209 Fatty Acid Oxidation in PLN R14del Cardiomyopathy 8 treatment. Briefly, cells were loaded 30 min in FluoroBrite DMEM Media (Thermo Fisher) supplemented with 1.25 µM Cal-520 (Abcam) and 0.02% Pluronic F-127 solution (Sigma Aldrich). Spontaneous calcium transients were recorded between two and three weeks after plating. Video streams of Cal-520 (the green channel) lasting 10 seconds at 33 fps were automatically scanned by a Leica Thunder microscope. Image analysis was conducted using Cyteseer (Vala Sciences, California, USA) as previously described.18 The physiological parameters; peak value (normalized area under the peak trace), rise time and decay time were automatically calculated for each time series. Data tables were analyzed with Microsoft Excel and drug responses and bar plots were generated with GraphPad Prism 9 software. Characterization of hiPSC-CMs by immunofluorescence staining. HiPSC-CMs were cultured on Matrigel-coated coverslips, fixed in paraformaldehyde (4%), and permeabilized in blocking/permeabilization buffer (5% BSA/0,3% Triton-X-100 in PBS) for 30 minutes. Primary antibodies were added and incubated in 1:5 diluted blocking/permeabilization buffer DPBS overnight at 4°C. Cells were washed four times with PBS (5 mins each time) and incubated with Alexa-conjugated secondary antibodies diluted in 1:5 blocking/permeabilization buffer in the dark at room temperature for 1 hr. Cells were washed as previously described and nuclei were stained using 1 µg/ml Hoechst (Life Technologies) for 15 mins. Coverslips were mounted using Fluoromount-G (Southern Biotech) and images were acquired using a Leica SP8X confocal microscope. Immunofluorescence stainings of Nile-red were quantified by the lipid mask macro of Image J, displaying the number of lipids/nuclei and were quantified using one-way ANOVA (n=3/3 coverslips/images per well, n=3 biological replicates, n=>10 wells per condition). Details of antibodies were shown in Table S8. RESULTS Identification of histone acetylation changes in PLN-R14del cardiac tissue H3K27ac histone acetylation regulates gene transcription and contributes to phenotypic responses in heart diseases.25 Therefore, we performed H3K27ac ChIP-seq to study histone acetylation changes in 6 PLN-R14del versus 4 control hearts (Fig.1A and Table S1A). We identified 28,149±9,538, and 182 25,721±8,460 H3K27ac enriched regions within PLN-R14del and control hearts, respectively. We subsequently combined regions that were identified in at least two independent samples into a set of 23,356 regions to assess differentially acetylated regions between control and PLN-R14del groups. In total, 2,107 autosomal regions showed differential H3K27ac levels between PLN-R14del and control hearts (Fig.1B and Table S2A). Compared to controls, regions with higher H3K27ac levels in PLN-R14del hearts are referred to as hyperacetylated regions (n=1,149) and regions with lower H3K27ac levels in PLN-R14del hearts are referred to as hypoacetylated regions (n=958, Fig.1C and Fig.S1).
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