Journal: The Journal of Biological Chemistry

Article Title: Putative malignant hyperthermia mutation Ca V 1.1-R174W is insufficient to trigger a fulminant response to halothane or confer heat stress intolerance

doi: 10.1016/j.jbc.2023.104992

Figure Lengend Snippet: Ca V 1.1-R174W is not sufficient to confer intolerance to heat stress or halothane anesthesia. A , all mice were tested while restrained under elevated ambient temperature (38 ° C) as described in to assess heat stress intolerance until they triggered with fulminant MH or for a maximum time of 60 min. B , a separate cohort of mice were induced with 2% halothane at room temperature followed by maintenance anesthesia with 1.5% halothane on a bed maintained at 37 to 38 °C for up to 60 min or until they triggered with fulminant MH. RyR1-T4826I and RyR1-R163C MHS mice served as positive controls for heat stress and halothane intolerance assays, respectively. In panels A and B , core body temperatures were monitored (1 reading/2 min) for up to 60 min. In panel A , temperature was monitored for up to 60 min in restraint at 38 °C and an additional 15 min (1 reading/5 min) when mice were returned to the home cage at 23 °C. All data are presented as mean ± SD from data taken from the number of mice indicated in each group. MH, malignant hyperthermia; MHS, malignant hyperthermia susceptibility.

Article Snippet: Membranes were then blocked with Odyssey blocking buffer (LI-COR Biosciences) with 0.1% Tween-20 for 1 h at RT and incubated overnight at 4 °C with primary antibodies: monoclonal RyR1(34C) 1/500 to 1/1000 (DSHB, Cat# 34C, RRID: AB_528457), monoclonal dihydropyridine receptor (M3F11) 1/200 to 1/600 (DSHB, Cat# M3F11, RRID: AB_1157868, Reno, NA), monoclonal FKBP12 1/500 to 1/1000 (FKBP12_H-5, Santa Cruz Biotechnology), CSQ 1/10,000 (Abcam, Anti-Calsequestrin 1 [EPR15227 (B)] antibody ab191564); polynoclonal GAPDH 1/1000 to 1/5000 (Millipore, Cat.# ABS16); Rabbit β-Actin Polyclonal Antibody 1/1000 (Invitrogen REF PA1-16889) in blocking buffer and then washed with Tris- buffered saline with 0.1% Tween-20.

Techniques:

Journal: The Journal of Biological Chemistry

Article Title: Putative malignant hyperthermia mutation Ca V 1.1-R174W is insufficient to trigger a fulminant response to halothane or confer heat stress intolerance

doi: 10.1016/j.jbc.2023.104992

Figure Lengend Snippet: Western blot analysis of triadic protein expression among genotypes. The same protein preparations used for [ 3 H]PN200-110 and [ 3 H]Ry binding activity assessments as shown in the Figures 4 and were used in the Western blot analysis. A , results of densitometric analysis of skeletal muscle dissected from individual mice from each of the three genotypes probed for ( A ) RyR1, ( B ) Ca V 1.1, ( C ) calsequestrin (CSQ), or ( D ) calstabin (FKBP12). Representative Western blots used for densitometric analysis are shown as insets with GAPDH to normalize for protein loading. Densitometric values and their statistical analysis are shown in the lower panels . For each Western experiment, preparations from three genotypes were run on the same gel, blotted, and probed. A total of N = 7 WT, N = 5 HET and N = 7 (N = 7) mice were used for the analysis. HET and HOM signals were normalized to GAPDH or -actin, then to the WT signal on the same blot ( Fig. S1 ). The scatter plots are the mean value with lower and upper 95% CI; SEM, SD values, the adjusted p values, ANOVA summary F statistic and p values are included in the inset tables. GraphPad Prism 9.0 was used for graph plot and statistical analysis. One-way ANOVA, Tukey’s multiple comparisons test was applied in the analysis. CI, confidence interval; CSQ, calsequestrin; HET, heterozygous; HOM, homozygous.

Article Snippet: Membranes were then blocked with Odyssey blocking buffer (LI-COR Biosciences) with 0.1% Tween-20 for 1 h at RT and incubated overnight at 4 °C with primary antibodies: monoclonal RyR1(34C) 1/500 to 1/1000 (DSHB, Cat# 34C, RRID: AB_528457), monoclonal dihydropyridine receptor (M3F11) 1/200 to 1/600 (DSHB, Cat# M3F11, RRID: AB_1157868, Reno, NA), monoclonal FKBP12 1/500 to 1/1000 (FKBP12_H-5, Santa Cruz Biotechnology), CSQ 1/10,000 (Abcam, Anti-Calsequestrin 1 [EPR15227 (B)] antibody ab191564); polynoclonal GAPDH 1/1000 to 1/5000 (Millipore, Cat.# ABS16); Rabbit β-Actin Polyclonal Antibody 1/1000 (Invitrogen REF PA1-16889) in blocking buffer and then washed with Tris- buffered saline with 0.1% Tween-20.

Techniques: Western Blot, Expressing, Binding Assay, Activity Assay

Journal: International Journal of Molecular Sciences

Article Title: Calpain-3 Is Not a Sodium Dependent Protease and Simply Requires Calcium for Activation

doi: 10.3390/ijms24119405

Figure Lengend Snippet: Ryanodine receptor (RyR1) protein is not affected by CAPN3 autolysis following exposure to either non-physiologically high [Na + ] with zero [K + ] or 500 µM Ca 2+ . ( A ): Representative Western blots showing RyR1, CAPN1 (first probe) and CAPN3 (second probe) full-length and autolysed forms in present in W/F sets (three Sprague-Dawley rat EDL fibre segments per sample) as described in A or fibre segments immediately collected in denaturing solution (SDS). Samples were run alongside a calibration curve of known amounts (i.e., 8–60 µg) of rat EDL homogenate that were prepared using the Na + -based solution with no K + . Note that CAPN3 blot shows the previous CAPN1 signal and CAPN2 signal because CAPN3 was probed for after first probing for CAPN1, and the CAPN3 antibody also detects CAPN2 in rodent skeletal muscle (see A). Myosin seen on the post-transfer Coomassie-stained gel visually indicates the total amount of protein in each sample. A space indicates non-contiguous lanes. These experiments were repeated several times with similar results ( n = 48 fibres) from 5 rats. ( B ): Representative Western blots of human muscle samples treated with <10 nM (Control) or 500 µM Ca 2+ for 60 min, showing RyR1, CAPN3, Junctophilin-1 (JP1, see ) and titin N-terminal fragments in the cytosolic (Cyt) and pellet (Pel) fractions, alongside the unfractionated whole/Wh muscle sample (see description in and ). After CAPN3 was imaged, the membrane was stripped for 15 min at 37 °C, then incubated in the mouse anti-JP1 antibody. ( C ): Representative Western blot showing CAPN1 from the same experiments as ( B ). Myosin on the Stain Free gel visually indicates total amount of protein in each sample. A dashed line in B and C separates the Cyt and Pel samples from the matching Wh sample. A space is provided between adjacent Control Wh and Ca-treated Cyt lanes to visually separate Control and Ca 2+ -treated samples. Experiments repeated several times with similar results ( n = 4 human muscle samples). Mean amount of RyR1 ( D ) and titin fragments ( E ) in whole muscles samples prepared under Control and Ca 2+ -treated conditions, normalized to the mean of the Control samples. Note that each protein density value was expressed relative to the total protein density value in the sample from the UV Stain Free image and then normalized to Control. ( F ): Mean amount of ~75 kDa JP1 band in whole muscles samples expressed as a mean (+SD) percentage of total JP1 detected (i.e., sum of 75 and 90 kDa bands). ( G ): Distribution of JP1 in Cyt and Pel fractions, expressed as mean (+SD) percentage of the total JP1 in all fractions (i.e., Cyt + Pel = 100%) under Control (white bars) and Ca 2+ -treated (grey bars) conditions. Same coloured symbol represents same individual in all panels. p -values are provided above each graph ( n = 4 individuals, paired t -test used in ( D – F ), and two-way ANOVA used in ( G )).

Article Snippet: The primary antibodies used were: mouse anti-CAPN1 (C0355, Sigma-Aldrich), rabbit anti-CAPN2 (C3989, Sigma-Aldrich), mouse anti-CAPN3 (12A2, Novocastra), rabbit anti-SERCA2a (A010-20, Badrilla), mouse anti-GAPDH (Ab8245, Abcam), mouse anti-RyR1 (34C, Developmental Studies Hybridoma Bank), mouse anti-JSRP1 (NB120-2875 clone VF1c, Novus Biologicals) and mouse anti-titin N-terminal fragments (H00007273-M06, Abnova), which is designed to target the Z-disk region of titin [ ].

Techniques: Western Blot, Staining, Incubation

Journal: Life Science Alliance

Article Title: The role of Limch1 alternative splicing in skeletal muscle function

doi: 10.26508/lsa.202201868

Figure Lengend Snippet: Immunofluorescence staining of RYR1, FM 4–64 (T-tubule membrane), and BIN1 in Limch1 6exKO and WT age–matched control myofibers. Scale bar, 15 μM. WT, wild-type; HOM, homozygous; T-tubule, transverse tubule.

Article Snippet: Fixed myofibers were washed with PBS three times and permeabilized using 0.1% Triton X-100 (Sigma-Aldrich) in PBS for 10 min. Myofibers were then blocked in 5% FBS, 0.1% Triton X-100 in PBS at room temperature for 1 h and incubated overnight in 5% FBS and 0.1% Triton X-100 in PBS at 4°C with the following antibodies: anti-LIMCH1 (1:200, catalog number HPA063840; Sigma-Aldrich), anti-mLIMCH1 (1:200; Biomatik), anti-RYR1 (1:200, catalog number 34C; Developmental Studies Hybridoma Bank), and anti-BIN1 (1:200, catalog number 14647-1-AP; Proteintech).

Techniques: Immunofluorescence, Staining

Journal: International Journal of Molecular Sciences

Article Title: Searching for Mechanisms Underlying the Assembly of Calcium Entry Units: The Role of Temperature and pH

doi: 10.3390/ijms24065328

Figure Lengend Snippet: Sarcomeric localization of STIM1 and Orai1 before and after an ex vivo protocol performed at 30 °C and pH 7.4. Representative immunofluorescence images of EDL fibers showing RyR1 and STIM1 ( A , B ) and RyR1-Orai1 ( C , D ) double-staining. Each panel contains also a fluorescence intensity profile along three sarcomeres (see dashed line in ( A )) and the Pearson’s correlation coefficient value, i.e., a method of measuring the covariance of pixel intensities, given as the mean ± SEM. * p < 0.01, compared to fibers from control mice; n = number of images analyzed. Scale bar: 2.5 µm (insets: 1 µm).

Article Snippet: Primary antibodies used (a) mouse monoclonal anti-RyR1/RyR3 (34C antibody, 1:30, Developmental Studies Hybridoma Bank, IA, USA); (b) rabbit polyclonal anti-stromal-interacting molecule-1 (STIM1) (1:100, Sigma Aldrich, St. Louis, OH, USA); and (c) rabbit polyclonal anti-Orai1, (1:20, Thermo Scientific, Waltham, MA, USA).

Techniques: Ex Vivo, Immunofluorescence, Double Staining, Fluorescence

Journal: Oxidative Medicine and Cellular Longevity

Article Title: Estrogens Protect Calsequestrin-1 Knockout Mice from Lethal Hyperthermic Episodes by Reducing Oxidative Stress in Muscle

doi: 10.1155/2017/6936897

Figure Lengend Snippet: Assessment of muscle damage and blood levels of CK K + , and Ca 2+ following exposure to heat stress protocol. (a–h) Histological (a–d) and immunofluorescence of EDL fibers labeled with anti-RYR1 antibody (e–h) examination of EDL muscles after exposure to the heat stress protocol in male and female CASQ1-null mice, either untreated or treated with Premarin (males) and leuprolide (females). (i) Quantitative analysis of EDL fibers presenting structural damage and contractures. See also Table S4. (j–l) Blood levels of CK in serum (j), K + , and Ca 2+ in plasma (k and l) following heat stress protocol. Data are given as mean ± SEM; ∗ p < 0.05; n = number of mice. Scale bars in (a–e): 10 μ m (insets 5 μ m).

Article Snippet: Briefly, samples were first exposed to a mouse monoclonal primary antibody which recognizes both RYR1 and RYR3 (34C, 1 : 20; Developmental Studies Hybridoma Bank, University of Iowa) and then to a Cy3 goat anti-mouse IgG secondary antibody (Jackson ImmunoResearch Laboratories, West Grove, PA, USA).

Techniques: Immunofluorescence, Labeling

Journal: Oxidative Medicine and Cellular Longevity

Article Title: Estrogens Protect Calsequestrin-1 Knockout Mice from Lethal Hyperthermic Episodes by Reducing Oxidative Stress in Muscle

doi: 10.1155/2017/6936897

Figure Lengend Snippet: Assessment of muscle damage and blood levels of CK K + , and Ca 2+ following exposure to heat stress protocol. (a–h) Histological (a–d) and immunofluorescence of EDL fibers labeled with anti-RYR1 antibody (e–h) examination of EDL muscles after exposure to the heat stress protocol in male and female CASQ1-null mice, either untreated or treated with Premarin (males) and leuprolide (females). (i) Quantitative analysis of EDL fibers presenting structural damage and contractures. See also Table S4. (j–l) Blood levels of CK in serum (j), K + , and Ca 2+ in plasma (k and l) following heat stress protocol. Data are given as mean ± SEM; ∗ p < 0.05; n = number of mice. Scale bars in (a–e): 10 μ m (insets 5 μ m).

Article Snippet: Briefly, samples were first exposed to a mouse monoclonal primary antibody which recognizes both RYR1 and RYR3 (34C, 1 : 20; Developmental Studies Hybridoma Bank, University of Iowa) and then to a Cy3 goat anti-mouse IgG secondary antibody (Jackson ImmunoResearch Laboratories, West Grove, PA, USA).

Techniques: Immunofluorescence, Labeling

Journal: International Journal of Nanomedicine

Article Title: Evaluation of adipose-derived stem cells for tissue-engineered muscle repair construct-mediated repair of a murine model of volumetric muscle loss injury

doi: 10.2147/IJN.S101955

Figure Lengend Snippet: TEMR–ADSC construct-repaired LD muscles display structural and functional hallmarks reminiscent of native muscles. Notes: LD muscles repaired with TEMR–ADSC constructs were retrieved 2 months postimplantation and analyzed for morphology and new tissue formation by IHC staining on paraffin-embedded histological sections. IHC staining demonstrated positivity for structural proteins, myosin heavy chain (MF20; A ) and titin (9D10; B ). Similarly IHC staining revealed the presence of functional proteins, Junctophilin (Jp1; C ) and ryanodine receptor 1 (RyR1; D ). Abbreviations: TEMR, tissue-engineered muscle repair; ADSC, adipose-derived stem cell; LD, latissimus dorsi; IHC, immunohistochemistry.

Article Snippet: MF-20, titin, and RyR1 antibodies were acquired from Developmental Studies Hybridoma Bank, created by the National Institute of Child Health and Human Development of the National Institutes of Health and maintained at The University of Iowa, Department of Biology, Iowa City, IA, USA.

Techniques: Construct, Functional Assay, Immunohistochemistry, Derivative Assay