253 Unfolded Protein Response in PLN R14del Cardiomyopathy 9 UPR Activity Protects Contractile Function in hiPSC-CMs Harboring PLN-R14del In principle, UPR could have a pathological or beneficial effect on the PLN R14del cardiomyocytes. To establish the role of the UPR, we carried out a loss-of-function experiment in which we attenuated the main transducers of the UPR (IRE1, ATF6, and PERK; Figure 4A). These interconnected signaling branches of the UPR provide the cells with an adaptive response to ER stress to restore proteostasis.22 Under baseline conditions, targeting each of the 3 branches by siRNA did not significantly affect the contractility in either PLN WT (HD and patient R14del corrected) or PLN R14del (patient and HD R14del introduced) hiPSC-CMs, although a trend toward decreased function (≈10%) was observed with siRNA against IRE1 and PERK in PLN R14del (Figure 4B and 4C) hiPSC-CMs. Given that long-term adrenergic stimulation dramatically increased UPR fluxes in hiPSC-CMs harboring PLN R14del (Figure 2I), we next evaluated the impact of the loss of function in the presence of isoproterenol. Under adrenergic stress, the contractility of PLN WT hiPSC-CMs remained unchanged after the siRNA-mediated knockdown of IRE1 and ATF6, but we observed significantly decreased contractile function after PERK knockdown (14.3±3.7%; Figure 4D). In contrast, we observed a significant contractility deficit in PLN R14del hiPSC-CMs upon knockdown of each of the 3 UPR arms compared with control siRNA treated (siRNA against IRE1, −15.5±4.2%; siRNA against ATF6, −16.7±4.1%; siRNA against PERK, −36.6±5%), indicating that the mutant hiPSC-CMs were sensitized to a loss of UPR signaling in a genotype-specific manner (Figure 4E). Taken together, these data suggest that activation of the UPR in PLN R14del hiPSC-CMs preserves cell function and therefore plays a protective role in alleviating ER stress and potentially blunts disease pathogenesis. Pharmacological Targeting the UPR in PLNR14del Ameliorates Contractility Next, we evaluated whether pharmacologically stimulating the UPR pathway in PLN R14del hiPSC-CMs beyond basal levels would have a positive effect on cardiomyocyte function. BiP protein inducer X (BiX) is a small molecule proteostasis regulator that increases expression of the BiP protein to consequently induce UPR and protect neurons from ER stress.23,24 In both PLN R14del (patient and HD R14del introduced) and PLN WT (HD and patient R14del corrected) hiPSC-CMs, treatment with BiX induced a dose-dependent increase in a reporter for XBP1-splicing (indicative of activated UPR). The maximal effect was higher in PLN R14del than in corresponding isogenic control hiPSC-CMs (Emax=33.1±7% versus 56.8±7% XBP-1 splice positive cells in normal versus R14del hiPSC-CMs, respectively; Figure IVb and IVc in the Data Supplement). BiX (0.1 µmol/L) significantly enhanced contractility in PLN R14del and PLN WT hiPSC-CMs as detected in 2-dimensional cell sheets (Figure 4G and 4H). In the EHTs, this translated to an increase in force from about 20 µN in untreated EHTs to 40 µN in treated EHTs, corresponding to a level statistically indistinguishable from isogenic control EHTs (Figure 4I and 4J). Remarkably, the increase in peak contractility was more pronounced in the PLN R14del hiPSC-CMs, and BiX treatment (0.1 µmol/L) led to a nearly complete restoration of
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