245 Unfolded Protein Response in PLN R14del Cardiomyopathy 9 siRNAs (Silencer Select, Ambion) with 0.2 µL Lipofectamine RNAiMax (Invitrogen) 20 nM final concentration. Prior to analysis, the iPSC-CMs cells were loaded with Tetramethylrhodamine methyl ester dye (TMRM, 400 nM) and fluorescent time-lapse images were acquired automatically using the IC200 KIC instrument (Vala Sciences) at an acquisition frequency of 100 Hz for a duration of 10s using a 20x objective (0.75 NA). The contractility image analysis was performed using custom particle image velocity software as previously described45. XBP1-splicing reporter / AAV production. The F-XBP1 and F-XBP1ΔDBD7 were a kind gift from Dr. Miura (University of Tokyo). The two XBP1 splicing reporters were cloned into the pAAV-CMV vector (Takara). HEK293T cells were co-transfected (Lipofectamine 2000, Thermo) with pAAV-F-XBP1 (or pAAV-F-XBP1ΔDBD), pRC2, and pHelper (Takara). ). After 3 days the cells were collected and the AAV particles extracted from the cell pellets using the AAVpro extraction solutions (Takara). The AAV2 particles were incubated at 37 °C for 30 minutes with Cryonase Cold-active Nuclease (2 U/μl) and purified with a resin-based approach according to manufacturer’s instructions (AAVpro purification kit, Takara). The purity was evaluated by the SDS-PAGE and the amount of viral genome was quantified using a real-time qPCR assay using the ITR sequence of AAV2 as a target according to the manufacturer’s protocol (Takara). The hiPSC-CMs were seeded in a 384-well at 20,000 cells per well and infected with 5x103 viral genomes per cell. Fluorescent images were acquired with the IC200 KIC (Vala Sciences, San Diego, CA). For quantification, nuclear expression (F-XBP1) was calculated as a percentage of positive nuclei to total nuclei (Hoechst 33342, Invitrogen), and cytoplasmic expression (F-XBP1ΔDBD) was calculated as integrated fluorescent density Patient RNA sequencing for UPR genes. Human cardiac tissue collection. This study was approved by the Biobank Research Ethics Committee, University Medical Center Utrecht, Utrecht, the Netherlands (protocol number WARB 12/387). Written informed consent was obtained or in certain cases waived by the ethics committee when obtaining informed consent was not possible due to the death of the individual. Heart samples collected at autopsy or transplantation were obtained from a homogeneous cohort of patients who all carried the same pathogenic PLN R14del mutation (n = 6). Four control hearts obtained from rejected organ donors (n = 3) or from autopsy (n =1) were used as a reference. Human heart tissue immunohistochemistry. Human heart tissue was obtained from the local tissue biobanks of the Departments of Pathology from the University Medical Center Utrecht and the University Medical Center Groningen, The Netherlands. Three explanted hearts from heart failure patients carrying the heterozygous pathogenic PLN R14del variant and autopsy heart tissue from three sudden death patients with the PLN R14del variant expected to harbor PLN aggregates in cardiomyocytes were included. Immunohistochemistry
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