Introduction
Older adults suffer from age-related, uncontrollable loss of skeletal muscle mass and strength known as sarcopenia (Walston, 2012). The usual onset of sarcopenia occurs in the fourth decade of life and progresses to the point where adults in their eighth decade have only half of the skeletal muscle mass that they used to have (Metter et al., 1997). Skeletal muscle is an important metabolically active tissue, which is why its gradual involuntary loss is associated with profound physiological, psychological, and social consequences for older adults. It has been found that even a 5% loss can translate into a higher chance of morbidity (Attaway et al., 2021). Moreover, sarcopenia is associated with higher nursing institutionalization and mortality rates (Roubenoff, 2011). Indeed, muscle mass means strength that implies independence at later stages of life. It is not at all surprising that negative subjective health status and loss of functionality are reliable predictors of depression in older adults (Padayachey et al., 2017).
Today, Canada has a rapidly aging population; it is projected that by 2030, 9.5 million Canadians, or almost a quarter of the total population, will be aged 65 or older (Government of Canada, 2014). The demographic shift will mean a higher prevalence of sarcopenia since it is an age-related condition. Therefore, there is a need for evidence-based interventions that could dramatically improve senior citizens’ quality of life and prolong their independence. Existing literature suggests that resistance training is the only treatment that reliably reverses the muscle effects or sarcopenia (Rolland et al., 2008). Dietary and pharmacological approaches are understudied, which needs to be rectified since muscle loss and decreased protein metabolism require well-rounded complex solutions (Rolland et al., 2008).
Therefore, this paper proposes an experimental study whose goal is to investigate the effects of omega-3 polyunsaturated fatty acids (n3-PUFA) on muscle mitochondrial physiology, anabolic response to resistance exercise, and skeletal muscle protein synthesis. The alternative hypothesis for the present study is that the inclusion of dietary n3-PUFA will result in the desired adaptations within skeletal muscle in the elderly. The primary outcomes are lower mitochondrial ROS production, enhanced muscle protein synthesis rates, and improved anabolic response to resistance exercise. The secondary outcomes include improved muscle strength and subjective health status in older adults.
Methods
Participants
For this study, the research team will recruit a small group of young (18-30 years old) and older (65-85) adults of both genders. The eligibility criteria include tolerance to fish and shellfish and the physical ability to participate in the exercise portion of the research. Therefore, persons with allergies and debilitating musculoskeletal or pulmonary diseases could not qualify for the study. Other health conditions that immediately guarantee exclusion are anemia (hemoglobin <11 g/dL for females and <12 g/dL for males), diabetes (or fasting blood glucose ≥126 mg/dL), unmanaged liver, renal, or thyroid disease, and cardiovascular disease. As for lifestyle-related exclusion criteria, the study will not accept pregnant and breastfeeding participants. Nor will it include smokers and persons currently taking medications affecting muscle metabolisms, such as opiates, beta-blockers, corticosteroids, benzodiazepines, tricyclic antidepressants, and barbiturates.
Experimental Design
Experimental design will include initial screening, measurements of the predetermined indicators of skeletal muscle health, administration of amino-acid supplements, and repeated measurements for comparison. The study will start with a screening and a two-day inpatient observation whose goals will be to assess muscle protein synthesis, responsiveness to acute resistance exercise, and mitochondrial physiology in both young and older adults. In particular, the screening will comprise:
- blood tests (glucose, insulin, Hba1c, ALT, AST, bilirubin, creatinine, and lipid panel);
- whole-body peak oxygen uptake through indirect calorimetry on a stationary cycle ergometer;
- dual-energy X-ray absorptiometry (DEXA) to determine indicators, such as whole-body fat mass, fat-free mass (FFM), and body fat mass;
- maximal knee extensor strength.
After the initial screening, the older participants will communicate their dietary preferences to a hired dietician. For the next three days, they will eat according to the pre-established meal plan with the macronutrient distribution of 20% protein, 50% carbohydrate, and 30% fat (Solon-Biet et al., 2014). On the fourth day, the participants will be offered breakfast, after which the research team will administer a primed (1.5 mg·kg FFM−1), continuous infusion (1.5 mg·kg FFM−1·hr−1) of 13C6-Phenylalanine for tracing. The participants will perform unilateral leg extension on the left side; they will complete a total of six sets of ten repetitions at around 70% of their muscle extensor strength determined during the screening stage. There will be short rests in-between the sets; the exercise will be supervised by a member of the research team. Muscle biopsies from the right vastus lateralis will be administered using a Bergstrom needle under local anesthesia. It is expected that the participants will be ready to return home in the afternoon of the fourth day.
The effects in older adults will be studied at a four-month point after open-label consumption of dietary n3-PUFA (3.9g/day). The participants will take 1200 mg of fat in the form of n3-PUFA (3.9g/day) soft gels. This amount is chosen due to evidence showing its benefits for the anabolic response of skeletal muscle (Smith et al., 2011). By the end of the intervention, the participants will return to the clinic to undergo the same screening and muscle strength tests as described in the previous paragraph. The young participants will serve the role of a comparison group for determining to which extent age affects skeletal muscle dysfunction with aging; they will not be subject to any interventions.
Analytical Approach
Muscle biopsy will serve the purpose of studying muscle mitochondria function and muscle protein synthesis rates. Muscle mitochondria function manifests itself through two indicators: muscle mitochondrial oxidative capacity and mitochondrial reactive oxygen species (ROS) production (Lanza & Nair, 2009). As described by Lanza and Nair (2009), mitochondria isolation will be done through differential centrifugation followed by suspension in a respiration buffer. Respiration of isolated mitochondria will be determined by using high-resolution respirometry in compliance with a stepwise protocol evaluating the diverse aspects of the electron transport system (Lanza & Nair, 2009). The research team will measure hydrogen peroxide production in isolated mitochondria through the observation and monitoring oxidation of Amplex Red (Lanza et al., 2013).
As for muscle protein synthesis rates, the research team will prioritize taking note of isotopic enrichment in muscle tissue fluid and muscle protein pools. For this purpose, HPLC and tandem mass spectrometry are likely to be of use (Lanza et al., 2013). Measuring muscle protein synthesis rates will require isolating the total mixed muscle, myofibrillar, mitochondrial, and sarcoplasmic (cytosolic), as well as tissue fluid-free amino acid fractions (Lanza et al., 2013). Aside from that, the chosen analytical approach will include quantitative real-time PCR and mRNA sequencing. Spectrophotometry will be applied to determine RNA concentration and purity, and Applied Biosystems will help to reverse-transcribe RNA to cDNA as per the manufacturer’s instructions.
Statistical Analysis
Quantitative research design handles objective, numerical data and applies statistical, mathematical, and computational methods. Statistical tests will be carried out using software like SPSS or JMP Software or Python programming language in an integrated development environment suitable for bioinformatics. Descriptive statistics (mean, median, standard deviation, percentile) can provide an insight into the demographic characteristics of the sample as well as continuous variables. Unpaired Student’s t-tests will be used to compare various parameters (fat-free mass, whole-body peak oxygen uptake, knee extensor strength, and others) between the young and old participants. In turn, paired t-tests are appropriate for measuring the effect size caused by the intervention in the old group. For variables that are not normally distributed, Student’s t-test will be substituted with Wilcoxon signed-rank test.
Impact
Due to the demographic shifts in the Canadian population, it is expected that age-related diseases, such as sarcopenia, will become more prevalent and, hence, burdensome for the healthcare system. In Canada, physical inactivity translates into $5.3 billion costs, or 2.6% of total health care expenses (Morley et al., 2014). At the moment, research concerning interventions against skeletal muscle loss is in process. Dietary changes may be an important addition to treatment plans, for which the scientific community requires more reliable evidence. The proposed study promises to add to the growing body of evidence indicating the reversibility of muscle loss in older adults. If translated into practice, the study’s findings may promote muscle mass and strength preservation that are key to a high quality of life for older adults.
References
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