Skeletal muscle mitochondrial dysfunction in contemporary antiretroviral therapy: a single cell analysis

Abstract

Objective: To quantify mitochondrial function in skeletal muscle of people treated with contemporary antiretroviral therapy.

Design: Cross-sectional observational study.

Methods: 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.

Results: 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-naive 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.

Conclusion: 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.

Figure 1. Quantitative multiplex immunofluorescence analysis of mitochondrial dysfunction in individual myofibres.

(A) Representative examples of multiplex immunofluorescence for mitochondrial respiratory chain complex 1 (CI, NDUFB8), complex 4, (CIV, MTCO1), mitochondrial mass (VDAC1) and laminin in an ART-treated PWH, an HIV negative control, and an ART-naïve PWH. The yellow arrow indicates a myofibre deficient in CI and CIV, with hyperintensity of VDAC1, indicating compensatory mitochondrial proliferation. The pink arrow indicates a myofibre deficient in CI but not CIV. Scale bars are 100μm. (B) Example 2D dot plots of CI z-scores (x-axis) and CIV z-scores (y-axis) of individual myofibres from two ART-treated PWH. Dotted lines are at z-score of 0, i.e. the expected mean. Box outlined in upper right quadrant of each panel contains myofibres with normal CI and CIV abundance. Each dot represents an individual myofibre and is coloured based on VDAC1 intensity (mitochondrial mass) category: light blue = ‘low’ (z -3 to -2), cream = ‘normal’ (z -2 to +2), orange = ‘high’ (z +2 to +3), and red = ‘very high’ (z > +3).

Figure 2. Mitochondrial respiratory chain deficiency in single skeletal myofibres.

Mitochondrial complex I (CI) (A) and complex IV (CIV) (B) and mass (C) according to HIV and ART status (x-axes). CI and CIV expressed as proportion of myofibres showing respiratory chain deficiency. Mitochondrial mass (porin / VDAC1) expressed as mean z-score. Mitochondrial CI deficiency by age (D), ART duration (E), and current CD4 cell count (F). ** p <0.01; dots represent individual subjects; dotted line on y-axis, lower limit of detection of assay.

Figure 3. Skeletal muscle mitochondrial respiratory chain deficiency by ART class exposure.

Mitochondrial complex 1 (CI) deficiency in ART-treated PWH. PI, protease inhibitor (current exposure); NNRTI, non-nucleoside reverse transcriptase inhibitor (current exposure); INSTI, integrase inhibitor (current exposure); mNRTI, nucleoside reverse transcriptase inhibitor with systemic mitochondrial toxicity (prior exposure). Dotted line on y-axis is limit of detection of assay. Dots are individual subjects.

Figure 4. mtDNA analysis of single myofibres.

qPCR analysis of individual CI-deficient, CI-intermediate and CI-positive myofibres from ART-treated PWH. Each dot represents an individual myofibre. (A). mtDNA content (SQ, arbitrary units). Dotted line is 5^th centile of SQ for CI-positive myofibres. (B). Large-scale mtDNA deletions. Mutation loads are expressed as δδC_t (difference in MT-ND1 and MT-ND4 C_t (cycle threshold) values relative to undeleted assay control). Deletions in the major arc of the mtDNA genome delete MT-ND4 and are shown as negative δδC_t values. Minor arc deletions delete MT-ND1 and are shown as positive δδC_t values. Thin dotted lines show 2 standard deviations from the mean for CI-positive myofibres.