The Unfolded Protein Response as a Compensatory Mechanism and Potential Therapeutic Target in PLN R14del Cardiomyopathy
Authors: Folkert W Asselbergs
Michelle Vu
Albert J Suurmeijer
Ting-Hsuan Wu
Wouter P Te Rijdt
Prashila L Amatya
Joost P G Sluijter
Magdalena Harakalova
Jennifer Arthur Ataam
Isaac Perea-Gil
Aryan Vink
Logan Dunkenberger
Maricela Prado
Jiayi Pei
Karim Sallam
Ioannis Karakikes
Yuan Zhang
Dries A M Feyen
Mark Mercola
Nirmal Vadgama
Alexandra A Gavidia
Renee G C Maas
Journal: Circulation (2021)
Discipline: CLINICAL MEDICINE
Keywords: cardiomyopathy, dilated; induced pluripotent stem cells; models, biological; phospholamban; sequence analysis, RNA
Affiliations: Cardiovascular Institute, Stanford University, Stanford, CA;  Department of Medicine, Stanford University School of Medicine, Stanford, CA, Cardiovascular Institute, Stanford University, Stanford, CA; Department of Cardiothoracic Surgery, Stanford University School of Medicine, Stanford, CA, Department of Cardiology, Division Heart and Lungs, University Medical Center Utrecht, University of Utrecht, Utrecht, The Netherlands, Department of Cardiothoracic Surgery, Stanford University School of Medicine, Stanford, CA, Department of Pathology, University Medical Center Utrecht, University of Utrecht, Utrecht, The Netherlands, Deptment of Pathology, University Medical Center Groningen, University of Groningen, Groningen, The Netherlands, Netherlands Heart Institute, Utrecht, The Netherlands; Department of Genetics, University Medical Center Groningen, University of Groningen, Groningen, The Netherlands, Cardiovascular Institute, Stanford University, Stanford, CA; Department of Medicine, Stanford University School of Medicine, Stanford, CA, Department of Cardiology, Division Heart and Lungs, University Medical Center Utrecht, University of Utrecht, Utrecht, The Netherlands; Institute of Cardiovascular Science, Faculty of Population Health Sciences, University College London, London, United Kingdom; Health Data Research UK and Institute of Health Informatics, University College London, London, United Kingdom

Abstract:  Background: Phospholamban (PLN) is a critical regulator of calcium cycling and contractility in the heart. The loss of arginine at position 14 in PLN (R14del) is associated with dilated cardiomyopathy with a high prevalence of ventricular arrhythmias. How the R14 deletion causes dilated cardiomyopathy is poorly understood, and there are no disease-specific therapies. Methods: We used single-cell RNA sequencing to uncover PLN R14del disease mechanisms in human induced pluripotent stem cells (hiPSC-CMs). We used both 2-dimensional and 3-dimensional functional contractility assays to evaluate the impact of modulating disease-relevant pathways in PLN R14del hiPSC-CMs. Results: Modeling of the PLN R14del cardiomyopathy with isogenic pairs of hiPSC-CMs recapitulated the contractile deficit associated with the disease in vitro. Single-cell RNA sequencing revealed the induction of the unfolded protein response (UPR) pathway in PLN R14del compared with isogenic control hiPSC-CMs. The activation of UPR was also evident in the hearts from PLN R14del patients. Silencing of each of the 3 main UPR signaling branches (IRE1, ATF6, or PERK) by siRNA exacerbated the contractile dysfunction of PLN R14del hiPSC-CMs. We explored the therapeutic potential of activating the UPR with a small molecule activator, BiP (binding immunoglobulin protein) inducer X. PLN R14del hiPSC-CMs treated with BiP protein inducer X showed a dose-dependent amelioration of the contractility deficit in both 2-dimensional cultures and 3-dimensional engineered heart tissues without affecting calcium homeostasis. Conclusions: Together, these findings suggest that the UPR exerts a protective effect in the setting of PLN R14del cardiomyopathy and that modulation of the UPR might be exploited therapeutically. 

Retracted: No

PubPeer comments:
 figure 1.b. of the paper In the patient knock-in of the WT allele, the heterozygous position witnessing correct insertion of ssODN sequence is the first not shown but early green line could indicate a solo A base (blue arrow). Authors might have included this position to confirm heterozygous status of the clone (and not potential hemizygosity due to loss of one allele - the WT here). In addition, the black line with green filling at position 5 is surprising (green arrow). In the healthy donor knock-in of delArg14: If it is correctly edited the silent C to A change in ssODN should have left 2 A bases at position 8 (red arrow) on the electropherogram. This is not the case; the sequence seems to be a true heterozygous carrier rather than an edited one. However the redrawing of peaks is evident on the zoomed panel. Finaly, there is apparent copy-past of part of the figure with redrawings. ![file] ![file] ,