Mitochondrial PKD Activates Mitochondrial Fission and Proliferative Signaling in Cardiac Fibroblasts

• Published in: The FASEB journal Vol. 36; no. S1
• Main Authors: Zhou, Xiaoxu, Adhikari, Neeta, Cypress, Michael W., Polina, Iuliia, Landherr, Maria, Chaput, Isabel, Suckow, Mark A., Choudhary, Gaurav, O‐Uchi, Jin, Jhun, Bong Sook
• Format: Journal Article
• Language: English
• Published: United States The Federation of American Societies for Experimental Biology 01-05-2022
• ISSN: 0892-6638; 1530-6860
• DOI: 10.1096/fasebj.2022.36.S1.R6274

Introduction
Cardiac fibrosis, characterized by wall stiffening, reduced contractility, and impaired overall heart performance, is a self‐reinforcing process in response to injury (e.g., myocardial infarction) and pressure overload (e.g., systemic hypertension and pulmonary arterial hypertension [PAH]). The severity of cardiac fibrosis is associated with cardiac dysfunction, increased risk of arrhythmia, and mortality. However, there is a lack of effective therapies designed to inhibit or reverse cardiac fibrosis. Hyperproliferation of cardiac fibroblasts (CFs) and their differentiation into myofibroblasts are the key contributors to cardiac fibrosis. We previously showed that mitochondrial reactive oxygen species (mROS) are one of the regulators for activating proliferative signaling in rat neonatal CFs. Moreover, we reported that phosphorylation of dynamin‐related protein 1 (DRP1) by a stress‐responsive protein kinase D (PKD) at the outer mitochondrial membrane (OMM) promotes mROS generation via increased mitochondrial fission in H9c2 cardiac myoblasts.
Hypothesis
Mitochondrial PKD‐DRP1 signaling increases mitochondrial fission leading to increased mROS levels, which activates proliferative signaling.
Methods
Primary adult CFs from human ventricles and the right ventricles (RVs) of a SU5415/hypoxia‐induced rat PAH model were used. Mitochondrial morphology and mROS levels were measured by confocal microscopy.
Results
Overexpression of PKD1 increased DRP1 phosphorylation at Ser637, and activates proliferative signaling pathways including ERK1/2 and p38 in human CFs, but did not promote their differentiation into myofibroblasts, assessed by the amount of a‐smooth muscle actin. To specifically identify the involvement of OMM‐localized PKD activity in these processes, we generated an OMM‐targeted dominant‐negative PKD1 (termed mt‐PKD‐DN) by adding an OMM‐target sequence (amino acid 1‐33 from human TOM20) at the N‐terminus of PKD1‐K612W. Importantly, mt‐PKD‐DN was able to inhibit mitochondrial fission, decrease mROS, hyperpolarize mitochondrial membrane potential, and ultimately decrease proliferation in human CFs. To further understand the role of PKD‐DRP1 signaling in vivo, we employed a preclinical PAH animal model. Immunofluorescence staining of RVs from PAH rats revealed that PKD activity and DRP1 phosphorylation increased only in CFs, but not in ventricular myocytes. RV‐CFs isolated from PAH rats exhibited increased mitochondrial fission, mROS levels, and proliferative signaling compared to RV‐CFs from control rats, and these changes were abolished by adenoviral expression of mt‐PKD‐DN.
Conclusion
Mitochondrial mROS elevation via PKD‐DRP1‐dependent mitochondrial fission promotes CF proliferation. Targeting mitochondrial PKD may be a potential therapeutic strategy to reduce cardiac fibrosis under pathological conditions such as PAH.