A combination of major histocompatibility complex (MHC) I overexpression and type I interferon induce mitochondrial dysfunction in human skeletal myoblasts

Abstract

The overexpression of major histocompatibility complex (MHC) I on the surface of muscle fibers is a characteristic hallmark of the idiopathic inflammatory myopathies (IIMs), collectively termed myositis. Alongside MHC-I overexpression, subtypes of myositis, display a distinct type I interferon (IFN) signature. This study examined the combinational effects of elevated MHC-I and type I IFNs (IFNα/β) on mitochondrial function, as mitochondrial dysfunction is often seen in IIMs. Human skeletal muscle myoblasts were transfected with an MHC-I isoform using the mammalian HLA-A2/K^b vector. Mitochondrial respiration, mitochondrial membrane potential, and reactive oxygen/nitrogen species generation were assessed with or without IFNα and IFNβ. We show that MHC-I overexpression in human skeletal muscle myoblasts led to decreased basal glycolysis and mitochondrial respiration, cellular spare respiratory capacity, adenosine triphosphate-linked respiration, and an increased proton leak, which were all exaggerated by type I IFNs. Mitochondrial membrane depolarization was induced by MHC-I overexpression both in absence and presence of type I IFNs. Human myoblasts overexpressing MHC-I showed elevated nitric oxide generation that was abolished when combined with IFN. MHC-I on its own did not result in an increased reactive oxygen species (ROS) production, but IFN on their own, or combined with MHC-I overexpression did induce elevated ROS generation. Surprisingly, we observed no gross changes in mitochondrial reticular structure or markers of mitochondrial dynamics. We present new evidence that MHC-I overexpression and type I IFNs aggravate the effects each has on mitochondrial function in human skeletal muscle cells, providing novel insights into their mechanisms of action and suggesting important implications in the further study of myositis pathogenesis.

Keywords: idiopathic inflammatory myopathies; major histocompatibility complex I; mitochondria; myositis; reactive and nitric oxygen species; type I interferon.

Figure 1

Mitochondrial function of human myoblasts treated with interferon‐α (IFNα). Non‐mitochondrial respiration, basal respiration, maximal respiration, spare respiratory capacity, proton leak, and ATP‐linked respiration, normalized to protein content following 18 h (a–f) and 24 h (g–l) incubation with IFNα at indicated doses (n = 6). Data represent mean ± SEM, *p ≤ 0.05 compared to control. ATP, adenosine triphosphate; SEM, standard error of the mean.

Figure 2

Mitochondrial function of human myoblasts treated with interferon‐β (IFNβ). Non‐mitochondrial respiration, basal respiration, maximal respiration, spare respiratory capacity, proton leak, and ATP‐linked respiration, normalized to protein content following 18 h (a–f) and 24 h (g–l) incubation with IFNβ at indicated doses (n = 6). Data represent mean ± SEM, *p ≤ 0.05, **p < 0.01 compared to control. ATP, adenosine triphosphate; SEM, standard error of the mean.

Figure 3

In‐vitro overexpression of major histocompatibility complex‐I. Representative phase contrast and single‐channel fluorescent images of human skeletal muscle myoblasts transfected with human leukocyte antigen(HLA)‐A2/K^b or empty vector (EV) in presence or absence of type I interferons stained with anti‐HLA‐I (green) and 4′,6′‐diamidino‐2‐phenylindole dihydrochloride (blue). Images captured at 20× magnification. Scale bar = 100 μm. Quantification of HLA Class I fluorescence intensity level normalized to nuclei number. (c) HLA Class I gene expression. Data represent mean ± SEM, *p ≤ 0.05, ***p < 0.001 compared to EV or to HLA I‐overexpressing cells. SEM, standard error of the mean.

Figure 4

Mitochondrial function of human leukocyte antigen‐I‐transfected human myoblasts treated with or without type I interferons. (a) Real‐time measurements of oxygen consumption rate (OCR) following the sequential injection of oligomycin, FCCP, and a mixture of rotenone/antimycin A. (b) Real‐time measurements of extracellular acidification rate (ECAR) and (c) baseline ECAR values. (d) Bioenergetic profile expressed as OCR versus ECAR measured under basal condition. (e–j) Mitochondrial function parameters; nonmitochondrial respiration, basal respiration, maximal respiration, spare respiratory capacity, ATP‐linked respiration, and proton leak, normalized to protein content (n = 8 per group). Data represent mean ± SEM, *p ≤ 0.05, **p < 0.01, ***p < 0.001 compared to empty vector. ATP, adenosine triphosphate; FCCP, carbonyl cyanide‐p‐trifluoromethoxyphenylhydrazone; SEM, standard error of the mean.

Figure 5

Mitochondrial mass and membrane potential of human leukocyte antigen‐I‐transfected human myoblasts treated with or without type I interferons. (a) Mitochondrial membrane potential expressed as JC‐1 aggregates (red fluorescence) to JC‐1 monomers (green fluorescence) ratio normalized to protein content (n = 6). (b–d) Fluorescence intensity changes of MitoTracker Red CMXRos and tetramethylrhodamine (TMRM) indicative of changes in mitochondrial membrane potential of active mitochondria only, as well as of MitoTracker Green, representing changes in mitochondrial mass. (e) Fluorescence intensity of TMRM normalized to MitoTracker Green. Data represent mean ± SEM, *p ≤ 0.05, **p < 0.01, ***p < 0.001 compared to empty vector. SEM, standard error of the mean.

Figure 6

RONS generation in human leukocyte antigen (HLA)‐I‐transfected human myoblasts treated with or without type I interferons (IFNs). (a and b) Fluorescence intensity levels of MitoSOX Red, an indicator of mitochondrial superoxide generation, induced by type I IFNs or major histocompatibility complex‐I overexpressing cells with or without type I IFNs (n = 3–8), and (c) DAF‐FM DA, showing cellular nitric oxide generation (n = 4). (d) Release of hydrogen peroxide from cells as measured by Amplex Red assay. All data were normalized to protein content. Data represent mean ± SEM, *p ≤ 0.05, **p < 0.01, ***p < 0.001 compared to empty vector or HLA I‐overexpressing cells. DAF‐FM DA, 4‐amino‐5‐methylamino‐2',7'‐difluorofluorescein diacetate; RONS, reactive oxygen & nitrogen species; SEM, standard error of the mean.

Figure 7

Immunofluorescent imaging of human skeletal muscle myoblasts transfected with empty vector (a) human leukocyte antigen‐A2/K^b (b) in presence or absence of inteferon alpha (c) or interferon beta (d), with MitoTracker Red dye and 4′,6′‐diamidino‐2‐phenylindole dihydrochloride, with quantification of reticular structure using mitochondrial network analysis. reporting mitochondrial footprint (e), branch length (f) and branch count (g). Data are presented as mean ± SEM, (n = 4).

Figure 8

Quantitative polymerase chain reaction data showing changes in gene expression of PPARGC1 (a), MFN1 (b), MFN2 (c), OPA1 (d), TFAM (e) and Fis1 (f), expressed as fold change, in human skeletal muscle myoblasts transfected with human leukocyte antigen‐A2/K^b or empty vector in presence or absence of type I interferons. Data are presented as mean ± SEM, (n = 4). SEM, standard error of the mean.