Mortality, bone density and grip strength: lessons from the past and hope for the future?

Objectives: Therapeutic advances in the management of osteoporosis and sarcopenia have occurred at different rates over the last 2 decades. Here we examine associations between grip strength and BMD with subsequent all-cause and cause-specific mortality in a UK community-dwelling cohort. Methods: Data from 495 men and 414 women from the Hertfordshire Cohort Study were analysed. Grip strength was assessed by grip dynamometry, femoral neck BMD was ascertained using DXA and deaths were recorded from baseline (1998-2004) until 31 December 2018. Grip strength and BMD in relation to mortality outcomes (all-cause, cardiovascular-related, cancer-related and mortality due to other causes) were examined using Cox regression with adjustment for age and sex. Results: The mean baseline age of participants was 64.3 years (s.d. 2.5) and 65.9 years (s.d. 2.6) in men and women, respectively. Lower grip strength was associated with increased risk of all-cause mortality [hazard ratio (HR) 1.30 (95% CI 1.06, 1.58), P = 0.010] and cardiovascular-related mortality [HR 1.75 (95% CI 1.20, 2.55), P = 0.004]. In contrast, BMD was not associated with any of the mortality outcomes (P > 0.1 for all associations). Conclusion: We report strong relationships between grip strength and mortality compared with BMD. We hypothesize that this may reflect better recognition and treatment of low BMD in this cohort.

Adiposity is associated with widespread transcriptional changes and downregulation of longevity pathways in aged skeletal muscle.

BACKGROUND: Amongst healthy older people, a number of correlates of impaired skeletal muscle mass and function have been defined. Although the prevalence of obesity is increasing markedly in this age group, information is sparse about the particular impacts of obesity on ageing skeletal muscle or the molecular mechanisms that underlie this and associated disease risk. METHODS: Here, we examined genome-wide transcriptional changes using RNA sequencing in muscle biopsies from 40 older community-dwelling men from the Hertfordshire Sarcopenia Study with regard to obesity (body mass index [BMI] >30 kg/m2 , n = 7), overweight (BMI 25-30, n = 19), normal weight (BMI < 25, n = 14), and per cent and total fat mass. In addition, we used EPIC DNA methylation array data to investigate correlations between DNA methylation and gene expression in aged skeletal muscle tissue and investigated the relationship between genes within altered regulatory pathways and muscle histological parameters. RESULTS: Individuals with obesity demonstrated a prominent modified transcriptional signature in muscle tissue, with a total of 542 differentially expressed genes associated with obesity (false discovery rate ≤0.05), of which 425 genes were upregulated when compared with normal weight. Upregulated genes were enriched in immune response (P = 3.18 × 10-41 ) and inflammation (leucocyte activation, P = 1.47 × 10-41 ; tumour necrosis factor, P = 2.75 × 10-15 ) signalling pathways and downregulated genes enriched in longevity (P = 1.5 × 10-3 ) and AMP-activated protein kinase (AMPK) (P = 4.5 × 10-3 ) signalling pathways. Furthermore, differentially expressed genes in both longevity and AMPK signalling pathways were associated with a change in DNA methylation, with a total of 256 and 360 significant cytosine-phosphate-guanine-gene correlations identified, respectively. Similar changes in the muscle transcriptome were observed with respect to per cent fat mass and total fat mass. Obesity was further associated with a significant increase in type II fast-fibre area (P = 0.026), of which key regulatory genes within both longevity and AMPK pathways were significantly associated. CONCLUSIONS: We provide for the first time a global transcriptomic profile of skeletal muscle in older people with and without obesity, demonstrating modulation of key genes and pathways implicated in the regulation of muscle function, changes in DNA methylation associated with such pathways and associations between genes within the modified pathways implicated in muscle regulation and changes in muscle fibre type.

Epigenome-wide association study of sarcopenia: findings from the Hertfordshire Sarcopenia Study (HSS).

BACKGROUND: Sarcopenia is the age-related loss of muscle mass, strength, and function. Epigenetic processes such as DNA methylation, which integrate both genetic and environmental exposures, have been suggested to contribute to the development of sarcopenia. This study aimed to determine whether differences in the muscle methylome are associated with sarcopenia and its component measures: grip strength, appendicular lean mass index (ALMi), and gait speed. METHODS: Using the Infinium Human MethylationEPIC BeadChip, we measured DNA methylation in vastus lateralis muscle biopsies of 83 male participants (12 with sarcopenia) with a mean (standard deviation) age of 75.7 (3.6) years from the Hertfordshire Sarcopenia Study (HSS) and Hertfordshire Sarcopenia Study extension (HSSe) and examined associations with sarcopenia and its components. Pathway, histone mark, and transcription factor enrichment of the differentially methylated CpGs (dmCpGs) were determined, and sodium bisulfite pyrosequencing was used to validate the sarcopenia-associated dmCpGs. Human primary myoblasts (n = 6) isolated from vastus lateralis muscle biopsies from male individuals from HSSe were treated with the EZH2 inhibitor GSK343 to assess how perturbations in epigenetic processes may impact myoblast differentiation and fusion, measured by PAX7 and MYHC immunocytochemistry, and mitochondrial bioenergetics determined using the Seahorse XF96. RESULTS: Sarcopenia was associated with differential methylation at 176 dmCpGs (false discovery rate ≤ 0.05) and 141 differentially methylated regions (Stouffer ≤ 0.05). The sarcopenia-associated dmCpGs were enriched in genes associated with myotube fusion (P = 1.40E-03), oxidative phosphorylation (P = 2.78E-02), and voltage-gated calcium channels (P = 1.59E-04). ALMi was associated with 71 dmCpGs, grip strength with 49 dmCpGs, and gait speed with 23 dmCpGs (false discovery rate ≤ 0.05). There was significant overlap between the dmCpGs associated with sarcopenia and ALMi (P = 3.4E-35), sarcopenia and gait speed (P = 4.78E-03), and sarcopenia and grip strength (P = 7.55E-06). There was also an over-representation of the sarcopenia, ALMi, grip strength, and gait speed-associated dmCpGs with sites of H3K27 trimethylation (all P ≤ 0.05) and amongst EZH2 target genes (all P ≤ 0.05). Furthermore, treatment of human primary myoblasts with the EZH2 inhibitor GSK343 inhibitor led to an increase in PAX7 expression (P ≤ 0.05), decreased myotube fusion (P = 0.043), and an increase in ATP production (P = 0.008), with alterations in the DNA methylation of genes involved in oxidative phosphorylation and myogenesis. CONCLUSIONS: These findings show that differences in the muscle methylome are associated with sarcopenia and individual measures of muscle mass, strength, and function in older individuals. This suggests that changes in the epigenetic regulation of genes may contribute to impaired muscle function in later life.

Associations of osteoporosis and sarcopenia with frailty and multimorbidity among participants of the Hertfordshire Cohort Study.

BACKGROUND: Ageing is commonly associated with sarcopenia (SP) and osteoporosis (OP), both of which are associated with disability, impaired quality of life, and mortality. The aims of this study were to explore the relationships between SP, OP, frailty, and multimorbidity in community-dwelling older adults participating in the Hertfordshire Cohort Study (HCS) and to determine whether coexistence of OP and SP was associated with a significantly heavier health burden. METHODS: At baseline, 405 participants self-reported their comorbidities. Cut-offs for low grip strength and appendicular lean mass index were used according to the EWSGOP2 criteria to define SP. OP was diagnosed when T-scores of < -2.5 were present at the femoral neck or the participant reported use of the anti-OP medications including hormone replacement therapy (HRT), raloxifene, or bisphosphonates. Frailty was defined using the standard Fried definition. RESULTS: One hundred ninety-nine men and 206 women were included in the study. Baseline median (interquartile range) age of participants was 75.5 (73.4-77.9) years. Twenty-six (8%) and 66 (21.4%) of the participants had SP and OP, respectively. Eighty-three (20.5%) reported three or more comorbidities. The prevalence of pre-frailty and frailty in the study sample was 57.5% and 8.1%, respectively. Having SP only was strongly associated with frailty [odds ratio (OR) 8.28, 95% confidence interval (CI) 1.27, 54.03; P = 0.027] while the association between having OP alone and frailty was weaker (OR 2.57, 95% CI 0.61, 10.78; P = 0.196). The likelihood of being frail was substantially higher in the presence of coexisting SP and OP (OR 26.15, 95% CI 3.13, 218.76; P = 0.003). SP alone and OP alone were both associated with having three or more comorbidities (OR 4.71, 95% CI 1.50, 14.76; P = 0.008 and OR 2.86, 95% CI 1.32, 6.22; P = 0.008, respectively) although the coexistence of SP and OP was not significantly associated with multimorbidity (OR 3.45, 95% CI 0.59, 20.26; P = 0.171). CONCLUSIONS: Individuals living with frailty were often osteosarcopenic. Multimorbidity was common in individuals with either SP or OP. Early identification of SP and OP not only allows implementation of treatment strategies but also presents an opportunity to mitigate frailty risk.

Multi-Morbidity and Polypharmacy in Older People: Challenges and Opportunities for Clinical Practice.

Multi-morbidity and polypharmacy are common in older people and pose a challenge for health and social care systems, especially in the context of global population ageing. They are complex and interrelated concepts in the care of older people that require early detection and patient-centred shared decision making underpinned by multi-disciplinary team-led comprehensive geriatric assessment (CGA) across all health and social care settings. Personalised care plans need to remain responsive and adaptable to the needs and wishes of the patient, enabling the individual to maintain their independence. In this review, we aim to give an up-to-date account of the recognition and management of multi-morbidity and polypharmacy in the older person.

Mitochondrial oxidative capacity and NAD+ biosynthesis are reduced in human sarcopenia across ethnicities.

The causes of impaired skeletal muscle mass and strength during aging are well-studied in healthy populations. Less is known on pathological age-related muscle wasting and weakness termed sarcopenia, which directly impacts physical autonomy and survival. Here, we compare genome-wide transcriptional changes of sarcopenia versus age-matched controls in muscle biopsies from 119 older men from Singapore, Hertfordshire UK and Jamaica. Individuals with sarcopenia reproducibly demonstrate a prominent transcriptional signature of mitochondrial bioenergetic dysfunction in skeletal muscle, with low PGC-1α/ERRα signalling, and downregulation of oxidative phosphorylation and mitochondrial proteostasis genes. These changes translate functionally into fewer mitochondria, reduced mitochondrial respiratory complex expression and activity, and low NAD+ levels through perturbed NAD+ biosynthesis and salvage in sarcopenic muscle. We provide an integrated molecular profile of human sarcopenia across ethnicities, demonstrating a fundamental role of altered mitochondrial metabolism in the pathological loss of skeletal muscle mass and function in older people.

A Stab in the Dark: A Case Report of an Atypical Presentation of Giant Cell Arteritis (GCA).

Background: Giant cell arteritis/temporal arteritis (GCA) is an inflammatory condition that affects large to medium vessels such as the aorta and its primary branches. Patients classically present with fatigue, fever, headache, jaw claudication and in severe cases, may suffer either transient (amaurosis fugax) or permanent visual loss. The reference standard for diagnosis is the temporal artery biopsy (TAB) and the mainstay of treatment is with immunosuppression. Our patient JG, presented with a range of non-specific symptoms that mimicked generalised sepsis, but was ultimately diagnosed with GCA through effective, methodical multi-disciplinary team (MDT) work. Clinical case: JG, an 81 year old gentleman, presented acutely with a 3-4 weeks history of fatigue, lethargy, pyrexia and a marked inflammatory response suggestive of a sepsis but without a clear primary source or clinical features of vasculitis. His inflammatory markers were markedly raised although his erythrocyte sedimentation rate (ESR) was not elevated. He was initially treated for sepsis of unknown origin however, body imaging after admission suggested a possible infection around a previous aortic graft site. This was refuted in subsequent 18F-fluorodeoxyglucose-positron emission tomography (FDG-PET/CT) scanning. Microbiological, parasitic, as well as autoimmune assays were unremarkable. He underwent a TAB which was diagnostic for GCA and as a result, was started on oral corticosteroids with immediate symptom relief. He was discharged and followed up on an outpatient basis. Conclusions: This case highlights how a vasculitis can present with a range of non-specific symptoms that may resemble a fever of unknown origin (FUO)/sepsis that can lead to a delay in making the correct diagnosis. It also highlights the importance of considering a diagnosis of vasculitis in patients who present with a FUO where there is no clear focus of infection. Delays in diagnosis and management of these conditions can potentially lead to significant irreversible morbidity.

Learning from older peoples' reasons for participating in demanding, intensive epidemiological studies: a qualitative study.

BACKGROUND: Recruitment rates of older people in epidemiological studies, although relatively higher than in clinical trials, have declined in recent years. This study aimed to explore motivating factors and concerns among older participants in an intensive epidemiological study (Hertfordshire Sarcopenia Study - HSS) and identify those that could aid future recruitment to epidemiological studies and clinical trials. METHODS: Participants of the HSS fasted overnight and travelled several hours each way to the research facility at an English hospital for extensive diet/lifestyle questionnaires and investigations to assess muscle including blood tests and a muscle biopsy. We conducted semi-structured interviews with 13 participants (ten women) at the research facility in May-October 2015. The interviews were audio-taped, transcribed verbatim, coded and analysed thematically by three researchers. RESULTS: We identified personal motives for participation (potential health benefit for self and family; curiosity; comparing own fitness to others; socialising). Altruistic motives (benefit for other people; belief in importance of research) were also important. Participants voiced a number of external motives related to the study uniqueness, organisation and safety record; family support; and just 'being asked'. Anxiety about the biopsy and travel distance were the only concerns and were alleviated by smooth and efficient running of the study. CONCLUSIONS: Personal and altruistic reasons were important motivators for these older people to participate in demanding, intensive research. They valued belonging to a birth cohort with previous research experience, but personal contact with the research team before and after consent provided reassurance, aided recruitment to HSS and could be readily replicated by other researchers. Any fears or concerns related to certain aspects of a demanding, intensive study should ideally be explored at an early visit to establish a good relationship with the research team.

Increased expression of H19/miR-675 is associated with a low fat-free mass index in patients with COPD.

BACKGROUND: Loss of muscle mass and strength is a significant comorbidity in patients with chronic obstructive pulmonary disease (COPD) that limits their quality of life and has prognostic implications but does not affect everyone equally. To identify mechanisms that may contribute to the susceptibility to a low muscle mass, we investigated microRNA (miRNA) expression, methylation status, and regeneration in quadriceps muscle from COPD patients and the effect of miRNAs on myoblast proliferation in vitro. The relationships of miRNA expression with muscle mass and strength was also determined in a group of healthy older men. METHODS: We identified miRNAs associated with a low fat-free mass (FFM) phenotype in a small group of patients with COPD using a PCR screen of 750 miRNAs. The expression of two differentially expressed miRNAs (miR-675 and miR-519a) was determined in an expanded group of COPD patients and their associations with FFM and strength identified. The association of these miRNAs with FFM and strength was also explored in a group of healthy community-dwelling older men. As the expression of the miRNAs associated with FFM could be regulated by methylation, the relative methylation of the H19 ICR was determined. Furthermore, the proportion of myofibres with centralized nuclei, as a marker of muscle regeneration, in the muscle of COPD patients was identified by immunofluorescence. RESULTS: Imprinted miRNAs (miR-675 and from a cluster, C19MC which includes miR-519a) were differentially expressed in the quadriceps of patients with a low fat-free mass index (FFMI) compared to those with a normal FFMI. In larger cohorts, miR-675 and its host gene (H19) were higher in patients with a low FFMI and strength. The association of miR-519a expression with FFMI was present in male patients with severe COPD. Similar associations of miR expression with lean mass and strength were not observed in healthy community dwelling older men participating in the Hertfordshire Sarcopenia Study. Relative methylation of the H19 ICR was reduced in COPD patients with muscle weakness but was not associated with FFM. In vitro, miR-675 inhibited myoblast proliferation and patients with a low FFMI had fewer centralized nuclei suggesting miR-675 represses regeneration. CONCLUSIONS: The data suggest that increased expression of miR-675/H19 and altered methylation of the H19 imprinting control region are associated with a low FFMI in patients with COPD but not in healthy community dwelling older men suggesting that epigenetic control of this loci may contribute to a susceptibility to a low FFMI.

Human Vastus Lateralis Skeletal Muscle Biopsy Using the Weil-Blakesley Conchotome.

Percutaneous muscle biopsy using the Weil-Blakesley conchotome is well established in both clinical and research practice. It is a safe, effective and well tolerated technique. The Weil-Blakesley conchotome has a sharp biting tip with a 4 - 6 mm wide hollow. It is inserted through a 5 - 10 mm skin incision and can be maneuvered for controlled tissue penetration. The tip is opened and closed within the tissue and then rotated through 90 -180° to cut the muscle. The amount of muscle obtained following repeated sampling can vary from 20 mg to 290 mg which can be processed for both histology and molecular studies. The wound needs to be kept dry and vigorous physical activity kept to a minimum for approximately 72 hr although normal levels of activity can restart immediately following the procedure. This procedure is safe and effective when close attention is paid to the selection of subjects, full asepsis and post procedure care.  Both right and left vastus lateralis are suitable for biopsy dependent on participant preference.

Lean mass, muscle strength and gene expression in community dwelling older men: findings from the Hertfordshire Sarcopenia Study (HSS).

Sarcopenia is associated with adverse health outcomes. This study investigated whether skeletal muscle gene expression was associated with lean mass and grip strength in community-dwelling older men. Utilising a cross-sectional study design, lean muscle mass and grip strength were measured in 88 men aged 68-76 years. Expression profiles of 44 genes implicated in the cellular regulation of skeletal muscle were determined. Serum was analysed for circulating cytokines TNF (tumour necrosis factor), IL-6 (interleukin 6, IFNG (interferon gamma), IL1R1 (interleukin-1 receptor-1). Relationships between skeletal muscle gene expression, circulating cytokines, lean mass and grip strength were examined. Participant groups with higher and lower values of lean muscle mass (n = 18) and strength (n = 20) were used in the analysis of gene expression fold change. Expression of VDR (vitamin D receptor) [fold change (FC) 0.52, standard error for fold change (SE) ± 0.08, p = 0.01] and IFNG mRNA (FC 0.31; SE ± 0.19, p = 0.01) were lower in those with higher lean mass. Expression of IL-6 (FC 0.43; SE ± 0.13, p = 0.02), TNF (FC 0.52; SE ± 0.10, p = 0.02), IL1R1 (FC 0.63; SE ± 0.09, p = 0.04) and MSTN (myostatin) (FC 0.64; SE ± 0.11, p = 0.04) were lower in those with higher grip strength. No other significant changes were observed. Significant negative correlations between serum IL-6 (R = -0.29, p = 0.005), TNF (R = -0.24, p = 0.017) and grip strength were demonstrated. This novel skeletal muscle gene expression study carried out within a well-characterized epidemiological birth cohort has demonstrated that lower expression of VDR and IFNG is associated with higher lean mass, and lower expression of IL-6, TNF, IL1R1 and myostatin is associated with higher grip strength. These findings are consistent with a role of proinflammatory factors in mediating lower muscle strength in community-dwelling older men.

Muscle size, strength, and physical performance and their associations with bone structure in the Hertfordshire Cohort Study.

Sarcopenia is associated with a greater fracture risk. This relationship was originally thought to be explained by an increased risk of falls in sarcopenic individuals. However, in addition, there is growing evidence of a functional muscle-bone unit in which bone health may be directly influenced by muscle function. Because a definition of sarcopenia encompasses muscle size, strength, and physical performance, we investigated relationships for each of these with bone size, bone density, and bone strength to interrogate these hypotheses further in participants from the Hertfordshire Cohort Study. A total of 313 men and 318 women underwent baseline assessment of health and detailed anthropometric measurements. Muscle strength was measured by grip strength, and physical performance was determined by gait speed. Peripheral quantitative computed tomography (pQCT) examination of the calf and forearm was performed to assess muscle cross-sectional area (mCSA) at the 66% level and bone structure (radius 4% and 66% levels; tibia 4% and 38% levels). Muscle size was positively associated with bone size (distal radius total bone area ß = 17.5 mm2 /SD [12.0, 22.9]) and strength (strength strain index (ß = 23.3 mm3 /SD [18.2, 28.4]) amongst women (p < 0.001). These associations were also seen in men and were maintained after adjustment for age, height, weight-adjusted-for-height, limb-length-adjusted-for-height, social class, smoking status, alcohol consumption, calcium intake, physical activity, diabetes mellitus, and in women, years since menopause and estrogen replacement therapy. Although grip strength showed similar associations with bone size and strength in both sexes, these were substantially attenuated after similar adjustment. Consistent relationships between gait speed and bone structure were not seen. We conclude that although muscle size and grip strength are associated with bone size and strength, relationships between gait speed and bone structure and strength were not apparent in this cohort, supporting a role for the muscle-bone unit.

Hertfordshire sarcopenia study: design and methods.

BACKGROUND: Sarcopenia is defined as the loss of muscle mass and strength with age. Although a number of adult influences are recognised, there remains considerable unexplained variation in muscle mass and strength between older individuals. This has focused attention on influences operating earlier in life. Our objective for this study was to identify life course influences on muscle mass and strength in an established birth cohort and develop methodology for collection of muscle tissue suitable to investigate underlying cellular and molecular mechanisms. METHODS: One hundred and five men from the Hertfordshire Cohort Study (HCS), born between 1931 and 1939 who have historical records of birth weight and weight at one year took part in the Hertfordshire Sarcopenia Study (HSS). Each participant consented for detailed characterisation of muscle mass, muscle function and aerobic capacity. In addition, a muscle biopsy of the vastus lateralis using a Weil-Blakesley conchotome was performed. Data on muscle mass, function and aerobic capacity was collected on all 105 participants. Muscle biopsy was successfully carried out in 102 participants with high rates of acceptability. No adverse incidents occurred during the study. DISCUSSION: The novel approach of combining epidemiological and basic science characterisation of muscle in a well established birth cohort will allow the investigation of cellular and molecular mechanisms underlying life course influences on sarcopenia.