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Dysregulated ribonucleoprotein granules promote cardiomyopathy in RBM20 gene-edited pigs

Nature Medicine volume 26, pages 1788–1800 (2020)Cite this article

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An Author Correction to this article was published on 02 June 2021

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Abstract

Ribonucleoprotein (RNP) granules are biomolecular condensates—liquid–liquid phase-separated droplets that organize and manage messenger RNA metabolism, cell signaling, biopolymer assembly, biochemical reactions and stress granule responses to cellular adversity. Dysregulated RNP granules drive neuromuscular degenerative disease but have not previously been linked to heart failure. By exploring the molecular basis of congenital dilated cardiomyopathy (DCM) in genome-edited pigs homozygous for an RBM20 allele encoding the pathogenic R636S variant of human RNA-binding motif protein-20 (RBM20), we discovered that RNP granules accumulated abnormally in the sarcoplasm, and we confirmed this finding in myocardium and reprogrammed cardiomyocytes from patients with DCM carrying the R636S allele. Dysregulated sarcoplasmic RBM20 RNP granules displayed liquid-like material properties, docked at precisely spaced intervals along cytoskeletal elements, promoted phase partitioning of cardiac biomolecules and fused with stress granules. Our results link dysregulated RNP granules to myocardial cellular pathobiology and heart failure in gene-edited pigs and patients with DCM caused by RBM20 mutation.

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Fig. 1: Sarcoplasmic RBM20 misaccumulation, contractile dysfunction and molecular pathobiology in humanized RBM20 gene-edited pigs at postnatal day 7.
Fig. 2: Neonatal mortality, cardiomegaly, sarcoplasmic RBM20 misaccumulation and molecular pathobiology in humanized RBM20 gene-edited pigs at 16 weeks of age.
Fig. 3: Misaccumulation of RBM20 in the sarcoplasm of cardiomyocytes from an RBM20R636S allele carrier.
Fig. 4: Subcellular localization and biochemical, biophysical and ultrastructural features of RBM20R636S biomolecular condensates and dysregulated RNP granules.
Fig. 5: Cytoskeleton coupling and biomolecular partitioning by RBM20 RNP granules dysregulated in cardiomyocyte sarcoplasm.
Fig. 6: Pathological RNP granules in HMZ pig myocardium are linked to stress granule fusion and cytoskeletal disassembly in cell models.

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Data availability

Additional information and detailed protocols regarding all experimental methodologies are available upon request from J.W.S. The RBM20R636S genetically engineered pig is commercially available through Recombinetics (D.F.C.) and/or through collaboration with Discovery Engine at Mayo Clinic (J.W.S. or T.J.N.; timothy.nelson@mayo.edu). Data on the human iPSCs and iPSC-derived cardiomyocytes used in this study, unique Mayo Clinic identifiers 5RCM1 and 4RCM1, unrelated patients with DCM heterozygous for the R636S variant (described in ref. 1) and normal control hiPSCs and derived cardiomyocytes (81HLH4) are available upon request from J.W.S. and T.J.N. (timothy.nelson@mayo.edu) through the Mayo Clinic Discovery Engine and ReGen Theranostics (Rochester, Minnesota). The myocardial biopsy materials used in this study (from the patients described in ref. 1) are available upon request from T.M.O. (timothy.olson@mayo.edu) at the Mayo Clinic Discovery Engine. For the human and animal studies, all approved IRB, informed consent and IACUC animal treatment and husbandry protocols are available upon request from J.W.S. at Mayo Clinic or Recombinetics. Data on the plasmid DNA and all primer DNA sequences used in this study are available from J.W.S. upon request. There are no restrictions on data availability for any part of this paper. Source data are provided with this paper.

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Acknowledgements

We thank the patients who contributed cells and tissues to this study, E. Olson and J. Hill for support during the transition of J.W.S. from the University of Texas Southwestern Medical Center to Mayo Clinic, and J. Dearani and A. Terzic at Mayo Clinic. We also thank C. Riggs and N. Kedersha (Harvard Medical School) for sharing FRAP plasmids, and M. Rosen and S. McKnight (University of Texas Southwestern Medical Center) for sharing resources and helpful discussions. We also acknowledge grant support from NIH/NHLBI U01HL100404 (Progenitor Cell Biology Consortium) and NIH U54HD087351 (Wellstone Center for Muscular Dystrophy Research) to J.W.S., American Heart Association grant 19TPA34830072 to W.G., JSPS KAKENHI Grant Number JP20K21385 and Nanken-Kyoten, TMDU to H.K. and Steinmetz Cardiomyopathy Fund to L.M.S.

Author information

Author notes

  1. Wei Guo

    Present address: Department of Animal Science, University of Wisconsin-Madison, Madison, WI, USA

  2. These authors jointly supervised this work: Daniel F. Carlson, Timothy J. Nelson.

Authors and Affiliations

  1. Discovery Engine/Todd and Karen Wanek Family Program for Hypoplastic Left Heart Syndrome, Mayo Clinic, Rochester, MN, USA

    Jay W. Schneider, Saji Oommen, Muhammad Y. Qureshi, David R. Pease, Rhianna S. Sundsbak, Kimberly A. Holst, Brooks S. Edwards, Nikolas Newville, Matthew A. Hathcock, Timothy M. Olson & Timothy J. Nelson

  2. Hamon Center for Regenerative Science and Medicine, Department of Medicine/Cardiology, University of Texas Southwestern Medical Center, Dallas, TX, USA

    Jay W. Schneider & Sean C. Goetsch

  3. Department of Animal Science, University of Wyoming, Laramie, WY, USA

    Wei Guo & Mingming Sun

  4. Department of Genetics and Genome Center, Stanford University, Stanford, CA, USA

    Han Sun, Francesca Briganti, Wu Wei & Lars M. Steinmetz

  5. Laboratory of Gene Expression, Medical Research Institute, Tokyo Medical and Dental University, Tokyo, Japan

    Hidehito Kuroyanagi

  6. Recombinetics, St. Paul, MN, USA

    Dennis A. Webster, Alexander W. Coutts, Tamene Melkamu, Scott C. Fahrenkrug & Daniel F. Carlson

  7. Genome Biology Unit, European Molecular Biology Laboratory, Heidelberg, Germany

    Francesca Briganti & Lars M. Steinmetz

  8. Department of Neurosciences, Biomedicine and Movement Sciences, University of Verona, Verona, Italy

    Maria G. Romanelli

  9. Department of Chemistry, University of Texas at San Antonio, San Antonio, TX, USA

    Doug E. Frantz

  10. Department of General Internal Medicine, Mayo Clinic, Rochester, MN, USA

    Timothy J. Nelson

  11. Department of Molecular Pharmacology, Mayo Clinic, Rochester, MN, USA

    Timothy J. Nelson

  12. Todd and Karen Wanek Family Program for Hypoplastic Left Heart Syndrome, Mayo Clinic, Rochester, MN, USA

    Susana Cantero Peral, Sarah Edgerton, Joan Wobig & Boyd Rasmussen

  13. Department of Comparative Medicine, Mayo Clinic, Rochester, MN, USA

    Jodi A. Scholz & Craig S. Frisk

  14. Department of Pediatric Cardiology, Mayo Clinic, Rochester, MN, USA

    Frank Cetta

  15. Department of Cardiovascular Surgery, Mayo Clinic, Rochester, MN, USA

    Scott H. Suddendorf, Joanne M. Pedersen & Steve Krage

  16. Echocardiography Laboratory, Mayo Clinic, Rochester, MN, USA

    Chelsea L. Reece, Angela R. Miller, Sara E. Martineau, Rebecca K. Johnson, Amanda L. Breuer & Janell K. Fox

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  1. Jay W. Schneider
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Consortia

Wanek Program Preclinical Pipeline

  • Susana Cantero Peral
  • , Sarah Edgerton
  • , Joan Wobig
  • , Boyd Rasmussen
  • , Jodi A. Scholz
  • , Frank Cetta
  • , Scott H. Suddendorf
  • , Joanne M. Pedersen
  • , Steve Krage
  • , Craig S. Frisk
  • , Chelsea L. Reece
  • , Angela R. Miller
  • , Sara E. Martineau
  • , Rebecca K. Johnson
  • , Amanda L. Breuer
  • , Janell K. Fox
  • , Jay W. Schneider
  • , Saji Oommen
  • , Muhammad Y. Qureshi
  • , David R. Pease
  • , Rhianna S. Sundsbak
  • , Kimberly A. Holst
  • , Brooks S. Edwards
  • , Nikolas Newville
  • , Matthew A. Hathcock
  • , Timothy M. Olson
  •  & Timothy J. Nelson

Contributions

T.J.N., D.F.C., J.W.S., S.C.G., D.A.W., S.C.F., L.M. S. and T.M.O. conceived of the project. J.W.S., D.R.P., S.O., M.Y.Q., S.C.G., R.S.S., W.G., M.S., H.S., D.A.W., K.A.H., B.S.E., M.A.H., F.B., W.W., N.N., A.W.C., T.M., M.G.R., D.E.F. and H.K. provided crucial materials, performed experiments and/or analyzed data. T.M. and A.W.C. managed animal husbandry at Recombinetics. The Wanek Program Preclinical Pipeline managed animal husbandry at Mayo Clinic. J.W.S. wrote the manuscript.

Corresponding authors

Correspondence to Jay W. Schneider or Daniel F. Carlson.

Ethics declarations

Competing interests

Generation of the RBM20R636S pig by gene-editing technology represents a collaborative partnership between Recombinetics (St. Paul, Minnesota), a commercial entity and Mayo Clinic in Rochester (Minnesota). T.M., A.W.C., D.A.W., D.F.C. and S.C.F. are shareholders in Recombinetics (St. Paul, Minnesota), which seeks to commercialize the RBM20R636S pig. The Todd and Karen Wanek Family Program for Hypoplastic Left Heart Syndrome at Mayo Clinic—the co-inventor of the RBM20R636S pig—maintains a profit-sharing agreement with Recombinetics regarding this gene-edited pig.

Additional information

Peer review information Michael Basson was the primary editor on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data

Extended Data Fig. 1 Human RBM20-R636S germline genome editing strategy validated by cardiac MRI.

a, Cartoon depicting campaign to produce humanized R636S HTZ pigs by TALENs/HDR genome editing in WT pig fibroblasts followed by SCNT of gene-edited fibroblast nuclei into oocytes, implanted into surrogate sows. Crossbreeding of HTZ R636S germline gene-edited pigs produced HMZ F1 progeny with severe DCM/HF, the focus of this study. WT and R636S alleles signified by green and red color, respectively. A presentative piglet litter from this study is shown. b, Representative cardiac MRI images of 8 week-old HMZ pig showing end-diastolic and end-systolic frames in long and short axis with volumetrically calculated EF from ESV 60 ml and EDV 80 ml of 25% in this example. Yellow arrowhead, small pericardial effusion. c, Graph series comparing average heart rate (HR), calculated LVEF and corresponding LVEDV and LVESV (normalized to body surface area) by cardiac MRI at 8, 12 and 16 weeks of age in WT and HMZ pigs reported as median, Q1, Q3 and range with outliers as dots and n=number of individual pigs. Note that robust difference between WT and HMZ pigs at 8 weeks did not achieve statistical significance because of WT n=2 while later time points showed no statistical difference between WT and HMZ pigs. HMZ LVEDV increases with age but LVESV decreases even more explaining the normalization of LVEF, despite ventricular enlargement, in HMZ from 8 to 16 weeks.

Extended Data Fig. 2 Demonstration of endocardial fibroelastosis and abnormal sarcomere organization in HMZ gene-edited pigs.

a, Gross anatomical apical segments of same hearts in (Fig. 1g) showing endocardial fibroelastosis (circled area, whitish layer) in dilated HMZ but not WT heart at postnatal day-3. Scale bar, cm. b, Study of highly focal and HMZ-specific endocardial fibroelastosis, as in circled area (Extended Data Fig. 2a) above, by Masson’s trichrome and Verhoeff-Van Gieson’s histochemical staining. Scale bar, μm. c, TEM study comparing sarcomere striation patterns in WT (upper panel) versus HMZ (middle and lower panels) 7-day pig myocardium and graph of measured Z-disc to Z-disc intervals demonstrating shorter sarcomere length and swollen Z-discs consistent with impaired myofibril biogenesis in HMZ versus WT pig myocardium. Scale bar, μm.

Extended Data Fig. 3 Decreased postnatal mitotic cell cycle activity and TEM survey in HMZ pig myocardium.

a, Analogous to (Fig. 1i), upper panels compare LV myocardial proliferative activity in WT and HMZ pigs at day-7 assessed by condensed chromatin/mitotic figures by H&E histochemical staining (upper panels) or pH3 F-IHC with DAPI co-stain (lower panels), individual mitotic figures highlighted by yellow circles. These results are representative of multiple repeat experiments in different animals. Scale bars, μm. b, Survey of HMZ pig myocardium at day-7 by TEM at increasing magnification from top to bottom, 2,500X-to-50,000X, respectively, highlighting multifaceted ultrastructural cellular pathology. Scale bars, μm.

Extended Data Fig. 4 Pathological hypertrophy, polyploidy and abnormal mitochondria in HMZ pig myocardium.

a, Graphical comparison of body weight, heart weight, heart weight/body weight ratio and LV free wall thickness (hypertrophy) in WT and HMZ pigs at 16 weeks of age reported as graphs showing median, Q1, Q3 and range. N=number of individual pigs studied and significance calculated by two-sided Student’s t-test. b, Fluorescence in situ hybridization (FISH) for pig X-chromosome in HMZ male (XY) pig myocardium. Yellow circles highlight X-chromosome signals in nuclei with greater than diploid (one dot) genomic DNA content. This result is representative of three repeat FISH studies. Scale bar, μm. c, Time course study of H&E stained HMZ pig LV-myocardium at day-1, day-7 and 16-weeks highlighting progression of pathological hypertrophy, nuclear abnormalities, cellular disarray and fibrosis. Scale bar, μm. d, Analogous to heat map data presented in Fig. 2g (see blue arrow), RNA-seq alternative splicing scatter plot analysis of IMMT (mitofilin) comparing WT (green), HTZ (black) and HMZ (red) pig myocardium at 16 weeks, clustered dots (circled) highlight genotype-specific alternative splicing of IMMT pre-mRNA, n=number of individual pigs. Right hand panel, correlating with IMMT missplicing, TEM comparing mitochondrial ultrastructure highlighting abnormally shaped cristae (arrows) in HMZ versus WT myocardium at day-7 by TEM, with similar myocardial mitochondrial morphological abnormalities observed in three different HMZ piglets. Scale bar, μm.

Extended Data Fig. 5 Sarcoplasmic misaccumulation of RBM20-R636S in inherited RBM20-DCM and splicing speckle amplification in ischemic DCM.

a, Expanded views of (Fig. 3a, third panel) demonstrating RBM20 IHC at increasing magnification power (10x, 20X and 40X) highlighting intense sarcoplasmic and nuclear expression in myocardium of DCM patient, an R636S mutant allele carrier, analogous to the HTZ gene-edited RBM20 pig (Fig. 1c, central panel). Automated IHC repeated three times in this patient’s myocardium yielded results. Scale bars, μm. b, Human end-stage ischemic cardiomyopathy myocardium stained by H&E histochemistry (left hand panel) comparing size of cardiomyocyte nuclei (white arrows) to non-cardiomyocytes (yellow arrow), FISH for human chromosomes 6 (green) and 8 (red) with DAPI highlighting advanced HF-associated polyploidy (central panel) and, correspondingly, amplification of transcriptionally-active RBM20 nuclear splicing speckles, each demonstrating a TTN gene locus on a human chromosome 2 by IF-IHC (right hand panel). Each result is representative of experiments repeated three times. Scale bars, μm.

Extended Data Fig. 6 Sarcoplasmic RBM20-R636S rosary beads compared with TDP-43 myo-granules in RBM20-DCM myocardium.

a, Expanding on the results shown in (Fig. 3c), RBM20 IF-IHC confocal microscopy of human RBM20-DCM myocardium highlighting rosary beads-on-a-string staining pattern when confocal plane is focused on sarcomeres in longitudinal orientation. Repeated three times, this experiment produced identical results. Scale bar, μm. b, Double IF-IHC confocal microscopy for TARDBP (TDP-43), the driver of myo-granules in skeletal muscle, and RBM20 in HMZ pig myocardium highlighting distinctive (non-coincident) linear staining patterns of these two RNA-binding proteins. Repeated three times, this experiment produced identical results. Scale bar, μm. c, Corroborating the results observed in (b) above, GFP-RBM20-R636S transfected human iPSC-CMs highlighting distinctive fibrillary TARDBP by ICC and droplet-like RBM20-R636S RNP granules by confocal microscopy. Scale bar, μm.

Extended Data Fig. 7 RBM20-R636S liquid droplets reconstructed by recombinant protein expression in U2OS cells.

Transfection of GFP-RBM20-R636S in U2OS cells co-imaged with DAPI and ICC for cytoskeletal elements, microtubules (α-tubulin), F-actin microfilaments (phalloidin) or intermediate filaments (vimentin), highlighting the liquid behavior of GFP-RBM20-R636S droplets “beading-up” and fusing caused by the nuclear envelope’s surface tension. Experiment repeated numerous times with identical results. Scale bars, 50 μm for all panels.

Extended Data Fig. 8 RBM20-R636S RNP granules (R3Gs) at confocal microscopy super-resolution, laden with mRNA and associated with cytoskeletal elements in iPSC-CMs.

a, Additional images of GFP-RBM20 WT (upper panels) and GFP-RBM20-R636S (lower panels) R3Gs by super-resolution confocal microscopy, at increasing magnification from left-to-right, demonstrating SG-like stable core and dynamic shell sub-structural organization. Experiment repeated three times with identical results. Scale bars, μm. b, Additional image panel depicting poly-A+ mRNA FISH in GFP-RBM20 transfected hPSC-CMs demonstrating co-localization of mRNA signal with RBM20 RNP granules (in this case “overflow” RBM20 WT granules) in CM sarcoplasm. Scale bars, μm. c, Another example as in (b above). Three independent poly-A+ mRNA FISH plus RBM20 ICC co-localization experiments produced identical results. Scale bar, μm. d, Co-linearity of R3Gs and α-tubulin signal in R636S patient-derived iPSC-CMs suggesting association of R3Gs with microtubules in these cells. Similar results obtained in three independent ICC experiments, see (Fig. 6c) for TUBB co-staining. Scale bar, μm.

Extended Data Fig. 9 B-isox affinity chromatography evidence of ACTC1 associated with R3Gs in HMZ myocardium and R3G antagonized SG assembly in U2OS cells.

a, B-isox affinity chromatography proteomics demonstrating cardiomyopathy gene enrichment analysis and specifically ACTC1 pulldown from R3G-laden HMZ gene-edited pig myocardium, see (Fig. 4d, e). Repeated three times, this experiment consistently produced similar results. Proteomics statistical analysis as described in (Fig. 4d legend). b, Double IHC for RBM20 and ACTC1 in 7-day HMZ pig myocardium in transverse section confirming reciprocal staining intensity of R3Gs and ACTC1, see (Fig. 6a) for analogous longitudinal sections. This myocardial IHC experiment repeated in three different HMZ piglets produced identical results. Scale bar, μm. c, GFP signal and SG marker, G3BP1, by ICC with DAPI in NaAsO2 treated GFP-RBM20-R636S transfected U2OS cells, demonstrating that cells expressing R3Gs fail to construct G3BP1 SGs, consistent R3G sequestration of G3BP1, a protein critical for SG assembly. Similar results observed in multiple independent U2OS cell transfection ICC experiments. Scale bars, μm.

Extended Data Fig. 10 Workflow for FTT analysis of GFP-RBM20 transfected hPSC-CMs and model of the liquid droplet organizational logic of cardiomyocytes.

a, Workflow for quantitative analysis of decreased striated patterning associated with R3G accumulation in GFP-RBM20 transfected hPSC-CMs, see Methods for description of protocol. The symbol ||e|| in graph stands for ‘norm of residuals’ (square root of the sum of squares of residuals), which is a measure of ‘goodness of fit’ in regression analysis: lower numbers equal better fit. b, Infographic of RBM20 liquid pipeline for transfer of genetic information from nuclear transcription/splicing factories to sarcoplasmic Z-discs, the command center of myofibril biogenesis, across an elaborate sarcoplasmic network of cytoskeletal-linked biomolecular condensates. Conceptually, this liquid condensate pipeline organizes and manages the flow of genetic information from the cardiac genome-to-message-to-sarcomeres with RBM20 returning to the nucleus to pick-up new cargo.

Supplementary information

Reporting Summary

Supplementary Video 1

Echocardiogram of ventricular function in a WT pig. Short-axis echo loop showing the contractile function of a WT pig at day 21, corresponding to Fig. 1f (left-hand panel).

Supplementary Video 2

Echocardiogram of ventricular dysfunction in a HMZ pig. Short-axis echo loop at the mid-ventricular level of a critically ill HMZ pig (in cardiogenic shock with high BNP and ANP) at day 21, corresponding to Fig. 1f (right-hand panel).

Supplementary Video 3

Echocardiogram of mitral regurgitation by color Doppler in a HMZ pig. Echo loop highlighting systolic contractile dysfunction and moderate-to-severe mitral regurgitation in a HMZ piglet at the time of birth. Note that by Mayo Clinic convention, the four-chamber view has the atria on the top and the ventricles on the bottom, with the left ventricle, mitral valve and associated mitral regurgitation on the viewer’s right.

Supplementary Video 4

Echocardiogram of tricuspid regurgitation by color Doppler in a HMZ pig. Echo loop highlighting systolic contractile dysfunction and moderate-to-severe tricuspid regurgitation in a HMZ piglet at the time of birth. For orientation, see the note for Supplementary Video 3.

Supplementary Video 5

Serial echocardiograms of contractile function in a neonatal HMZ pig. Echo loops of an individual HMZ pig, highlighting the progressive decline of LVEF from ~50–55% at birth (Supplementary Video 5) to <10% at postnatal day 21 (Supplementary Video 8), with Supplementary Videos 6 and 7 at days 2 and 13, respectively. A representative result is shown. Echo, clinical observation and necropsy confirmed this scenario across the majority of HMZ piglets, corresponding to high neonatal mortality (Fig. 2a).

Supplementary Video 6

Serial echocardiograms of contractile function in a neonatal HMZ pig. Echo loops of an individual HMZ pig, highlighting the progressive decline of LVEF from ~50–55% at birth (Supplementary Video 5) to <10% at postnatal day 21 (Supplementary Video 8), with Supplementary Videos 6 and 7 at days 2 and 13, respectively. A representative result is shown. Echo, clinical observation and necropsy confirmed this scenario across the majority of HMZ piglets, corresponding to high neonatal mortality (Fig. 2a).

Supplementary Video 7

Serial echocardiograms of contractile function in a neonatal HMZ pig. Echo loops of an individual HMZ pig, highlighting the progressive decline of LVEF from ~50–55% at birth (Supplementary Video 5) to <10% at postnatal day 21 (Supplementary Video 8), with Supplementary Videos 6 and 7 at days 2 and 13, respectively. A representative result is shown. Echo, clinical observation and necropsy confirmed this scenario across the majority of HMZ piglets, corresponding to high neonatal mortality (Fig. 2a).

Supplementary Video 8

Serial echocardiograms of contractile function in a neonatal HMZ pig. Echo loops of an individual HMZ pig, highlighting the progressive decline of LVEF from ~50–55% at birth (Supplementary Video 5) to <10% at postnatal day 21 (Supplementary Video 8), with Supplementary Videos 6 and 7 at days 2 and 13, respectively. A representative result is shown. Echo, clinical observation and necropsy confirmed this scenario across the majority of HMZ piglets, corresponding to high neonatal mortality (Fig. 2a).

Supplementary Video 9

Serial MRIs of contractile function in a HMZ pig. Cardiac MRI loops highlighting progressive improvement in systolic contractile function, despite DCM, starting at week 8 (the earliest time point possible for cardiac MRI in pigs; Supplementary Video 9) through weeks 12 (Supplementary Video 10) and 16 (Supplementary Video 11), corresponding to group data in Fig. 2d. Note that although cardiac MRI LVEF is normalized, the ventricular mass of the HMZ pig at 16 weeks is around twice that of a normal pig, corresponding to massive cardiomegaly (Fig. 2b,c and Extended Data Fig. 4a). A representative result is shown. Clinical observation, serological analysis, cardiac MRI and necropsy confirmed this scenario across all HMZ survivors (Fig. 2a).

Supplementary Video 10

Serial MRIs of contractile function in a HMZ pig. Cardiac MRI loops highlighting progressive improvement in systolic contractile function, despite DCM, starting at week 8 (the earliest time point possible for cardiac MRI in pigs; Supplementary Video 9) through weeks 12 (Supplementary Video 10) and 16 (Supplementary Video 11), corresponding to group data in Fig. 2d. Note that although cardiac MRI LVEF is normalized, the ventricular mass of the HMZ pig at 16 weeks is around twice that of a normal pig, corresponding to massive cardiomegaly (Fig. 2b,c and Extended Data Fig. 4a). A representative result is shown. Clinical observation, serological analysis, cardiac MRI and necropsy confirmed this scenario across all HMZ survivors (Fig. 2a).

Supplementary Video 11

Serial MRIs of contractile function in a HMZ pig. Cardiac MRI loops highlighting progressive improvement in systolic contractile function, despite DCM, starting at week 8 (the earliest time point possible for cardiac MRI in pigs; Supplementary Video 9) through weeks 12 (Supplementary Video 10) and 16 (Supplementary Video 11), corresponding to group data in Fig. 2d. Note that although cardiac MRI LVEF is normalized, the ventricular mass of the HMZ pig at 16 weeks is around twice that of a normal pig, corresponding to massive cardiomegaly (Fig. 2b,c and Extended Data Fig. 4a). A representative result is shown. Clinical observation, serological analysis, cardiac MRI and necropsy confirmed this scenario across all HMZ survivors (Fig. 2a).

Supplementary Video 12

3D video projection of RBM20R636S signal in human myocardium by confocal microscopy. RBM20 IHC and confocal microscopy highlighting Z-disc localization in 3D video projection (see Fig. 3c). A representative video image is shown. Multiple confocal microscopy IHC experiments yielded similar results.

Supplementary Video 13

LCI of R3Gs in U2OS cells. Video loops highlighting dynamic liquid droplet fusion and fission events of GFP–R3Gs expressed in U2OS cells. Representative FRAP video loops are shown. Multiple confocal microscopy LCI experiments yielded similar results (see Fig. 4g, graph).

Supplementary Video 14

LCI of R3Gs in U2OS cells. Video loops highlighting dynamic liquid droplet fusion and fission events of GFP–R3Gs expressed in U2OS cells. Representative FRAP video loops are shown. Multiple confocal microscopy LCI experiments yielded similar results (see Fig. 4g, graph).

Supplementary Video 15

LCI of R3Gs in U2OS cells. Video loops highlighting dynamic liquid droplet fusion and fission events of GFP–R3Gs expressed in U2OS cells. Representative FRAP video loops are shown. Multiple confocal microscopy LCI experiments yielded similar results (see Fig. 4g, graph).

Supplementary Video 16

FRAP analysis of GFP–RBM20R636S droplets assembled in U2OS cells. Representative videos of FRAP in fixed (Supplementary Video 16; corresponding to Fig. 4g (inset)) and live (Supplementary Video 17; corresponding to Fig. 4g) GFP–RBM20R636S-transfected U2OS cells. The graph in Fig. 4g is compiled from numerous such videos.

Supplementary Video 17

FRAP analysis of GFP–RBM20R636S droplets assembled in U2OS cells. Representative videos of FRAP in fixed (Supplementary Video 16; corresponding to Fig. 4g (inset)) and live (Supplementary Video 17; corresponding to Fig. 4g) GFP–RBM20R636S-transfected U2OS cells. The graph in Fig. 4g is compiled from numerous such videos.

Supplementary Video 18

Super-resolution confocal microscopy 3D video projection of GFP–RBM20-tagged nuclear splicing speckles. Super-resolution confocal microscopy imaging of nuclear GFP–RBM20 in hPSC-CMs, highlighting the worm-like cylinder morphology of RBM20 bound to nascent pre-mRNA molecules emerging from the >300-kilobase human TTN gene and other genes of the trans-interacting chromatin domain/splicing factories at low magnification (Supplementary Video 18) and each individual factory at high magnification (Supplementary Videos 19 and 20) (see Fig. 4h, left and central panels). Representative examples are shown. Super-resolution confocal microscopy confirmed similar results in numerous GFP–RBM20-transfected hPSC-CMs (Fig. 4g, graph).

Supplementary Video 19

Super-resolution confocal microscopy 3D video projection of GFP–RBM20-tagged nuclear splicing speckles. Super-resolution confocal microscopy imaging of nuclear GFP–RBM20 in hPSC-CMs, highlighting the worm-like cylinder morphology of RBM20 bound to nascent pre-mRNA molecules emerging from the >300-kilobase human TTN gene and other genes of the trans-interacting chromatin domain/splicing factories at low magnification (Supplementary Video 18) and each individual factory at high magnification (Supplementary Videos 19 and 20) (see Fig. 4h, left and central panels). Representative examples are shown. Super-resolution confocal microscopy confirmed similar results in numerous GFP–RBM20-transfected hPSC-CMs (Fig. 4g, graph).

Supplementary Video 20

Super-resolution confocal microscopy 3D video projection of GFP–RBM20-tagged nuclear splicing speckles. Super-resolution confocal microscopy imaging of nuclear GFP–RBM20 in hPSC-CMs, highlighting the worm-like cylinder morphology of RBM20 bound to nascent pre-mRNA molecules emerging from the >300-kilobase human TTN gene and other genes of the trans-interacting chromatin domain/splicing factories at low magnification (Supplementary Video 18) and each individual factory at high magnification (Supplementary Videos 19 and 20) (see Fig. 4h, left and central panels). Representative examples are shown. Super-resolution confocal microscopy confirmed similar results in numerous GFP–RBM20-transfected hPSC-CMs (Fig. 4g, graph).

Supplementary Video 21

Super-resolution confocal microscopy of RNP granules in GFP–RBM20R636S-transfected hPSC-CMs. Super-resolution confocal microscopy imaging of sarcoplasmic GFP–RBM20R636S granules, highlighting the internal core/shell substructure analogous to stress granules (see Fig. 4h, right-hand panel and inset). Representative examples are shown. Super-resolution confocal microscopy confirmed similar results in numerous GFP–RBM20R636S-transfected hPSC-CMs (Fig. 4g, graph).

Supplementary Video 22

LCI of cytoskeleton-linked GFP–RBM20R636S granules in beating hPSC-CMs. GFP–RBM20 (WT or R636S) expressed in hPSC-CMs, highlighting the constellation of granules stably docked at precisely spaced intervals along cytoskeletal elements by LCI beating in concert with the cardiomyocyte’s native rhythmicity. Supplementary Video 22 corresponds to Fig. 5a (left-hand panel) and Supplementary Video 23 shows a second independent example.

Supplementary Video 23

LCI of cytoskeleton-linked GFP–RBM20R636S granules in beating hPSC-CMs. GFP–RBM20 (WT or R636S) expressed in hPSC-CMs, highlighting the constellation of granules stably docked at precisely spaced intervals along cytoskeletal elements by LCI beating in concert with the cardiomyocyte’s native rhythmicity. Supplementary Video 22 corresponds to Fig. 5a (left-hand panel) and Supplementary Video 23 shows a second independent example.

Supplementary Video 24

LCI of mobile RNP granules in GFP–RBM20R636S-transfected hPSC-CMs. GFP–RBM20 (WT or R636S)-overexpressing hPSC-CMs, highlighting mobile granules rapidly darting about the sarcoplasm. Supplementary Video 24 corresponds to Fig. 5a (right-hand panel) and Supplementary Videos 25 and 26 are additional examples.

Supplementary Video 25

LCI of mobile RNP granules in GFP–RBM20R636S-transfected hPSC-CMs. GFP–RBM20 (WT or R636S)-overexpressing hPSC-CMs, highlighting mobile granules rapidly darting about the sarcoplasm. Supplementary Video 24 corresponds to Fig. 5a (right-hand panel) and Supplementary Videos 25 and 26 are additional examples.

Supplementary Video 26

LCI of mobile RNP granules in GFP–RBM20R636S-transfected hPSC-CMs. GFP–RBM20 (WT or R636S)-overexpressing hPSC-CMs, highlighting mobile granules rapidly darting about the sarcoplasm. Supplementary Video 24 corresponds to Fig. 5a (right-hand panel) and Supplementary Videos 25 and 26 are additional examples.

Supplementary Video 27

Confocal microscopy 3D video projection of nuclear versus sarcoplasmic GFP–RBM20 accumulation in hPSC-CMs. Confocal microscopy imaging of GFP–RBM20 (WT) overexpressing H9 hPSC-CMs, highlighting actin cytoskeletal disorganization (shown by fluorescence-tagged phalloidin staining) associated with sarcoplasmic RNP granules but not with nuclear-only accumulation of RBM20. Supplementary Videos 27 and 28 are lower- and higher-magnification videos focusing on cytoskeletal organization and RNP granule–actin microfilament interaction, respectively.

Supplementary Video 28

Confocal microscopy 3D video projection of nuclear versus sarcoplasmic GFP–RBM20 accumulation in hPSC-CMs. Confocal microscopy imaging of GFP–RBM20 (WT) overexpressing H9 hPSC-CMs, highlighting actin cytoskeletal disorganization (shown by fluorescence-tagged phalloidin staining) associated with sarcoplasmic RNP granules but not with nuclear-only accumulation of RBM20. Supplementary Videos 27 and 28 are lower- and higher-magnification videos focusing on cytoskeletal organization and RNP granule–actin microfilament interaction, respectively.

Source data

Source Data Fig. 1

a, Genetically engineered BglII restriction fragment length polymorphism RBM20-R636S myocardial RT-PCR genotyping strategy in pigs with original gel. b, Unprocessed western blots for Fig. 1h.

Source Data Fig. 2

a, Unprocessed western blots for Fig. 2f. b, Unprocessed vertical agarose gel electrophoresis with Coomassie blue staining of pig myocardial protein extracts demonstrating genotype-specific alternative splicing of titin with the largest isoform, TTN-N2BA-G migrating at ~3,900,000 Daltons.

Source Data Fig. 3

Whole slide scan of RBM20 IHC in R636S carrier patient and control myocardium.

Source Data Fig. 4

a, Unprocessed western blots of recombinant RBM20 proteins shown in Fig. 4b. b, Unprocessed western blot of RBM20 isolated two different HMZ pigs by B-isox affinity chromatography, an essential step for enriching this protein.

Source Data Fig. 4

Gene list from proteomics analysis of biotinylated isoxazole affinity chromatography of HMZ RBM20-R636S pig myocardium and analysis for enrichment by DAVID Functional Annotation Clustering (FAC) with P-values calculated by Fisher’s exact test, see Methods for details

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Schneider, J.W., Oommen, S., Qureshi, M.Y. et al. Dysregulated ribonucleoprotein granules promote cardiomyopathy in RBM20 gene-edited pigs. Nat Med 26, 1788–1800 (2020). https://doi.org/10.1038/s41591-020-1087-x

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