(T1330-02-09) Age-Dependent Skeletal Muscle Response to Long-Term Clofazimine Treatment

Purpose: Consequences of skeletal muscle (SM) aging include structural and functional loss. These age-related changes can be influenced by different exposures, including medications. Our objective was to investigate how age affects SM structure, its ability to sequester drugs, and its immune responses following treatment with the lipophilic antimicrobial riminophenazine dye, clofazimine (CFZ).

Methods: Two groups of C57BL/6J male mice based on age were studied. The first group consisted of young mice (4 weeks old) and the second group of old mice (61 weeks old). Mice in each group were randomized to receive either CFZ (44mg/kg/day), a known immune-stimulating molecule, or vehicle (equivalent sesame oil) for an 8-week period. Whole blood, major drug-sequestering organs, and tricep (TRI) and quadricep (QUAD) muscles were collected at the end of treatment. CFZ concentration was measured in blood samples and isolated organs by LC-MS or UV-Vis. QUAD cryosections were trichrome stained to gain muscle structure data and were also analyzed via Raman-microscopy to assess CFZ distribution. Lastly, cytokine array assays (40 measured analytes) were used to study the SM immune response in whole TRI samples.

Results: CFZ treatment in young (Y+) and old (O+) mice did not result in different CFZ concentrations (mean ± SD) in the blood, 3.05 ± 0.66 µg/mL and 3.40 ± 0.76 µg/mL, respectively (p=0.25).  Based on isolated drug measurements that were verified by Raman microscopy, both groups accumulated 3-times more CFZ-HCl than CFZ-freebase (Fig. 1). CFZ-HCl, is the form of CFZ created via ion-trapping in the lysosome of macrophages, a compartment that digests foreign material.  However, CFZ distribution patterns were different in Y+ and O+ mice. O+ mice had 142% more CFZ in SM compared to Y+. Specifically, O+ had more CFZ per gram of SM (58.46 ± 19.21 µg/g) compared to Y+ (26.72 ±5 .80 µg/g) (p < 0.0001). Unlike Y+, O+ also had a 24% increase in QUAD collagen levels compared to its vehicle control O-; p=0.03. This is usually seen in older or injured tissue. However, Y+ and O+ had a 17% decrease (p=0.04, 0.006) in SM fiber diameter and a 14% decrease (p < 0.0001) in SM mass compared to negative controls, Y- and O- (Fig. 2,3), respectively. In addition to the different structural changes that occurred in the CFZ-treated groups, the SM also showed trends in CFZ-induced cytokine responses.  Y+ experienced upregulation in 98% (39/40) of the measured cytokines, and O+ only experienced upregulation in 48% (19/40) of the measured cytokines.

Conclusion: Older mice (61 weeks old) experienced greater accumulation of CFZ in SM, which was accompanied by a trend towards downregulation in a number of SM cytokine levels compared to young mice (4 weeks old). These results are important because they reveal that age is a variable that affects SM response to a long-term CFZ treatment and that macrophage lysosomal occupancy may underlie some of the loss in muscle structure and function typically associated with aging.

Fig 1. Clofazimine (CFZ) distribution in mouse quadricep (QUAD) muscle. Both young (Y+) and old (O+) CFZ treated mice had more CFZ-HCL than CFZ-freebase in QUAD muscle. Data are the mean ± SD of 5 animals/group (Student’s t test. *p=0.02, ***p=0.0007).



Fig 2. Skeletal muscle (SM) fiber diameter was altered by clofazimine (CFZ) treatment. Both young (Y+) and old (O+) CFZ treated mice had smaller muscle fiber diameter compared with young (Y-) and old (O-) control mice, respectively. Data are the mean ± SD of 5-6 animals/group (one-way ANOVA, *p=0.04, **p=0.006).



Fig 3. Clofazimine (CFZ) treatment reduced total isolated skeletal muscle (SM) mass. Both young (Y+) and old (O+) CFZ treated mice had lower total muscle mass (sum of quadricep (QUAD) and tricep (TRI)) compared with young (Y-) and old (O-) control mice, respectively. Data are the mean ± SD of 10-19 summed pairs/group (one-way ANOVA, ****p < 0.0001)​.