Vamorolone treatment improves skeletal muscle outcome in a critical illness myopathy rat model

Abstract

Aim: Critical illness myopathy (CIM) is a consequence of modern critical care, leading to skeletal muscle atrophy/paralysis with negative consequences for mortality/morbidity and health care costs. Glucocorticoids (GCs) have been proposed to trigger CIM. Here, we compare outcomes of two GCs, the commonly used prednisolone and the newly developed dissociative vamorolone in response to the intensive care unit (ICU) condition for 5 days, ie, sedation, immobilization, and mechanical ventilation.

Methods: Rats were divided into a 0-day sham-operated control group, and three groups exposed to 5 days ICU condition during treatment with prednisolone (PRED) or vamorolone (VAM) or none of these GCs (ICU-group). Survival, body and muscle weights, cytokine concentrations, regulation of muscle contraction in single fast- and slow-twitch muscle fibres, myofibrillar protein expression and protein degradation pathways were studied.

Results: Critical illness myopathy geno- and pheno-types were confirmed in the ICU group. However, VAM and PRED groups showed reduced atrophy/weakness than the ICU group, and muscle specific differences with more severe negative effects on fast-twitch muscle fibres in the PRED than the other groups.

Conclusion: These results show that vamorolone provides a GC intervention superior to typical GCs in improving CIM outcomes. Further, the findings do not support the notion that moderate-dose GC treatment represents a factor triggering CIM.

Keywords: Glucocorticoids; ICU; muscle wasting; prednisolone; vamorolone.

FIGURE 1

Survival, body, and muscle weights. A, Kaplan-Meier’s survival plot excluding mortality before the initial 2 days of ICU intervention (N = 8, 7, and 7, ICU, PRED, and VAM respectively). B, Relative changes in body weight (BW) before and after adjustment for fluid balance (FB). C, Soleus and D, EDL muscle weights expressed as per cent of BW. In B, C, D, N = 6, 5, 4 and 5, for Control, ICU, PRED, and VAM respectively. Controls are sham-operated rats; ICU are exposed to 5 days of sedation, pharmacological paralysis and mechanical ventilation with no glucocorticoid treatment; PRED, with prednisolone treatment and VAM with vamorolone treatment. Values are means ± SEM. T test was used to compare Control and ICU and one-way ANOVA to compare ICU, PRED and VAM. Asterisk (*) represents P < 0.05, obelisk (†) P < 0.01 and diesis (‡) P < 0.001

FIGURE 2

Single muscle fibre measurements. Box plots representing cross-sectional area (CSA) at a defined sarcomere length (A, D), maximal force (P₀) (B, E), and specific force (SF) (C, F) in soleus and EDL muscles. Filled circles are the 95th percentiles. Asterisk (*) represents P < 0.05, obelisk (†) P < 0.01 and diesis (‡) P < 0.001

FIGURE 3

Contractile proteins. Fold change (FC) in mRNA expression of dominant MyHC isoforms (A, C), and actin (B, D) in soleus and EDL muscles respectively. Myosin:actin protein ratio (E, F). Values are means ± SEM. Asterisk (*) represents P < 0.05 and obelisk (†) P < 0.01

FIGURE 4

Protein degradation. Quantitative PCR expression levels as fold change (FC) of MuRF1 (A, E), atrogin-1 (B, F), SMART (C, G), and FBOX31 (D, H) in soleus and EDL muscles respectively. Values are means ± SEM. Asterisk (*) represents P < 0.05, obelisk (†) P < 0.01 and diesis (‡) P < 0.001

FIGURE 5

Western blot analysis. Protein levels in the soleus and EDL, respectively, of MuRF1 (A, G), atrogin-1 (B, H), and LC3B I (C, I), ratio of LC3B II:I (D, J) and calpain 1 full-length (E, K) and truncated calpain 1 (F, L). Values are means ± SEM of protein blot signal intensities normalized to Coomassie staining. Asterisk (*) represents P < 0.05