Structured Review

Developmental Studies Hybridoma Bank ryr3
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 <t>anti-RYR1</t> 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
Ryr3, supplied by Developmental Studies Hybridoma Bank, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/ryr3/product/Developmental Studies Hybridoma Bank
Average 93 stars, based on 1 article reviews
Price from $9.99 to $1999.99
ryr3 - by Bioz Stars, 2022-12
93/100 stars

Images

1) Product Images from "Estrogens Protect Calsequestrin-1 Knockout Mice from Lethal Hyperthermic Episodes by Reducing Oxidative Stress in Muscle"

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

Journal: Oxidative Medicine and Cellular Longevity

doi: 10.1155/2017/6936897

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
Figure Legend 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

Techniques Used: Immunofluorescence, Labeling, Mouse Assay

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    Developmental Studies Hybridoma Bank mouse monoclonal anti ryr1
    Generation of spega/b knockout zebrafish. (A) Schematic diagram showing the workflow of generating Speg single and double CRISPR-Cas9 mutants. gRNAs (against spega or spegb ) showing high editing efficiency [as determined by high-resolution melting (HRM)] were co-injected with Cas9 mRNA into one-cell-stage embryos <t>(AB</t> strain), and the resulting larvae were raised to adulthood. These F0 zebrafish were then outcrossed to WT AB, and resulting embryos screened for germline mutation by HRM and Sanger sequencing. Larvae that showed nucleotide changes leading to premature stop codons were raised to adulthood (F1), i.e. spega single KO ( spega Δ 10 ) or spegb single KO ( spegb Δ 17 ). To dilute off-target effects, F1 adults were outcrossed to AB WTs to generate F2 embryos and adults. Double KO lines (e.g. spega Δ 10;spegb Δ 17 ) were then generated by crossing spega F2 heterozygous (+/−) adults to spegb F2 heterozygous (+/−) adults. (B) Genotypes and predicted protein products (if any) of the single mutant lines. spega -V2046H_fsTer9: V2046 > H, frame shift and terminates nine amino acids downstream. spegb -S2210I_fsTer16: S2210 > I, frame shift and terminates 16 amino acids downstream. (C) RT-qPCR analysis shows significant reductions (∼50%) in both spega (black bars) and spegb (white bars) mRNA transcript levels in spega Δ 10;spegb Δ 17 . Unpaired two-tailed Student's t -test: * P
    Mouse Monoclonal Anti Ryr1, supplied by Developmental Studies Hybridoma Bank, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/mouse monoclonal anti ryr1/product/Developmental Studies Hybridoma Bank
    Average 93 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    mouse monoclonal anti ryr1 - by Bioz Stars, 2022-12
    93/100 stars
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    88
    Developmental Studies Hybridoma Bank ryr1
    Ca 2+ redistribution preserves force generation. ( A ) Ca 2+ content (per fiber volume) of the major subcellular compartments in WT, HET, and HOM <t>RYR1</t> KI mice and CSQ1 null mouse muscle, shown as cumulative histograms (data from Figs. 2 and 4 and fig. S6). ( B ) Averaged records of force responses to a single stimulation of whole EDL muscle from WT ( n = 4), HET ( n = 7), and HOM ( n = 4) RYR1 KI animals. ( C ) Twitch force responses generated using our mathematical model. Ca 2+ released in WT, HET, and HOM muscles was 300, 270, and 130 μM, respectively. The model underestimated the duration of relaxation in the HOM muscles (solid red trace); slower relaxation would occur if either the duration of the Ca 2+ transient was increased (dashed line) or the SR Ca 2+ pump was slower (dotted line). Note that all the modeled time courses are faster than those of the experimental records due to the absence of a series elastic component in the model, which slows twitch time course in muscles. ( D ) Modeled Ca 2+ transients underlying the force responses in (C). The Ca 2+ transient for HOM fibers is much smaller than those of the other genotypes, indicating impaired Ca 2+ release. The dashed and dotted lines correspond to the use of prolonged Ca 2+ release pulse and slower SR Ca 2+ pump, as in (C).
    Ryr1, supplied by Developmental Studies Hybridoma Bank, used in various techniques. Bioz Stars score: 88/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/ryr1/product/Developmental Studies Hybridoma Bank
    Average 88 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    ryr1 - by Bioz Stars, 2022-12
    88/100 stars
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    Image Search Results


    Generation of spega/b knockout zebrafish. (A) Schematic diagram showing the workflow of generating Speg single and double CRISPR-Cas9 mutants. gRNAs (against spega or spegb ) showing high editing efficiency [as determined by high-resolution melting (HRM)] were co-injected with Cas9 mRNA into one-cell-stage embryos (AB strain), and the resulting larvae were raised to adulthood. These F0 zebrafish were then outcrossed to WT AB, and resulting embryos screened for germline mutation by HRM and Sanger sequencing. Larvae that showed nucleotide changes leading to premature stop codons were raised to adulthood (F1), i.e. spega single KO ( spega Δ 10 ) or spegb single KO ( spegb Δ 17 ). To dilute off-target effects, F1 adults were outcrossed to AB WTs to generate F2 embryos and adults. Double KO lines (e.g. spega Δ 10;spegb Δ 17 ) were then generated by crossing spega F2 heterozygous (+/−) adults to spegb F2 heterozygous (+/−) adults. (B) Genotypes and predicted protein products (if any) of the single mutant lines. spega -V2046H_fsTer9: V2046 > H, frame shift and terminates nine amino acids downstream. spegb -S2210I_fsTer16: S2210 > I, frame shift and terminates 16 amino acids downstream. (C) RT-qPCR analysis shows significant reductions (∼50%) in both spega (black bars) and spegb (white bars) mRNA transcript levels in spega Δ 10;spegb Δ 17 . Unpaired two-tailed Student's t -test: * P

    Journal: Disease Models & Mechanisms

    Article Title: Characterization of a novel zebrafish model of SPEG-related centronuclear myopathy

    doi: 10.1242/dmm.049437

    Figure Lengend Snippet: Generation of spega/b knockout zebrafish. (A) Schematic diagram showing the workflow of generating Speg single and double CRISPR-Cas9 mutants. gRNAs (against spega or spegb ) showing high editing efficiency [as determined by high-resolution melting (HRM)] were co-injected with Cas9 mRNA into one-cell-stage embryos (AB strain), and the resulting larvae were raised to adulthood. These F0 zebrafish were then outcrossed to WT AB, and resulting embryos screened for germline mutation by HRM and Sanger sequencing. Larvae that showed nucleotide changes leading to premature stop codons were raised to adulthood (F1), i.e. spega single KO ( spega Δ 10 ) or spegb single KO ( spegb Δ 17 ). To dilute off-target effects, F1 adults were outcrossed to AB WTs to generate F2 embryos and adults. Double KO lines (e.g. spega Δ 10;spegb Δ 17 ) were then generated by crossing spega F2 heterozygous (+/−) adults to spegb F2 heterozygous (+/−) adults. (B) Genotypes and predicted protein products (if any) of the single mutant lines. spega -V2046H_fsTer9: V2046 > H, frame shift and terminates nine amino acids downstream. spegb -S2210I_fsTer16: S2210 > I, frame shift and terminates 16 amino acids downstream. (C) RT-qPCR analysis shows significant reductions (∼50%) in both spega (black bars) and spegb (white bars) mRNA transcript levels in spega Δ 10;spegb Δ 17 . Unpaired two-tailed Student's t -test: * P

    Article Snippet: The primary antibodies used were as follows: rabbit polyclonal anti-Speg (1:100; PA553875, Invitrogen), mouse monoclonal anti-RyR1 (1:100; 34C, DSHB) , rabbit polyclonal anti-CACNA1S (1:100; ab203662, Abcam; which labels the DHPR protein), mouse monoclonal anti-SERCA1a (1:200; ab2819, Abcam) , rabbit polyclonal anti-Desmin (1:100; D8281, Sigma-Aldrich) , rabbit polyclonal anti-Dnm2 (1:100; GTX127330, GeneTex) ( ) and mouse monoclonal anti-α-Actinin (1:100; A7811, Sigma-Aldrich) ( ).

    Techniques: Knock-Out, CRISPR, Injection, Mutagenesis, Sequencing, Generated, Quantitative RT-PCR, Two Tailed Test

    Desmin subcellular localization and protein levels in zebrafish models of CNM. (A-D) Myofibers were isolated at 5 dpf ( speg -DKO), 7 dpf ( mtm1 -KO) and 3 dpf ( DNM2 overexpression), and stained with anti-Desmin (green; D8281, Sigma-Aldrich) and DAPI (blue, nucleus). (A) At 5 dpf, Desmin is normally localized to the sarcolemma, perinucleus and the sarcomeric Z-disks (as transverse striations). (B) In speg -DKO (5 dpf), Desmin is predominantly localized to the perinucleus (orange arrowhead). (C) In mtm1 -KO (7 dpf), Desmin localization appears similar to that of WT siblings. (D,E) WT DNM2 -EGFP-overexpressing myofibers show similar Desmin staining pattern to that of non-transgenic WTs (D), while DNM2 -S619L DNM2 -EGFP-overexpressing myofibers show loss of Desmin in transverse striations, with Desmin localization predominantly at the perinucleus (orange arrowhead) (E). Scale bars: 10 µm. (F-H′) Western blot analysis using whole-zebrafish lysates shows Desmin upregulation in 5 dpf speg -DKO (by 2- to 3-fold) (F,F′) and in 7 dpf mtm1 -KO (by 5- to 10-fold) (G,G′) compared to WT siblings, but not in 3 dpf DNM2 -S619L zebrafish (H,H′) compared to DNM2 -WT controls. Each lane (F,G,H) or each dot (F′,G′,H′) represents n =25 zebrafish (40 µg of total proteins), four lanes represent four independent experiments. Densitometry was measured using Fiji ImageJ. Desmin protein levels were normalized to β-actin loading controls in speg -DKO and DNM2 , or to REVERT total protein stains in mtm1 -KO (as β-actin level is changed by the lack of Mtm1). Data are mean±s.e.m. Unpaired two-tailed Student's t -test: * P

    Journal: Disease Models & Mechanisms

    Article Title: Characterization of a novel zebrafish model of SPEG-related centronuclear myopathy

    doi: 10.1242/dmm.049437

    Figure Lengend Snippet: Desmin subcellular localization and protein levels in zebrafish models of CNM. (A-D) Myofibers were isolated at 5 dpf ( speg -DKO), 7 dpf ( mtm1 -KO) and 3 dpf ( DNM2 overexpression), and stained with anti-Desmin (green; D8281, Sigma-Aldrich) and DAPI (blue, nucleus). (A) At 5 dpf, Desmin is normally localized to the sarcolemma, perinucleus and the sarcomeric Z-disks (as transverse striations). (B) In speg -DKO (5 dpf), Desmin is predominantly localized to the perinucleus (orange arrowhead). (C) In mtm1 -KO (7 dpf), Desmin localization appears similar to that of WT siblings. (D,E) WT DNM2 -EGFP-overexpressing myofibers show similar Desmin staining pattern to that of non-transgenic WTs (D), while DNM2 -S619L DNM2 -EGFP-overexpressing myofibers show loss of Desmin in transverse striations, with Desmin localization predominantly at the perinucleus (orange arrowhead) (E). Scale bars: 10 µm. (F-H′) Western blot analysis using whole-zebrafish lysates shows Desmin upregulation in 5 dpf speg -DKO (by 2- to 3-fold) (F,F′) and in 7 dpf mtm1 -KO (by 5- to 10-fold) (G,G′) compared to WT siblings, but not in 3 dpf DNM2 -S619L zebrafish (H,H′) compared to DNM2 -WT controls. Each lane (F,G,H) or each dot (F′,G′,H′) represents n =25 zebrafish (40 µg of total proteins), four lanes represent four independent experiments. Densitometry was measured using Fiji ImageJ. Desmin protein levels were normalized to β-actin loading controls in speg -DKO and DNM2 , or to REVERT total protein stains in mtm1 -KO (as β-actin level is changed by the lack of Mtm1). Data are mean±s.e.m. Unpaired two-tailed Student's t -test: * P

    Article Snippet: The primary antibodies used were as follows: rabbit polyclonal anti-Speg (1:100; PA553875, Invitrogen), mouse monoclonal anti-RyR1 (1:100; 34C, DSHB) , rabbit polyclonal anti-CACNA1S (1:100; ab203662, Abcam; which labels the DHPR protein), mouse monoclonal anti-SERCA1a (1:200; ab2819, Abcam) , rabbit polyclonal anti-Desmin (1:100; D8281, Sigma-Aldrich) , rabbit polyclonal anti-Dnm2 (1:100; GTX127330, GeneTex) ( ) and mouse monoclonal anti-α-Actinin (1:100; A7811, Sigma-Aldrich) ( ).

    Techniques: Isolation, Over Expression, Staining, Transgenic Assay, Western Blot, Two Tailed Test

    Dnm2 protein is upregulated in both speg -DKO and mtm1 -KO zebrafish. (A-C) Myofibers were isolated at 5 dpf ( speg -DKO) and 7 dpf ( mtm1 -KO), and stained with anti-Dnm2 (GTX127330, GeneTex). (A) In WT myofibers (5 dpf), Dnm2 is localized to the triads (transverse striations). (B,C) Similar striated patterns can be observed for Dnm2 in speg -DKO (B) and mtm1 -KO (C), with occasional Dnm2 aggregations observed along the striations (yellow arrowheads). Scale bars: 10 µm. (D-E′) Western blot analysis shows increased Dnm2 protein levels in 5 dpf speg -DKO (D,D′) and 7 dpf mtm1 -KO (E,E′) compared to WT. Each lane (D,E) or each dot (D′,E′) represents n =25 zebrafish (40 µg total protein). Densitometry was accomplished using Fiji ImageJ. Dnm2 protein level was normalized to β-actin loading controls in speg -DKO, or to REVERT total protein stains in mtm1 -KO (as β-actin level is changed by the lack of Mtm1). Student's t -test: * P

    Journal: Disease Models & Mechanisms

    Article Title: Characterization of a novel zebrafish model of SPEG-related centronuclear myopathy

    doi: 10.1242/dmm.049437

    Figure Lengend Snippet: Dnm2 protein is upregulated in both speg -DKO and mtm1 -KO zebrafish. (A-C) Myofibers were isolated at 5 dpf ( speg -DKO) and 7 dpf ( mtm1 -KO), and stained with anti-Dnm2 (GTX127330, GeneTex). (A) In WT myofibers (5 dpf), Dnm2 is localized to the triads (transverse striations). (B,C) Similar striated patterns can be observed for Dnm2 in speg -DKO (B) and mtm1 -KO (C), with occasional Dnm2 aggregations observed along the striations (yellow arrowheads). Scale bars: 10 µm. (D-E′) Western blot analysis shows increased Dnm2 protein levels in 5 dpf speg -DKO (D,D′) and 7 dpf mtm1 -KO (E,E′) compared to WT. Each lane (D,E) or each dot (D′,E′) represents n =25 zebrafish (40 µg total protein). Densitometry was accomplished using Fiji ImageJ. Dnm2 protein level was normalized to β-actin loading controls in speg -DKO, or to REVERT total protein stains in mtm1 -KO (as β-actin level is changed by the lack of Mtm1). Student's t -test: * P

    Article Snippet: The primary antibodies used were as follows: rabbit polyclonal anti-Speg (1:100; PA553875, Invitrogen), mouse monoclonal anti-RyR1 (1:100; 34C, DSHB) , rabbit polyclonal anti-CACNA1S (1:100; ab203662, Abcam; which labels the DHPR protein), mouse monoclonal anti-SERCA1a (1:200; ab2819, Abcam) , rabbit polyclonal anti-Desmin (1:100; D8281, Sigma-Aldrich) , rabbit polyclonal anti-Dnm2 (1:100; GTX127330, GeneTex) ( ) and mouse monoclonal anti-α-Actinin (1:100; A7811, Sigma-Aldrich) ( ).

    Techniques: Isolation, Staining, Western Blot

    spega/b deficiency in zebrafish disrupts triad protein organization and triad ultrastructure, leading to reduced triad numbers. (A-D′) IF staining was performed on 2 dpf (A,B,C,D) and 5 dpf (A′,B′,C′,D′) isolated myofibers. Confocal images show disrupted transverse pattern of RyR1 (A,A′), DHPR (B,B′) and SERCA1a (D,D′) in speg -DKO starting from 2 dpf, while α-Actinin (D,D′) is not affected. Scale bars: 50 µm. (E-F′) Electron micrographs of 7 dpf WT and speg -DKO muscles. (E) In WT zebrafish skeletal muscle, normal triads are physically above the sarcomeric Z-disks and composed of centrally-located T-tubules flanked by terminal sarcoplasmic reticulum (yellow arrowhead). (E′) Triads in speg -DKO appear structurally disrupted, losing the obvious terminal cisternae of the sarcoplasmic membrane (tSR)/T-tubule/tSR pattern (red arrowhead). (F,F′) More importantly, the majority of sarcomeric Z-disks in speg -DKO do not have adjacent triads (yellow arrows). Scale bars: 0.5 µm (E,E′); 1 µm (F,F′). (G) The total number of triads per 60 µm 2 (under an electron microscope) was significantly reduced in speg -DKO. Each dot represents the average of technical triplicates, and three biological replicates are included. Data are mean±s.e.m. Unpaired two-tailed Student's t -test: * P

    Journal: Disease Models & Mechanisms

    Article Title: Characterization of a novel zebrafish model of SPEG-related centronuclear myopathy

    doi: 10.1242/dmm.049437

    Figure Lengend Snippet: spega/b deficiency in zebrafish disrupts triad protein organization and triad ultrastructure, leading to reduced triad numbers. (A-D′) IF staining was performed on 2 dpf (A,B,C,D) and 5 dpf (A′,B′,C′,D′) isolated myofibers. Confocal images show disrupted transverse pattern of RyR1 (A,A′), DHPR (B,B′) and SERCA1a (D,D′) in speg -DKO starting from 2 dpf, while α-Actinin (D,D′) is not affected. Scale bars: 50 µm. (E-F′) Electron micrographs of 7 dpf WT and speg -DKO muscles. (E) In WT zebrafish skeletal muscle, normal triads are physically above the sarcomeric Z-disks and composed of centrally-located T-tubules flanked by terminal sarcoplasmic reticulum (yellow arrowhead). (E′) Triads in speg -DKO appear structurally disrupted, losing the obvious terminal cisternae of the sarcoplasmic membrane (tSR)/T-tubule/tSR pattern (red arrowhead). (F,F′) More importantly, the majority of sarcomeric Z-disks in speg -DKO do not have adjacent triads (yellow arrows). Scale bars: 0.5 µm (E,E′); 1 µm (F,F′). (G) The total number of triads per 60 µm 2 (under an electron microscope) was significantly reduced in speg -DKO. Each dot represents the average of technical triplicates, and three biological replicates are included. Data are mean±s.e.m. Unpaired two-tailed Student's t -test: * P

    Article Snippet: The primary antibodies used were as follows: rabbit polyclonal anti-Speg (1:100; PA553875, Invitrogen), mouse monoclonal anti-RyR1 (1:100; 34C, DSHB) , rabbit polyclonal anti-CACNA1S (1:100; ab203662, Abcam; which labels the DHPR protein), mouse monoclonal anti-SERCA1a (1:200; ab2819, Abcam) , rabbit polyclonal anti-Desmin (1:100; D8281, Sigma-Aldrich) , rabbit polyclonal anti-Dnm2 (1:100; GTX127330, GeneTex) ( ) and mouse monoclonal anti-α-Actinin (1:100; A7811, Sigma-Aldrich) ( ).

    Techniques: Staining, Isolation, Microscopy, Two Tailed Test

    Schematic diagrams of excitation–contraction coupling (ECC) at the triad, and SPEG domains and pathogenic variants. (A) The triads are made of transverse tubules (T-tubules) flanked by terminal cisternae of the sarcoplasmic reticulum (SR). Junctional membranes (JM) are connected by the interaction of the ryanodine receptor (RyR1) at the SR, and dihydropyridine receptor (DHPR) at the T-tubules, forming the core components of the ECC machinery. ECC starts when a neuronal action potential arrives via T-tubules and causes a conformational change in DHPR, allowing it to interact with RyR1, leading to its activation. As a result, calcium (Ca 2+ ) leaves the SR via RyR1 channel opening and moves into the cytosol, promoting sarcomeric contraction. Finally, RyR1 is closed and the Ca 2+ transporter sarco/endoplasmic reticulum Ca 2+ -ATPase [SERCA; also known as ATP2A; regulated by phospholamban (PLN)] returns Ca 2+ into the SR, where it is largely bound to calsequestrin (C). The terminal SR also contains RyR1 modulators, such as junctophilin-1 (JPH1), FK506-binding protein 1A (F), junctin (J) and triadin (T). Striated muscle enriched protein kinase (SPEG) is localized at the triad, but its role remains elusive. (B) SPEG contains immunoglobulin domains (Ig; light purple shaded), fibronectin domains (Fn; light green shaded) and two kinase domains (yellow shaded). SPEG directly binds to myotubularin 1 (MTM1; dark green; SPEG 2530-2674 a.a.) ( Agrawal et al., 2014 ) and desmin (DES; magenta; SPEG 2200-2960 a.a.) ( Luo et al., 2020 ) at the inter-kinase domain. Pathogenic variants in SPEG span different regions of the gene and are typically nonsense mutations that result in decreased SPEG levels. The variants exist either in compound heterozygosity (e.g. K359Vfs*35; R1467*), or homozygosity (e.g. T544Dfs*48). SPEG mutations can cause a skeletal muscle disorder only (i.e. centronuclear myopathy or CNM, blue font), a cardiomyopathy only (e.g. dilated cardiomyopathy, red font) or both (black font), with no clear genotype–phenotype correlation. Note that patient Q2233* died before a cardiac evaluation. Illustrations were made using Illustrator for Biological Sciences (IBS) ( Liu et al., 2015 ).

    Journal: Disease Models & Mechanisms

    Article Title: Characterization of a novel zebrafish model of SPEG-related centronuclear myopathy

    doi: 10.1242/dmm.049437

    Figure Lengend Snippet: Schematic diagrams of excitation–contraction coupling (ECC) at the triad, and SPEG domains and pathogenic variants. (A) The triads are made of transverse tubules (T-tubules) flanked by terminal cisternae of the sarcoplasmic reticulum (SR). Junctional membranes (JM) are connected by the interaction of the ryanodine receptor (RyR1) at the SR, and dihydropyridine receptor (DHPR) at the T-tubules, forming the core components of the ECC machinery. ECC starts when a neuronal action potential arrives via T-tubules and causes a conformational change in DHPR, allowing it to interact with RyR1, leading to its activation. As a result, calcium (Ca 2+ ) leaves the SR via RyR1 channel opening and moves into the cytosol, promoting sarcomeric contraction. Finally, RyR1 is closed and the Ca 2+ transporter sarco/endoplasmic reticulum Ca 2+ -ATPase [SERCA; also known as ATP2A; regulated by phospholamban (PLN)] returns Ca 2+ into the SR, where it is largely bound to calsequestrin (C). The terminal SR also contains RyR1 modulators, such as junctophilin-1 (JPH1), FK506-binding protein 1A (F), junctin (J) and triadin (T). Striated muscle enriched protein kinase (SPEG) is localized at the triad, but its role remains elusive. (B) SPEG contains immunoglobulin domains (Ig; light purple shaded), fibronectin domains (Fn; light green shaded) and two kinase domains (yellow shaded). SPEG directly binds to myotubularin 1 (MTM1; dark green; SPEG 2530-2674 a.a.) ( Agrawal et al., 2014 ) and desmin (DES; magenta; SPEG 2200-2960 a.a.) ( Luo et al., 2020 ) at the inter-kinase domain. Pathogenic variants in SPEG span different regions of the gene and are typically nonsense mutations that result in decreased SPEG levels. The variants exist either in compound heterozygosity (e.g. K359Vfs*35; R1467*), or homozygosity (e.g. T544Dfs*48). SPEG mutations can cause a skeletal muscle disorder only (i.e. centronuclear myopathy or CNM, blue font), a cardiomyopathy only (e.g. dilated cardiomyopathy, red font) or both (black font), with no clear genotype–phenotype correlation. Note that patient Q2233* died before a cardiac evaluation. Illustrations were made using Illustrator for Biological Sciences (IBS) ( Liu et al., 2015 ).

    Article Snippet: The primary antibodies used were as follows: rabbit polyclonal anti-Speg (1:100; PA553875, Invitrogen), mouse monoclonal anti-RyR1 (1:100; 34C, DSHB) , rabbit polyclonal anti-CACNA1S (1:100; ab203662, Abcam; which labels the DHPR protein), mouse monoclonal anti-SERCA1a (1:200; ab2819, Abcam) , rabbit polyclonal anti-Desmin (1:100; D8281, Sigma-Aldrich) , rabbit polyclonal anti-Dnm2 (1:100; GTX127330, GeneTex) ( ) and mouse monoclonal anti-α-Actinin (1:100; A7811, Sigma-Aldrich) ( ).

    Techniques: Activation Assay, Binding Assay

    speg deficiency in zebrafish disrupts triad protein organization and triad ultrastructure, leading to reduced triad numbers. Immunofluorescence staining was performed on 2 dpf ( A-D ) and 5 dpf ( A’-D’ ) isolated myofibres. Average projections of confocal Z-stacks show disrupted transverse pattern of RyR1 ( A and A’ ), DHPR ( B and B’ ), and SERCA1a ( D ad D’ ) in speg -DKO starting from 2 dpf, while α-Actinin ( D and D’ ) is not affected. IF images - scale bars : 50 μm. ( E-F ) Electron micrographs of 7 dpf WT and speg -DKO muscles. ( E ) In WT zebrafish skeletal muscle, normal triads are physically above the sarcomeric Z-disks, and composed of centrally-located T-tubules flanked by terminal sarcoplasmic reticulum ( yellow arrows ). ( E’ ) Triads in speg -DKO appear structurally disrupted, losing the obvious tSR/T-tubule/tSR pattern ( red arrows ). More importantly, ( F ) the majority of sarcomeric Z-disks in speg -DKO do not have adjacent triads ( yellow arrows ). EM images – scale bars: (E) – 0.5 μm; (F) – 1 μm. ( G ) The total number of triads per 60 μm 2 (under electron microscopy) was significantly reduced in speg -DKO. Each dot represents the average of technical triplicates, and three biological replicates are included. Error bars are shown as Mean ± SEM. Two-tailed Student’s t -test: *, P

    Journal: bioRxiv

    Article Title: A novel zebrafish model of SPEG-related centronuclear myopathy (CNM): characterization and comparison with other CNM model zebrafish

    doi: 10.1101/2021.12.22.473918

    Figure Lengend Snippet: speg deficiency in zebrafish disrupts triad protein organization and triad ultrastructure, leading to reduced triad numbers. Immunofluorescence staining was performed on 2 dpf ( A-D ) and 5 dpf ( A’-D’ ) isolated myofibres. Average projections of confocal Z-stacks show disrupted transverse pattern of RyR1 ( A and A’ ), DHPR ( B and B’ ), and SERCA1a ( D ad D’ ) in speg -DKO starting from 2 dpf, while α-Actinin ( D and D’ ) is not affected. IF images - scale bars : 50 μm. ( E-F ) Electron micrographs of 7 dpf WT and speg -DKO muscles. ( E ) In WT zebrafish skeletal muscle, normal triads are physically above the sarcomeric Z-disks, and composed of centrally-located T-tubules flanked by terminal sarcoplasmic reticulum ( yellow arrows ). ( E’ ) Triads in speg -DKO appear structurally disrupted, losing the obvious tSR/T-tubule/tSR pattern ( red arrows ). More importantly, ( F ) the majority of sarcomeric Z-disks in speg -DKO do not have adjacent triads ( yellow arrows ). EM images – scale bars: (E) – 0.5 μm; (F) – 1 μm. ( G ) The total number of triads per 60 μm 2 (under electron microscopy) was significantly reduced in speg -DKO. Each dot represents the average of technical triplicates, and three biological replicates are included. Error bars are shown as Mean ± SEM. Two-tailed Student’s t -test: *, P

    Article Snippet: The primary antibodies used were: rabbit polyclonal anti-SPEG (1:100; PA553875, Invitrogen), mouse monoclonal anti-RyR1 (1:100; 34C, DSHB), rabbit polyclonal anti-CACNA1S (1:100; ab203662, Abcam; which labels the DHPR protein), mouse monoclonal anti-SERCA1a (1:200; ab2819; Abcam), rabbit polyclonal anti-DES (1:100; D8281, Sigma), rabbit polyclonal anti-DNM2 (1:100; GTX127330; GeneTex), and mouse monoclonal anti-α-actinin (1:100; A7811; Sigma).

    Techniques: Immunofluorescence, Staining, Isolation, Electron Microscopy, Two Tailed Test

    Schematic diagrams of (A) excitation-contraction coupling (ECC) at the triad and (B) SPEG domains and pathogenic variants. ( A ) The triads are made of transverse tubules (T-tubules) flanked by terminal sarcoplasmic reticulum (tSR). Junctional membranes (JM) are connected by the interaction of the ryanodine receptor (RyR1) at the tSR, and dihydropyridine receptor (DHPR) at the T-tubules, forming the core components of the ECC machinery. ECC starts when a neuronal action potential arrives via T-tubules and causes a conformational change in DHPR, allowing it to interact with RyR1, causing its activation. As a result, calcium (Ca 2+ ) leaves the SR via RyR1 channel opening and moves into the cytosol, promoting sarcomeric contraction. Finally, RyR1 is closed and the calcium transporter SERCA (sarco/endoplasmic reticulum Ca2+-ATPase, regulated by PLN or phospholamban) returns calcium into the SR, where it is largely bound to calsequestrin (C). The terminal SR also contains RyR1 modulators, such as junctophilin-1 (JPH1), FK506-binding protein 1A (F), junctin (J); and triadin (T). SPEG (striated muscle enriched protein kinase) is localized at the triad, but its role remains elusive. ( B ) SPEG contains immunoglobulin domains (Ig, light purple shaded ), fibronectin domains (Fn, light green shaded ), and two kinase domains ( yellow shaded ). SPEG directly binds to Myotubularin 1 (MTM1, dark green ; SPEG 2530∼2674 a.a.) (Agrawal et al., 2014), and Desmin (DES, magenta ; SPEG 2200∼2960 a.a.) (Luo et al., 2021) at the inter-kinase domain. Pathogenic variants in SPEG span different regions of the gene and are typically nonsense mutations that result in decreased SPEG levels. The variants exist either in compound heterozygosity (e.g. K359Vfs*35; R1467*), or homozygosity (e.g. T544Nfs*48). SPEG mutations can cause a skeletal muscle disorder only (i.e. centronuclear myopathy or CNM, blue fonts ), a cardiomyopathy only (e.g. dilated cardiomyopathy, red fonts ), or both ( black fonts ), with no clear genotype-phenotype correlation. Illustrations were made using Illustrator for Biological Sciences (IBS) ( Liu et al., 2015 ).

    Journal: bioRxiv

    Article Title: A novel zebrafish model of SPEG-related centronuclear myopathy (CNM): characterization and comparison with other CNM model zebrafish

    doi: 10.1101/2021.12.22.473918

    Figure Lengend Snippet: Schematic diagrams of (A) excitation-contraction coupling (ECC) at the triad and (B) SPEG domains and pathogenic variants. ( A ) The triads are made of transverse tubules (T-tubules) flanked by terminal sarcoplasmic reticulum (tSR). Junctional membranes (JM) are connected by the interaction of the ryanodine receptor (RyR1) at the tSR, and dihydropyridine receptor (DHPR) at the T-tubules, forming the core components of the ECC machinery. ECC starts when a neuronal action potential arrives via T-tubules and causes a conformational change in DHPR, allowing it to interact with RyR1, causing its activation. As a result, calcium (Ca 2+ ) leaves the SR via RyR1 channel opening and moves into the cytosol, promoting sarcomeric contraction. Finally, RyR1 is closed and the calcium transporter SERCA (sarco/endoplasmic reticulum Ca2+-ATPase, regulated by PLN or phospholamban) returns calcium into the SR, where it is largely bound to calsequestrin (C). The terminal SR also contains RyR1 modulators, such as junctophilin-1 (JPH1), FK506-binding protein 1A (F), junctin (J); and triadin (T). SPEG (striated muscle enriched protein kinase) is localized at the triad, but its role remains elusive. ( B ) SPEG contains immunoglobulin domains (Ig, light purple shaded ), fibronectin domains (Fn, light green shaded ), and two kinase domains ( yellow shaded ). SPEG directly binds to Myotubularin 1 (MTM1, dark green ; SPEG 2530∼2674 a.a.) (Agrawal et al., 2014), and Desmin (DES, magenta ; SPEG 2200∼2960 a.a.) (Luo et al., 2021) at the inter-kinase domain. Pathogenic variants in SPEG span different regions of the gene and are typically nonsense mutations that result in decreased SPEG levels. The variants exist either in compound heterozygosity (e.g. K359Vfs*35; R1467*), or homozygosity (e.g. T544Nfs*48). SPEG mutations can cause a skeletal muscle disorder only (i.e. centronuclear myopathy or CNM, blue fonts ), a cardiomyopathy only (e.g. dilated cardiomyopathy, red fonts ), or both ( black fonts ), with no clear genotype-phenotype correlation. Illustrations were made using Illustrator for Biological Sciences (IBS) ( Liu et al., 2015 ).

    Article Snippet: The primary antibodies used were: rabbit polyclonal anti-SPEG (1:100; PA553875, Invitrogen), mouse monoclonal anti-RyR1 (1:100; 34C, DSHB), rabbit polyclonal anti-CACNA1S (1:100; ab203662, Abcam; which labels the DHPR protein), mouse monoclonal anti-SERCA1a (1:200; ab2819; Abcam), rabbit polyclonal anti-DES (1:100; D8281, Sigma), rabbit polyclonal anti-DNM2 (1:100; GTX127330; GeneTex), and mouse monoclonal anti-α-actinin (1:100; A7811; Sigma).

    Techniques: Activation Assay, Binding Assay

    Immunofluorescence and EM analysis of EDL fibers from aged-control mice. (A,B) Representative immunofluorescence images obtained from aged mice, double-labeled for RYR1 (red) and STIM1 (green) in panel (A) and RYR1 (red) and ORAI1 in panel (B) . Raw images for the individual fluorescence channel used to construct these overlays are shown in Supplementary Figure 1 . (C,D) Representative EM images of longitudinal (C) and transversal (D) sections with TAs false-labeled in green (arrows point to TTs stained with ferrocyanide) and TTs stained with ferrocyanide (dark precipitate). Black arrows in panel (C,D) point to TTs within the interior of the TA. Inset in panel (D) small bridges, pointed by small arrows, are visible between membranes of adjacent cross-sectioned tubes. Scale bars: (A,B) , 5 μm (insets 2 μm); (C,D) , 1 μm (inset 0.1 μm).

    Journal: Frontiers in Physiology

    Article Title: Long-Term Exercise Reduces Formation of Tubular Aggregates and Promotes Maintenance of Ca2+ Entry Units in Aged Muscle

    doi: 10.3389/fphys.2020.601057

    Figure Lengend Snippet: Immunofluorescence and EM analysis of EDL fibers from aged-control mice. (A,B) Representative immunofluorescence images obtained from aged mice, double-labeled for RYR1 (red) and STIM1 (green) in panel (A) and RYR1 (red) and ORAI1 in panel (B) . Raw images for the individual fluorescence channel used to construct these overlays are shown in Supplementary Figure 1 . (C,D) Representative EM images of longitudinal (C) and transversal (D) sections with TAs false-labeled in green (arrows point to TTs stained with ferrocyanide) and TTs stained with ferrocyanide (dark precipitate). Black arrows in panel (C,D) point to TTs within the interior of the TA. Inset in panel (D) small bridges, pointed by small arrows, are visible between membranes of adjacent cross-sectioned tubes. Scale bars: (A,B) , 5 μm (insets 2 μm); (C,D) , 1 μm (inset 0.1 μm).

    Article Snippet: Small bundles of fixed EDL fibers were washed three times in PBS containing 1% [PBS/bovine serum albumin (BSA)] and incubated in blocking solution (PBS/BSA with 10% goat serum and 0.5% Triton X-100) for 1 h at RT, followed by an overnight incubation at 4°C with one of the following primary antibodies: (a) mouse monoclonal anti-RYR1 (34C antibody, 1:30, Developmental Studies Hybridoma Bank University of Iowa, Iowa City, Iowa); (b) rabbit polyclonal anti-STIM1 (1:100, Sigma–Aldrich, Milan, Italy); and (c) rabbit polyclonal anti-ORAI1 (1:20, Thermo Fisher Scientific, Waltham, MA, United States).

    Techniques: Immunofluorescence, Mouse Assay, Labeling, Fluorescence, Construct, Staining

    Ca 2+ redistribution preserves force generation. ( A ) Ca 2+ content (per fiber volume) of the major subcellular compartments in WT, HET, and HOM RYR1 KI mice and CSQ1 null mouse muscle, shown as cumulative histograms (data from Figs. 2 and 4 and fig. S6). ( B ) Averaged records of force responses to a single stimulation of whole EDL muscle from WT ( n = 4), HET ( n = 7), and HOM ( n = 4) RYR1 KI animals. ( C ) Twitch force responses generated using our mathematical model. Ca 2+ released in WT, HET, and HOM muscles was 300, 270, and 130 μM, respectively. The model underestimated the duration of relaxation in the HOM muscles (solid red trace); slower relaxation would occur if either the duration of the Ca 2+ transient was increased (dashed line) or the SR Ca 2+ pump was slower (dotted line). Note that all the modeled time courses are faster than those of the experimental records due to the absence of a series elastic component in the model, which slows twitch time course in muscles. ( D ) Modeled Ca 2+ transients underlying the force responses in (C). The Ca 2+ transient for HOM fibers is much smaller than those of the other genotypes, indicating impaired Ca 2+ release. The dashed and dotted lines correspond to the use of prolonged Ca 2+ release pulse and slower SR Ca 2+ pump, as in (C).

    Journal: Science Advances

    Article Title: Ryanodine receptor leak triggers fiber Ca2+ redistribution to preserve force and elevate basal metabolism in skeletal muscle

    doi: 10.1126/sciadv.abi7166

    Figure Lengend Snippet: Ca 2+ redistribution preserves force generation. ( A ) Ca 2+ content (per fiber volume) of the major subcellular compartments in WT, HET, and HOM RYR1 KI mice and CSQ1 null mouse muscle, shown as cumulative histograms (data from Figs. 2 and 4 and fig. S6). ( B ) Averaged records of force responses to a single stimulation of whole EDL muscle from WT ( n = 4), HET ( n = 7), and HOM ( n = 4) RYR1 KI animals. ( C ) Twitch force responses generated using our mathematical model. Ca 2+ released in WT, HET, and HOM muscles was 300, 270, and 130 μM, respectively. The model underestimated the duration of relaxation in the HOM muscles (solid red trace); slower relaxation would occur if either the duration of the Ca 2+ transient was increased (dashed line) or the SR Ca 2+ pump was slower (dotted line). Note that all the modeled time courses are faster than those of the experimental records due to the absence of a series elastic component in the model, which slows twitch time course in muscles. ( D ) Modeled Ca 2+ transients underlying the force responses in (C). The Ca 2+ transient for HOM fibers is much smaller than those of the other genotypes, indicating impaired Ca 2+ release. The dashed and dotted lines correspond to the use of prolonged Ca 2+ release pulse and slower SR Ca 2+ pump, as in (C).

    Article Snippet: The primary antibodies used and the assigned dilutions were RyR1 [mouse, 1:100; Developmental Studies Hybridoma Bank (DSHB), IA, USA, 34C], DHPR (mouse, 1:400; DSHB, IIID5EI), PMCA (mouse, 1:1000; Abcam, Cambridge, UK, ab2825), optic atrophy 1 (OPA1; mouse, 1:1000; BD Biosciences, San Jose, CA, USA, #612607), MFN2 (rabbit, 1:2000), dynamic-related protein 1 (DRP1; mouse, 1:1000; Cell Signaling Technology, MA, USA, #8570), oxidative phosphorylation (OXPHOS; mouse, 1:1000; Abcam, ab110411), MiD49 (rabbit, 1:500), actin (rabbit, 1:500; Sigma Aldrich, #A2066), NADH:Ubiquinone Oxidoreductase Subunit A9 (NDUFA9; rabbit, 1:1000), and cytochrome c oxidase subunit IV (COXIV; rabbit, 1:1000; Cell Signaling Technology, #4844), as described previously ( ).

    Techniques: Mouse Assay, Generated

    Increased [Ca 2+ ] cyto raises mitochondrial Ca 2+ content. ( A ) Resting [Ca 2+ ] mito versus [Ca 2+ ] cyto in RYR1 KI muscle. ( B ) Mitochondrial Ca 2+ content (expressed per fiber volume) in muscle from male and female RYR1 KI mice and CSQ1 null mice. Genotypes were compared using a pooled one-way ANOVA with Tukey’s multiple comparisons ( P

    Journal: Science Advances

    Article Title: Ryanodine receptor leak triggers fiber Ca2+ redistribution to preserve force and elevate basal metabolism in skeletal muscle

    doi: 10.1126/sciadv.abi7166

    Figure Lengend Snippet: Increased [Ca 2+ ] cyto raises mitochondrial Ca 2+ content. ( A ) Resting [Ca 2+ ] mito versus [Ca 2+ ] cyto in RYR1 KI muscle. ( B ) Mitochondrial Ca 2+ content (expressed per fiber volume) in muscle from male and female RYR1 KI mice and CSQ1 null mice. Genotypes were compared using a pooled one-way ANOVA with Tukey’s multiple comparisons ( P

    Article Snippet: The primary antibodies used and the assigned dilutions were RyR1 [mouse, 1:100; Developmental Studies Hybridoma Bank (DSHB), IA, USA, 34C], DHPR (mouse, 1:400; DSHB, IIID5EI), PMCA (mouse, 1:1000; Abcam, Cambridge, UK, ab2825), optic atrophy 1 (OPA1; mouse, 1:1000; BD Biosciences, San Jose, CA, USA, #612607), MFN2 (rabbit, 1:2000), dynamic-related protein 1 (DRP1; mouse, 1:1000; Cell Signaling Technology, MA, USA, #8570), oxidative phosphorylation (OXPHOS; mouse, 1:1000; Abcam, ab110411), MiD49 (rabbit, 1:500), actin (rabbit, 1:500; Sigma Aldrich, #A2066), NADH:Ubiquinone Oxidoreductase Subunit A9 (NDUFA9; rabbit, 1:1000), and cytochrome c oxidase subunit IV (COXIV; rabbit, 1:1000; Cell Signaling Technology, #4844), as described previously ( ).

    Techniques: Mouse Assay

    RyR1 Ca 2+ leak decreases SR Ca 2+ content. ( A ) Endogenous and non-SR (noncaffeine releasable) Ca 2+ content of male (M) and female (F) RYR1 KI and CSQ1 null fibers. ( B ) Maximum Ca 2+ content of male (M) and female (F) RYR1 KI and CSQ1 null fibers. ( C ) Endogenous SR Ca 2+ content versus RyR1 Ca 2+ leak in male and female RYR1 KI and CSQ1 null fibers. Data from all graphs were compared using a pooled one-way ANOVA with Tukey’s multiple comparisons test to compare genotype. In (A), symbols denote # = 3 significant comparisons and θ = 2 significant comparisons. Significant differences between genotypes in each panel for endogenous and non–SR Ca 2+ were found between WT and both HOM and CSQ1 null, HET and both HOM and CSQ1 null, as well as between HOM and CSQ1 null (all P

    Journal: Science Advances

    Article Title: Ryanodine receptor leak triggers fiber Ca2+ redistribution to preserve force and elevate basal metabolism in skeletal muscle

    doi: 10.1126/sciadv.abi7166

    Figure Lengend Snippet: RyR1 Ca 2+ leak decreases SR Ca 2+ content. ( A ) Endogenous and non-SR (noncaffeine releasable) Ca 2+ content of male (M) and female (F) RYR1 KI and CSQ1 null fibers. ( B ) Maximum Ca 2+ content of male (M) and female (F) RYR1 KI and CSQ1 null fibers. ( C ) Endogenous SR Ca 2+ content versus RyR1 Ca 2+ leak in male and female RYR1 KI and CSQ1 null fibers. Data from all graphs were compared using a pooled one-way ANOVA with Tukey’s multiple comparisons test to compare genotype. In (A), symbols denote # = 3 significant comparisons and θ = 2 significant comparisons. Significant differences between genotypes in each panel for endogenous and non–SR Ca 2+ were found between WT and both HOM and CSQ1 null, HET and both HOM and CSQ1 null, as well as between HOM and CSQ1 null (all P

    Article Snippet: The primary antibodies used and the assigned dilutions were RyR1 [mouse, 1:100; Developmental Studies Hybridoma Bank (DSHB), IA, USA, 34C], DHPR (mouse, 1:400; DSHB, IIID5EI), PMCA (mouse, 1:1000; Abcam, Cambridge, UK, ab2825), optic atrophy 1 (OPA1; mouse, 1:1000; BD Biosciences, San Jose, CA, USA, #612607), MFN2 (rabbit, 1:2000), dynamic-related protein 1 (DRP1; mouse, 1:1000; Cell Signaling Technology, MA, USA, #8570), oxidative phosphorylation (OXPHOS; mouse, 1:1000; Abcam, ab110411), MiD49 (rabbit, 1:500), actin (rabbit, 1:500; Sigma Aldrich, #A2066), NADH:Ubiquinone Oxidoreductase Subunit A9 (NDUFA9; rabbit, 1:1000), and cytochrome c oxidase subunit IV (COXIV; rabbit, 1:1000; Cell Signaling Technology, #4844), as described previously ( ).

    Techniques:

    RyR1 Ca 2+ leak increases with RYR1 mutations and absence of CSQ1. ( A ) Schematic depicting the detection of RyR1 Ca 2+ leak using rhod-5N trapped in the sealed t-system. The presence of functional RyR1 (+RyR1) allows Ca 2+ to leak into the JS and increase [Ca 2+ ] JS above that of the bulk cytoplasm. [Ca 2+ ] JS directly influences PMCA activity and [Ca 2+ ] t-sys . When RyR1 leak is blocked with Tet (−RyR1), [Ca 2+ ] t-sys drops. ( B ) [Ca 2+ ] t-sys ( t ) in a WT fiber and ( C ) in an HOM fiber during SOCE activation in caffeine, followed by caffeine washout and addition of standard solution with 200 nM [Ca 2+ ] cyto in the absence, then presence, of Tet. ( D ) Steady-state [Ca 2+ ] t-sys versus [Ca 2+ ] cyto in all genotypes. Intersection of dotted line and curves fitted to [Ca 2+ ] t-sys indicates the [Ca 2+ ] cyto where physiological [Ca 2+ ] t-sys is reached. ( E ) Example of the determination of Δ[Ca 2+ ] t-sys in +RyR1 and −RyR1 conditions in the presence of 200 nM [Ca 2+ ] cyto (HOM fiber). The difference between the histograms (Δ[Ca 2+ ] t-sys ) is RyR1 Ca 2+ leak (text on panel). ( F ) RyR1 Ca 2+ leak in each genotype. ( G to J ) RyR1 Ca 2+ leak in genotypes by sex. Data are presented as means ± SEM and fitted by Hill curves. Statistics and n value details are in Materials and Methods. Two-way analysis of variance (ANOVA) [in (F)]: # = 4 significant results and Ψ = 3 significant results. Unpaired t test [in (G to J)]: * P

    Journal: Science Advances

    Article Title: Ryanodine receptor leak triggers fiber Ca2+ redistribution to preserve force and elevate basal metabolism in skeletal muscle

    doi: 10.1126/sciadv.abi7166

    Figure Lengend Snippet: RyR1 Ca 2+ leak increases with RYR1 mutations and absence of CSQ1. ( A ) Schematic depicting the detection of RyR1 Ca 2+ leak using rhod-5N trapped in the sealed t-system. The presence of functional RyR1 (+RyR1) allows Ca 2+ to leak into the JS and increase [Ca 2+ ] JS above that of the bulk cytoplasm. [Ca 2+ ] JS directly influences PMCA activity and [Ca 2+ ] t-sys . When RyR1 leak is blocked with Tet (−RyR1), [Ca 2+ ] t-sys drops. ( B ) [Ca 2+ ] t-sys ( t ) in a WT fiber and ( C ) in an HOM fiber during SOCE activation in caffeine, followed by caffeine washout and addition of standard solution with 200 nM [Ca 2+ ] cyto in the absence, then presence, of Tet. ( D ) Steady-state [Ca 2+ ] t-sys versus [Ca 2+ ] cyto in all genotypes. Intersection of dotted line and curves fitted to [Ca 2+ ] t-sys indicates the [Ca 2+ ] cyto where physiological [Ca 2+ ] t-sys is reached. ( E ) Example of the determination of Δ[Ca 2+ ] t-sys in +RyR1 and −RyR1 conditions in the presence of 200 nM [Ca 2+ ] cyto (HOM fiber). The difference between the histograms (Δ[Ca 2+ ] t-sys ) is RyR1 Ca 2+ leak (text on panel). ( F ) RyR1 Ca 2+ leak in each genotype. ( G to J ) RyR1 Ca 2+ leak in genotypes by sex. Data are presented as means ± SEM and fitted by Hill curves. Statistics and n value details are in Materials and Methods. Two-way analysis of variance (ANOVA) [in (F)]: # = 4 significant results and Ψ = 3 significant results. Unpaired t test [in (G to J)]: * P

    Article Snippet: The primary antibodies used and the assigned dilutions were RyR1 [mouse, 1:100; Developmental Studies Hybridoma Bank (DSHB), IA, USA, 34C], DHPR (mouse, 1:400; DSHB, IIID5EI), PMCA (mouse, 1:1000; Abcam, Cambridge, UK, ab2825), optic atrophy 1 (OPA1; mouse, 1:1000; BD Biosciences, San Jose, CA, USA, #612607), MFN2 (rabbit, 1:2000), dynamic-related protein 1 (DRP1; mouse, 1:1000; Cell Signaling Technology, MA, USA, #8570), oxidative phosphorylation (OXPHOS; mouse, 1:1000; Abcam, ab110411), MiD49 (rabbit, 1:500), actin (rabbit, 1:500; Sigma Aldrich, #A2066), NADH:Ubiquinone Oxidoreductase Subunit A9 (NDUFA9; rabbit, 1:1000), and cytochrome c oxidase subunit IV (COXIV; rabbit, 1:1000; Cell Signaling Technology, #4844), as described previously ( ).

    Techniques: Functional Assay, Activity Assay, Activation Assay

    T-system Ca 2+ permeability is dependent on chronic RyR1 Ca 2+ leak. ( A ) [Ca 2+ ] t-sys ( t ) in a WT fiber during activation of SOCE via caffeine-dependent SR Ca 2+ depletion, followed by establishment of steady-state [Ca 2+ ] t-sys , in 200 nM [Ca 2+ ] cyto (black line). T-system Ca 2+ flux is overlaid in red, and peak t-system Ca 2+ extrusion flux is indicated by an arrow. ( B ) [Ca 2+ ] t-sys ( t ) and t-system Ca 2+ flux in a CSQ1 null fiber using the same experimental protocol as in (A). Note the different scale of the right-hand y axes in (A) and (B). SOCE and t-system Ca 2+ uptake are mu ch more rapid events in CSQ1 null fibers than in WT. ( C ) Summary of peak t-system Ca 2+ uptake rate in WT, HET, and HOM RYR1 KI mice and CSQ1 null mouse fibers. ( D ) Bmax of peak t-system Ca 2+ uptake plotted against the RyR1 Ca 2+ leak determined for each genotype (from Fig. 1F ). A two-way ANOVA with Tukey’s multiple comparisons was used to determine significant differences in peak t-system Ca 2+ uptake flux by both genotype [ F (3) = 122.06, P

    Journal: Science Advances

    Article Title: Ryanodine receptor leak triggers fiber Ca2+ redistribution to preserve force and elevate basal metabolism in skeletal muscle

    doi: 10.1126/sciadv.abi7166

    Figure Lengend Snippet: T-system Ca 2+ permeability is dependent on chronic RyR1 Ca 2+ leak. ( A ) [Ca 2+ ] t-sys ( t ) in a WT fiber during activation of SOCE via caffeine-dependent SR Ca 2+ depletion, followed by establishment of steady-state [Ca 2+ ] t-sys , in 200 nM [Ca 2+ ] cyto (black line). T-system Ca 2+ flux is overlaid in red, and peak t-system Ca 2+ extrusion flux is indicated by an arrow. ( B ) [Ca 2+ ] t-sys ( t ) and t-system Ca 2+ flux in a CSQ1 null fiber using the same experimental protocol as in (A). Note the different scale of the right-hand y axes in (A) and (B). SOCE and t-system Ca 2+ uptake are mu ch more rapid events in CSQ1 null fibers than in WT. ( C ) Summary of peak t-system Ca 2+ uptake rate in WT, HET, and HOM RYR1 KI mice and CSQ1 null mouse fibers. ( D ) Bmax of peak t-system Ca 2+ uptake plotted against the RyR1 Ca 2+ leak determined for each genotype (from Fig. 1F ). A two-way ANOVA with Tukey’s multiple comparisons was used to determine significant differences in peak t-system Ca 2+ uptake flux by both genotype [ F (3) = 122.06, P

    Article Snippet: The primary antibodies used and the assigned dilutions were RyR1 [mouse, 1:100; Developmental Studies Hybridoma Bank (DSHB), IA, USA, 34C], DHPR (mouse, 1:400; DSHB, IIID5EI), PMCA (mouse, 1:1000; Abcam, Cambridge, UK, ab2825), optic atrophy 1 (OPA1; mouse, 1:1000; BD Biosciences, San Jose, CA, USA, #612607), MFN2 (rabbit, 1:2000), dynamic-related protein 1 (DRP1; mouse, 1:1000; Cell Signaling Technology, MA, USA, #8570), oxidative phosphorylation (OXPHOS; mouse, 1:1000; Abcam, ab110411), MiD49 (rabbit, 1:500), actin (rabbit, 1:500; Sigma Aldrich, #A2066), NADH:Ubiquinone Oxidoreductase Subunit A9 (NDUFA9; rabbit, 1:1000), and cytochrome c oxidase subunit IV (COXIV; rabbit, 1:1000; Cell Signaling Technology, #4844), as described previously ( ).

    Techniques: Permeability, Activation Assay, Mouse Assay

    Mitochondrial morphology changes in RYR1 KI mouse muscle. Ca 2+ content. ( A , A′ , B , and B′ ) Electron micrographs of muscle from WT and HET, respectively, show normal sarcomeric and triad structure. ( C and C′ ) Less defined triads and circular morphology of mitochondria in HOM. Images in (A′), (B′), and (C′) represent higher magnification images of areas in (A), (B), and (C) indicated by asterisk. Normal triad structures are denoted by parentheses in (A′) and (B′). Scale bars, 2 μm (A to C), 500 nm (A′ to C′). Mitochondrial ultrastructural parameters in WT, HET, and HOM were as follows: ( D ) Volume [as a % of cytoplasm, V(v)]. Relative volume to WT (in %) indicated above column. Colored dots are from male (blue) and female (red) mice, and column shows average value. ( E ) Surface area (μm 2 ) calculated by measuring surface-to-volume ratio (Sv) multiplied by volume in (D). Relative surface area to WT (in %) indicated above column. ( F ) Mitochondrial diameter (nm; means ± SD, one-way ANOVA). Blue and red circles represent individual measurements from male and female mice. Statistical analysis was conducted across genotypes. **** P

    Journal: Science Advances

    Article Title: Ryanodine receptor leak triggers fiber Ca2+ redistribution to preserve force and elevate basal metabolism in skeletal muscle

    doi: 10.1126/sciadv.abi7166

    Figure Lengend Snippet: Mitochondrial morphology changes in RYR1 KI mouse muscle. Ca 2+ content. ( A , A′ , B , and B′ ) Electron micrographs of muscle from WT and HET, respectively, show normal sarcomeric and triad structure. ( C and C′ ) Less defined triads and circular morphology of mitochondria in HOM. Images in (A′), (B′), and (C′) represent higher magnification images of areas in (A), (B), and (C) indicated by asterisk. Normal triad structures are denoted by parentheses in (A′) and (B′). Scale bars, 2 μm (A to C), 500 nm (A′ to C′). Mitochondrial ultrastructural parameters in WT, HET, and HOM were as follows: ( D ) Volume [as a % of cytoplasm, V(v)]. Relative volume to WT (in %) indicated above column. Colored dots are from male (blue) and female (red) mice, and column shows average value. ( E ) Surface area (μm 2 ) calculated by measuring surface-to-volume ratio (Sv) multiplied by volume in (D). Relative surface area to WT (in %) indicated above column. ( F ) Mitochondrial diameter (nm; means ± SD, one-way ANOVA). Blue and red circles represent individual measurements from male and female mice. Statistical analysis was conducted across genotypes. **** P

    Article Snippet: The primary antibodies used and the assigned dilutions were RyR1 [mouse, 1:100; Developmental Studies Hybridoma Bank (DSHB), IA, USA, 34C], DHPR (mouse, 1:400; DSHB, IIID5EI), PMCA (mouse, 1:1000; Abcam, Cambridge, UK, ab2825), optic atrophy 1 (OPA1; mouse, 1:1000; BD Biosciences, San Jose, CA, USA, #612607), MFN2 (rabbit, 1:2000), dynamic-related protein 1 (DRP1; mouse, 1:1000; Cell Signaling Technology, MA, USA, #8570), oxidative phosphorylation (OXPHOS; mouse, 1:1000; Abcam, ab110411), MiD49 (rabbit, 1:500), actin (rabbit, 1:500; Sigma Aldrich, #A2066), NADH:Ubiquinone Oxidoreductase Subunit A9 (NDUFA9; rabbit, 1:1000), and cytochrome c oxidase subunit IV (COXIV; rabbit, 1:1000; Cell Signaling Technology, #4844), as described previously ( ).

    Techniques: Mouse Assay