Aging Effects of Caenorhabditis elegans Ryanodine Receptor Variants Corresponding to Human Myopathic Mutations

Abstract

Delaying the decline in skeletal muscle function will be critical to better maintenance of an active lifestyle in old age. The skeletal muscle ryanodine receptor, the major intracellular membrane channel through which calcium ions pass to elicit muscle contraction, is central to calcium ion balance and is hypothesized to be a significant factor for age-related decline in muscle function. The nematode Caenorhabditis elegans is a key model system for the study of human aging, and strains were generated with modified C. elegans ryanodine receptors corresponding to human myopathic variants linked with malignant hyperthermia and related conditions. The altered response of these strains to pharmacological agents reflected results of human diagnostic tests for individuals with these pathogenic variants. Involvement of nerve cells in the C. elegans responses may relate to rare medical symptoms concerning the central nervous system that have been associated with ryanodine receptor variants. These single amino acid modifications in C. elegans also conferred a reduction in lifespan and an accelerated decline in muscle integrity with age, supporting the significance of ryanodine receptor function for human aging.

Keywords: Caenorhabditis elegans; aging; malignant hyperthermia; muscle; ryanodine receptor.

Figure 1

Comparison of the rate of locomotion of unc-68 variant strains and wild-type C. elegans in increasing concentrations of halothane or caffeine. Mean body bends per minute for 50 individuals in the presence of various concentrations of halothane (A) or caffeine (B) are presented for each strain. C. elegans strains corresponding to malignant hyperthermia (MH)-associated variants are in shades of blue, central core disease (CCD)-associated variants are in shades of green, exertional heat illness (EHI)-associated variant is in red, and late-onset axial myopathy (LOAM)-associated variant is in purple. In the key, strain names are provided, with the nature of the RyR1 variant and whether the wild-type fosmid (WT) was also present indicated in brackets. Transgenic strains generated using only a variant fosmid are represented by solid bars, while strains generated using a variant fosmid and the wild-type fosmid are represented by striped bars, in adjacent corresponding pairs. The solid gray bars are for the wild-type N2 while the dashed gray bars are for the control transgenic strain, UL4140, generated with just the wild-type unc-68. Error bars are SEM. Significant differences between variant strains and UL4140 are indicated: * P < 0.05, ** P < 0.01, *** P < 0.001 (one-way ANOVA).

Figure 2

Locomotion of C. elegans expressing the LOAM associated variant of unc-68 is specifically increasingly stimulated by caffeine with age. The locomotion of the strain for the RyR1 variants G341R (UL4141), R2163H (UL4147), A4940T (UL4157), R163C (UL4155), and K3452Q (UL4168) are compared with the strain transgenic for only the wild-type unc-68 (UL4140) and the standard wild-type strain (N2). Mean body bends per minute in the presence of increasing concentrations of caffeine are presented for 50 individuals at 0, 3, 7, 10, and 14 d of adulthood. The color coding from Figure 1 is retained with, broadly, blue for MH, green for CCD, red for EHI, and purple for LOAM. Error bars are SEM.

Figure 3

Single amino acid changes in UNC-68 shorten C. elegans lifespan. The percentage of animals surviving on successive days of adulthood is presented for each strain: strains transgenic for different unc-68 variants; UL4140, transgenic for just the wild-type unc-68 (light gray); and the nontransgenic wild-type N2 (dark gray). In the key, strain names are provided, with the nature of the RyR1 variant indicated in brackets. The color coding from Figure 1 is retained with, broadly, blue for MH, green for CCD, red for EHI, and purple for LOAM.

Figure 4

Examples illustrating the five grades in the muscle disorganization scoring scale. The myosin::gfp fusion protein is localized to the thick filaments and so distribution of the fluorescence reports on the regularity in the arrangement of the sarcomeres. Images captured by fluorescence microscopy. (A) Typical structure of a grade 1 muscle score; myosin filaments are linear and well organized. (B) Typical structure of a grade 2 muscle score; myosin filaments are starting to show more bends but the pattern is still well organized. (C) Typical structure of a grade 3 muscle score; myosin filaments are more fragmented and there are apparently overlapping filaments. (D) Typical structure of a grade 4 muscle score; myosin filaments are further fragmented and the regularity of pattern is no longer clear. (E) Typical example of grade 5 muscle score; the pattern of myosin filaments is severely disorganized. Figure compiled by Matt Pipe.

Figure 5

Comparison of increase in myofilament disorganization with age, for each unc-68 variant. The whole-body scores for extent of disorganization of the sarcomeric structure from 0 to 14 d of adulthood are presented for the strain transgenic for wild-type unc-68 (UL4140) (A), and strains transgenic for unc-68 with amino acid changes equivalent to RyR1 variants associated with: MH in blue (G341R (B), R2454H (C), R2163H (E), and R2458H (F); EHI in red (R163C) (D); CCD in green (A4940T) (G) and (R4861H) (H); and LOAM in purple (K3452Q) (I). Each boxplot represents the median, interquartile range, and minimum and maximum for the whole-body scores.

Figure 6

The locomotive response to caffeine that is modified by amino acid changes in UNC-68 is dependent upon the chemosensory neuron–specific genes che-3 and osm-3. The locomotion of strains transgenic for the wild-type unc-68 (UL4140), for the EHI-associated variant R163C (UL4155), and for the LOAM-associated variant K3452Q (UL4168) was recorded upon RNAi knockdown of che-3 or osm-3 or in precisely equivalent blank control RNAi experiments. Mean body bends per minute in the presence of increasing concentrations of caffeine are presented for 50 individuals at 0 and 7 d of adulthood, i.e., young and old adults respectively. The color coding from Figure 1 is retained with, broadly, red for EHI and purple for LOAM. Error bars are SEM. Equivalent results for other strains are presented in Figure S1.