Background/Purpose: The aim of this study was to investigate whether in immune-mediated necrotising myopathy (IMNM), anti-HMGCR antibodies interfere with HMGCR activity and have a myopathic effect.

Methods: To obtain polyclonal anti-HMGCR autoantibodies, the 6 peptides identified as the epitopes of anti-HMGCR antibodies were used to immunise a white New Zealand rabbit. Antibodies from a non-immunised animal served as control. Plasma of anti-HMGCR (n=5) and anti-SRP (n=2) IMNM patients were obtained from plasmapheresis eluates. Rabbit and human anti-HMGCR antibodies were purified by affinity chromatography. Inhibition of HMGCR was measured in vitro by spectrophotometry in the presence of different concentrations of autoantibodies. Pravastatin was used as a negative control. Human autoantibodies were electroporated into human myotubes: creatine-kinase levels (CK) were measured in the supernatant, and hematoxylin/eosin (H&E) as well as oil red-O stainings were performed.Thirty-four patients with inflammatory myopathies (IM) according to the EULAR/ACR 2017 criteria, not taking immunomodulators or statins, were included (anti-HMGCR: n=10; other IM: n=24) as well as 3 patients without neuromuscular diseases (no NMD). Histological sections of deltoid muscle taken at the time of diagnosis were immunostained with an anti-human IgG antibody. Oil red-O staining was used to study the accumulation of lipid droplets, quantified (extension score 0-4) by two myopathologists blinded to the diagnosis.

Results: To test whether anti-HMGCR antibodies inhibit the function of their target, HMGCR enzymatic activity was assessed in vitro in the presence of anti-HMGCR, control antibodies and pravastatin. A dose-dependent inhibition of HMGCR activity was observed in the presence of anti-HMGCR (Figure 1a). Rabbit anti-HMGCR 5 µg/ml and human anti-HMGCR 2 µg/ml had similar effects to that of pravastatin 0.5 μM (Figure 1a, 1b). IgG from the non-immunised rabbit and plasma from anti-SRP patients did not affect HMGCR activity (Figure 1a, 1b). To test whether anti-HMGCR internalisation in myofibres exerts a myopathic effect, human myotubes were electroporated with human purified anti-HMGCR, control IgG or pravastatin. Antibody presence in the cytoplasm 4 days after electroporation was confirmed. CK levels were 2.4-fold higher in the supernatant of myotubes electroporated with anti-HMGCR compared to those electroporated with control IgG (p=0.0001). H&E staining showed necrotic myotubes after electroporation with purified anti-HMGCR and pravastatin, but not with control IgG. Oil red-O staining revealed a lipid droplet accumulation in myotubes electroporated with purified anti-HMGCR and pravastatin, but not with control IgG (Figure 1c). Lipid droplet accumulation score was 10-fold higher in anti-HMGCR IMNM patients compared to other IM and no NMD (3.2 ± 0.6 vs. 0.3 ± 0.5; vs. 0.3 ± 0.6, respectively, p=0.0001) and a score ≥ 2 was a hallmark of anti-HMGCR IMNM (Figure 1d, 1e).

Conclusion: Together, these data demonstrate that anti-HMGCR antibodies inhibit HMGCR activity, leading to the sarcoplasmic accumulation of lipid droplets and myofibres necrosis. These findings could have implications for both the diagnosis and treatment of IMNM.

ACR Meeting Abstracts - https://acrabstracts.org/abstract/in-immune-mediated-necrotising-myopathy-anti-hmgcr-antibodies-inhibit-hmgcr-activity-leading-to-the-sarcoplasmic-accumulation-of-lipid-droplets-and-myofibres-necrosis/