Impairment of the ER/mitochondria compartment in human cardiomyocytes with PLN p.Arg14del mutation
2021; Springer Nature; Volume: 13; Issue: 6 Linguagem: Inglês
10.15252/emmm.202013074
ISSN1757-4684
AutoresFriederike Cuello, Anika E. Knaust, Umber Saleem, Malte Loos, Janice Raabe, Diogo Mosqueira, Sandra D. Laufer, Michaela Schweizer, Petra van der Kraak, Frederik Flenner, Bärbel Ulmer, Ingke Braren, Xiaoke Yin, Konstantinos Theofilatos, Jorge Ruiz‐Orera, Giannino Patone, Birgit Klampe, Thomas G. Schulze, Angelika Piasecki, Yigal M. Pinto, Aryan Vink, Norbert Hübner, Siân E. Harding, Manuel Mayr, Chris Denning, Thomas Eschenhagen, Arne Hansen,
Tópico(s)Genetic Neurodegenerative Diseases
ResumoArticle16 May 2021Open Access Source DataTransparent process Impairment of the ER/mitochondria compartment in human cardiomyocytes with PLN p.Arg14del mutation Friederike Cuello Friederike Cuello orcid.org/0000-0003-1612-1715 Department of Experimental Pharmacology and Toxicology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany German Center for Heart Research (DZHK), Kiel, Germany These authors contributed equally to this work Search for more papers by this author Anika E Knaust Anika E Knaust Department of Experimental Pharmacology and Toxicology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany German Center for Heart Research (DZHK), Kiel, Germany These authors contributed equally to this work Search for more papers by this author Umber Saleem Umber Saleem Department of Experimental Pharmacology and Toxicology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany German Center for Heart Research (DZHK), Kiel, Germany Search for more papers by this author Malte Loos Malte Loos Department of Experimental Pharmacology and Toxicology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany German Center for Heart Research (DZHK), Kiel, Germany Search for more papers by this author Janice Raabe Janice Raabe Department of Experimental Pharmacology and Toxicology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany German Center for Heart Research (DZHK), Kiel, Germany Search for more papers by this author Diogo Mosqueira Diogo Mosqueira orcid.org/0000-0002-2306-3915 Division of Cancer & Stem Cells, Biodiscovery Institute, University of Nottingham, Nottingham, UK Search for more papers by this author Sandra Laufer Sandra Laufer Department of Experimental Pharmacology and Toxicology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany German Center for Heart Research (DZHK), Kiel, Germany Search for more papers by this author Michaela Schweizer Michaela Schweizer Electron Microscopy Unit, Center for Molecular Neurobiology Hamburg, University Medical Center Hamburg-Eppendorf, Hamburg, Germany Search for more papers by this author Petra van der Kraak Petra van der Kraak Department of Pathology, University Medical Center Utrecht, Utrecht University, Utrecht, The Netherlands Search for more papers by this author Frederik Flenner Frederik Flenner Department of Experimental Pharmacology and Toxicology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany German Center for Heart Research (DZHK), Kiel, Germany Search for more papers by this author Bärbel M Ulmer Bärbel M Ulmer Department of Experimental Pharmacology and Toxicology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany German Center for Heart Research (DZHK), Kiel, Germany Search for more papers by this author Ingke Braren Ingke Braren German Center for Heart Research (DZHK), Kiel, Germany Vector Core Unit, University Medical Center Hamburg-Eppendorf, Hamburg, Germany Search for more papers by this author Xiaoke Yin Xiaoke Yin King’s British Heart Foundation Centre of Research Excellence, King's College London, London, UK Search for more papers by this author Konstantinos Theofilatos Konstantinos Theofilatos King’s British Heart Foundation Centre of Research Excellence, King's College London, London, UK Search for more papers by this author Jorge Ruiz-Orera Jorge Ruiz-Orera Cardiovascular and Metabolic Sciences, Max Delbrück Center for Molecular Medicine in the Helmholtz Association (MDC), Berlin, Germany Search for more papers by this author Giannino Patone Giannino Patone Cardiovascular and Metabolic Sciences, Max Delbrück Center for Molecular Medicine in the Helmholtz Association (MDC), Berlin, Germany Search for more papers by this author Birgit Klampe Birgit Klampe Department of Experimental Pharmacology and Toxicology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany German Center for Heart Research (DZHK), Kiel, Germany Search for more papers by this author Thomas Schulze Thomas Schulze Department of Experimental Pharmacology and Toxicology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany German Center for Heart Research (DZHK), Kiel, Germany Search for more papers by this author Angelika Piasecki Angelika Piasecki Department of Experimental Pharmacology and Toxicology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany German Center for Heart Research (DZHK), Kiel, Germany Search for more papers by this author Yigal Pinto Yigal Pinto Department of Experimental Cardiology, Academic Medical Center, Amsterdam, The Netherlands Search for more papers by this author Aryan Vink Aryan Vink Department of Pathology, University Medical Center Utrecht, Utrecht University, Utrecht, The Netherlands Search for more papers by this author Norbert Hübner Norbert Hübner Cardiovascular and Metabolic Sciences, Max Delbrück Center for Molecular Medicine in the Helmholtz Association (MDC), Berlin, Germany DZHK (German Centre for Cardiovascular Research), Berlin, Germany Charité -Universitätsmedizin, Berlin, Germany Berlin Institute of Health (BIH), Berlin, Germany Search for more papers by this author Sian Harding Sian Harding British Heart Foundation Centre of Research Excellence, NHLI, Imperial College London, London, UK Search for more papers by this author Manuel Mayr Manuel Mayr King’s British Heart Foundation Centre of Research Excellence, King's College London, London, UK Search for more papers by this author Chris Denning Chris Denning Division of Cancer & Stem Cells, Biodiscovery Institute, University of Nottingham, Nottingham, UK Search for more papers by this author Thomas Eschenhagen Thomas Eschenhagen Department of Experimental Pharmacology and Toxicology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany German Center for Heart Research (DZHK), Kiel, Germany Search for more papers by this author Arne Hansen Corresponding Author Arne Hansen [email protected] orcid.org/0000-0003-2628-0453 Department of Experimental Pharmacology and Toxicology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany German Center for Heart Research (DZHK), Kiel, Germany Search for more papers by this author Friederike Cuello Friederike Cuello orcid.org/0000-0003-1612-1715 Department of Experimental Pharmacology and Toxicology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany German Center for Heart Research (DZHK), Kiel, Germany These authors contributed equally to this work Search for more papers by this author Anika E Knaust Anika E Knaust Department of Experimental Pharmacology and Toxicology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany German Center for Heart Research (DZHK), Kiel, Germany These authors contributed equally to this work Search for more papers by this author Umber Saleem Umber Saleem Department of Experimental Pharmacology and Toxicology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany German Center for Heart Research (DZHK), Kiel, Germany Search for more papers by this author Malte Loos Malte Loos Department of Experimental Pharmacology and Toxicology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany German Center for Heart Research (DZHK), Kiel, Germany Search for more papers by this author Janice Raabe Janice Raabe Department of Experimental Pharmacology and Toxicology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany German Center for Heart Research (DZHK), Kiel, Germany Search for more papers by this author Diogo Mosqueira Diogo Mosqueira orcid.org/0000-0002-2306-3915 Division of Cancer & Stem Cells, Biodiscovery Institute, University of Nottingham, Nottingham, UK Search for more papers by this author Sandra Laufer Sandra Laufer Department of Experimental Pharmacology and Toxicology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany German Center for Heart Research (DZHK), Kiel, Germany Search for more papers by this author Michaela Schweizer Michaela Schweizer Electron Microscopy Unit, Center for Molecular Neurobiology Hamburg, University Medical Center Hamburg-Eppendorf, Hamburg, Germany Search for more papers by this author Petra van der Kraak Petra van der Kraak Department of Pathology, University Medical Center Utrecht, Utrecht University, Utrecht, The Netherlands Search for more papers by this author Frederik Flenner Frederik Flenner Department of Experimental Pharmacology and Toxicology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany German Center for Heart Research (DZHK), Kiel, Germany Search for more papers by this author Bärbel M Ulmer Bärbel M Ulmer Department of Experimental Pharmacology and Toxicology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany German Center for Heart Research (DZHK), Kiel, Germany Search for more papers by this author Ingke Braren Ingke Braren German Center for Heart Research (DZHK), Kiel, Germany Vector Core Unit, University Medical Center Hamburg-Eppendorf, Hamburg, Germany Search for more papers by this author Xiaoke Yin Xiaoke Yin King’s British Heart Foundation Centre of Research Excellence, King's College London, London, UK Search for more papers by this author Konstantinos Theofilatos Konstantinos Theofilatos King’s British Heart Foundation Centre of Research Excellence, King's College London, London, UK Search for more papers by this author Jorge Ruiz-Orera Jorge Ruiz-Orera Cardiovascular and Metabolic Sciences, Max Delbrück Center for Molecular Medicine in the Helmholtz Association (MDC), Berlin, Germany Search for more papers by this author Giannino Patone Giannino Patone Cardiovascular and Metabolic Sciences, Max Delbrück Center for Molecular Medicine in the Helmholtz Association (MDC), Berlin, Germany Search for more papers by this author Birgit Klampe Birgit Klampe Department of Experimental Pharmacology and Toxicology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany German Center for Heart Research (DZHK), Kiel, Germany Search for more papers by this author Thomas Schulze Thomas Schulze Department of Experimental Pharmacology and Toxicology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany German Center for Heart Research (DZHK), Kiel, Germany Search for more papers by this author Angelika Piasecki Angelika Piasecki Department of Experimental Pharmacology and Toxicology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany German Center for Heart Research (DZHK), Kiel, Germany Search for more papers by this author Yigal Pinto Yigal Pinto Department of Experimental Cardiology, Academic Medical Center, Amsterdam, The Netherlands Search for more papers by this author Aryan Vink Aryan Vink Department of Pathology, University Medical Center Utrecht, Utrecht University, Utrecht, The Netherlands Search for more papers by this author Norbert Hübner Norbert Hübner Cardiovascular and Metabolic Sciences, Max Delbrück Center for Molecular Medicine in the Helmholtz Association (MDC), Berlin, Germany DZHK (German Centre for Cardiovascular Research), Berlin, Germany Charité -Universitätsmedizin, Berlin, Germany Berlin Institute of Health (BIH), Berlin, Germany Search for more papers by this author Sian Harding Sian Harding British Heart Foundation Centre of Research Excellence, NHLI, Imperial College London, London, UK Search for more papers by this author Manuel Mayr Manuel Mayr King’s British Heart Foundation Centre of Research Excellence, King's College London, London, UK Search for more papers by this author Chris Denning Chris Denning Division of Cancer & Stem Cells, Biodiscovery Institute, University of Nottingham, Nottingham, UK Search for more papers by this author Thomas Eschenhagen Thomas Eschenhagen Department of Experimental Pharmacology and Toxicology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany German Center for Heart Research (DZHK), Kiel, Germany Search for more papers by this author Arne Hansen Corresponding Author Arne Hansen [email protected] orcid.org/0000-0003-2628-0453 Department of Experimental Pharmacology and Toxicology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany German Center for Heart Research (DZHK), Kiel, Germany Search for more papers by this author Author Information Friederike Cuello1,2, Anika E Knaust1,2, Umber Saleem1,2, Malte Loos1,2, Janice Raabe1,2, Diogo Mosqueira3, Sandra Laufer1,2, Michaela Schweizer4, Petra Kraak5, Frederik Flenner1,2, Bärbel M Ulmer1,2, Ingke Braren2,6, Xiaoke Yin7, Konstantinos Theofilatos7, Jorge Ruiz-Orera8, Giannino Patone8, Birgit Klampe1,2, Thomas Schulze1,2, Angelika Piasecki1,2, Yigal Pinto9, Aryan Vink5, Norbert Hübner8,10,11,12, Sian Harding13, Manuel Mayr7, Chris Denning3, Thomas Eschenhagen1,2 and Arne Hansen *,1,2 1Department of Experimental Pharmacology and Toxicology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany 2German Center for Heart Research (DZHK), Kiel, Germany 3Division of Cancer & Stem Cells, Biodiscovery Institute, University of Nottingham, Nottingham, UK 4Electron Microscopy Unit, Center for Molecular Neurobiology Hamburg, University Medical Center Hamburg-Eppendorf, Hamburg, Germany 5Department of Pathology, University Medical Center Utrecht, Utrecht University, Utrecht, The Netherlands 6Vector Core Unit, University Medical Center Hamburg-Eppendorf, Hamburg, Germany 7King’s British Heart Foundation Centre of Research Excellence, King's College London, London, UK 8Cardiovascular and Metabolic Sciences, Max Delbrück Center for Molecular Medicine in the Helmholtz Association (MDC), Berlin, Germany 9Department of Experimental Cardiology, Academic Medical Center, Amsterdam, The Netherlands 10DZHK (German Centre for Cardiovascular Research), Berlin, Germany 11Charité -Universitätsmedizin, Berlin, Germany 12Berlin Institute of Health (BIH), Berlin, Germany 13British Heart Foundation Centre of Research Excellence, NHLI, Imperial College London, London, UK *Corresponding author. Tel: +49 40 741057207; E-mail: [email protected] EMBO Mol Med (2021)13:e13074https://doi.org/10.15252/emmm.202013074 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract The phospholamban (PLN) p.Arg14del mutation causes dilated cardiomyopathy, with the molecular disease mechanisms incompletely understood. Patient dermal fibroblasts were reprogrammed to hiPSC, isogenic controls were established by CRISPR/Cas9, and cardiomyocytes were differentiated. Mutant cardiomyocytes revealed significantly prolonged Ca2+ transient decay time, Ca2+-load dependent irregular beating pattern, and lower force. Proteomic analysis revealed less endoplasmic reticulum (ER) and ribosomal and mitochondrial proteins. Electron microscopy showed dilation of the ER and large lipid droplets in close association with mitochondria. Follow-up experiments confirmed impairment of the ER/mitochondria compartment. PLN p.Arg14del end-stage heart failure samples revealed perinuclear aggregates positive for ER marker proteins and oxidative stress in comparison with ischemic heart failure and non-failing donor heart samples. Transduction of PLN p.Arg14del EHTs with the Ca2+-binding proteins GCaMP6f or parvalbumin improved the disease phenotype. This study identified impairment of the ER/mitochondria compartment without SR dysfunction as a novel disease mechanism underlying PLN p.Arg14del cardiomyopathy. The pathology was improved by Ca2+-scavenging, suggesting impaired local Ca2+ cycling as an important disease culprit. Synopsis The disease mechanism linking the phospholamban (PLN) p.Arg14del mutation to dilated cardiomyopathy is incompletely understood. In this study, patient-derived human induced pluripotent stem cell-cardiomyocytes were used to elucidate this molecular mechanism. Sarcoplasmic reticulum function remained unaltered in PLN p.Arg14del human cardiomyocytes. Impairment of the interface between endoplasmic reticulum and mitochondria was discovered as a novel disease phenotype. Cytoplasmic calcium-scavenging improved the cardiomyopathy phenotype and revealed the role for cytoplasmic calcium in disease development. The paper explained Problem The mutation p.Arg14del in the gene encoding for human phospholamban (hPLN) causes dilated cardiomyopathy in patients. The precise molecular disease mechanisms remain incompletely understood. PLN is an important regulator of cytoplasmic calcium import into its internal stores, the sarcoplasmic (SR) and endoplasmic (ER) reticulum. Results Dermal fibroblasts from a PLN p.Arg14del mutation carrier were rederived to human-induced pluripotent stem cells (hiPSCs). The mutation was corrected by CRISPR/Cas9 technology. Control and PLN p.Arg14del hiPSC lines were differentiated into hiPSC cardiomyocytes (hiPSC-CMs) and analyzed in a three-dimensional engineered heart tissue organoid format. PLN p.Arg14del hiPSC-CM revealed significantly lower force development and the occurrence of an irregular beating pattern. Inter-organelle dysfunction between the ER and mitochondria was observed by RNA-seq and proteomic analyses and further validated by independent methodologies. Interestingly, cytoplasmic calcium lowering achieved by viral-mediated expression of calcium-binding proteins improved the cardiomyopathy phenotype. Impact We modeled PLN p.Arg14del cardiomyopathy in hiPSC-CM. This in vitro disease model allowed the demonstration of cytosolic calcium scavenging as a novel, promising therapeutic approach for future individualised precision medicine. Introduction Dilated cardiomyopathy (DCM) is a common cause of severe heart failure (HF). Mutations in several genes central to cardiomyocyte biology have been described to cause DCM (McNally & Mestroni, 2017). The disease relevance of mutations in the gene encoding for phospholamban (PLN) was first discovered in 2003 (Haghighi et al, 2003; Schmitt et al, 2003). PLN is a 6.1 kDa protein localized in the membrane of the sarcoplasmic and endoplasmic reticulum (SR/ER). It is regulated by protein kinase-mediated phosphorylation and acts as a brake on the sarcoplasmic/endoplasmic reticulum Ca2+ ATPase (SERCA2). In its non-phosphorylated state, PLN inhibits Ca2+ re-uptake into the SR/ER by forming a hetero-dimer with SERCA2. Upon phosphorylation by cAMP (PKA)- or cGMP (PKG)-dependent protein kinase at serine 16 or Ca2+/calmodulin-dependent protein kinase II (CaMKII) at threonine 17, PLN dissociates from SERCA2 and forms a homo-pentamer, resulting in increased SERCA2 Ca2+ affinity and facilitated Ca2+ re-uptake into the SR/ER. Hence, PLN acts as a key regulator at the interphase between Ca2+ homoeostasis and force production. Overexpression of PLN in mice resulted in reduced SR Ca2+ uptake, diminished Ca2+ transient amplitude, and impaired left ventricular function (Kadambi et al, 1996). In contrast, heterozygous or homozygous PLN knockout mouse models demonstrated improved SR Ca2+ import and increased contractility (Luo et al, 1994, 1996; Hoit et al, 1995), suggesting a close correlation between PLN protein abundance and contractile function in mice (Lorenz & Kranias, 1997). In recent years, several human DCM mutations in PLN have been discovered. PLN p. Leu39 results in expression of a truncated unstable PLN protein. In cellular expression systems, the PLN p. Leu39stop mutation leads to a maximally active SERCA2a pump. Heterozygous and homozygous mutant carriers exhibit severe cardiomyopathy (Haghighi et al, 2003). Importantly, substantial species differences exist regarding the disease outcome of PLN deficiency. While beneficial effects resulted from PLN ablation in mice, the absence of PLN protein in patients was associated with severe cardiomyopathy (Haghighi et al, 2003). PLN p. Arg9Leu and PLN p. Arg9His mutations potentially led to altered interaction with PKA and abnormal phosphorylation of PKA substrate proteins (Medeiros et al, 2011). Finally, PLN p. Arg9Cys, PLN p. Arg25Cys, and PLN p. Arg14del were described to exert super-inhibitory effects on SERCA2a activity (Schmitt et al, 2003; Haghighi et al, 2012; Liu et al, 2015). PLN p. Arg14del was first identified in a greek family (Haghighi et al, 2006) and subsequently identified as a DCM founder mutation in the Netherlands, where this mutation is accounting for up to 15% of all DCM cases (Van Der Zwaag et al, 2012). Clinically, PLN p. Arg14del mutation carriers present with left ventricular dysfunction and dilation and malignant ventricular arrhythmia. Transgenic overexpression of PLN p. Arg14del in wild-type (WT) mice resulted in the development of cardiomyopathy and reduced survival. SR preparations from PLN p. Arg14del overexpressing mice showed a small, but significant rightward shift of the SR Ca2+-uptake rate, suggesting that PLN p. Arg14del, similar to the other mutant forms, acts as a super-inhibitor of SERCA2a (Haghighi et al, 2006). The effect was shown to depend on the presence of WT PLN, because transgenic overexpression of PLN p. Arg14del in a PLN knockout mouse model did not alter Ca2+ re-uptake kinetics (Haghighi et al, 2012). Of note, normal SR Ca2+ uptake in this model was associated with a similarly severe DCM phenotype, indicating that mechanisms distinct from abnormal SR Ca2+ cycling contribute to the pathology of PLN p. Arg14del cardiomyopathy. Homozygous PLN p. Arg14del knock-in mice exhibited a DCM phenotype, with cardiac dilation and fibrosis, accompanied by contractile dysfunction, and arrhythmia (Eijgenraam et al, 2020). It is difficult to elucidate disease mechanisms exerted by PLN mutations in mice. This is exemplified by the antithetic effects observed in response to complete PLN deletion, which relates to the lower chronotropic reserve of the mouse versus the human heart (Kranias & Hajjar, 2012). This limitation emphasizes the necessity for human-based studies to unravel disease mechanisms associated with PLN p. Arg14del. Histological analysis of explanted human hearts and left ventricular biopsies from PLN p. Arg14del heart failure (HF) patients showed dense perinuclear globular PLN-positive aggregates as a disease-specific characteristic (te Rijdt et al, 2016, 2017). Human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) from a DCM patient carrying a PLN p. Arg14del mutation revealed a significantly higher spontaneous beating frequency, irregular Ca2+ transients, and lower force development than respective isogenic controls (Karakikes et al, 2015; Stillitano et al, 2016). The authors concluded that the phenotype was compatible with super-inhibition of SERCA2a by PLN p. Arg14del. However, the higher caffeine-induced Ca2+ transient amplitude in PLN p. Arg14del cardiomyocytes is difficult to reconcile with super-inhibition of SERCA2a and indicative of other, yet unknown consequences of PLN p. Arg14del. The present study aimed at shedding new light on the molecular disease mechanisms evoked by the PLN p. Arg14del mutation and involved: (i) establishment of a patient-specific PLN p. Arg14del hiPSC line and a respective repaired isogenic control; (ii) phenotypical and functional characterization of hiPSC-CM in a three-dimensional engineered heart tissue (EHT) format showing preserved SR function; (iii) proteomic and RNA-seq analysis suggesting impairment of the ER/mitochondria compartment; (iv) confirmation by histology of end-stage HF patient and non-failing human heart samples; and (v) improvement of the disease phenotype by heterologous expression of Ca2+ scavengers. Taken together, the data suggest that PLN p. Arg14del cardiomyopathy is associated with functional alterations in the ER/mitochondria compartment with improvement by Ca2+ scavenging. Results Clinical profile and hiPSC derivation A female 31-year-old DCM patient was identified as a carrier of a heterozygous PLN p. Arg14del mutation. The patient was diagnosed with DCM at the age of 28 years and presented clinically with HF symptoms and ventricular arrhythmia. Her mother was also identified as a PLN p. Arg14del mutation carrier and diagnosed with DCM (Fig 1A). Patient-derived dermal fibroblasts were reprogrammed to hiPSC, and a respective isogenic control hiPSC line (PLNic) was generated by CRISPR/Cas9 technology (Fig 1B). Pluripotency was demonstrated by immunofluorescence (IF) staining for Oct-4A or TRA-1-60. G-banding demonstrated a normal karyotype (Appendix Fig S1A–E). No off-target effects after CRISPR/Cas9 genome editing were detected by sequencing the 10 most likely off-target loci (Appendix Fig S1F). Cardiomyocytes from PLN p. Arg14del (78 ± 15% positive for cTNT; mean ± SD, n = 9 batches from two clones) and PLNic hiPSC (80 ± 16% positive for cTNT; mean ± SD, n = 9 batches from two clones) were differentiated, and hiPSC-CMs were cultured in two-dimensional (2D) and three-dimensional (3D, engineered heart tissue; EHT) format. Quantitative RT–PCR revealed no detectable PLN p. Arg14del mRNA in PLNic hiPSC-CM and approximately 50% expression of both, PLN WT and p. Arg14del mRNA, in PLN p. Arg14del hiPSC-CM (Fig 1C). Analysis of total RNA from left ventricular samples obtained from two independent DCM patients carrying a heterozygous PLN p. Arg14del mutation revealed a ~1:1 ratio of PLN WT and p. Arg14del mRNA (Fig 1D). IF analysis of 2D hiPSC-CM revealed similar perinuclear PLN and SERCA2 protein localization in both genotypes, albeit staining was weaker in PLN p. Arg14del (Fig 1E, Appendix Fig S1G and H). Western blot analysis of EHTs showed lower abundance of monomeric PLN protein in PLN p. Arg14del and PLNic versus human non-failing heart (NFH), with no difference in the pentameric PLN form (Fig 2A and B). The close proximity between PLN p. Arg14 and PLN p. Ser16, the phosphorylation target site, raised the question, whether the response to the β-adrenoceptor agonist isoprenaline (ISO) was altered in PLN p. Arg14del. Western immunoblot analysis showed that ISO significantly enhanced band intensity of the pentameric pSer16 PLN only for PLNic, suggesting lower ISO-mediated phosphorylation of PLN p. Arg14del (Fig 2C and D). Functionally, ISO exerted a positive inotropic (Force PLNic: +61%, PLN p. Arg14del: +64%) and lusitropic effect (relaxation time PLNic: −7%, PLN p. Arg14del: −11%), without apparent differences between genotypes (Fig 2E and F, Appendix Fig S2A). This showed that despite lower ISO-mediated phosphorylation in PLN p. Arg14del, the canonical inotropic and lusitropic force responses to ISO remained unchanged. Figure 1. HiPSC line derivation and validation A. Pedigree of the patient family. Arrow: Index patient; index patient and mother are PLN p.Arg14del mutation carriers and were diagnosed with DCM. B. Sequencing of PLN patient-derived hiPSC (PLN p.Arg14del) and isogenic controls (PLNic) to confirm CRISPR/Cas9-mediated gene correction. Red box indicates codon for Arg14, and blue box indicates silent PAM (protospacer adjacent motif) mutation. C, D. Relative PLN transcript level (normalized to ACTN2 and PLNic wt+mt) with PCR primers amplifying PLN wild type (wt) and p.Arg14del (mt), only wt or only p.Arg14del (mt). (C) Analysis of PLNic (n = 9 EHTs from 5 batches, each replicate consists of a pool of 1–2 EHTs from separate batches) and PLN p.Arg14del (n = 9 EHTs from 3 batches, each replicate consists of a pool of 2–4 EHTs from separate batches) EHTs, mean ± SEM. (D) Analysis of PLN p.Arg14del EHTs (n = 9 EHTs from 3 batches, each replicate consists of a pool of 2–4 EHTs from separate batches) and two independent failing heart patient samples from heterozygous PLN p.Arg14del mutation carriers (each n = 1 RNA sample, 2 separate PCR analysis), mean ± SEM. E. Immunofluorescence of 2D hiPSC-CM from each genotype with antibodies against PLN (red) and SERCA2 (green) and DAPI staining of nuclei (blue); scale bar 20 µm. Download figure Download PowerPoint Figure 2. PLN expression profile and response to isoprenaline A. Western immunoblot analysis of total PLN in non-failing heart tissue (NFH), PLNic, and PLN p.Arg14del EHTs. Pentameric (PM) and monomeric (MM) form of PLN under boiled (B) and non-boiled (NB) conditions. B. PLN protein quantification of PM and MM forms under non-boiled condition. NFH (n = 5 protein samples from two different donor hearts), PLNic (n = 8, each replicate consists of a pool of 3 EHTs from three different separate batches), and PLN p.Arg14del (n = 8, each replicate consists of a pool of 3–4 EHTs from three different separate batches). Loading was normalized to α-actinin. One-way ANOVA of PM and MM forms with Tukey’s post-test, mean ± SEM, * P < 0.05. C. Western immunoblot analysis of PLN pSer16 MM and PM in PLNic and p.Arg14del EHTs (non-boiled) in the absence and presence of isoprenaline (ISO; 100 nM, loading was normalized to α-actinin). D. PLN pSer16 quantification of PM and MM form (non-boiled; loading was normalized to α-actinin). PLNic ± isoprenaline (n = 8, each replicate consists of a pool of 3–4 EHTs from three different separate batches) and PLN p.Arg14del ± isoprenaline (n = 8, each replicate consists of a pool of 3–4 EHTs from three different separate batches). One-way ANOVA of PM and MM forms with Sidak’s post-test, *P < 0.05. Mean ± SEM. E, F. Force (E) and relaxation time (RT) (F) of PLNic and PLN p.Arg14del EHTs in Tyrode's solution (Ca2+ 1.8 mM), Tyrode’s solution (EC50 [Ca2+]: PLNic: 0.4–0.6 mM, PLN p.Arg14del: 0.7–0.8 mM), in the presence of ISO (100 nM) or carbachol (10 µM); PLNic (n = 27 EHTs from 4 batches), PLN p.Arg14del EHTs (n = 19 EHTs from 3 batches), mean ± SEM, one-way ANOVA for PLNic or PLN p.Arg14del EHTs with Tukey’s post hoc t
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