Future Physiology 2019

Contemporary Issues in an Ageing Society and Ageing Research

Healthy ageing is essential to prevent overburdening health care facilities and to capture the potential of an increasingly older population. Since 2001, commitments to achieving healthy ageing have featured heavily in government policy and directives. However, almost 20 years later, healthy ageing for all remains elusive. As a consequence, we have an urgent duty to reduce lifestyle-linked chronic ill health, if we are to enable healthy ageing for all. To deliver the goal of increasing healthy, active ageing, we must first understand what causes ageing, if we are to develop means to influence the processes. To achieve this goal, we need to study the ageing process in order to determine, manipulate and model functional, physiological, cellular and molecular adaptations with age. Using cellular (human skeletal muscle stem cells and C2C12 muscle cells), molecular, biochemical and human (age, exercise, disuse and cancer populations) intervention studies, it has been a career goal to develop useful, adoptable, translatable models to understand aspects of mal/adaptation with age. The purpose of this presentation will be to provide oversight of the work undertaken and career pathway followed to facilitate questions, challenge dogmas and enable collaborations in the highly important field of healthy ageing.

The Role of Epigenetics in Regulating Ageing Skeletal Muscle

The deterioration of skeletal muscle into old age (sarcopenia) poses a significant risk to human health and well-being. Sarcopenia is not a uniform condition, with large inter-species variability in the intensity and progression of sarcopenia observed. With underlying genetics not able to adequately explain such variability in the condition, it leads one to suggest that environmental encounters, at least in part, regulate the sarcopenic milieu. In this regard, epigenetics, referring to gene expression changes not caused by underlying genetic variation, becomes a plausible candidate for the regulation of skeletal muscle during environmental insult. Indeed, recent work from has begun to characterise the role of epigenetic modifications in mammalian skeletal muscle during periods of both anabolism and catabolism. For example, we have previously shown an important role for promotor associated DNA methylation of atro-genes during periods of acute muscular atrophy and recovery (Fisher et al., 2017) and global methylome changes during both acute and chronic resistance training induced muscular anabolism (Seaborne et al., 2018). Furthermore, classical research has shown the mammalian organism to be susceptible to early life stress encounters inducing adverse life-long phenotypic outcomes, referred to as developmental/foetal programming. Such a model has also been linked to epigenetics, where underlying modifications are maintained over time, leading to long-term regulation of the phenotype (Holland et al., 2016; Sharples, Stewart and Seaborne, 2016). We have recently shown a similar molecular memory mechanism in skeletal muscle (Seaborne et al., 2018). Epigenetic modifications may therefore act as the link between environmental insult and adverse transcriptomic and phenotypic outcome in skeletal muscle. We will therefore detail our current understanding of skeletal muscle epigenetics, the epigenetic aberrations associated with the sarcopenic niche and how exercise may alleviate these adverse outcomes.

Individual and Combined Genetic Variations are Associated with in vivo and in vitro Muscle Damage in Humans: a Genetic Approach to Elucidate the Mechanisms Underpinning the Response to Exercise-Induced Muscle Damage

Introduction We aimed to test the hypothesis that a combination of genetic variations forming a polygenic profile could estimate high and low responders to exercise-induced muscle damage (EIMD). Methods Sixty-five young female and male untrained Caucasians performed 120 maximal eccentric knee-extensions to induce EIMD. Maximal quadriceps strength, range of motion (ROM), muscle soreness and serum blood biomarkers were assessed before, directly after and 48 h after the EIMD intervention and participants were genotyped for 20 candidate single nucleotide polymorphisms (SNPs). Significant SNPs were further investigated regarding the muscle recovery following an artificial wound healing assay in vitro. SNPs that showed a gene-intervention interaction in vivo, were used to calculate a total genotype score in respect to the acute response following EIMD (TGS-A) and the cohort was then divided into a “preferential” (PG), “moderate” (MG), and “non-preferential” (NPG) genetic group. Results Four SNPs, which showed an interaction/main effect with the EIMD-intervention in vivo, also demonstrated changes in muscle stem cell characteristics in vitro. Seven SNPs demonstrated significant interactions in vivo, and these candidate SNPs were used to compute the TGS-A. There was a main effect for isometric and isokinetic MVC torque regarding TGS-A (both P<0.001). Individuals of the NPG and MG group were consistently weaker compared to the PG group (P=0.005), and NPGs demonstrated higher muscle soreness (P=0.003) and decreased ROM (P=0.006) following the EIMD-intervention, respectively. Conclusion Seven SNPs have been associated with EIMD and recovery. When SNPs were combined, NPG not only demonstrated poorer recovery following EIMD but this group was also generally weaker than PG, which could have clinical implications in terms of exercise prescription for reducing injury risk in those who are predisposed to greater muscle damage following strenuous exercise. Further, the associations between four of these seven SNPs with in vitro damage/regeneration suggest novel gene-cell-skeletal muscle mechanisms explaining the individual response to EIMD.

Single-Cell Transcriptomic Analysis Revealed molecular signature of Pnmt-derived Cardiomyocytes

Rationale: Phenylethanolamine N-methyl transferase (PNMT) is the enzyme that catalyses the conversion of noradrenaline into adrenaline in endocrine cells. Our group previously reported a new type of cardiomyocytes that historically express PNMT during development stage. We call these cells as Pnmt-derived cardiomyocytes (PdCMs)1. PdCMs have unique left heart localisation in adult heart1. However, so far little is known about their molecular signature and their potential additional function in physiological or pathophysiological conditions comparing to conventional cardiomyocytes due to their neuronal origin. . Methods: single cardiac cells were isolated from either C57BL neonatal mouse hearts (aged 3 days, n=20) or adult (12 weeks old) YFP-ChR2/Cre-Pnmt mouse hearts for Single-cell RNA sequencing (scRNAseq). For neonatal cells, 10X Genomics scRNAseq was used. Single cell suspensions were converted to barcoded scRNA-seq libraries with the Chromium Single Cell 3′ kit with 10X Genomics platform, aiming for an estimated 10,000 cells per library. The libraries were sequenced using HiSeq400. Expression matrices were generated using CellRanger and analysed by Seurat package. Dimensionality reduction using principle component analysis was applied to identify major cell types and their subtypes. For adult myocytes, SORT- RNAseq was used. Results: In neonatal heart samples, a total of 10550 transcripts were sequenced at single cell resolution. Major cell types were identified by leveraging single-cell transcriptomics analysis. We found Pnmt expressing cells are exclusively cardiomyocytes in neonatal heart. Rspo3 and Bmp2 are the most differentially expressed genes in Pnmt expressing cardiomyocytes compared to conventional cardiomyocytes. The clustering of Pnmt expressing cells are mainly presented in cardiac conduction system (CCS), left atrium and left ventricle. In adult mice, 192 YFP-Pnmt negative and 192 YFP-Pnmt positive cells were sequenced. Expression of Pnmt gene was significantly declined and even disappeared in most PdCMs. However, in both neonatal and adult Pnmt+ cells, a high expression of exocytosis-related genes including CPLX2, SLC12A2, SLC6A4, SLC22A1, RAB31, RAB35, RAP2B, STX7, VAMP3, VAMP4 have been identified. Conclusions: Single-cell RNA sequencing revealed molecular signature of Pnmt-derived myocytes. Their high expression of exocytosis-related gene expression and special localization suggest their potential role in localized endocrine signaling and regulation.

A transcriptomic insight into the mechanism underlying the decrease in atrial ICa-L in heart failure

In heart failure (HF) impaired ventricular relaxation means the atrial component of ventricular filling becomes more important. However, the amplitude of the Ca2+ transient in the atria, which underlies contraction, is decreased in HF. We have shown that a decrease in atrial L-type Ca2+ current (ICa-L) in HF underlies the decrease in Ca2+ transient amplitude but the mechanism behind the decrease in ICa-L in HF is not fully understood. Since transverse (t)-tubules, the deep invaginations of the cell membrane on which ICa-L resides, are almost completely lost in HF we investigated if ICa-L expression is decreased in the HF atria. HF was induced in sheep by rapid ventricular pacing at 210 beats per minute for ~6 weeks. A pacing lead was implanted in the endocardial apex of the right ventricle using a minimally invasive transvenous approach under fluoroscopic guidance. Surgical plane anaesthesia was maintained under isoflurane (3-5%) mixed with of oxygen (4.5-6 L.min-1). HF progression was monitored via echocardiography. We have employed a transcriptomic based approach through RNA-sequencing and qPCR of atrial tissue in control and HF in order to determine the causes of reduced Ica-L in HF. RNA was isolated from time-matched control and HF tissue using a Trizol method and column-size isolation. Following quality control, samples (normalised to 1 µg), with an RNA integrity number of ≈8, were synthesised into a cDNA library by polyadenylation enrichment and sequenced on an Illumina HiSeq4000 at the Wellcome trust for Human Genetics, Oxford. Samples were pooled and run across three lanes at a length of 75 base pairs with paired-end reads. Subsequent BAM files were analysed through DESeq2 at QFAB. Despite T-tubules loss in HF no change in the LTCC pore forming unit, CACNA1C, was detected through RNA-Seq or qPCR. Secondly, the associated subunits that form the LTCC together with CACNA1C were investigated. Expressional changes (P<0.05) were detected in three subunits, CACNB1, CACNA2D1 and CACNG6, none of which would be expected to decrease ICa-L . Finally, external modulators of ICa-L were considered. Phosphodiesterases (PDEs) are known to modulate ICa-L through protein kinases A and G (PKA/G) and were identified as potential candidates via PANTHER. Six PDEs were found to change in HF; PDEs 3B,3A,8A,6D and 12 were upregulated whilst 1B is downregulated. Of these six PDE3B is thought the most likely to co-localise to the LTCC nanodomain at the cell membrane. Increased expression of PDE3B was confirmed by qPCR and thus may be responsible for the decrease in ICa-L in HF. In conclusion we show the decrease in atrial ICa-L in HF is not brought about by a decrease in mRNA encoding the channel subunits. Our data suggests alterations in ICa-L regulators such as PRKAR1A and an increase in PDEs may play a role in decreased ICa-L function and warrant further investigation.

A proteomic insight into liver dysfunction and metabolism during obesity

Obesity and insulin resistance are characterised by altered metabolism and function in various tissues. In the liver, obesity contributes to liver-related metabolic diseases including insulin resistance and non-alcoholic fatty liver disease (Kitade et al., 2017). The ob/ob mouse, which produces a truncated inactive leptin protein, is widely used as a model of obesity-induced metabolic disease (Drel et al., 2006). Whilst the physiological phenotype of ob/ob mice has been well characterised, the underlying alterations in protein content and function remain relatively understudied. Using a deep-proteomic approach, we have identified divergent proteins and pathways that may underpin the mechanistic basis of obesity-induced liver dysfunction. Liver samples were harvested from four-month old ob/ob mice and lean littermates (n = 4 per group, C57BL/6J background) following a four-hour fast. Liver samples were lysed in a 4% SDS buffer and processed according to the MED-FASP protocol using Lys-C and trypsin. Peptides were separated on an Easy nano-flow HPLC system coupled to a LTQ Orbitrap mass spectrometer (HFX) via a nanoelectrospray source (Thermo Fisher Scientific). MS and MS/MS spectra were acquired in a data-dependent manner and analysed using MaxQuant software. Downstream data analysis was performed in Perseus software using label-free quantification (LFQ) intensities. Proteomics analysis of liver from lean and ob/ob mice led to the quantification of 5551 proteins. 330 proteins were differentially regulated with obesity, of which 172 and 158 proteins were up- or downregulated, respectively, in ob/ob mice. Gene Ontology annotations related to fatty acid metabolism were upregulated in the liver of ob/ob mice as well as the KEGG pathways PPAR signalling and peroxisome, indicating an adaptation to excess fatty acid availability. Furthermore, Gene Ontology cellular components related to the extracellular matrix were also upregulated in obese livers. Conversely, cellular component terms related to the endoplasmic reticulum were enriched in the downregulated proteins. The cytochrome p450 family, involved in the metabolism of arachidonic acid (a membrane phospholipid) and lipocalins, specifically the major urinary protein (MUP) isoforms, were also significantly downregulated in ob/ob liver. Overall, we provide a deep-proteomic analysis of liver from ob/ob and wild-type mice, identifying known and emerging regulators of insulin sensitivity within the liver. Further analyses will provide additional mechanistic insight into the development of liver dysfunction during obesity, in particular into the dysregulation of the extracellular matrix.

Muscle adaptation induced by endurance activity involves changes to protein abundance that are regulated by protein degradation as well as protein synthesis.

Muscle adapts to exercise through changes in the abundance of specific contractile and metabolic proteins. Exercise training also increases muscle protein turnover, but it is not known how synthesis and degradation contribute to exercise-induced changes in the abundance of individual proteins. We used stable isotope (deuterium oxide) labelling in combination with chronic low-frequency stimulation (CLFS) in vivoto investigate the synthesis, abundance and degradation of individual proteins during exercise-induced muscle adaptation. Surgery was performed on 4 independent groups of rats (n = 3 in each) under aseptic conditions. Pre-operativeanalgesia 0.05 mg Buprenorphine /kg body mass was provided and anaesthesia was induced by4 % isoflurane-O2adjusted to 1-2 % isoflurane during surgery.Unilateral CLFS (10 Hz, 24 h/d) and deuterium oxide were administered for 0 d, 10 d, 20 d or 30 d. Rats were humanely killed and the extensor digitorium longus (EDL) was isolated from stimulated (Stim) and non-stimulated (Ctrl) legs. Proteomic analysis encompassed 30 myofibrillar and 47 soluble proteins. Protein fractional synthesis rate (FSR) was calculated from peptide mass spectrometry data. Absolute synthesis rates (ASR) were derived from FSR and abundance data and used to calculate protein degradation rates. Data are mean ± SD compared by within-animal paired t-test. CLFS tended (P = 0.145) to increase the synthesis of mixed myofibrillar proteins from Ctrl (FSR, 4.7 ± 0.3 %/d; ASR, 8.6 ± 0.3 pg/d) to Stim (FSR, 5 ± 0.1 %/d; ASR, 10.3 ± 1.6 pg/d). Whereas the synthesis of mixed soluble proteins increased (P = 0.001) from Ctrl (FSR, 5.2 ± 0.04%/d; ASR, 43.7 ± 0.1 pg/d) to Stim (FSR, 5.4 ± 0.4 %/d; ASR, 60 ± 0.7 pg/d). Protein turnover responses differed on a protein-by-protein basis. The abundances of glycogen phosphorylase(PYGB), beta-enolase(ENOB) and troponin T, fast (TNNT3) significantly (P<0.05) decreased (-11 ± 3-fold) after CLFS. The decrease in abundance of ENOB was partially accounted for by a lesser synthesis in Stim muscle. The decrease in PYGB abundance was entirely accounted for by the decrement in its synthesis rate, whereas the abundance of TNNT3 decreased without a detectable change in synthesis. Myosin regulatory light chain 2 (MLRS) was resolved as 2 separate proteoforms (a and b). MLRSa was uniquely phosphorylated at S20 and significantly (P< 0.05) decreased (62 %) in abundance, whereas MLRSb tended (P = 0.100) to increase (16 %) after 30 days of CLFS. There was no difference in synthesis rate between Stim and Ctrl muscle of either MLRS proteoform. Therefore, we attribute the decrease in MLRSa abundance to proteoform-specific degradation. In conclusion, muscle adaptation in response to CLFS is underpinned by protein-specific changes in degradation as well as synthesis.

Physiological and structural changes in skeletal muscle and nerve-muscle interactions: the effects of ROS and ageing

With an ever growing population, and the fastest growing group being elderly, we need to understand and treat those factors which affect quality of life in later years. It is well established that there is an age-related decline in muscle mass and function with increasing age (termed sarcopenia) and this is associated with increased falls and hospital admissions. There are many factors which have been associated with sarcopenia. Substantial alteration of motor neurons and changes in reactive oxygen species (ROS) with advancing age are widely described in rodent models. We have utilised several mouse models, deficient in the antioxidant enzyme superoxide dismutase 1 (SOD1), to examine to role of aberrant redox homeostasis on nerve-muscle interactions. Whole body knock out of Sod1 in mice causes accelerated age-related muscle wasting, a decline in force production and alterations in neuromuscular junctions (NMJs). The NMJ is composed of the pre-synaptic nerve terminal, the acetylcholine receptor clusters on the muscle and terminal Schwann cells that encase this region. It is a dynamic structure and multiple cycles of denervation and re-innervation throughout life are thought to result in structural changes which accumulate. When re-innervation fails the muscle fibre remains denervated and the incidence of denervated fibres is increased with advancing age which correlates with reduced muscle mass and function. Using a surgical model of nerve transection we have shown that there is an increase in mitochondrial generation of hydrogen peroxide and other peroxides in denervated muscle fibres which also occurs in neighbouring fibres which have a “normal” NMJ structure. We speculate that the initial increase in peroxide production may stimulate adaptations to protect the muscle fibre. More chronic production of peroxides has also been shown to activate several degenerative processes preceding loss of muscle mass. Recent in vivo imaging of HyPer2 transduced skeletal muscle from our lab has also drawn strong links between innervation status and peroxide production in nerve crush models Ongoing work seeks to understand the complex relationship between the redox status of motor nerves and skeletal muscle in the Sod1KO mouse model and in ageing of wild type mice together with the role that Schwann cells, which are responsible for NMJ stability, are playing in these situations.

MicroRNA control of the circadian rhythm in heart rate

Background: There is a circadian rhythm in heart rate and conversely, bradyarrhythmias occur at night. Recent work from our lab has demonstrated heart rate rhythmicity to be underscored by a diurnal fluctuation in the key pacemaking channel HCN4, and its corresponding funny current If, likely driven by a local clock transcription factor BMAL1 (1). We have also previously shown that microRNAs (miRs) regulate sinus node pacemaking by direct negative regulation of Hcn4 transcription (2). Here we tested whether rhythmic miR expression explains Hcn4 and Bmal1 rhythmicity in the sinus node. Methods: Sinus node biopsies were collected from nocturnal adult male C57BL/6J mice at zeitgeber time (ZT) 0, ZT6, ZT12 and ZT18 (n=7/8). TaqMan Array MicroRNA A+B Cards were used to measure the expression of 750 miRs and 6 reference transcripts. JTK_Cycle (3) and sine wave fitting were used to identify rhythmic miRs (exhibiting a ~24 h periodicity) and in silico prediction algorithms RNA22, TargetScan and PITA applied to identify potential 3′ untranslated region (3′UTR) target sites. Functionality of predicted sites was tested by dual luciferase reporter gene assay; recombinant plasmids, in which a luciferase coding sequence was fused to (i) Hcn4 3’UTR and (ii) Bmal1 3’UTR, were co-transfected individually with (i) 19 and (ii) 11 miR mimics in H9C2 rat myoblast cells and compared to control plasmid. Firefly luciferase was measured and normalised to Renilla luciferase in 7 independent batches of cells. Results: JTK_Cycle analysis identified 159 rhythmic microRNAs (Bonferroni adjusted P<0.05). Sine wave fitting was carried out on this dataset to identify false positives, after which 56 miRs were found to exhibit day-night rhythms in the sinus node. Of these miRs, 39 and 11, respectively, were expressed antiphase to Hcn4 and Bmal1, indicating potential for negative regulation. Bioinformatics analyses identified 19 miRs with binding sites on the Hcn4 3’UTR whereas 11 miRs were predicted for Bmal1 3’UTR binding. Compared to control, 6/19 miRs predicted to target Hcn4, and 2/11 miRs predicted to target Bmal1, resulted in a significant reduction in reporter bioluminescence vs. control (P<0.05, unpaired t test). Compared to co-transfection with negative control C.elegans miR-67, miRs-146a-5p, -128-3p, -150-5p and -203-3p significantly reduced Hcn4 3′UTR activity (P<0.05, unpaired t test). Sine curve analysis of these miRs determined them to be expressed antiphase (peak ~ZT9) to Hcn4 (peak ~ZT21). Conclusions: This is the first report of rhythmic miR expression in the sinus node. miRs-146a-5p, -128-3p, -150-5p and -203-3p are novel regulators of Hcn4 transcription and ongoing studies are examining their physiological relevance for the circadian rhythm in sinus node pacemaking. This could be the first step towards chronotherapeutic targets for the treatment of heart rhythm disturbances.

3D Segmentation of the Cardiac Mitochondria and Nuclei from the Greenland Shark (Somniosus microcephalus) Insights Into Extreme Longevity

The Greenland shark (Somniosus microcephalus) live up to 392 ± 120 years, making it the world’s oldest-living vertebrate [1]. Because cardiovascular diseases are synonymous with age in humans, we aimed to understand how the heart of this vertebrate can beat since Shakespearian times without failing. Our objective was to elucidate morphological characteristics of organelles associated with natural aging, the mitochondria and the nuclei. Heart tissue samples from the compact region of the Greenland shark ventricle were collected from a ∼200 year old female Greenland shark and processed for serial blockface scanning electron microscopy according to the Ellisman protocol [2]. Serial images were collected using Gatan 3View and analysed with IMOD. Heart tissue samples from female Greenland shark (aged 108-220 years-old) preserved in formalin were processed following immunohistochemistry procedures. Image analysis was performed using ImageJ. Approximately 1,200 mitochondria were reconstructed providing a mitochondrial volume density of 69% which is higher than that found in other polar fishes, and similar to that found in highly aerobic muscles such as billfish heater cells. The mitochondrial volume density observed in the Greenland shark may reflect aerobic need relative to its cold environment [3]. This high mitochondrial content could have happened through mitochondrial biogenesis through a molecular pathway contributing to longevity in a variety of species [4]. We observed mitochondrial syncitia which are clues for mitochondrial fusion. Mitochondrial morphology is shaped by mitochondrial dynamics, including mitochondrial fusion which is essential to maintain a normal cardiac function [5]. The shape of the cardiomyocyte nuclei and the heterochromatic structure further support a phenotype resilient to age. We conclude that the subcellular characteristics of the cardiac myocyte in the Greenland shark reflect a healthy and youthful phenotype. In the future, our dataset will be complemented with an increased sampling size, comparisons with juvenile Greenland shark cardiac myocytes and molecular assessments of mitochondrial dynamics.

The impact of replicative ageing and dietary flavonoids on C2C12 muscle cell mitochondrial function

Dietary flavonoids possess distinct antioxidant and anti-inflammatory properties and may also regulate mitochondrial function. With advancing age and physical inactivity, skeletal muscle mitochondrial function is compromised. No studies have investigated the potential for flavonoids to modulate mitochondrial function in young and replicatively ‘aged’ C2C12 skeletal muscle cells. Therefore, the objective of this study was to examine the impact of acute dietary flavonoid treatment on mitochondrial function in control (P5-8) and replicatively aged (P49-51) C2C12 myoblasts and myotubes. Undifferentiated myoblasts (0-24 h) and differentiated myotubes (96-120 h) were treated with flavonoids (Quercetin, Q; Epigallocatechin Gallate, EGCG; and (-)-Epicatechin, EPI) at four doses (0, 1, 5 and 10 µM). After 24 h treatment, mitochondrial bioenergetics were examined using an extracellular flux analyser (Seahorse XFe96). Mitochondrial and antioxidant related gene expression in young myoblasts was also determined by RT-PCR in response to Q, EGCG and EPI treatment (0, 5 and 10 µM) over 24 and 48 h. PCG1a gene expression was augmented 2.2- and 1.2-fold over control at 48 h by 5 and 10 µM EPI (P = 0.011) and EGCG (P = 0.030), respectively. Similarly, at 48 h, 10 µM EPI and 5 µM EGCG increased SOD2 expression by 1.8-fold (P = 0.018) and 2.2-fold (P = 0.010), respectively vs control. A significant main effect of age on basal respiration (BR), ATP production, proton leak (PL) and coupling efficiency (CE) was evident in myoblasts (P < 0.05), with aged cells demonstrating 35, 43, 27 and 6 % higher values over control, respectively. Compared to untreated myoblasts, BR was ~30% lower in EPI treated (1 and 5 µM) aged myoblasts (P < 0.05). EPI treatment (10 µM) also lowered PL in aged myoblasts vs. untreated control (P = 0.020). In myotubes, there was a significant main effect of age (P < 0.005) on maximal respiration, PL, ATP generation, spare respiratory capacity % and CE, with young controls exhibiting 190 % greater (P < 0.001), 38 % lower (P = 0.090), 20 % higher (P = 0.534), 62 % higher (P < 0.001) and 13 % higher (P < 0.001) values over aged controls, respectively. There was generally no significant effect of flavonoid treatment, although, EGCG (1 µM) lowered BR and PL in comparison to untreated aged myotubes (P < 0.05). In summary, we demonstrate flavonoids may upregulate the transcription of mitochondrial associated genes in myoblasts. Moreover, we show flavonoids may offer therapeutic potential in aged skeletal muscle cells via actions on mitochondrial bioenergetics. Our findings also suggest replicative ageing has divergent effects on mitochondrial function in myoblasts and myotubes. Whilst aged myoblasts show no evidence of mitochondrial deficiency over control cells, aged myotube bioenergetics seem impaired following differentiation vs unaged control myotubes.

Reduction in Multiple Cellular Mechanisms During Early Replicatively Aged C2C12Differentiation Results in Subsequent Lack of Myotube Formation

Introduction:The ability to retain skeletal muscle mass and function declines with ageing (sarcopenia). Myoblast fusion is integral to the efficient regeneration and growth of muscle. Using a relevant model, we investigated mechanisms during early differentiation involved in preventing aged myoblast fusion. The hypotheses being challenged were: 1) during early differentiation, aged cells have impairments in cellular signalling and gene expression of markers of myoblast fusion, 2) aged cells have decreased abundance and turnover of specific proteins that are associated with inhibited cell fusion. Methods:Control and replicatively aged (do not fuse but are not senescent) murine C2C12myoblasts were cultured for 96 h and myotube formation was assessed morphologically and biochemically (creatine kinase (CK)). During early myoblast differentiation (0-24 h) intracellular signalling (Akt, mTOR, ERK, p38) and gene expression (IGF-I, myogenin, ID3) were assessed. Differentiatingaged and control myoblasts were grown in the absence and presence of deuterium oxide (D20). Samples were lysed at 0 h and 24 h and analysed using LC-MS/MS to measure changes in the protein abundance and to determine fractional synthesis rate (FSR) on a protein by protein basis. Experiments were repeated 3 times in duplicate. Data were analysed using one-way ANOVA. Results: Control cells exhibited significant fusion (P < 0.05) with time, whereas aged myoblasts did not fuse and displayed significant reductions in CK (6-fold: P < 0.05) vs. control at 96 h. Akt and mTOR activation over 24 h were significantly decreased (8-fold and 3-fold respectively: both P < 0.05) vs. control. Significant suppression of myogenin, IGF-I (1000-fold and 10-fold; both P < 0.05) and a significant increase in ID3 expression (4-fold: P < 0.05) were observed at 24 h vs. control. Aged myoblasts had 33 ribosomal proteins with significantly lower (P < 0.01: FDR 10 %) abundance vs. control myoblasts. Ten metabolic enzymes with significantly greater (P < 0.01: FDR 10 %) abundance in aged myoblasts vs. control myoblasts. The average synthesis rate of proteins in aged myoblasts (0.47 ± 0.34 %/h) was significantly less (1.3-fold; P < 0.05) vs. controls (0.59 ± 0.28 %/h) and there were 157 individual proteins with relative differences in FSR. Conclusion:Replicatively aged myoblasts failed to fuse, illustrated morphologically and confirmed biochemically. Suppressed levels of Akt and mTOR activation, together with reductions in myogenin and IGF-I and increases in ID3 may underpin this process. Aged myoblasts had reduced abundance of ribosomal proteins and altered energy demands, with reduced protein turnover compared to control. Therefore, adaptations in multiple pathways during the initial 24 h of differentiation appear to underpin the inability of aged cells to fuse.

Lifestyle related diseases, aging and cardiovascular impairments

A well-functioning cardiovascular system is key in keeping lifelong health. Impairments to the cardiovascular system, as observed with ageing and lifestyle related diseases, drastically shortens the healthy lifespan with great impact on quality of life. Key components of the cardiovascular system are the heart, the larger conduit arteries, the smaller resistance arteries and the capillaries. The heart ensures a perfusion pressure sufficient to drive oxygenated blood to all regions of the body and especially the elastic and contractile properties of the heart are central elements of optimal cardiac function. The larger arteries serve as highways for blood delivery but importantly also as a pressure reservoir that lessens the workload of the heart. Thus, elasticity of the arterial wall is of outmost importance in absorbing the bulk of blood ejected from the left ventricle during systole and in gently propagating this pressurized blood down the arterial tree. The smaller arteries govern to which regions blood is being delivered and holds a key role in maintaining blood pressure within the optimal range. This function is mediated by a thick layer of smooth muscle cells arranged circumferentially giving the smaller arteries potential to control diameter within a very large range. The smallest vessels of the body, the capillaries are the site of delivery of oxygen and nutrients and removal of waste products. This exchange with the surrounding tissue is secured by the capillary wall structure, only one cell thick and with gaps, slits and transporters, allowing for diffusion and transport of smaller and larger molecules. Functional or structural impairments at any level of this delivery chain from heart to capillary bed affects functionality of the cardiovascular system as they are all reliant on one another. Impairment introduced at one level by ageing or poor lifestyle may lead to conditions like hypertension that affects all levels and in turn can lead to even more severe conditions like heart failure and stroke. Normal ageing has structural and functional impact on the heart and the vasculature that causes cardiovascular function to decrease as we age. These ageing induced changes include decreased elastic and contractile properties of the myocardium and the arterial wall, impaired control of vascular smooth muscle cells and thickening of the delicate capillary structure. Interestingly, lifestyle related diseases, like hypertension and diabetes mellitus, impose similar impairments to all components of the cardiovascular system, even at a very young age. Ultimately, this changes the health trajectory towards earlier morbidity and mortality. Fortunately, detrimental changes to the cardiovascular system can be prevented, stopped or even reversed by physical activity. The focus of this talk will be on cellular mechanisms of ageing and lifestyle related diseases connected to impaired cardiovascular function and how these are affected by physical activity. Classic and novel data from invasive human studies will be presented in order to establish knowledge of how far this field of cardiovascular research has come. Furthermore, the talk will bring focus on future research strings that may have great potential in translating cellular mechanism into lifelong health strategies.

Adipose Tissue Remodelling in Response to Obesity, Exercise Training and Psychological Stress

Pathological remodelling of adipose tissue in obesity; including the occurrence of enlarged adipocyte, is often as a result of leptin insensitivity, the mechanism of which is still poorly understood. Leptin is produced in proportion to fat mass to increase energy expenditure. However, the enlarged adipocyte displays an altered metabolic capacity and gene expression profile despite the elevated circulating leptin levels. We have studied the adipose tissue remodelling in Zucker (fa/fa) rats, proposed to be the most appropriate rodent model for the early onset of human obesity, due to a mutation in the long form of the leptin receptor. We determined the effects of obesity, exercise and/or psychological stress on visceral adipose tissue (VAT) phenotype in Zucker rats. Lean (LZR) and obese (OZR) Zucker rats (male, 8-9 weeks old, n=64) were divided into (1) Controls, (2) unpredictable chronic mild stress (UCMS), (3) treadmill exercise (Ex), or (4) UCMS+Ex, 8 rats per strain in each experimental group for 8 weeks (Brooks et al., 2018). VAT was sectioned and stained with haematoxylin and eosin (H&E) staining. Total RNA was extracted with TRIzol reagent as described (Toni et al., 2018). Values are means ± S.E.M., compared by ANOVA. To determine the adipose tissue phenotype, tissue cellularity and gene expression profile in VAT were analysed. Analysis of tissue cellularity using H&E staining showed that obesity increased adipocyte size in the VAT of the OZR compared with LZR (7728±286.7, 3089±160.3µm2; p< 0.001; n≥4). Neither exercise and/or psychological stress had any effect on the tissue cellularity. Leptin mRNA was quantified using quantitative polymerase chain reaction (qPCR). One of the features of OZR is the elevated circulating leptin levels compared with LZR. Contrary to the increased adipocyte size in OZR, obesity caused downregulation of leptin mRNA in OZR compared with LZR (1.2±0.2, 4.8±1.1; p<0.05; n≥6). To further elucidate our findings, the mRNA of peroxisome proliferator-activated receptor-γ (PPAR-γ) was quantified using qPCR method. PPAR-γ is a transcription factor that positively regulate adipocyte differentiation and lipid storage. The data showed that obesity downregulated PPAR-γ mRNA when compared OZR to LZR (0.8±0.1, 1.8±0.3; p<0.05, n≥6). However, the interventions of exercise, and/or psychological stress did not affect the mRNA of leptin and PPAR-γ. Our study demonstrates that obesity increases adipocyte size but downregulated the gene expression of leptin. We demonstrate the impaired regulation of adipocyte differentiation in visceral adipose tissue of OZR group. However, exercise and/or psychological stress had no effect on tissue cellularity and, gene expression in both LZR and OZR. Further studies are needed to establish the extent to which leptin signalling links VAT to the development of metabolic and cardiovascular diseases.

Fibre and sex specific differences in mitochondrial content and subcellular distribution and morphology of lipid droplets in skeletal muscle biopsies obtained from lean, obese and type 2 diabetes patients.

Intramuscular triglycerides (IMTG) are stored in lipid droplets (LD) in skeletal muscle, and it is thought that fibre type distribution, subcellular location, size and number of LDs relate to insulin sensitivity (IS) more so than IMTG content, however this is yet to be investigated in human skeletal muscle. Oxidative capacity plays an important role in IS. In skeletal muscle LD are predominantly located adjacent to the mitochondria with greater abundance in the oxidative type I fibres. Reduced mitochondrial content may reduce oxidative capacity which hinders capacity to utilise IMTG as a fuel source. The aim of the study was to identify fibre and sex specific differences in the content of both LD and mitochondria as well as the subcellular distribution and morphology of LD in human skeletal muscle of lean, obese and type 2 diabetes (T2D) patients. Muscle biopsies were obtained from 48 male (n=24) and female (n=24) participants, categorised into groups based on metabolic health; lean (n=16), obese (n=16) and people with type 2 diabetes T2D (n=16). Cryosections (5μm) were stained using appropriate antibodies to identify fibre type and mitochondria. LD were stained using bodipy and cell membranes were labelled using WGA. Images were obtained using confocal immunofluorescence and widefield immunofluorescence microscopy, and analysed using Image Pro Plus. In both sexes, there was a hierarchical distribution (T1 > T2a >T2x) of both LD content (area fraction stained) and number (P < 0.001) and were located more in the central region than the peripheral region (5µm below cell membrane) of the cell (P < 0.001). Males had more LD content than females (P<0.001) in all fibre types due to both larger LDs (P<0.001) and a greater number of LDs (P=0.001). In type IIa fibres T2D have more LD than lean and obese participants which was driven primarily by an increased size of LD rather than increased number of LD (P T2) (P<0.001) in both males and females regardless of IS. The study demonstrates that males have more LD than females which is contrary to previous literature which reports greater LD stores in females. However the females in the present study were postmenopausal therefore may have had impaired lipid metabolism due to the reduced oestrogen concentrations that occur at this time. The increased LD content in males compared to females alongside no difference in mitochondrial density may suggest that the LD-mitochondria interactions are coupled better in females leading to a greater oxidative capacity. The increased LD content specifically in type IIa fibres of T2D may suggest a dysregulation of LD turnover (synthesis and lipolysis) which is supported by the lower mitochondria content in these fibres. More investigation is needed to understand whether the different LD-mitochondria ratio in males compared to females explains why women remain more IS than men for a given body mass.

Skeletal muscle weakness, atrophy, and abnormal blood flow response in an animal model of heart failure with preserved ejection (HFpEF)

Heart failure (HF) induces skeletal muscle alterations that impair physical function and quality of life. However, most studies have been performed in HF with a reduced ejection fraction (HFrEF) whereas skeletal muscle remodelling in HF with preserved ejection fraction (HFpEF) remains poorly explored. The present study, therefore, used an obese cardio-metabolic model to investigate functional, morphological, and vascular alterations that occur in the skeletal muscles of HFpEF rats. Methods: Lean (n=8) and obese (n=8) diabetic Zucker fatty/spontaneously hypertensive heart failure F1 hybrid (ZSF1) rats were compared at 20 weeks of age and cardio-metabolic function assessed (i.e. body weight, blood glucose levels and mean arterial pressure). Tibialis anterior (TA) fibre size (stained with haematoxylin and eosin) was evaluated alongside contractility measures for in vitro soleus function (specific force, shortening velocity and power) and in situ extensor digitorum longus (EDL) function (force and fatigability) following stimulation across a range of frequencies. Anaesthesia was induced with 4% isoflurane in 100% oxygen and maintained throughout experiments by constant jugular infusion (30-35mg kg-1 hr-1) of alfaxalone (Jurox, Crawley, UK). Femoral artery blood flow at rest and during stimulation was quantified using Transonic (0.7PSB; Ithaca, NY, USA) perivascular flowprobes. Data were compared by unpaired Student t test and are presented as mean±SEM. Results: HFpEF rats were obese (424.50±11 vs. 549.50±11 g body weight; P<0.05), hyperglycaemic (8.50±0.80 vs. 16.80±0.50 mmol.L-1; P<0.05) and hypertensive (151.34±3.14 vs. 177.44±4.39 mmHg; P<0.05) compared to lean controls. HFpEF rats developed ~40% fibre atrophy in the TA (3172±527 vs. 5667±332 µm2; P<0.05). Despite no differences (P>0.05) in soleus specific forces or shortening velocities, maximal power was lower in HFpEF compared to controls by ~30% (0.95±0.04 vs. 1.33±0.11 W cm-2; P<0.05). Compared to controls, both EDL twitch and maximal tetanic absolute forces were impaired in HFpEF by ~30% (57±5 vs. 42±4 g and 225±27 vs. 151±16 g, respectively; P<0.05), yet hindlimb blood flow was higher at rest (1.75±0.20 vs. 2.84±0.47 ml min-1; P<0.05) but lower during contractions (5.44±0.70 vs. 3.74±47 ml min-1; P<0.05). Conclusions: HFpEF induced skeletal muscle weakness, fibre atrophy and contractile dysfunction alongside impaired blood flow response during contractions. While the mechanisms underlying these deficits remain unclear, these data suggest that vascular, functional, and structural impairments of skeletal muscle contribute to life-limiting symptoms observed in patients with HFpEF.

Evaluation of the effects of WISP-1 on cardiac fibrosis mediated by cardiac fibroblasts via MMPs and TIMPs

Wnt signalling plays a key role in the physiology of cardiac development and pathogenesis of cardiovascular disease. Cardiac fibrosis is characterised by the net accumulation of extracellular matrix proteins in the heart, and has a detrimental effect on cardiac function. Wnt1-inducible signalling pathway protein-1 (WISP-1) is a crucial downstream growth factor of Wnt signalling which is involved in fibrotic remodelling. In order to investigate whether the deletion of WISP-1 attenuates angiotensin II (Ang II) induced cardiac fibrosis, male Apo-E-/- WISP-1+/+ mice (single knockout mice, SKO mice, n=7) and male Apo-E-/- WISP-1-/- mice (double knockout mice, DKO mice, n=6) were infused with Ang II (1000 ng/kg/min) using mini-osmotic pumps for 4 weeks. Proteoglycan and collagen content in the heart sections of the mice were measured using alcian blue staining and immunohistochemical staining respectively. Human cardiac fibroblasts (HCFs) were used in vitro to demonstrate whether WISP-1 upregulates collagen synthesis. mRNA levels were analysed using quantitative PCR. Protein levels in cell lysates and concentrated conditioned media of cultured HCFs were assessed using Western blotting analysis. Values are presented as means ± SEM, compared by Mann-Whitney U test or Kruskal-Wallis H tests. Increased proteoglycan content (4.97±0.87 vs. 0.89±0.40, p<0.05) and collagen type 1 content (1.07±0.27 vs. 0.29±0.06, p<0.05) were detected in the SKO + Ang II mice compared to the SKO controls. However, WISP-1 deletion attenuated the profibrotic effect of Ang II (proteoglycan content: 2.56±1.27 (DKO + Ang II) vs. 4.97±0.87 (SKO + Ang II), p<0.05). Treatment of HCFs with WISP-1 did not alter the collagens expression in cell lysates, whereas an additional form of collagen with smaller molecular weight was observed in concentrated conditioned media due to collagens being cleaved. A broad spectrum MMP inhibitor abrogated the collagen cleavage induced by WISP-1. In addition, WISP-1 treatment significantly decreased TIMP-2 mRNA level (0.88±0.04 vs. 1.00±0.00, n=6) compared to the control group. In conclusion, WISP-1 is a pivotal mediator in Ang II induced cardiac fibrosis. These effects are dependent on the regulation of cardiac fibroblasts collagen processing, possibly via the mediation of expression and activity of matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs).

A combination of major histocompatibility complex (MHC) I overexpression and Type I interferon (IFN) signature induces profound mitochondrial dysfunction in human skeletal muscle myoblasts

Myositis is a group of rare autoimmune diseases with progressive skeletal muscle weakness and dysfunction. Morphological features of the disease include inflammatory infiltration (CD4+/CD8+ T-cells), the presence of circulating autoantibodies, and interferons (IFN) upregulation in muscle cells (1). Specifically, patients with dermatomyositis (DM) have shown strong Type I IFN-alpha and -beta signature (2). Overexpression of major histocompatibility complex (MHC) I in muscle fibres is a key pathological feature, which is observed in the absence of inflammation or overt disease, while muscle weakness persists (3); this has been attributed to other non-immune mediated mechanisms, including mitochondrial abnormalities (4). Even though both IFN signature and MHC I have been identified to play an important role in DM, their downstream combinational effects remain to be revealed. The aim of this study was to examine the impact of MHC I overexpression in presence or absence of Type I IFNs, on mitochondrial function. Human skeletal muscle myoblasts were transfected with a mammalian HLA-A2/K^b overexpression vector, a MHC I isoform, and treated with Type I IFNs (100 ng/ml) for 18 hours. Mitochondrial and function, membrane potential and superoxide generation were assessed using seahorse extracellular flux analysis (n=4), JC-1 (n=6), and MitoSOX Red (n=8), respectively. Statistical analysis was performed by ANOVA with Dunnett’s post-hoc test. MHC I overexpression results in decreased mitochondrial respiration, including maximal, basal, and ATP-linked respiration, alongside with a decline in overall reserve capacity of cells; which was significantly compounded in the presence of Type I IFNs. Specifically, MHC I overexpressed myoblasts treated with IFN-beta show significant decrease in maximal respiration (p=0.0223), ATP production (p=0.02), and proton leak (p=0.01). Myoblasts overexpressing MHC I and treated with IFN-alpha show significant reduction in maximal respiration (p=0.0403) and proton leak (p=0.01). The reduction in proton leak is further supported by mitochondrial membrane hyperpolarisation, which is significantly observed in presence of Type I IFNs (IFN-alpha, p=0.007; IFN-beta, p<0.001). Moreover, cells overexpressing MHC I induce significant increase in mitochondrial superoxide generation (p=0.0448). Data suggest that MHC I and Type I IFNs have a strong combinational effect on mitochondrial function, providing further insights into DM pathogenesis.

The effects of age and acute aerobic and resistance exercise on circulating T lymphocyte vitamin D receptor (VDR) expression in healthy males.

A low vitamin D status has been identified as an association risk factor in the etiology of numerous chronic diseases, with older adults identified as generally more deficient than younger populations. Exercise has been shown to act as a direct and indirect stimulus on key vitamin D metabolites, specifically the vitamin D receptor (VDR) (1, 2), however investigations are limited to murine model studies. The primary aim of the study was to determine whether a single bout of exercise upregulated VDR expression in circulating systemic T cells. Thirty-five recreationally active males were included in the study (means ± SD: age 44 ± 17 y, body mass 82.5 ± 11.4 kg, height 1.79 ± 0.08 m, BMI 25.7 ± 3.1 kg/m2), separated into three age groups: 18-30 y (n=12), 31-45 y (n=11), and 60-75 y (n=12). Participants completed three trials: control (CON; 60 min rest), aerobic exercise (AE; 60 min cycling at 55% work load max) and resistance exercise (RE; repeated maximal voluntary isometric contractions), with intravenous blood samples collected pre- and post-CON/AE/RE (Pre, 0 h, 1 h, 3 h). T cells were isolated and analysed for cell-surface and intracellular T cell (CD3+, CD4+, and CD8+ T cell subsets) VDR expression via flow cytometry analysis. Values are means ± S.D., compared by ANOVA. Intracellular VDR expression (CD3+ T cells) was elevated following both AE (P<0.001) and RE (P=0.001) immediately upon cessation of the exercise, with AE promoting a greater increase compared to RE (1.5 fold vs 1.25 fold increase, respectively; P=0.048). VDR expression returned to baseline level after 3 h post-exercise. There was no difference between T cell subsets. The response was unaffected by age despite baseline VDR expression at rest declining with age (geomean: 882 ± 274 vs 796 ± 243 vs 594 ± 174; P=0.015). Moreover, there was no correlation between change in T cells and change in VDR expression in response to exercise. The results indicate that performing a single bout of exercise is an effective means to increase intracellular VDR expression in T cells. Interestingly, the upregulation in VDR protein expression may be independent of T cell mobilisation in response to exercise, and independent of age.

Absence of an ageing-related increase in fibre type grouping in master athletes and non-athletes

Guy A.M. Messa1, Mathew Piasecki2, Jörn Rittweger3,4, Jamie S. McPhee5, Erika Koltai6, Zsolt Radak6, Bostjan Simunic7, Ari Heinonen8, Harri Suominen8, Marko T. Korhonen9, Hans Degens1,9,10 1 Department of Life Sciences, Research Centre for Musculoskeletal Science & Sports Medicine, Manchester Metropolitan University, Manchester, UK; 2 MRC-ARUK Centre for Musculoskeletal Ageing Research and National Institute for Health Research (NIHR) Nottingham Biomedical Research Centre, University of Nottingham, UK; 3 Division Space Physiology, Institute of Aerospace Medicine, German Aerospace Center, Cologne, Germany; 4 Department of Paediatric and Adolescent Medicine, University of Cologne, Cologne, Germany 5 Department of Sport and Exercise Sciences, Manchester Metropolitan University, Manchester, UK; 6 Research Institute of Sport Science, University of Physical Education, Budapest, Hungary; 7 Science and Research Centre Koper, Institute for Kinesiology Research, Slovenia; 8 Gerontology Research Centre, Faculty of Sport and Health Sciences, University of Jyväskylä, Finland; 9 Institute of Sport Science and Innovations, Lithuanian Sports University, Kaunas, Lithuania; 10 University of Medicine and Pharmacy of Targu Mures, Rumania Abstract Background The ageing-related loss of muscle mass may be partly attributable to motor neuron loss and motor unit remodelling, where cycles of denervation and reinnervation may result in fibre type grouping in older muscles. The purpose of this study was to examine fibre type grouping in athletes and non-athletes of a wide age range (19-85 years) and to evaluate to which extent any observed grouping is explicable by the fibre type composition of the muscle. Since regular physical activity may stimulate reinnervation, we hypothesised that fibre groups are larger in master athletes than in age-matched non-athletes. Methods Fibre type grouping was assessed in m. vastus lateralis biopsies from 22 young (19-27 years) and 35 healthy older (66-82 years) non-athletes, and 14 young (20-29 years), 51 middle-aged (38-65 years) and 31 older (66-85 years) athletes. An ‘enclosed fibre’ was any muscle fibre of a particular type surrounded by fibres of the same type only. A group was defined as a group of fibres with at least one enclosed fibre. Results Only type II fibre cross-sectional area (FCSA) showed an age-related decrement that was greater in athletes (p < 0.001) than in non-athletes (p = 0.012). There was no significant agerelated effect on group size or group number in athletes or non-athletes, and the observed grouping was similar to that expected from the fibre type composition. Conclusions At face value these observations do 1) neither show evidence for an age-related loss and remodelling of motor units nor that 2) improved reinnervation with regular physical activity, but 3) histological examination may not reveal the full extent of ageing-related motor unit remodelling.

Are older adults at greater risk of the adverse musculoskeletal consequences of physical inactivity?

Introduction: Ageing and physical inactivity are independent risk factors for musculoskeletal decline. The aim of the current study was to assess the influence of age on the musculoskeletal changes incurred during short-term physical inactivity. Methods: 26 young (28 ± 8 y) and 21 older (60 ± 6 y) habitually active (>10,000 steps per day) participants were recruited. Musculoskeletal assessments, including dual-energy x-ray absorptiometry to quantify lean mass and bone mineral density (BMD), magnetic resonance spectroscopy to quantify mitochondrial function and intramuscular adipose tissue (IMAT) and a leg press protocol to assess muscle function/strength were performed at baseline, following 14 days of step reduction (~1,500 steps per day) and after 14 days of returning to habitual activity. Statistical analysis was performed using two-way repeated measures ANCOVA, and data are presented as mean±SD. Results: Average daily step count reduced by 10752 ± 2943 steps/day in the young (83 ± 7%) and 10204 ± 2338 steps/day in the older group (84 ± 7%), with concomitant increases in sedentary time (P<0.001). The intervention induced significant losses in total body lean mass (Young 0.43±0.78kg vs. older 0.08±0.76kg; P=0.024;), lean leg mass (young 0.18±0.33kg vs. older 0.08±0.33kg; P=0.003) and muscle strength per unit of lean muscle mass (muscle quality) (young -0.33±0.92kg vs. older 0.33±0.51kg; P<0.001), which were not significantly different between age groups (P>0.05). Correspondingly, IMAT increased (young 0.67±2.84% vs. older 2.73±3.97%; P=0.04) with no difference between groups (P=0.27). Serum markers of bone resorption (CTX) were elevated (P=0.002) in response to the inactivity period to similar extents in both age groups (P=0.54). Cardiorespiratory fitness and mitochondrial function significantly declined during the inactivity period, indicating reductions in whole body (VO2 peak: young 1.99±3.68ml.min.kg -1 vs. older 2.75±4.12ml.min.kg -1; P<0.001) and skeletal muscle oxidative capacity (rate constant: young -0.08 ± 0.39ks-1 vs. older 0.38±0.36ks-1; P=0.007). Importantly, these reductions were significantly greater in older adults (P>0.05 ). Conclusion: Results suggest that even short-term modest reductions in physical inactivity induces substantial impairments in musculoskeletal decline, regardless of age. Reductions in oxidative capacity were more pronounced within older adults indicating an increased vulnerability to physical inactivity compared with young. This highlights the importance of maintaining levels of physical activity throughout the lifespan.

Comparison of two Home-Based HIIT protocols with ‘60HIIT’ eliciting greater health benefits in VO2peak compared to ‘30HIIT’.

Currently 40% of the UK do not meet the physical activity guidelines with ‘lack of time’ the most commonly cited barrier to physical activity. High intensity interval training (HIIT) has been shown to be an effective time-efficient alternative to moderate intensity continuous training (MICT). However, public health researchers have criticised current HIIT research as exercise is often supervised and many protocols employ expensive equipment available only in laboratories or gym, creating additional barriers to exercise. As such, HIIT protocols using simple body weight exercises that can be performed in one’s home (home-based HIIT) are becoming more popular. However, the effect of interval duration has not been investigated using a home-based HIIT intervention. Therefore our aim was to investigate if two popular HIIT protocols (30 or 60 second interval durations (30HIIT; 60HIIT) induce similar improvements in aerobic capacity, arterial stiffness, and body composition when performed using simple body weight exercises. Twenty-six, previously sedentary men (n=9) and women (n=17) were randomised to complete either 6 weeks of 30HIIT (n=15; 29±3 y, BMI 25±0.8 kg.m-2)) (4-8 30s intervals with 120s rest) or 60HIIT (n=11; 28±4 y, BMI 26±1.4 kg.m-2)) (6-10 60s intervals with 60s rest). Both training protocols used simple body weight exercises, and training sessions were completed without supervision in a place of the participants choosing, 3 times per week. Training adherence and intensity (Heart rate (HR)) were monitored using the Polar Beat mobile app. VO2peak, body composition (Bio-impedance), and arterial stiffness (aortic pulse wave velocity (aPWV)) were assessed pre and post training. VO2peak increased post intervention in 60HIIT (33±7 to 35±7 ml.min-1.kg-1) (P<0.05) with no difference for 30HIIT (P=0.51). There was no significant difference in BMI (P=0.5), weight (P=0.3), and VAT (P=0.4) for 60HIIT. There was no significant difference for 30HIIT in BMI (P=0.052), weight (P=0.057), and VAT (P=0.062). There was no significant difference in arterial stiffness (aPWV) post intervention in 30HIIT (P=0.16) or 60HIIT (P=0.2). 6 weeks of 60HIIT induce improvements in aerobic capacity but not in body composition or arterial stiffness with 6 weeks of 30HIIT eliciting no changes. In both home-based HIIT protocols participants self-selected bodyweight exercise intensities, without researcher encouragement. Therefore, this data suggests that 60-second interval interventions could be used to improve health outcomes in a sedentary population in the real world, with 30-second interval interventions needing further research.

Can a Weekly Multi-Modal Exercise Class Preserve Motor and Non-Motor Function in Parkinson’s?

Introduction: Parkinson’s Disease (PD) is a chronic neurodegenerative disease that can lead to disability and disengagement with active lifestyles. Single modality exercise is effective at improving and sustaining cognitive and/or physical function in people with Parkinson’s (PwP) (1,2). However, multi-modal (MM) exercise (e.g. circuit training), including cognitive challenge, may be more beneficial. Only a few studies have assessed long-term effects of regular MM exercises in PD and there is a need to compare these results to non-exercise-attendees with PD (na-PD) and healthy older adults (HOA). We established a weekly community-based MM exercise session for PD (EX) to evaluate its effects on physical function, cognition and wellbeing outcomes over the period of 1, 2 and 3 years. Methods: 25 participants (20 male, 5 female; age 64±8 years; Hoehn and Yahr (H&Y) scores≤III, indicating mild to moderate PD) attended a once-a-week MM group exercise session (60 minutes) for over one-year. A battery of health and functional assessments were completed at the start and every four months for one (n=25), two (=18) and three years (n=8). Additionally, a battery of cognitive function assessments plus Older People’s Quality of Life Questionnaire (OPQOL-Brief) were measured. Results were compared to an aged-matched group of 20 HOA (8 male, 12 female; age 61±6 years) and 20 na-PD (12 male, 8 female; age 68±7 years; H&Y scores≤III) to evaluate the rate of functional and cognitive decline not influenced by the exercise session. Results: At baseline, no health-related between-group differences were observed. EX scores for six-minute walking test (6MWT), timed up and go (TUG) and bilateral grip strength (GS) did not significantly decrease across 1, 2 or 3 years, and the number of 1-minute sit-to-stands (STS) increased during 1 year between baseline and after the first four months (from 21 to 23; P=0.001). Scores for Clock Drawing Test (CDT), Trail Making Test A (TMT-A) and B (TMT-B), and OPQOL-Brief did not significantly change across four different assessments (i.e. 1 year). Mini-Mental Parkinson’s (MMP) increased between baseline and after 8 months (from 26.67 to 29.38; P=0.004). After 1 year of exercise, EX group’s 6MWT scores were significantly lower than HOA (P<0.001) but TUG, STS and GS were not significantly different from HOA, while na-PD showed lower 6MWT, TUG and STS scores than HOA (P=0.000, 0.006 and 0.003, respectively). Conclusion: A once-a-week MM exercise programme for PD showed an improvement in STS and MMP scores and no other significant changes (i.e. no decline) in health, cognition and physical function over 1, 2 and 3 years. Also, exercise reduced the difference in outcome scores between PwP and HOA. That functional and cognitive performance were slightly increased or maintained is a positive outcome given the progressive nature of Parkinson’s.

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Neurophysiological Bases of Human Movement 2025

Tuesday 17 December - Wednesday 18 December 2019

Future Physiology 2019

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Karyn Esser, University of Florida, US

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