Altered Ca2+ homeostasis in the skeletal muscle of DJ-1 null mice

Abstract

Loss-of-function mutations in DJ-1 are associated with early-onset of Parkinson's disease. Although DJ-1 is ubiquitously expressed, the functional pathways affected by it remain unresolved. Here we demonstrate an involvement of DJ-1 in the regulation of Ca(2+) homeostasis in mouse skeletal muscle. Using enzymatically dissociated flexor digitorum brevis muscle fibers from wild-type (wt) and DJ-1 null mice, we examined the effects of DJ-1 protein on resting, cytoplasmic [Ca(2+)] ([Ca(2+)](i)) and depolarization-evoked Ca(2+) release in the mouse skeletal muscle. The loss of DJ-1 resulted in a more than two-fold increase in resting [Ca(2+)](i). While there was no alteration in the resting membrane potential, there was a significant decrease in depolarization-evoked Ca(2+) release from the sarcoplasmic reticulum in the DJ-1 null muscle cells. Consistent with the role of DJ-1 in oxidative stress regulation and mitochondrial functional maintenance, treatments of DJ-1 null muscle cells with resveratrol, a mitochondrial activator, or glutathione, a potent antioxidant, reversed the effects of the loss of DJ-1 on Ca(2+) homeostasis. These results provide evidence of DJ-1's association with Ca(2+) regulatory pathways in mouse skeletal muscle, and suggest the potential benefit of resveratrol to functionally compensate for the loss of DJ-1.

Fig. 1

Increased [Ca²⁺]_i in DJ – 1 null muscle fibers. Recording of intracellular Ca²⁺ concentration ([Ca²⁺]_i) and resting membrane potential (V_m) with double-barreled microelectrodes in enzymatically dissociated flexor digitorum muscle fibers. A. Representative Ca²⁺ potential records (V_Ca) from a wt (gray) and a DJ – 1 null (black) muscle fibers. The initial portion of each trace was obtained prior to impalement of the cells. Penetration and removal of microelectrode was accompanied by immediate downward and upward deflection, respectively. B. Resting [Ca²⁺]_i in wt (n = 12, gray), DJ – 1 null (n = 16, black) muscle cells determined from V_Ca after calibration of recording microelectrodes. DJ – 1 null cells exhibited a greater then two-fold elevation in [Ca²⁺]_i. C. Resting V_m recorded from the same cells in panel B. Cells from DJ – 1 null animals exhibited V_m similar to those form the wt animals (p > 0.5, t-test). Muscle fibers were prepared from 8 to 12 months old mice. Asterisk indicates statistical significance (p < 0.001, t-test).

Fig. 2

Reduced Ca²⁺ release in DJ – 1 null muscle fibers. A. Mean Ca²⁺ transient from wt (gray) (n = 17) and DJ – 1 null (black) (n = 22) FDB muscle fibers measured in response to 1 ms action potential stimuli. B–C. Peak amplitude (ΔF/F) and decay time constant (τ, ms) of Ca²⁺ transients from wt and DJ – 1 null muscle fibers from the same cells presented in panel A, respectively. Asterisk indicates statistical significance (p < 0.05).

Fig. 3

DJ – 1 does not affect the protein expression of RyR1 and SERCA1. A. Immunoblots of RyR1 and SERCA1 protein levels from hamstring muscle of wt and DJ – 1 null mice. The membrane was reprobed for α-actin, which was used as loading control. Bottom panel demonstrates that there was no detectable DJ – 1 protein in the DJ – 1 null muscle. B–C. Quantitative analysis confirmed that there were no significant alterations in the steady-state expression levels of RyR1 (B) and SERCA1 (C) in the DJ – 1 null muscle.

Fig. 4

Resveratrol treatment reverses [Ca²⁺]_i changes and improves Ca²⁺ release in DJ – 1 null muscle cells A. Resting [Ca²⁺]_i measurements performed with Ca²⁺ selective microelectrodes in wt and DJ – 1 null muscle cells treated for 18 h with 10 μM (n_wt = 5, n_{DJ–1 null} = 17) or 50 μM (n_wt = 5, n_{DJ–1 null} = 8) resveratrol (RSV). Resveratrol treatment did not have an effect on [Ca²⁺]_i in wt cells, but lowered [Ca²⁺]_i in DJ – 1 null cells in dose-dependent manner. B. Mean Fluo-4 fluorescence transient from untreated DJ – 1 null muscle fibers (left) (n = 15) and DJ – 1 null muscle cells treated for 12 h with 50 μM resveratrol (right) (n = 16). Ca²⁺ transients were elicited by 1 ms action potential stimuli. Resveratrol treatment increased peak amplitudes of Ca²⁺ transients in DJ – 1 null cells. C. Resting [Ca²⁺]_i measurements performed with Ca²⁺ selective microelectrodes in wt and DJ – 1 null muscle cells treated for 18 h with 5 mM glutathione (GSH). GSH treatment did not have an effect on [Ca²⁺]_i in wt cells (n = 4), but lowered [Ca²⁺]_i in DJ – 1 null cells (n_{DJ–1 null} = 6, n_{DJ–1 null+GSH} = 10). Asterisk indicates statistical significance (p < 0.05).