Targeting unfolded protein response reverts ER stress and ER Ca2+ homeostasis in cardiomyocytes expressing the pathogenic variant of Lamin A/C R321X

Abstract

Background We previously demonstrated that an Italian family affected by a severe dilated cardiomyopathy (DCM) with history of sudden deaths at young age, carried a mutation in the Lmna gene encoding for a truncated variant of the Lamin A/C protein (LMNA), R321X. When expressed in heterologous systems, such variant accumulates into the endoplasmic reticulum (ER), inducing the activation of the PERK-CHOP pathway of the unfolded protein response (UPR), ER dysfunction and increased rate of apoptosis. The aim of this work was to analyze whether targeting the UPR can be used to revert the ER dysfunction associated with LMNA R321X expression in HL-1 cardiac cells. Methods HL-1 cardiomyocytes stably expressing LMNA R321X were used to assess the ability of 3 different drugs targeting the UPR, salubrinal, guanabenz and empagliflozin to rescue ER stress and dysfunction. In these cells, the state of activation of both the UPR and the pro-apoptotic pathway were analyzed monitoring the expression levels of phospho-PERK, phospho-eIF2α, ATF4, CHOP and PARP-CL. In addition, we measured ER-dependent intracellular Ca2+ dynamics as indicator of proper ER functionality. Results We found that salubrinal and guanabenz increased the expression levels of phospho-eIF2α and downregulated the apoptosis markers CHOP and PARP-CL in LMNA R321X-cardiomyocytes, maintaining the so-called adaptive UPR. These drugs also restored ER ability to handle Ca2+ in these cardiomyocytes. Interestingly, we found that empagliflozin downregulated the apoptosis markers CHOP and PARP-CL shutting down the UPR itself through the inhibition of PERK phosphorylation in LMNA R321X-cardiomyocytes. Furthermore, upon empagliflozin treatment, ER homeostasis, in terms of ER ability to store and release intracellular Ca2+ was also restored in these cardiomyocytes. Conclusions We provided evidence that the different drugs, although interfering with different steps of the UPR, were able to counteract pro-apoptotic processes and to preserve the ER homeostasis in R321X LMNA-cardiomyocytes. Of note, two of the tested drugs, guanabenz and empagliflozin, are already used in the clinical practice, thus providing preclinical evidence for ready-to-use therapies in patients affected by the LMNA R321X associated cardiomyocytes.

Multi-omics analysis reveals attenuation of cellular stress by empagliflozin in high glucose-treated human cardiomyocytes

“…Proteomics results evidenced mainly a modulation and involvement of ER-associated proteins. In this regard, in addition to mitochondrial dysfunction, ER stress has been associated with diabetic cardiomyopathy and different anti-glycemic drugs classes have been reported to possess the ability to reduce ER stress, such as metformin, GLP-1 agonists as well as SGLT-2i [ 61 – 63 ]. Among statistically significant proteins, EMPA was able to revert the effect of HG-mediated reduction of the small GTPase Rab1A (RAB1A), which is involved in autophagy.…”

Neuroprotective Effect of Antiapoptotic URG7 Protein on Human Neuroblastoma Cell Line SH-SY5Y

“…A dysregulation of the balance Ca 2+ /ROS between the ER and the mitochondria is an established etiopathogenetic factor of various disorders, including protein misfolding pathologies [ 17 , 41 , 42 , 43 ]. Moreover, tunicamycin, by altering the homeostasis of the ER and triggering the UPR, induces an alteration in the homeostasis of Ca 2+ , which itself contributes to the UPR [ 14 , 44 ]. Therefore, the Ca 2+ level within the ER under a stress condition, chemically induced by tunicamycin, was studied.…”

Cardiovascular protection of YiyiFuzi powder and the potential mechanisms through modulating mitochondria-endoplasmic reticulum interactions

“…The ER stress response and the UPR, triggered by ER stress, play crucial roles in maintaining cardiomyocyte homeostasis ( Table 3 ; Figure 4 ), which are important for cardiovascular health ( Zhang et al, 2017 ; Ren et al, 2021 ; Pietrafesa et al, 2023 ). Excessive ER stress disrupts secretory pathways and contributes to the progression of CVD ( Zhang et al, 2017 ; Fu and Doroudgar, 2022 ).…”