218 Chapter 8 cardiac tissue than in the control (Fig.5A). Transmission electron microscopy revealed a significantly lower mitochondrial length (aspect ratio) and a significantly higher accumulation of intracellular lipid droplets in PLN-R14del versus control hearts (Fig.5B and Fig.5C), suggesting impaired mitochondrial FAO. In line with the hearts, highly accumulated intracellular lipid droplets were observed in PLN-R14del versus control hiPSC-CMs, regardless of different culturing media (Fig.5D). Combined, these findings suggested intracellular lipid droplet accumulation is a key feature in PLN-R14del cardiomyopathy. CRISPR/Cas9-based correction of PLN-R14del attenuated intracellular lipid accumulation We used CRISPR/Cas9-based gene editing to correct PLN-R14del mutation28 and observed a significant reduction of intracellular lipid droplets in R14del-corrected hiPSC-CMs (isogenic control, Fig.6A). Interestingly, hiPSC-CMs derived from an asymptomatic PLN-R14del carrier and the homozygous hiPSC-CMs showed a comparable amount of the lipid droplets as in PLN-R14del hiPSC-CMs. These findings suggested a tight relationship between PLNR14del mutation and impaired lipid metabolism, thereby leading to intracellular lipid droplet accumulation. PPARA-targeted drug increased Ca2+ handling and mitochondrial trifunctional protein levels in PLN-R14del hiPSC-CMs. We also applied a PPARA agonist (bezafibrate) to further investigate PPARA-mediated FAO in PLN-R14del cardiomyocytes. First, we measured the Ca2+ transient in control and PLN-R14del hiPSC-CMs with and without bezafibrate treatment. We observed significantly increased rise and decay time in treated versus untreated PLN-R14del hiPSC-CMs (Fig.6B), whereas the treatment did not affect Ca2+ handling in control hiPSC-CMs. Next, we examined HADHA and HADHB levels, encoding the mitochondrial trifunctional protein involved in the FAO pathway, which showed suppressed histone acetylation and transcriptional levels in PLN-R14del versus control hearts (Fig.1I and Fig.2C). We confirmed the suppression of HADHA and HADHB in long-term cultured untreated PLN-R14del cardiomyocytes and further showed elevated HADHA and HADHB levels in bezafibrate-treated PLN-R14del cardiomyocytes (Fig.6C). Whereas, the treatment did not alter HADHA and HADHB levels in control hiPSC-CMs. Combined, these findings suggested disturbed HADHA/HADHB-involved FAO in PLN-R14del hiPSC-CMs, the potential association between PPARA-mediated FAO and Ca2+ handling, and the role of PPARA as a promising therapeutic target in PLN-R14del cardiomyopathy.
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