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Skeletal muscle mitochondrial dysfunction in contemporary antiretroviral therapy: a single cell analysis
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Hunt, Matthewa,b; Mcniff, Megan M.a; Vincent, Amy E.a; Sabin, Carolinec; Winston, Aland; Payne, Brendan A.I.a,e
AIDS: July 15, 2022

To quantify mitochondrial function in skeletal muscle of people treated with contemporary antiretroviral therapy.
Cross-sectional observational study.
Quantitative multiplex immunofluorescence was performed to determine mitochondrial mass and respiratory chain complex abundance in individual myofibres from tibialis anterior biopsies. Individual myofibres were captured by laser microdissection and mitochondrial DNA (mtDNA) content and large-scale deletions were measured by real-time PCR.
Cohort characteristics
We investigated skeletal muscle biopsies from 45 ART-treated PWH. Mean age was 57.6 years (SD 7.5) and well-matched to the HIV negative control group (mean age 59.7, SD 7.3). 93% were male and 98% were of white European ethnicity (Table 1). Mean duration of diagnosed HIV infection was 191 (SD 89) months and mean duration of ART was 125 (66) months. Half (51%) had history of prior exposure to NRTIs with systemic mitochondrial toxicity. All subjects had HIV-1 RNA plasma viral load (VL) <400 copies/mL and 81% had VL <50 copies/mL. Mean CD4 count was 619 (207) cells/μL and all subjects had CD4 count >200 cells/μL. Amongst the ART-naïve PWH, mean age was 36.8 years (SD 10.1), mean duration of diagnosed HIV 74 (56) months, and mean CD4 count 635 (414) cells/μL.
Forty five antiretroviral therapy (ART) treated people with HIV (PWH, mean age 58 years, mean duration of ART 125 months) were compared with 15 HIV negative age-matched controls. Mitochondrial complex I (CI) deficiency was observed at higher proportional levels in PWH than negative controls (P = 0.008). Myofibre mitochondrial mass did not differ by HIV status.
No ART class was significantly associated with mitochondrial deficiency, including prior exposure to historical NRTIs (nucleoside analogue reverse transcriptase inhibitors) associated with systemic mitochondrial toxicity.
To exclude an effect of untreated HIV, we also studied skeletal muscle from 13 ART-naïve PWH (mean age 37). These showed negligible CI defects, as well as comparable myofibre mitochondrial mass to ART-treated PWH.
Most CI-deficient myofibres contained mtDNA deletions. No mtDNA depletion was detected.
Here, we show that PWH treated with contemporary ART have mitochondrial dysfunction in skeletal muscle, exceeding that expected due to age alone. Surprisingly, this was not mediated by prior exposure to mitochondrially toxic NRTIs, suggesting novel mechanisms of mitochondrial dysfunction in contemporary ART-treated PWH. These findings are relevant for better understanding successful ageing in PWH.
Clinical and HIV-related predictors of mitochondrial dysfunction
None of age, current CD4 count, duration since HIV diagnosis, or duration on ART predicted proportional CI defect (age, Spearman rho -0.16, p 0.31; CD4 count, rho -0.07, p 0.7; HIV duration, rho -0.23, p 0.14; ART duration, rho -0.01, p 0.9; Figure 2D-F).
ART class and mitochondrial dysfunction
We next examined whether prior exposure to NRTIs with systemic mitochondrial toxicity (AZT, d4T, ddC, ddI) was associated with mitochondrial dysfunction in skeletal muscle. Surprisingly, there was no difference in proportional levels of CI deficient myofibres by prior
NRTI exposure (p 0.9, Figure 3).
We then investigated whether there was evidence of skeletal muscle mitochondrial dysfunction in PWH currently treated with ART from classes other than NRTIs. Here we saw no association between proportional level of CI deficiency and any ART class (PI, p 0.9; NNRTI, p 0.9; integrase inhibitor (INSTI), p 1.0, Figure 3).
Here we demonstrate defects of skeletal muscle mitochondrial function at the single cell level in PWH who have only ever been exposed to contemporary ART, despite this being regarded as largely free from mitochondrial toxicity. As mitochondrial dysfunction is known to increase with age [22, 23], we carefully controlled for this effect by age-matching the HIV negative control group. We can therefore conclude that mitochondrial dysfunction exceeds that expected for age in older ART-treated PWH.
These novel observations raise several mechanistic questions. As expected, we observed that the defects of respiratory chain complexes seen in single myofibres were explained mostly by mtDNA large-scale deletions. The pattern of molecular defects did not differ according to historical NRTI exposure. This is surprising as previous in vitro data suggest that contemporary ART does not inhibit the replication of mtDNA [9]. Nevertheless, it is conceivable that very prolonged exposure to a contemporary NRTI in vivo could be sufficient to promote mtDNA deletions [13]. Another possibility is that other ART classes might contribute to mitochondrial dysfunction in contemporary ART. For example, limited in vitro data suggest that the NNRTI efavirenz may impair mitochondrial function [16, 24]. We therefore examined for an effect of 3rd agent (NNRTI / PI / INSTI) exposure in our dataset, but this was not seen. Furthermore, the lack of any mitochondrial defects in the treatment- naïve group argues against a major effect of HIV itself. However, by necessity, this group was of younger age and had shorter duration of HIV infection than the ART-treated group. Overall, it seems most likely that that the observed skeletal muscle mitochondrial dysfunction in ART-treated PWH is multi-factorial, perhaps driven by factors known to be present even with suppressive ART, such as chronic inflammation or oxidative stress [25, 26]. These data underline the potential importance of skeletal muscle as an organ, and mitochondrial dysfunction as a mechanism, mediating adverse ageing phenotypes in PWH. Mechanistic studies such as ours may help lead to rational interventions for improving healthspan of older PWH (Supplementary material, http://links.lww.com/QAD/C594).

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