Sarco(endo)plasmic reticulum Ca2+-ATPase function is impaired in skeletal and cardiac muscles from young DBA/2J mdx mice

Figure 1. Impairments in Ca2+ uptake and maximal SERCA activity in gastrocnemius muscles from mdx mice are more prominent on the D2 background strain (A) Ca2+ uptake curves from gastrocnemius homogenates. (B) Area under the curve (AUC) of Ca2+ uptake curves. (C) Total Ca2+ uptake normalized to mg of protein loaded. (D) Ca2+-dependent SERCA activity curves in D2 and C57 -WT and -mdx gastrocnemius muscles. (E) Maximal SERCA activity expressed as μmol/g of protein/min. (F) The % reduction in maximal SERCA activity in C57- and D2 mdx mice. (G) pCa50 of SERCA activity. (H) Calpain activity in gastrocnemius muscles from D2 and C57 -WT and -mdx gastrocnemius muscles expressed as RFU min/mg of protein. Two-way ANOVAs were performed with a Sidak’s multiple comparisons test, ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, and ∗∗∗∗p < 0.0001 n = 4-6. Main effects and interactions expressed in bars over the graph. Values are represented as mean ± SEM. For F, a Student’s t-test was used, ∗p < 0.05.

Figure 2. Western blot analyses of SR Ca2+ handling proteins, total protein nitrosylation, and tyrosine nitration in gastrocnemius muscles from C57- and D2 WT and mdx mice (A and B) SERCA1 and SERCA2 content. (C) SLN content in C57 and D2 mdx gastrocnemius muscles. (D–F) RyR1, CSQ, and calstabin content in gastrocnemius muscles from D2 and C57 -WT and -mdx. (G) Protein nitrosylation measured via western blotting for nitrocysteine-adducted protein. (H) Protein tyrosine nitration measured via western blotting for nitrotyrosine-adducted protein. For (A–C) and (D–H), two-way ANOVAs were performed with a Sidak’s multiple comparisons test, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001 n = 6 per group. Main effects and interactions expressed in bars over the graph. For C, a Student’s t test was used, ∗p < 0.05, n = 6 per group. All protein contents are expressed as arbitrary units and relative to C57 WT. Values are represented as mean ± SEM.

Figure 3. Impairments in Ca2+ uptake and maximal SERCA activity in diaphragm muscles from mdx mice are more prominent on the D2 background strain (A) Ca2+ uptake curves from diaphragm homogenates. (B) Area under the curve (AUC) of Ca2+ uptake curves. (C) Ca2+ uptake rate in diaphragm homogenates from C57 WT and mdx mice measured via tangent analysis at 1000 nM free [Ca2+]. (D) Ca2+-dependent SERCA activity curves in D2 and C57 -WT and -mdx diaphragm muscles. (E) pCa50 of SERCA activity. (F) Maximal SERCA activity expressed as μmol/g of protein/min. (G) The % reduction in maximal SERCA activity in C57- and D2 mdx mice. For (B, E, and F), two-way ANOVAs were performed with a Sidak’s multiple comparisons test, ∗∗∗∗p < 0.0001 n = 5-6 per group. Main effects and interactions expressed in bars over the graph. Values are represented as mean ± SEM. For C and G, a Student’s t test was used, ∗p < 0.05.

Figure 4. Western blot analyses of SR Ca2+ handling proteins and total protein nitrosylation in diaphragm muscles from C57- and D2 WT and mdx mice (A and B) SERCA1 and SERCA2 content. (C) SLN content in C57 and D2 WT and mdx diaphragm muscles. (D–F) RyR1, CSQ, and calstabin content in diaphragm muscles from D2 and C57 -WT and -mdx. (G) Protein nitrosylation measured via western blotting for nitrocysteine-adducted protein. Two-way ANOVAs were performed with a Sidak’s multiple comparisons test, ∗∗p < 0.01, ∗∗∗∗p < 0.0001 n = 6 per group. Main effects and interactions expressed in bars over the graph. All protein contents are expressed as arbitrary units and relative to C57 WT. Values are represented as mean ± SEM.

Figure 5. Impairments in Ca2+ uptake in left ventricles from mdx mice are more prominent on the D2 background strain (A) Ca2+ uptake curves from left ventricle homogenates. (B) Area under the curve (AUC) of Ca2+ uptake curves. (C) Ca2+ uptake rate in left ventricle homogenates from C57 WT and mdx mice measured via tangent analysis at 1000 nM free [Ca2+]. (D) Ca2+-dependent SERCA activity curves in left ventricle homogenates from D2 and C57 -WT and -mdx mice. (E) pCa50 of SERCA activity. (F) Maximal SERCA activity expressed as μmol/g of protein/min. For (B, E, and F), two-way ANOVAs were performed with a Sidak’s multiple comparisons test, ∗∗∗∗p < 0.0001 n = 5-6 per group. Main effects and interactions expressed in bars over the graph. Values are represented as mean ± SEM. For C, a Student’s t test was used, ∗p < 0.05.

Figure 6. Western blot analyses of SR Ca2+ handling proteins and total protein nitrosylation in left ventricles from C57- and D2 WT and mdx mice (A) SERCA2 content in left ventricle homogenates. (B) PLN content in C57 and D2 WT and mdx diaphragm muscles. (C–F) RyR2, calstabin, and CSQ content in left ventricle homogenates from C57 and D2 -WT and -mdx mice. (G) Protein nitrosylation measured via western blotting for nitrocysteine-adducted protein. Two-way ANOVAs were performed with a Sidak’s multiple comparisons test, ∗p < 0.05, n = 6 per group. Main effects and interactions expressed in bars over the graph. All protein contents are expressed as arbitrary units and relative to C57 WT. Values are represented as mean ± SEM.