT-users (n = 42) or non-users (n = 47) HRT treatment, * p < 0.05. (SPPB, short physical performance battery; 4MWT, 4-m walk test; 5-STS, five-times chair sit-to-stand test).
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Figure 4. Mediation analysis showing the mediating effects of age (A), SMI (B), CRP (C), and 8-isoprostanes (D) on the relationship between CAF22 and HGS among HRT-user post-menopausal women, * p < 0.05. (CAF22, c-terminal agrin-fragment-22; SMI, skeletal muscle mass index; CRP, c-reactive protein).
Figure 4. Mediation analysis showing the mediating effects of age (A), SMI (B), CRP (C), and 8-isoprostanes (D) on the relationship between CAF22 and HGS among HRT-user post-menopausal women, * p < 0.05. (CAF22, c-terminal agrin-fragment-22; SMI, skeletal muscle mass index; CRP, c-reactive protein).
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Table 1. Age, number of comorbidities, body composition, and plasma profile of pre-menopausal and post-menopausal women, including HRT-users and non-users, * p < 0.05 vs. pre-menopause, # p < 0.05 vs. post-menopause non-users. Values are expressed as mean ± SD unless otherwise stated (BMI, body mass index; WBC, white blood cells; HbA1c, glycosylated hemoglobin; HDL-C, high-density lipoproteins-cholesterol).
Table 1. Age, number of comorbidities, body composition, and plasma profile of pre-menopausal and post-menopausal women, including HRT-users and non-users, * p < 0.05 vs. pre-menopause, # p < 0.05 vs. post-menopause non-users. Values are expressed as mean ± SD unless otherwise stated (BMI, body mass index; WBC, white blood cells; HbA1c, glycosylated hemoglobin; HDL-C, high-density lipoproteins-cholesterol).
Pre-Menopausal (n = 48)Post-Menopausal
Non-Users (n = 47)HRT-Users (n = 42)
Age (years)45.3 ± 3.155.4 ± 3.9 *56.7 ± 4.1 *
Number of comorbidities, n (%)
17 (14.6)7 (14.9)5 (11.9)
23 (6.2)4 (8.5)3 (7.4)
31 (2.1)2 (4.2)2 (4.7)
Body composition
BMI (kg/m2)23.5 ± 2.525.4 ± 3.226 ± 3.1
Lean body mass (kg)41.4 ± 3.838.2 ± 3.838.6 ± 3.4
Fat percentage38.3 ± 5.140.4 ± 5.541.4 ± 4.7
Bone mineral density (g/cm2)1.33 ± 0.121.22 ± 0.111.23 ± 0.13
Physical activity
Daily step count (×1000)7.03 ± 1.225.38 ± 1.096.17 ± 0.86
Plasma profile
Creatine kinase (IU/L)183.5 ± 25208.3 ± 27.2211.2 ± 24.8
Albumin (g/dL)4.27 ± 0.164.05 ± 0.154.11 ± 0.16
Total proteins (g/dL)6.46 ± 0.26.13 ± 0.27 *6.01 ± 0.29 *,#
WBC count (109/L)10.1 ± 1.49.7 ± 1.69.9 ± 1.9
HbA1c (%)5.84 ± 0.35.93 ± 0.45.88 ± 0.4
Total cholesterol (mg/dL)207.2 ± 41.2228.5 ± 39.9233.2 ± 34.7
HDL cholesterol (mg/dL)42.1 ± 3.841.6 ± 3.540.9 ± 3.3
Triglycerides (mg/dL)195.4 ± 37.2205.3 ± 34.9212.2 ± 38.5
Hemoglobin (g/dL)13.9 ± 1.512.2 ± 1.112 ± 1.4
Table 2. Pearson correlation coefficients of determinations of plasma CAF22 with markers of sarcopenia, physical capacity, and plasma biomarkers in pre-menopausal and post-menopausal HRT-users and non-users, * p < 0.05. (CAF22, c-terminal agrin-fragment-22; HGS, handgrip strength; SMI, skeletal muscle mass index; SPPB, short physical performance battery; 4MWT, 4-m walk test; 5-STS, five-times chair sit-to-stand test; CRP, C-reactive protein).
Table 2. Pearson correlation coefficients of determinations of plasma CAF22 with markers of sarcopenia, physical capacity, and plasma biomarkers in pre-menopausal and post-menopausal HRT-users and non-users, * p < 0.05. (CAF22, c-terminal agrin-fragment-22; HGS, handgrip strength; SMI, skeletal muscle mass index; SPPB, short physical performance battery; 4MWT, 4-m walk test; 5-STS, five-times chair sit-to-stand test; CRP, C-reactive protein).
Pre-Menopausal
(n = 48)		Post-Menopausal
No HRT (n = 47)HRT (n = 42)
Sarcopenia markers
HGS0.228 *0.16 *0.135 *
SMI0.0640.0720.039
Gait speed0.114 *0.187 *0.145 *
SPPB
Balance0.0640.0730.033
4MWT0.134 *0.0870.055
5-STS0.0290.0630.093
Total0.105 *0.0790.113 *
Plasma markers
CRP0.128 *0.0930.033
8-isoprostanes0.105 *0.112 *0.12 *
Table 3. Multiple linear regression analysis investigating the associations of plasma CAF22 with age, step count, and SMI in the study groups. (CAF22, c-terminal agrin-fragment-22; SMI, skeletal muscle mass index).
Table 3. Multiple linear regression analysis investigating the associations of plasma CAF22 with age, step count, and SMI in the study groups. (CAF22, c-terminal agrin-fragment-22; SMI, skeletal muscle mass index).
ParameterCoefficientpr2
Pre-menopausal
Age−1.0760.2990.034
Step count−0.0010.719
SMI−4.270.451
Post-menopausal (no HRT)
Age2.1170.1160.339
Step count−3.2570.002
SMI1.3790.174
Post-menopausal (HRT)
Age0.8010.1020.501
Step count−3.450.001
SMI−2.3660.023
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Abutair, M.; Elkhatib, S.; Flayyih, R.; Alarabid, D.; Nacer, M.; Qaisar, R.; Hussain, S.; Ashraf, A. Hormone Replacement Therapy Protects Skeletal Muscle by Stabilizing the Neuromuscular Junction in Post-Menopausal Women. J. Oman Med. Assoc. 2024, 1, 69-80. https://doi.org/10.3390/joma1010008

AMA Style

Abutair M, Elkhatib S, Flayyih R, Alarabid D, Nacer M, Qaisar R, Hussain S, Ashraf A. Hormone Replacement Therapy Protects Skeletal Muscle by Stabilizing the Neuromuscular Junction in Post-Menopausal Women. Journal of the Oman Medical Association. 2024; 1(1):69-80. https://doi.org/10.3390/joma1010008

Chicago/Turabian Style

Abutair, Maria, Sara Elkhatib, Rawan Flayyih, Dalal Alarabid, Mohamed Nacer, Rizwan Qaisar, Shah Hussain, and Areesha Ashraf. 2024. "Hormone Replacement Therapy Protects Skeletal Muscle by Stabilizing the Neuromuscular Junction in Post-Menopausal Women" Journal of the Oman Medical Association 1, no. 1: 69-80. https://doi.org/10.3390/joma1010008

APA Style

Abutair, M., Elkhatib, S., Flayyih, R., Alarabid, D., Nacer, M., Qaisar, R., Hussain, S., & Ashraf, A. (2024). Hormone Replacement Therapy Protects Skeletal Muscle by Stabilizing the Neuromuscular Junction in Post-Menopausal Women. Journal of the Oman Medical Association, 1(1), 69-80. https://doi.org/10.3390/joma1010008

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