Endogenously determined restriction of food intake overcomes excitation-contraction uncoupling in JP45KO mice with aging

Abstract

The decline in muscular strength with age is disproportionate to the loss in total muscle mass that causes it. Knocking out JP45, an integral protein of the junctional face membrane of the skeletal muscle sarcoplasmic reticulum (SR), results in decreased expression of the voltage-gated Ca(2+) channel, Ca(v)1.1; excitation-contraction uncoupling (ECU); and loss of muscle force (Delbono et al., 2007). Here, we show that Ca(v)1.1 expression, charge movement, SR Ca(2+) release, in vitro contractile force, and sustained forced running remain stable in male JP45KO mice at 12 and 18 months. They also exhibit the level of ECU reported for 3-4-month mice (Delbono et al., 2007). No further decline at later ages was recorded. Preserved ECC was not related to increased expression of any protein that directly or indirectly interacts with JP45 at the triad junction. However, maintained muscle force and physical performance were associated with ablation of JP45 expression in the brain, spontaneous and significantly diminished food intake and less tendency toward obesity when exposed to a high-fat diet compared to WT. We propose that (1) endogenously generated restriction in food intake overcomes the deleterious effects of JP45 ablation on ECC and skeletal muscle force mainly through downregulation of neuropeptide-Y expression in the hypothalamic arcuate nucleus; and (2) the JP45KO mouse constitutes an invaluable model to examine the mechanisms controlling food intake as well as skeletal muscle function with aging.

Figure 1. Skeletal muscle force, Ca_v1.1 charge movement and content in total SR membrane from JP45 KO and WT mice

A. Twitch, tetanic force, and specific force recorded in WT and JP45KO EDL and soleus muscles. The number of muscles tested was: EDL WT 3 [months] = 17; EWT12 = 14; EWT18=12; soleus [S] WT 3=15; SWT 12=13; SWT18 =9; EDL JP45KO [KO] 3=16; EKO12=14; EKO18=16; SKO3 =14; SKO12 =15; and SKO18=11. Data points are expressed as mean ± SD. *ANOVA test P <0.05. Data for 3-month-old WT and JP45KO mice are from (Delbono et al., 2007). B. Ca_v1.1 charge movement recorded after blocking the inward Ca²⁺ current. Data points were fitted to equation 1. Table 2 shows bestfitting parameters. C. [³H]-PN200-110 binding assay. R1 fraction proteins from 18-month JP45KO and WT mice were separated by 7.5% PAGE, blotted, and stained with a peroxidaseconjugated antibody against albumin (see Methods). Albumin content was measured by densitometry of the protein band. Data of 20–24 determinations carried out in three different total SR fraction preparations. Data for 3-month-old mice are from (Delbono et al., 2007). Data points represent mean ± S.E.M.

Figure 2. Age-dependent JP45 expression in WT and SR protein composition in JPKO mice

A. Western blot analysis of JP45 expression in microsomes from 3, 6, 12, and 18 monthold WT mice. B. JP45 expression at 6–18 months is represented as a percent at 3 months (n = 3). The asterisk indicates a statistically significant difference at 18 compared with 3 month-old mice (p < 0.05). Immunoblot analysis of SR fraction protein expression in 12- (C) and 18- (D) month JP45KO mice. Total SR (15μg) was separated on SDS gel and transferred to nitrocellulose membrane (n = 12–14 determinations in 2–3 SR membrane preparations). Data are expressed as a percent of albumin expression. Data for 3-month-old mice are from (Delbono et al., 2007). Values represent mean ± S.E.M.

Figure 3. Spontaneous and forced activity, body weight and food intake in JP45KO and WT mice

Dark phase (5 P.M.-5 A.M.) running distance assessed in 3- (A), 12- (B) and 18- (C) month JP45KO (triangles) and WT (squares) mice, housed and equipped individually. Speed events represent the running distance over 10 sec. Data points from 3 (n = 12–14), 12 (n = 7–8) and 18 (n = 9–10) month-old mice. D. Maximal running time was measured using a forced treadmill. Data are compared with the 2–3-month-old mouse cohort. Data are mean ± S.E.M. for 2–3 (n= 6), 12–14 (n=6), and 18 (n=5) month-old mice. E. Body weight recorded in 3-month WT (n = 11) and JP45KO (n = 12); 4-month WT (n = 13) and JP45KO (n = 15); 12-month WT (n = 30) and JP45KO (n = 11) mice; and 18-month WT (n = 9) and JP45KO (n = 16) mice. *ANOVA test P < 0.05. F. Food consumed over a 20-day period by 13 JP45KO and 14 WT mice at 12 months of age. * p< 0.05. G. Food consumed over a 20-day period by 10 JP45KO and 10 WT mice at 18 months of age. * p< 0.05. H. Mouse body weight recorded at 5 weeks and for the next 33 weeks. Data points represent mean ± S.E.M of 6 JP45KO and 5 WT mice. Differences between asterisks are statistically significant.

Figure 4. Fiber-type composition and fiber size in EDL and soleus muscle, and MHC fiber size distribution in soleus muscle from JP45 KO and WT mice

Myosin-heavy chain I (MHCI) composition in EDL and soleus muscles from (A, C) 12- and (B, D) 18-month-old mice. Fiber-type composition determined by myosin-heavy chain (MHC) immunocytochemistry in E[DL] WT 12 [months]= 4; EWT18 = 4; S[oleus]WT12 = 4; SWT18 = 4; E[JP45]KO12 = 4; EKO18 = 4; SKO12 = 4; and SKO18 = 4. Asterisks indicate statistically significant differences between JP45KO and WT mice (P < 0.05). EDL (E) and soleus (F) fiber size distribution and average minimal fiber Feret (G) in 12-month JP45KO and WT mice. EDL (H) and soleus (I) fiber size distribution and average minimal fiber Feret (J) in 18-month JP45KO and WT mice. The number of fibers analyzed was: EWT12 = 1026; EKO12 = 1213; SWT12 = 869; SKO12 = 844; EWT18 = 1372; EKO18 = 1460; SWT18 = 1171; and SKO18 = 1377. MHC I distribution in (K) 12- and (C) 18-month JP45KO and WT mice and MHC II distribution in (L) 12- and (D) 18-month JP45KO and WT mice. For 12-month WT and JP45KO mice, the number of MHC I fibers studied was 376 and 220, respectively (A); MHC II fibers, 493 and 319, respectively (B). For 18-month WT and JP45KO mice, the number of MHC I fibers was 417 and 594, respectively (M); MHC II, 759 and 778, respectively (N). Values represent mean ± S.E.M.

Figure 5. Serum leptin and insulin concentration in JP45KO and WT mice

Individual serum leptin concentration determination as a function of age (4–24 months) (A) or mouse weight (C). Serum insulin concentration as a function of age (B) or weight (D). Linear regression lines for JP45KO and WT mice are superimposed.

Figure 6. JP45 expression in hipothalamus, effect of JP45 ablation on hypothalamic neuropeptide expression and proposed model for neuropeptide network operation in JP45KO mice

Brain coronal sections showing JP45 expression in the arcuate nucleus of the hypothalamus of a WT mouse (A) and JP45KO mouse (B). Whole WT brain coronal sections in which the JP45 primary antibody was omitted (C) or replaced by rabbit IgG (D). Images are representative of brains from JP45KO (n = 3) and WT (n = 3) mice. (E) Expression of POMC, NPY and AGRP in arcuate hypothalamus (ARH). (F) Expression of TRH and CRH in paraventricular hypothalamus (PVN). Dissection of hypothalamic regions and measurement of gene expression have been described in Materials and Methods. Gene expression was normalized to 18S ribosomal RNA level. Data are expressed as mean ± S.E.M. G. JP45KO leads to decreased neuropeptide-Y (NPY) and agouti-related peptide (AgRP) but not proopiomelanocortin (POMC) secretion, which results in increased thyrotropin-releasing hormone (TRH) and corticotropin-releasing hormone (CRH) secretion in the paraventricular nucleus and reduced food intake.

Figure 7. Time course of excitation-contraction uncoupling and endogenous caloric restriction in JP45KO and WT mice