216 Chapter 8 basal OCR was significantly lower in PLN-R14del versus control hiPSC-CMs (Fig.4G), implying an impaired FAO in the PLN-R14del hiPSC-CMs at the beginning. Although OCRs of both PLNR14del and control hiPSC-CMs decreased after FAO- and glycolysis-blockages, OCRs remained significantly lower in PLN-R14del hiPSC-CMs than in the controls. These findings suggest that despite the presence of excess lipids in the medium, PLN-R14del hiPSC-CMs are less capable of utilizing their FAO metabolism to produce the required energy, therefore being more glycolysis-dependent. Higher glucose dependency in PLN-R14del hiPSC-CMs Glucose metabolism, another major metabolic program in cardiomyocytes (Fig.4B), was also studied. We measured the extracellular acidification rate (ECAR) to study the activity of the glycolytic pathway in PLN-R14del and wild-type hiPSC-CMs by manipulating the FAO and glucose metabolism using ETO and 2-DG, respectively (Fig.4D). In the maturation medium, basal ECAR levels were comparable between both groups, but it became significantly lower in PLN-R14del versus control hiPSC-CMs after FAO- and glycolysis-blockages (Fig.4H), indicating a higher glycolysis dependency of PLN-R14del hiPSC-CMs. A significantly higher glycolytic reserve was also observed in PLN-R14del hiPSC-CMs than in the controls. In the glucoserich medium, ECAR levels remained comparable between both groups at the basal level, after blocking FAO, and after blocking glycolysis (Fig.4I). However, the decline of ECAR after glycolysis-blockage was more profound in PLN-R14del versus control hiPSC-CMs, suggesting a higher glycolysis-dependence and a higher glycolytic reserve of PLN-R14del hiPSC-CMs. In the lipid-rich medium, basal ECAR levels were comparable between both groups (Fig.4J). Interestingly, a higher ECAR was shown in PLN-R14del versus control hiPSC-CMs after FAOblockage, which decreased profoundly after glycolysis-blockage, again, confirming the higher glycolysis-dependency in PLN-R14del hiPSC-CMs. These results imply our in vitro maturation-induced PLN-R14del hiPSC-CM mimics the heart failure-related metabolic alterations consisting of the energy production reduction by mitochondria through oxidative phosphorylation and an increase in (anaerobic) glycolysis. Intracellular lipid droplet accumulation is a key feature of PLN-R14del cardiomyopathy Data acquired from the cardiac tissues and/or hiPSC-CMs at DNA, RNA, protein, and functional levels consistently pointed towards altered metabolism in PLN-R14del versus control groups, particularly FAO. Since lipid accumulation is known as the hallmark of impaired FAO, we examined the lipid accumulation in cardiac tissues and hiPSC-CMs. First, we performed a digital quantification in heart slices assessing the percentage of adipose tissue, which showed an increased adipocyte deposition in PLN-R14del versus control hearts (Fig.S8). Next, we used Nile red to localise intracellular lipid droplets in snap-frozen and paraffin-embedded tissues and observed a more frequent perinuclear accumulation of lipid droplets in PLN-R14del
RkJQdWJsaXNoZXIy MTk4NDMw