Characterizing the Role of Palmitoylation in Cardiac Fibroblast Activation and Fibrosis

Abstract

Cardiac fibrosis is characterized by maladaptive accumulation of collagen and extracellular matrix (ECM) components in the heart, a condition that decreases cardiac function and accelerates heart failure. Cardiac fibroblasts, the principal cellular mediators of cardiac fibrosis, are normally quiescent, though injury-related signals trigger their activation and increases in their proliferation, migration, and synthesis and secretion of ECM materials. While a number of cardiac fibroblast activation pathways are established, much remains unknown about how these pathways are regulated, in part from the difficulty of their study in vitro posed by their spontaneous activation in non-physiologic stiffness conditions present in cell culture. The post-translational lipid modification known as palmitoylation, mediated by a family of enzymes called S-acyltransferases, has emerged as a critical regulator of disease signaling, though little is known of how palmitoylation influences pathways in the heart. Recently, pro-fibrotic signaling pathways have been shown to be regulated by palmitoylation in other cell types by the S-acyltransferases zDHHC3 and zDHHC7, including the GTPase Rac1, stressing the need to explore the possibility that palmitoylation is regulating adult cardiac fibroblast (ACF) signaling. We hypothesized that deletion of Zdhhc3, Zdhhc7, or their combined deletion in ACFs would result in blunted fibroblast activation and reduced fibrosis in vivo. Further, we hypothesized that ACFs with impaired Rac1 palmitoylation (Rac1 ConKI) would show reduced activation in vitro. To test these hypotheses, a physiologic-stiffness polydimethylsiloxane (PDMS) coverslip coating protocol was developed to maintain ACF quiescence in vitro. Zdhhc3 fl/fl mice and whole-body Zdhhc7 knockout (KO) mice were used to test for changes in ACF activation in vitro and cardiac fibrosis in vivo, and our newly developed Cre-inducible Rac1 palmitoylation-deficient mutant knock-in mouse model (Rac1 ConKI) was used to test for changes in ACF activation in vitro. Zdhhc3 KO ACFs in vitro responded comparably to controls in migration and levels of TGFβ1-induced α-smooth muscle actin (αSMA, hallmark activated fibroblast marker) expression, and levels of the fibroblast marker genes Postn and Tcf21. Using an in vivo pressure overload model of cardiac injury, no significant differences were observed in cardiac function, hypertrophy, or fibrosis in myofibroblast Zdhhc3 KO mice compared to controls. Zdhhc7 KO and Zdhhc3/7 KO ACFs in vitro exhibited reduced Postn expression in response to TGFβ1 compared to controls, though no significant differences were observed in collagen gene expression or nuclear factor of activated T cells (NFAT) transcription factor activity. A migratory defect was present in Zdhhc7 KO ACFs, and a downward trend was seen in Zdhhc3/7 KO ACFs compared to controls. In vivo, hypertrophy and contractility in response to chronic angiotensin II/phenylephrine (AngII/PE) infusion were unchanged in Zdhhc7 KO and cardiac fibroblast-specific Zdhhc3/7 KO mice compared to controls. Zdhhc7 and Zdhhc3/7 KO mice similarly exhibited comparable fibrotic gene mRNA levels in response to AngII/PE, and no significant differences were detected in ventricular fibrosis. Rac1 ConKI ACFs had increased levels of stress-inducible NFAT and SRF transcription factor activity, though showed no differences in αSMA expression or fibrotic gene mRNA levels compared to controls when treated with TGFβ1, nor were changes observed in migration. These data suggest that ACF zDHHC3 and zDHHC7 are not essential for the development of cardiac fibrosis and that TGFβ1-induced ACF activation is not dependent on Rac1 palmitoylation. Future experiments will focus on identifying key S-acyltransferases and palmitoylated substrates involved in the cardiac fibroblast activation process.