Figure 2

<p>(A) MitoSox red loaded single isolated skeletal muscle fiber, (i) Fluorescent image obtained using excitation at 405 nm, (ii) Fluorescent image obtained using excitation at 488 nm, (iii) merged images i and ii, (iv) bright field image, scale bar = 25 µm. Figures B and D show the relative fluorescence with time from skeletal muscle fibers loaded with MitoSox red (data presented from 405 nm excitation). Fibers were either non-stimulated or subjected to two periods of electrically stimulated contractions during the time periods denoted by a black bar. No significant effect of stimulation over the whole time course was found in the absence of L-NAME (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0096378#pone-0096378-g002" target="_blank">Fig. 2B</a>) whereas in the presence of L-NAME (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0096378#pone-0096378-g002" target="_blank">Fig. 2D</a>) a significant effect of stimulation was found (repeated measures, F = 5.3, P = 0.04) compared with non stimulated fibers. Figures C and E show the rate of change in relative fluorescence (derived from figures B and D ) between the indicated time points with the stimulation periods denoted by black bars. Data shown in Figures B and C were from untreated fibers, data in Figures D and E were from fibers in the presence of 100 µM L-NAME. *P<0.05 compared with non-stimulated fibers at the same time point (n = 6–7 for all groups).</p

Figure 1

<p>(A) Fluorescent confocal images of a single isolated FDB muscle fiber loaded with MitoSox red (125 nM) and Mitotracker green (20 nM). (i)Fluorescent image of fiber loaded with mitotracker, (ii) Fluorescent image of fiber loaded with MitoSox obtained using excitation at 405 nm, (iii) Fluorescent image of fiber loaded with MitoSox obtained using excitation at 488 nm, (iv) merge of bright field and fluorescent images, (v) merged fluorescent images i–iii, (vi) bright field image. Scale bar = 25 µm. (B) Fluorescence values from MitoSox red loaded fibers (n = 3) with time, prior to -, and after exposure to 10 µM antimycin A (denoted by the black bar). Data from excitation at 405 nm is claimed to reflect 2-hydroxy MitoSox and excitation at 488 nm monitors non-specific oxidation products of MitoSox.</p

Figure 4

<p>(A) DHE loaded single muscle fiber, (i) Fluorescent image obtained using excitation at 405 nm, (ii) Fluorescent image obtained using excitation at 488 nm, (iii) Fluorescent image obtained with 405 nm emission and 450/35 nm emission monitoring non-oxidised DHE, (iv) bright field image, scale bar = 50 µm, (v) merged figures i–iii. (B) Data from muscle fibers that were either stimulated (as denoted by black bar) or non-stimulated (n = 6 for all groups). Solid symbols show 2-HE fluorescence over time measured from nuclei showing the acute increases in fluorescence following contractions and open symbols show the “unoxidised” DHE fluorescence measured from the same muscle fibers. Cytosolic fluorescence from “unoxidised” DHE decreased by approximately 50% over sixty minutes in both experimental groups reflecting loss of DHE from the cell. (C) Rate of change in relative fluorescence with time for cytosolic DHE (derived from figure B). *P<0.05, compared with non stimulated fibers at the same time point.</p

Figure 3

<p>(A) DHE loaded single muscle fiber, (i) Fluorescent image obtained using excitation at 405 nm, (ii) Fluorescent image obtained using excitation at 488 nm, (iii) merged images i and ii, (iv) bright field image, scale bar = 50 µm. Figures B and D show relative fluorescence with time from skeletal muscle fiber nuclei (data presented from 405 nm excitation only, n = 8). Fibers were either at rest or subjected to muscle contraction as denoted by the black bar. Figures C and E show the rate of change in relative fluorescence (derived from figures B and D respectively) between indicated time points. Data shown in Figures B and C were from untreated fibers, data in Figures D and E were from fibers in the presence of 100 µM L-NAME. *P<0.05 compared with non-stimulated fibers at the same time point.</p

Figure 5

<p>(A) Cyto-<i>HyPer</i> transfected single muscle fiber, (i) Fluorescent image obtained using excitation at 405 nm, (ii) Fluorescent image obtained using excitation at 488 nm, (iii) bright field image, (iv) merged images i and ii, scale = 50 µm. (B) Non-transfected single muscle fiber, (i) Fluorescent image obtained using excitation at 405 nm, (ii) Fluorescent image obtained using excitation at 488 nm, (iii) bright field image, (iv) merged images i and ii, scale = 50 µm. (C) Ratio of fluorescence values at excitations of 488/405 nm from fibers transfected with <i>HyPer</i> that were either stimulated (as denoted by black bars) or non- stimulated (n = 7 for both groups). (D) Rate of change in ratio of fluorescence at excitations of 488/405 nm from fibers transfected with <i>HyPer</i>. *P<0.05, compared with non stimulated fibers at the same time point.</p

DataSheet1_Removal of p16INK4 Expressing Cells in Late Life has Moderate Beneficial Effects on Skeletal Muscle Function in Male Mice.docx

Aging results in the progressive accumulation of senescent cells in tissues that display loss of proliferative capacity and acquire a senescence-associated secretory phenotype (SASP). The tumor suppressor, p16INK4A, which slows the progression of the cell cycle, is highly expressed in most senescent cells and the removal of p16-expressing cells has been shown to be beneficial to tissue health. Although much work has been done to assess the effects of cellular senescence on a variety of different organs, little is known about the effects on skeletal muscle and whether reducing cellular senescent load would provide a therapeutic benefit against age-related muscle functional decline. We hypothesized that whole-body ablation of p16-expressing cells in the advanced stages of life in mice would provide a therapeutic benefit to skeletal muscle structure and function. Treatment of transgenic p16-3MR mice with ganciclovir (GCV) from 20 to 26 months of age resulted in reduced p16 mRNA levels in muscle. At 26 months of age, the masses of tibialis anterior, extensor digitorum longus, gastrocnemius and quadriceps muscles were significantly larger in GCV-treated compared with vehicle-treated mice, but this effect was limited to male mice. Maximum isometric force for gastrocnemius muscles was also greater in GCV-treated male mice compared to controls. Further examination of muscles of GCV- and vehicle-treated mice showed fewer CD68-positive macrophages present in the tissue following GCV treatment. Plasma cytokine levels were also measured with only one, granulocyte colony stimulating factor (G-CSF), out of 22 chemokines analyzed was reduced in GCV-treated mice. These findings show that genetic ablation of p16+ senescent cells provides moderate and sex specific therapeutic benefits to muscle mass and function.</p

Cognitive and psychiatric symptom trajectories 2–3 years after hospital admission for COVID-19: a longitudinal, prospective cohort study in the UK

Background: COVID-19 is known to be associated with increased risks of cognitive and psychiatric outcomes after the acute phase of disease. We aimed to assess whether these symptoms can emerge or persist more than 1 year after hospitalisation for COVID-19, to identify which early aspects of COVID-19 illness predict longer-term symptoms, and to establish how these symptoms relate to occupational functioning. Methods: The Post-hospitalisation COVID-19 study (PHOSP-COVID) is a prospective, longitudinal cohort study of adults (aged ≥18 years) who were hospitalised with a clinical diagnosis of COVID-19 at participating National Health Service hospitals across the UK. In the C-Fog study, a subset of PHOSP-COVID participants who consented to be recontacted for other research were invited to complete a computerised cognitive assessment and clinical scales between 2 years and 3 years after hospital admission. Participants completed eight cognitive tasks, covering eight cognitive domains, from the Cognitron battery, in addition to the 9-item Patient Health Questionnaire for depression, the Generalised Anxiety Disorder 7-item scale, the Functional Assessment of Chronic Illness Therapy Fatigue Scale, and the 20-item Cognitive Change Index (CCI-20) questionnaire to assess subjective cognitive decline. We evaluated how the absolute risks of symptoms evolved between follow-ups at 6 months, 12 months, and 2–3 years, and whether symptoms at 2–3 years were predicted by earlier aspects of COVID-19 illness. Participants completed an occupation change questionnaire to establish whether their occupation or working status had changed and, if so, why. We assessed which symptoms at 2–3 years were associated with occupation change. People with lived experience were involved in the study. Findings: 2469 PHOSP-COVID participants were invited to participate in the C-Fog study, and 475 participants (191 [40·2%] females and 284 [59·8%] males; mean age 58·26 [SD 11·13] years) who were discharged from one of 83 hospitals provided data at the 2–3-year follow-up. Participants had worse cognitive scores than would be expected on the basis of their sociodemographic characteristics across all cognitive domains tested (average score 0·71 SD below the mean [IQR 0·16–1·04]; p<0·0001). Most participants reported at least mild depression (263 [74·5%] of 353), anxiety (189 [53·5%] of 353), fatigue (220 [62·3%] of 353), or subjective cognitive decline (184 [52·1%] of 353), and more than a fifth reported severe depression (79 [22·4%] of 353), fatigue (87 [24·6%] of 353), or subjective cognitive decline (88 [24·9%] of 353). Depression, anxiety, and fatigue were worse at 2–3 years than at 6 months or 12 months, with evidence of both worsening of existing symptoms and emergence of new symptoms. Symptoms at 2–3 years were not predicted by the severity of acute COVID-19 illness, but were strongly predicted by the degree of recovery at 6 months (explaining 35·0–48·8% of the variance in anxiety, depression, fatigue, and subjective cognitive decline); by a biocognitive profile linking acutely raised D-dimer relative to C-reactive protein with subjective cognitive deficits at 6 months (explaining 7·0–17·2% of the variance in anxiety, depression, fatigue, and subjective cognitive decline); and by anxiety, depression, fatigue, and subjective cognitive deficit at 6 months. Objective cognitive deficits at 2–3 years were not predicted by any of the factors tested, except for cognitive deficits at 6 months, explaining 10·6% of their variance. 95 of 353 participants (26·9% [95% CI 22·6–31·8]) reported occupational change, with poor health being the most common reason for this change. Occupation change was strongly and specifically associated with objective cognitive deficits (odds ratio [OR] 1·51 [95% CI 1·04–2·22] for every SD decrease in overall cognitive score) and subjective cognitive decline (OR 1·54 [1·21–1·98] for every point increase in CCI-20). Interpretation: Psychiatric and cognitive symptoms appear to increase over the first 2–3 years post-hospitalisation due to both worsening of symptoms already present at 6 months and emergence of new symptoms. New symptoms occur mostly in people with other symptoms already present at 6 months. Early identification and management of symptoms might therefore be an effective strategy to prevent later onset of a complex syndrome. Occupation change is common and associated mainly with objective and subjective cognitive deficits. Interventions to promote cognitive recovery or to prevent cognitive decline are therefore needed to limit the functional and economic impacts of COVID-19. Funding: National Institute for Health and Care Research Oxford Health Biomedical Research Centre, Wolfson Foundation, MQ Mental Health Research, MRC-UK Research and Innovation, and National Institute for Health and Care Research.</p

Long term health outcomes in people with diabetes 12 months after hospitalisation with COVID-19 in the UK: a prospective cohort study

Background: People with diabetes are at increased risk of hospitalisation, morbidity, and mortality following SARS-CoV-2 infection. Long-term outcomes for people with diabetes previously hospitalised with COVID-19 are, however, unknown. This study aimed to determine the longer-term physical and mental health effects of COVID-19 in people with and without diabetes. Methods: The PHOSP-COVID study is a multicentre, long-term follow-up study of adults discharged from hospital between 1 February 2020 and 31 March 2021 in the UK following COVID-19, involving detailed assessment at 5 and 12 months after discharge. The association between diabetes status and outcomes were explored using multivariable linear and logistic regressions. Findings: People with diabetes who survived hospital admission with COVID-19 display worse physical outcomes compared to those without diabetes at 5- and 12-month follow-up. People with diabetes displayed higher fatigue (only at 5 months), frailty, lower physical performance, and health-related quality of life and poorer cognitive function. Differences in outcomes between diabetes status groups were largely consistent from 5 to 12-months. In regression models, differences at 5 and 12 months were attenuated after adjustment for BMI and presence of other long-term conditions. Interpretation: People with diabetes reported worse physical outcomes up to 12 months after hospital discharge with COVID-19 compared to those without diabetes. These data support the need to reduce inequalities in long-term physical and mental health effects of SARS-CoV-2 infection in people with diabetes. Funding: UK Research and Innovation and National Institute for Health Research. The study was approved by the Leeds West Research Ethics Committee (20/YH/0225) and is registered on the ISRCTN Registry (ISRCTN10980107)