Selective silencing of p38δ in the ex vivo human heart organotypic slices using hydrophobic conjugate-mediated delivery of chemically engineered siRNA

Introduction: The dose-dependent cardiotoxic effects of doxorubicin (DOX) limit the use of the widely used chemotherapy. p38 mitogen-activated kinases (p38 MAPKs) have been identified as critical regulators of DOX-induced cardiotoxicity. We previously reported that the p38δ systemic genetic deletion protected female mice from DOX-induced cardiotoxicity. However, p38δ-selective small-molecule inhibitors are not currently available. Hydrophobic conjugate-mediated delivery of chemically stabilized small interfering RNAs (siRNAs) provides a new avenue to achieve selective, non-toxic, durable, and transient gene silencing in the heart.

Materials and

Methods: Human left ventricular organotypic cardiac slices were generated from five donor human hearts unsuitable for transplantation. A panel of p38δ-selective siRNA sequences was screened, and the lead siRNA si-p38δ-1, with the highest silencing efficacy and potency in vitro, was identified. Cardiac slices (n = 3 per treatment group) were treated with 1 mM cholesterol- or docosanoic acid (DCA)-conjugated si-p38δ-1, non-targeting control siRNA
(NTC), or mock-treated with control culture medium (CNTL, M199 Medium, 2% penicillin-streptomycin, 1x ITS, 10 mM BDM) for 72 hours. p38δ protein and mRNA knockdown levels were assessed by western blot and QuantiGene Assay, respectively. Global changes in protein expression were evaluated via label-free quantification mass spectrometry.

Results, Conclusions, and Discussions: We observed 77% (p = 0.06) of protein and 71% (p = 0.05) of mRNA silencing of p38δ in si-p38δ-1-treated human cardiac slices after 72 hours, validating the efficacy of this approach in ex vivo human cardiac tissue. p38δ silencing resulted in the downregulation of nine proteins (encoded by ACTN3, KIF20B, DNAJA1, PFKFB2, MRPS7, GPD1, SQSTM1, MRPL46, and CCDC51 genes), which are involved in the regulation of the cytoskeletal, metabolic, ribosomal, and autophagy functions. In addition, p38d silencing led to the upregulation of five proteins (encoded by ADD1, TBCB, OBSL1, RPL18A, and EML1 genes) implicated in regulating cytoskeletal and ribosomal functions.

Acknowledgements (Optional): Hydrophobic conjugate-mediated delivery of chemically engineered siRNAs provides selective, non-toxic, transient, and durable silencing of p38δ in human left ventricular cardiac organotypic slices. Our findings support a role for p38δ in regulating the cytoskeletal, metabolic, autophagy, and ribosomal functions in the heart. Future studies will assess whether selective p38δ silencing is cardioprotective during DOX treatment in the human heart and elucidate the underlying mechanisms.