1. Biochemistry and Chemical Biology
Download icon

Myopalladin knockout mice develop cardiac dilation and show a maladaptive response to mechanical pressure overload

  1. Maria Carmela Filomena
  2. Daniel L Yamamoto
  3. Pierluigi Carullo
  4. Roman Medvedev
  5. Andrea Ghisleni
  6. Nicoletta Piroddi
  7. Beatrice Scellini
  8. Roberta Crispino
  9. Francesca D'Autilia
  10. Jianlin Zhang
  11. Arianna Felicetta
  12. Simona Nemska
  13. Simone Serio
  14. Chiara Tesi
  15. Daniele Catalucci
  16. Wolfgang A Linke
  17. Roman Polishchuk
  18. Corrado Poggesi
  19. Mathias Gautel
  20. Marie-Louise Bang  Is a corresponding author
  1. Institute of Genetic and Biomedical Research (IRGB) - National Research Council (CNR), Milan unit, Italy
  2. IRCCS Humanitas Research Hospital, Italy
  3. Department of Cardiac Surgery, University of Verona, Italy
  4. Randall Centre for Cell and Molecular Biophysics, King's College London BHF Centre of Research Excellence, United Kingdom
  5. Department of Experimental and Clinical Medicine, University of Florence, Italy
  6. Telethon Institute of Genetics and Medicine (TIGEM), Italy
  7. Department of Medicine, University of California, San Diego, United States
  8. Humanitas University, Italy
  9. Institute of Physiology II, University of Muenster, Germany
Research Article
Cite this article as: eLife 2021;10:e58313 doi: 10.7554/eLife.58313
10 figures, 2 tables and 2 additional files

Figures

Myopalladin (MYPN) directly binds to titin in the Z-line.

(A) Yeast two-hybrid (Y2H) assay showing binding of the C-terminal region of MYPN and its homologue palladin (PALLD) to the titin Ig domains Z4-Z5 (titin Ig Z4-Z5) in the Z-line. A culture plate with different combinations of Y2H co-transformations is shown as indicated in the table. (B) Microscale thermophoresis (MST) analysis of 450 nM labeled titin Ig Z4-Z5 incubated with increasing concentrations of MYPN and PALLD C-terminal domains. Due to precipitation, signal saturation was not reached. The dose/response curve is representative of two independent experiments (n = 17 and n = 16 runs). Fnorm, normalized fluorescence. (C) MST analysis of 400 nM labeled MYPN C-terminal domain incubated with increasing concentrations of titin Ig Z4-Z5. The dose/response curve is representative of four independent experiments (n = 7 runs).

Figure 1—source data 1

Microscale thermophoresis binding (MST) analysis of myopalladin (MYPN)/titin interaction.

https://cdn.elifesciences.org/articles/58313/elife-58313-fig1-data1-v2.xlsx
Figure 2 with 1 supplement
Histological and electron microscopy analyses of hearts from wild-type (WT) and myopalladin knockout (MKO) mice.

(A) Hematoxylin and eosin (H&E) (top) and Picro Sirius Red (bottom) stainings showing no morphological abnormalities or fibrosis in the heart of 6-month-old MKO male mice compared to WT mice (n = 3 per group). (B) Histograms and Gaussian distribution curves showing similar adult cardiomyocyte (CMC) length in 4-month-old WT and MKO male mice (n = 1292 CMCs from three WT mice and 1740 CMCs from three MKO mice). (C) Average CMC length in WT and MKO male mice. Data are represented as mean ± standard error of the mean (SEM). (D) Histograms and Gaussian distribution curves showing reduced adult CMC width in 4-month-old MKO male mice compared to WT mice (n = 1373 CMCs fromthree WT mice and 1193 CMCs from three MKO mice). (E) Average CMC width in WT and MKO male mice. Data are represented as mean ± SEM. ***p < 0.001; unpaired Student’s t-test. (F) Body weight to tibia length ratio (BW/TL) of 10-week-old WT and MKO male mice. Data are represented as mean ± SEM (n = 12–13 per group). ***p < 0.001; unpaired Student’s t-test. (G–H) Electron micrographs of papillary muscle from 8-month-old WT and MKO male mice, showing sarcomere (G) and intercalated disc (ICD) (H) structure. (I) Average ICD fold amplitude in WT and MKO mice. Data are represented as mean ± SEM (n = 11 ICDs from WT mice and 14 ICDs from MKO mice). *p < 0.05; unpaired Student’s t-test.

Figure 2—source data 1

Cardiomyocyte (CMC) size measurements in wild-type (WT) and myopalladin knockout (MKO) male mice.

https://cdn.elifesciences.org/articles/58313/elife-58313-fig2-data1-v2.xlsx
Figure 2—source data 2

Body weight to tibia length ratio (BW/TL) measurements in wild-type (WT) and myopalladin knockout (MKO) male mice.

https://cdn.elifesciences.org/articles/58313/elife-58313-fig2-data2-v2.xlsx
Figure 2—source data 3

Intercalated disc (ICD) fold amplitude measurements in wild-type (WT) and myopalladin knockout (MKO) male mice.

https://cdn.elifesciences.org/articles/58313/elife-58313-fig2-data3-v2.xlsx
Figure 2—figure supplement 1
Cardiomyocyte (CMC) size in 4-month-old wild-type (WT) and myopalladin knockout (MKO) male mice.

(A) Histograms and Gaussian distribution curves showing reduced adult CMC size in MKO mice compared to WT mice (n = 1274 CMCs from 3 WT mice and 1587 CMCs from 4 MKO mice). (B) Average CMC size in WT and MKO mice. Data are represented as mean ± SEM. ***p < 0.001; Student’s t-test.

Figure 3 with 1 supplement
Echocardiographic analyses and sarcomere-length-tension relationship in cardiac myofibrils from wild-type (WT) and myopalladin knockout (MKO) mice.

(A–H) Echocardiographic analysis of WT and MKO male mice at 10 weeks (10 W), 4 months (4 M), and 6 months (6 M) of age. LVID, left ventricular inner diameter; FS, fractional shortening; EF, ejection fraction; LVPW, left ventricular posterior wall thickness; IVS, interventricular septum thickness; RWT, relative wall thickness ((LVPWd + IVSd)/LVIDd); CO, cardiac output; d, diastole; s, systole. Data are represented as mean ± standard error of the mean (SEM) (n = 16–42 per group). *p < 0.05; **p < 0.01; ***p < 0.001; two-way analysis of variance (ANOVA) with Bonferroni’s multiple comparison test. (I) Representative echocardiographic short-axis M-mode images from hearts of 6-month-old WT and MKO male mice. (J–L) Body weight (BW) (J), heart weight (HW) (K), and heart weight to body weight ratio (HW/BW) (L) of WT and MKO male mice at 10 W, 10 M, and 6 M of age. Data are represented as mean ± SEM (n = 12–24 per group). *p < 0.05; **p < 0.01; ***p < 0.001; two-way ANOVA with Bonferroni’s multiple comparison test. (M) Ratio of tension measured at pCa 5.75 (P5.75, submaximal activation) vs. pCa 4.50 (P4.50, maximal activation) in WT and MKO myofibrils using Ca2+ jump protocols. Data are represented as mean ± SEM (n = 10 myofibrils from two WT mice and 12 myofibrils from two MKO mice). (N) Average sarcomere-length-tension relationship in cardiac myofibrils from the left ventricle of 4-month-old WT and MKO male mice. Each data point is represented as mean ± SEM from 10 myofibrils from three WT mice and 16 myofibrils from three MKO mice. *p < 0.05; **p < 0.01; ***p < 0.001; unpaired Student’s t-test. (O–S) Densitometric analysis for (O) titin (TTN) N2BA/N2B isoform ratio as determined by sodium dodecyl sulfate (SDS)-PAGE and Coomassie blue staining, (P) titin serine/threonine phosphorylation as determined by Western blot analysis using anti-phosphoserine/threonine antibody, and (Q–R) site-specific titin phosphorylation on Ser3991 (corresponding to human pTTN-Ser4010, phosphorylated by PKA and ERK2) (Q), Ser4080 (corresponding to human pTTN-Ser4099, phosphorylated by PKG) (R), and Ser12742 (corresponding to human pTTN-Ser11878, phosphorylated by PKCα) (S) using titin phosphosite-specific antibodies. Normalization was performed to total protein content as determined by Coomassie blue staining of each blot. Data are represented as mean ± SEM (n = 6 per group). *p < 0.05; unpaired Student’s t-test.

Figure 3—source data 1

Echocardiographic parameters of wild-type (WT) and myopalladin knockout (MKO) male mice at different ages.

https://cdn.elifesciences.org/articles/58313/elife-58313-fig3-data1-v2.docx
Figure 3—source data 2

Echocardiographic analyses and heart weight to body weight ratio (HW/BW) measurements on wild-type (WT) and myopalladin knockout (MKO) male mice under basal conditions.

https://cdn.elifesciences.org/articles/58313/elife-58313-fig3-data2-v2.xlsx
Figure 3—source data 3

Heart weight to body weight (HW/BW) measurements on wild-type (WT) and myopalladin knockout (MKO) male mice under basal conditions.

https://cdn.elifesciences.org/articles/58313/elife-58313-fig3-data3-v2.xlsx
Figure 3—source data 4

Sarcomere-length-tension relationship and calcium jump experiments in cardiac myofibrils from wild-type (WT) and myopalladin knockout (MKO) male mice.

https://cdn.elifesciences.org/articles/58313/elife-58313-fig3-data4-v2.xlsx
Figure 3—source data 5

Densitometry of titin blots.

https://cdn.elifesciences.org/articles/58313/elife-58313-fig3-data5-v2.xlsx
Figure 3—figure supplement 1
Titin isoform expression and phosphorylation in the left ventricle of 4-month-old wild-type (WT) and myopalladin knockout (MKO) male mice.

Titin (TTN) isoform expression was determined by SDS-PAGE and Coomassie blue staining. The TTN N2B isoform is the principal isoform in mouse heart. Titin phosphorylation was determined by Western blot analysis using anti-phosphoserine/threonine antibody (pTTN-Ser/Thr) and titin phosphosite-specific antibodies against Ser3991, Ser4080, and Ser12742. Total protein was determined by Coomassie blue staining of each blot.

Echocardiographic analyses of wild-type (WT) and myopalladin knockout (MKO) mice following transaortic constriction (TAC).

(A–H) Echocardiography analyses of WT and MKO male mice under basal conditions and 1, 2, and 4 weeks after TAC. Pressure gradient >70 mmHg. LVID, left ventricular inner diameter; FS, fractional shortening; EF, ejection fraction; LVPW, left ventricular posterior wall thickness; IVS, interventricular septum thickness; RWT, relative wall thickness ((LVPWd + IVSd)/LVIDd); CO, cardiac output; d, diastole; s, systole. Data are represented as mean ± standard error of the mean (SEM) (n = 8–19 per group). **p < 0.01; ***p < 0.001; linear mixed model (LMM) with Bonferroni’s multiple comparison test. (I) Representative echocardiographic short-axis M-mode images from hearts of WT and MKO male mice under basal conditions and 1 and 4 weeks after TAC. (J–L) Body weight (BW) (J), heart weight (HW) (K), and heart weight to body weight ratio (HW/BW) (L) of WT and MKO male mice 4 weeks after TAC or SHAM surgery. Data are represented as mean ± SEM (n = 9–11 per group). **p < 0.01; ***p < 0.001; two-way analysis of variance (ANOVA) with Bonferroni’s multiple comparison test.

Figure 4—source data 1

Echocardiographic parameters of 8-week-old wild-type (WT) and myopalladin knockout (MKO) male mice before and after transaortic constriction (TAC).

https://cdn.elifesciences.org/articles/58313/elife-58313-fig4-data1-v2.docx
Figure 4—source data 2

Echocardiographic analysis on wild-type (WT) and myopalladin knockout (MKO) male mice subjected to transaortic constriction (TAC) or SHAM.

https://cdn.elifesciences.org/articles/58313/elife-58313-fig4-data2-v2.xlsx
Figure 4—source data 3

Measurements of heart weight to body weight ratio (HW/BW) in wild-type (WT) and myopalladin knockout (MKO) male mice subjected to transaortic constriction (TAC) or SHAM.

https://cdn.elifesciences.org/articles/58313/elife-58313-fig4-data3-v2.xlsx
Histological and transmission electron microscopy analyses of hearts from wild-type (WT) and myopalladin knockout (MKO) male mice 4 weeks after transaortic constriction (TAC) or SHAM.

(A) Top, Hematoxylin and eosin (H&E) stainings of hearts from WT and MKO mice subjected to TAC or SHAM. Middle, Representative Picro Sirius Red stainings of the left ventricle, showing fibrosis in MKO mice after TAC. Bottom, Representative wheat germ agglutinin (WGA) stainings of the left ventricle (red). Nuclei are visualized by DAPI (blue). (B) Percent area of interstitial fibrosis in the left ventricle. Data are represented as mean ± standard error of the mean (SEM) (n = 3 per group). **p < 0.01; ***p < 0.001; two-way analysis of variance (ANOVA) with Bonferroni’s multiple comparison test. (C) Gaussian distribution curves from histograms of cardiomyocyte (CMC) size in the left ventricle of WT and MKO mice subjected to TAC or SHAM (n = 333 CMCs from three WT mice and 809 CMCs from three MKO mice subjected to SHAM; 423 CMCs from three WT mice and 665 CMCs from three MKO mice subjected to TAC). (D) Average CMC size in WT and MKO mice subjected to TAC or SHAM. Data are represented as mean ± SEM. **p < 0.01; ***p < 0.001; two-way ANOVA with Bonferroni’s multiple comparison test. (E–F) Electron micrographs of papillary muscle from WT and MKO mice 4 weeks after TAC, showing sarcomere (E) and intercalated disc (ICD) (F) structure. (G) Average ICD fold amplitude in WT and MKO mice. Data are represented as mean ± SEM (n = 11 ICDs from WT mice and n = 14 ICDs from MKO mice, n = 22 ICDs from WT mice, and n = 18 ICDs from MKO mice subjected to TAC). ***p < 0.001; two-way ANOVA with Bonferroni’s multiple comparison test.

Figure 5—source data 1

Measurements of fibrotic area in the left ventricle of wild-type (WT) and myopalladin knockout (MKO) male mice 4 weeks after transaortic constriction (TAC) or SHAM.

https://cdn.elifesciences.org/articles/58313/elife-58313-fig5-data1-v2.xlsx
Figure 5—source data 2

Cardiomyocyte (CMC) size measurements in wild-type (WT) and myopalladin knockout (MKO) male mice 4 weeks after transaortic constriction (TAC) or SHAM.

https://cdn.elifesciences.org/articles/58313/elife-58313-fig5-data2-v2.xlsx
Figure 5—source data 3

Intercalated disc (ICD) fold amplitude measurements in wild-type (WT) and myopalladin knockout (MKO) male mice 4 weeks after transaortic constriction (TAC).

https://cdn.elifesciences.org/articles/58313/elife-58313-fig5-data3-v2.xlsx
Figure 6 with 1 supplement
Quantitative real-time PCR (qRT-PCR) and Western blot analyses on wild-type (WT) and myopalladin knockout (MKO) mice at basal conditions and following transaortic constriction (TAC).

(A) qRT-PCR analysis for Mypn, Palld, and markers of cardiac remodeling on left ventricular RNA from WT and MKO male mice at basal conditions and 4 days (D) and 4 weeks (W) after TAC. B2m was used for normalization. Data are represented as mean ± standard error of the mean (SEM) (n = 3–8 per group performed in triplicate). *p < 0.05, **p < 0.01, **p < 0.001; two-way analysis of variance (ANOVA) with Bonferroni’s multiple comparison test. (B) Western blot analysis on left ventricular lysate from WT and MKO male mice at basal conditions and 4 days (D) and 4 weeks (W) after TAC. Representative blots are shown (n = 3–4 per group). (C) Densitometric analysis for proteins that were significantly altered in MKO mice. Normalization was performed to total protein content as assessed on TGX Stain-Free gels (Bio-Rad Laboratories). Data are represented as mean ± SEM (n = 3–4 per group). *p < 0.05, **p < 0.01, ***p < 0.001; two-way ANOVA with Bonferroni’s multiple comparison test.

Figure 6—source data 1

Quantitative real-time PCR (qRT-PCR) and densitometry analysis on wild-type (WT) and myopalladin knockout (MKO) male mice subjected to transaortic constriction (TAC) or SHAM.

https://cdn.elifesciences.org/articles/58313/elife-58313-fig6-data1-v2.xlsx
Figure 6—figure supplement 1
Immunofluorescence stainings of left ventricular tissue from the heart of 4-month-old wild-type (WT) and myopalladin knockout (MKO) male mice for palladin (PALLD) and myopalladin (MYPN)-interacting proteins.

Nuclei are visualized by DAPI (blue).

Functional analyses on adult cardiomyocytes (CMCs) from wild-type (WT) and myopalladin knockout (MKO) male mice 7 days after transaortic constriction (TAC) or SHAM surgery.

(A–C) Sarcomere shortening, time to 90% peak, and time to 50% relaxation (n = 62 cells from nine WT mice and 71 cells from seven MKO mice). (D–F) Amplitude of Ca2+ transient, time to 90% peak of Ca2+ transient, and time to 50% decay of Ca2+ transient (n = 29 cells from eight WT mice and 16 cells from six MKO mice). (G) Representative Ca2+ spark images from WT and MKO male mice subjected to TAC or SHAM for 7 days. (H–M) Ca2+ spark frequency, amplitude, velocity of Ca2+ release (derivative; dF/dt), time to peak, full width at half maximum (FWHM), and tau (n = 355 sparks from 33 cells from three WT SHAM mice, 403 sparks from 27 cells from three MKO SHAM mice, 462 sparks from 31 cells from three WT TAC mice, and 429 sparks from 33 cells from three MKO TAC mice). Data are represented as mean ± standard error of the mean (SEM) as determined by hierarchical analysis (Sikkel et al., 2017). *p < 0.05, **p < 0.01, ***p < 0.001; two- or three-level hierarchical testing with Bonferroni correction, as appropriate.

Figure 7—source data 1

Analysis of sarcomere shortening, Ca2+ transients, and Ca2+ sparks in cardiomyocytes (CMCs) from wild-type (WT) and myopalladin knockout (MKO) male mice subjected to transaortic constriction (TAC) or SHAM.

https://cdn.elifesciences.org/articles/58313/elife-58313-fig7-data1-v2.xlsx
Overview figure illustrating the phenotype of myopalladin knockout (MKO) mice under basal conditions and following transaortic constriction (TAC).

M, month; LV, left ventricle; SL, sarcomere length, ICD, intercalated disc.

Author response image 1
Author response image 2

Tables

Table 1
Tension generation and relaxation in ventricular myofibrils from wild-type (WT) and myopalladin knockout (MKO) male mice.
Myofibril batchResting conditionsTension generationRelaxation
Slow phaseFast phase
SL(µm)RT(mN/mm2)P0(mN/mm2)kACT(s–1)Duration(ms)kREL(s–1)kREL(s–1)
WT2.22 ± 0.02 (26)11.1 ± 1.4 (26)161 ± 11 (26)5.17 ± 0.29 (23)74 ± 4 (20)1.96 ± 0.18 (20)47 ± 4 (23)
MKO2.21 ± 0.01 (27)10.3 ± 1.3 (26)112 ± 6***(27)5.38 ± 0.40 (26)72 ± 3 (22)2.03 ± 0.15 (19)30 ± 4 (24)
  1. All values are presented as mean ± standard error of the mean (SEM). Numbers in parentheses are number of myofibrils. SL, sarcomere length, RT, resting tension, P0, maximum isometric tension; kACT, rate constant of tension rise following step-wise pCa decrease (8.0→4.5) by fast solution switching. Full tension relaxation kinetics were characterized by the duration and rate constant of tension decay of the isometric slow relaxation phase (slow kREL) and the rate constant of the fast relaxation phase (fast kREL). ***p < 0.001 vs. WT; unpaired Student’s t-test.

Appendix 1—key resources table
Reagent type (species) or resourceDesignationSource or referenceIdentifiersAdditional information
Strain, strain background (male Mus musculus)MYPN knockout (MKO) mice (in C57BL/6J background)Filomena et al., 2020N/A
Strain, strain background (Mus musculus)C57BL/6JThe Jackson LaboratoriesCat# 000664RRID:IMSR_JAX:000664
Strain, strain background (Escherichia coli)DH5α electrocompetent cellsNew England BioLabsCat# C2989K
Strain, strain background (Escherichia coli)BL21-CodonPlus (DE3)-RIPL strainAgilent TechnologiesCat# 230280
Strain, strain background (Saccharomyces cerevisiae)Y2H Gold yeast strainTakara BioCat# 630498
Strain, strain background (Saccharomyces cerevisiae)L40 yeast strainTakara BioN/A
AntibodyAnti-MYPN (rabbit polyclonal)Yamamoto et al., 2013N/A WB (1:1000)
AntibodyAnti-PALLD 621 (rabbit polyclonal)Pogue-Geile et al., 2006Kindly provided by Prof. Carol Otey, University of North Carolina, Chapel Hill, NC, USA WB (1:500) IF (1:30)
AntibodyAnti-NEBL (rabbit polyclonal)Mastrototaro et al., 2015N/A WB (1:500)
AntibodyAnti-ANKRD1/CARP (rabbit polyclonal)Miller et al., 2003N/A WB (1:200) IF (1:20)
AntibodyAnti-Cypher (rabbit polyclonal)Zhou et al., 2001Kindly provided by Prof. Ju Chen, University of California San Diego, La Jolla, CA, USA WB (1:500) IF (1:50)
AntibodyAnti-α-actinin (mouse monoclonal)Merck Life ScienceCat# A7811RRID:AB_476766 WB (1:50000) IF (1:250)
AntibodyAnti-desmin (rabbit polyclonal)AbcamCat# Ab8592RRID:AB_306653 IF (1:80)
AntibodyAnti-MKL1/MRTF-A (rabbit polyclonal)Merck Life ScienceCat# AV37504RRID:AB_1853972 IF (1:30)
AntibodyAnti-MKL1/MRTF-A (rabbit polyclonal)Immunological SciencesCat# AB-84312RRID:AB_2892156 WB (1:500)
AntibodyAnti-desmoplakin 1/2 (mouse monoclonal)Bio-Rad LaboratoriesCat# 2722–5204RRID:AB_619950 WB (1:750)
AntibodyAnti-α-E-catenin (mouse monoclonal)Santa Cruz BiotechnologyCat# sc-9988RRID:AB_626805 WB (1:1000)
AntibodyAnti-β-catenin (rabbit monoclonal)Cell Signaling TechnologyCat# 8480RRID:AB_11127855 WB (1:1000)
AntibodyAnti-γ-catenin (rabbit polyclonal)Immunological SciencesAB-90215RRID:AB_2892157 WB (1:1000)
AntibodyAnti-vinculin (mouse monoclonal)Merck Life ScienceCat# V9264RRID:AB_10603627 WB (1:2000)
AntibodyAnti-N-cadherin (mouse monoclonal)Cell Signaling TechnologyCat# 4061RRID:AB_10694647 WB (1:1000)
AntibodyAnti-SORBS2 (mouse monoclonal)Merck Life ScienceCat# SAB4200183RRID:AB_10638778 WB (1:750)
AntibodyAnti-connexin 43 (mouse monoclonal)Thermo Fisher ScientificCat# 35–5000RRID:AB_87322 WB (1:400)
AntibodyAnti-pAkt-Ser473 (rabbit monoclonal)Immunological SciencesMAB-94111RRID:AB_2892158 WB (1:500)
AntibodyAnti-Akt (rabbit polyclonal)Cell Signaling TechnologyCat# 9272RRID:AB_329827 WB (1:1000)
AntibodyAnti-pErk1/2-Thr202/Tyr204 (rabbit polyclonal)Cell Signaling TechnologyCat# 4370RRID:AB_2315112 WB (1:500)
AntibodyAnti-Erk1/2 (mouse monoclonal)Santa Cruz BiotechnologyCat# sc-514302RRID:AB_2571739 WB (1:1000)
AntibodyAnti-pP38-Tyr182 (mouse monoclonal)Santa Cruz BiotechnologyCat# sc-166182RRID:AB_2141746 WB (1:500)
AntibodyAnti-P38α/β (mouse monoclonal)Santa Cruz BiotechnologyCat# sc-7972RRID:AB_628079 WB (1:500)
AntibodyAnti-calcineurin B (rabbit polyclonal)Novus BiochemicalCat# NBP1-32720RRID:AB_2168483 WB (1:500)
AntibodyAnti-calsequestrin - (mouse monoclonal)BD Trasduction LabsCat# C16420 WB (1:5000)
AntibodyAnti-SERCA2A (mouse monoclonal)Affinity Bio Reagent (ABR)Cat# ma3-919RRID:AB_325502 WB (1:1000)
AntibodyAnti-pPLN-Thr16 (rabbit polyclonal)BadrillaCat# 010–12RRID:AB_2617047 WB (1:500)
AntibodyAnti-PLN (mouse monoclonal)Novus BiochemicalCat# NB300-582RRID:AB_10000946 WB (1:500)
AntibodyAnti-RyR2 (mouse monoclonal)Affinity Bio Reagent (ABR)Cat# ma3-916RRID:AB_2183054 WB (1:1000)
AntibodyAnti-pRyR-Ser2808 (rabbit polyclonal)BadrillaCat# A010-30APRRID:AB_2617052 WB (1:500)
AntibodyAnti-sodium calcium exchanger (NCX) (mouse monoclonal)Merck Life ScienceCat# N216RRID:AB_260750 WB (1:1000)
AntibodyAnti-CACNA1C (rabbit polyclonal)AbcamCat# Ab58552RRID:AB_879800 WB (1:500)
AntibodyAnti-CACNA1S (mouse monoclonal)AbcamCat# Ab2862RRID:AB_2069567 WB (1:500)
AntibodyAnti-phosphoserine/ threonine (rabbit polyclonal)ECM BiosciencesCat# PP2551RRID:AB_1184778 WB (1:500)
AntibodyAnti-pTTN-Ser3991 (rabbit polyclonal)Kötter et al., 2013N/A WB (1:500)
AntibodyAnti-pTTN-Ser4080 (rabbit polyclonal)Kötter et al., 2013N/A WB (1:500)
AntibodyAnti-pTTN-Ser12742 (rabbit polyclonal)Kötter et al., 2013N/A WB (1:500)
AntibodyAnti-actin (goat polyclonal)Santa Cruz BiotechnologyCat# sc-1615RRID:AB_630835 WB (1:10000)
AntibodyGoat anti-mouse IgG (H + L) secondary antibody Alexa Fluor 488-conjugated IgGThermo Fisher ScientificCat# A11029RRID:AB_138404 IF (1:500)
AntibodyGoat anti-rabbit IgG (H + L) secondary antibody Alexa Fluor 488-conjugated IgGThermo Fisher ScientificCat# A11034RRID:AB_2576217 IF (1:500)
AntibodyGoat anti-mouse IgG (H + L) Secondary antibody Alexa Fluor 568-conjugated conjugateThermo Fisher ScientificCat# A11031RRID:AB_144696 IF (1:500)
AntibodyGoat anti-rabbit IgG (H + L) Secondary antibody Alexa Fluor 568-conjugated conjugateThermo Fisher ScientificCat# A11036RRID:AB_10563566 IF (1:500)
AntibodyGoat anti-mouse IgG (H + L) Secondary antibody Alexa Fluor 647-conjugated conjugateThermo Fisher ScientificCat# A21236RRID:AB_2535805 IF (1:500)
AntibodyGoat anti-rabbit IgG (H + L) Secondary antibody Alexa Fluor 647-conjugated conjugateThermo Fisher ScientificCat# A21245RRID:AB_2535813 IF (1:500)
AntibodyGoat anti-rabbit IgG Horseradish Peroxidase (HRP)Thermo Fisher ScientificCat# 31460RRID:AB_228341 WB (1:5000)
AntibodyGoat anti-mouse IgG-HRPThermo Fisher ScientificCat# 31430RRID:AB_228307 WB (1:5000)
AntibodyDonkey anti-goat IgG-HRPSanta Cruz BiotechnologyCat# sc-2020RRID:AB_631728 WB (1:2000)
Recombinant DNA reagentModified pLexA vectorStenmark et al., 1995 N/A
Recombinant DNA reagentpGBKT7 DNA-BD vectorTakara BioCat# 630443
Recombinant DNA reagentpGADT7 AD vectorTakara BioCat# 630442
Recombinant DNA reagentHuman skeletal muscle cDNA library in the pGAD10 vectorClontech LaboratoriesHL4010AB
Recombinant DNA reagentpET-3d vectorMerck Life ScienceCat# 69421
Recombinant DNA reagentpETM-14Dümmler et al., 2005N/A
Recombinant DNA reagentpET-3d 6xHis human MYPN C-termThis paperN/A Cloning primers in Supplementary file 1
Recombinant DNA reagentpETM-14 human MYPN C-termThis paperN/A Cloning primers in Supplementary file 1
Recombinant DNA reagentpET-3d-6xHis human Titin Z4-Z5This paperN/A Cloning primers in Supplementary file 1
Recombinant DNA reagentpETM-14 human PALLD C-termThis paperN/A Cloning primers in Supplementary file 1
Recombinant DNA reagentpGBKT7 human Titin IgZ4-Z5This paperN/A Cloning primers in Supplementary file 1
Recombinant DNA reagentpGADT7 human MYPN full-lengthThis paperN/A Cloning primers in Supplementary file 1
Recombinant DNA reagentpGADT7 human MYPN Ig3-endThis paperN/A Cloning primers in Supplementary file 1
Recombinant DNA reagentpGADT7 human MYPN Ig5-endThis paperN/A Cloning primers in Supplementary file 1
Recombinant DNA reagentpGADT7 human MYPN Ig3-4This paperN/A Cloning primers in Supplementary file 1
Recombinant DNA reagentpGADT7 human MYPN Ig4-endThis paperN/A Cloning primers in Supplementary file 1
Recombinant DNA reagentpGADT7 human PALLD C-termThis paperN/A Cloning primers in Supplementary file 1
Recombinant DNA reagentpLexA-titin Z3-Z5This paperN/A Cloning primers in Supplementary file 1
Recombinant DNA reagentpLexA-titin Z4-Z5This paperN/A Cloning primers in Supplementary file 1
Sequence-based reagentqRT-PCR primersThis paperN/ASupplementary file 1
Commercial assay or kitIn-Fusion HD Cloning kitTakara BioCat# 639650
Commercial assay or kitMonolith NT Protein Labeling Kit RED-NHSNanoTemper TechnologiesCat# L001
Commercial assay or kitDC Protein Assay Kit IIBio-Rad LaboratoriesCat# 5000112
Commercial assay or kitFrozen-EZ Yeast Transformation II kitZymo ResearchCat# T2001
Chemical compound, drugAureobasidin ATakara BioCat# 630499
Chemical compound, drugX-α-GalTakara BioCat# 630,463
Chemical compound, drugHisTrap columnGE Healthcare
Chemical compound, drugHiLoad 16/600 Superdex 75 pg columnGE HealthcareCat# 28989333
Chemical compound, drugTRIzol ReagentThermo Fisher ScientificCat# 15596026
Chemical compound, drugHigh Capacity cDNA Reverse Transcription kitThermo Fisher ScientificCat# 4368814
Chemical compound, drugGoTaq qPCR Master MixPromegaCat# A6002
Chemical compound, drugCOmplete Protease Inhibitor Cocktail TabletsMerck Life ScienceCat# 11697498001
Chemical compound, drugPierce Phosphatase Inhibitor Mini TabletsThermo Fisher ScientificCat# A32957
Chemical compound, drugImmobilon Western Chemiluminescent HRP SubstrateMerck Life ScienceCat# WBKLS0500
Chemical compound, drugRhodamine phalloidinThermo Fisher ScientificCat# R415 IF (1:100)
Chemical compound, drugWheat Germ Agglutinin, Alexa Fluor 594 ConjugateThermo Fisher ScientificCat# W11262 IF (1:500)
Chemical compound, drugVECTASHIELD Vibrance Antifade Mounting Medium with DAPID.B.A. Italia Srl.Cat# H-1800–10
Chemical compound, drugCollagenase, Type 2Worthington Biochemical CorporationCat# LS004177
Chemical compound, drugFura-2, AM, cell permeantThermo Fisher ScientificCat# F1201
Chemical compound, drugFluo-4, AM, cell permeantThermo Fisher ScientificCat# F14201
Other4–20% Criterion TGX Stain Free protein gel,18 wellBio-Rad LaboratoriesCat# 5678094
Software, algorithmPrism, version 7.0GraphPad Software Inchttps://www.graphpad.com/scientific-software/prism/RRID:SCR_002798
Software, algorithmFiji (ImageJ) (analysis software, version 2.0.0-rc-69/1.52 p)National Institute of Health (NIH)https://fiji.sc/RRID:SCR_002285SparkMaster plugin used for Ca2+ spark analysis
Software, algorithmNT Affinity Analysis software, version 2.0.1334NanoTemper TechnologiesN/A
Software, algorithmIon Wizard, software, version 6.6.11IonOptix B.V.N/A

Additional files

Download links

A two-part list of links to download the article, or parts of the article, in various formats.

Downloads (link to download the article as PDF)

Download citations (links to download the citations from this article in formats compatible with various reference manager tools)

Open citations (links to open the citations from this article in various online reference manager services)