Characterization of fibroblasts from hypertrophied right ventricle of pulmonary hypertensive rats

Abstract

Pulmonary arterial hypertension (PAH), which is characterized by an elevation of pulmonary arterial resistance, leads to a lethal right heart failure. It is an urgent issue to clarify the pathogenesis of PAH-induced right heart failure. The present study aimed to elucidate the characteristics of cardiac fibroblasts (CFs) isolated from hypertrophied right ventricles of monocrotaline (MCT)-induced PAH model rats. CFs were isolated from the right ventricles of MCT-injected rats (MCT-CFs) and saline-injected control rats (CONT-CFs). Expression of α-smooth muscle actin and collagen type I in MCT-CFs was lower than that in CONT-CFs. On the other hand, proliferation, migration, and matrix metalloproteinase (MMP)-9 production were significantly enhanced in MCT-CFs. In MCT-CFs, phosphorylation of extracellular signal-regulated kinase (ERK) 1/2, c-Jun N-terminal kinase (JNK), and Ca/calmodulin-dependent protein kinase (CaMK) II was significantly enhanced. In addition to mRNA expression of Orai1, a Ca release-activated Ca channel, and stromal interaction molecules (STIM) 1, an endoplasmic reticulum Ca sensor, the associated store-operated Ca entry (SOCE) was significantly higher in MCT-CFs than CONT-CFs. Pharmacological inhibition of ERK1/2 pathway prevented the enhanced proliferation of MCT-CFs. The enhanced migration of MCT-CFs was prevented by a pharmacological inhibition of ERK1/2, JNK, CaMKII, or SOCE pathway. The enhanced MMP-9 production in MCT-CFs was prevented by a pharmacological inhibition of ERK1/2, CaMKII, or SOCE pathway but not JNK. The present results suggested that MCT-CFs exhibit proliferative and migratory phenotypes perhaps through multiple signaling pathways. This study for the first time determined the characteristics of CFs isolated from hypertrophied right ventricles of MCT-induced PAH model rats.

Periostin Mediates Right Ventricular Failure through Induction of Inducible Nitric Oxide Synthase Expression in Right Ventricular Fibroblasts from Monocrotaline-Induced Pulmonary Arterial Hypertensive Rats

“…POSTN, which is associated with the development of cardiac fibrosis [17,19], is known to promote the phenotypical change of fibroblasts into myofibroblasts with the increase of α-smooth muscle actin (α-SMA) expression and collagen type I production [10,37,38,39]. In contrast, we previously demonstrated that the phenotype of RVFbs isolated from MCT-injected rats is entirely different from that of myofibroblasts [23]. In the RVFbs, the proliferation, migration and matrix metalloproteinase-9 expression were increased, but the expression of α-SMA and collagen type I was decreased [23].…”

“…In contrast, we previously demonstrated that the phenotype of RVFbs isolated from MCT-injected rats is entirely different from that of myofibroblasts [23]. In the RVFbs, the proliferation, migration and matrix metalloproteinase-9 expression were increased, but the expression of α-SMA and collagen type I was decreased [23]. It is thus suggested that POSTN might be involved in the PAH-induced RVF of MCT-injected rats through the regulation of RVFbs functions other than the induction of differentiation into myofibroblasts.…”

“…RVFbs were isolated from RVs of MCT (3 weeks)-injected rats and CONT rats, and also from non-treated normal rats as described previously [23]. The heart excised from rats was enzymatically digested by 0.02% collagenase using a modified Langendorff apparatus.…”

“…RVFbs (passage 1–5) and H9c2 cardiomyoblasts (passage 16–28, ATCC, Manassas, VA, USA) were cultured in DMEM supplemented with 10% FBS and 1% antibiotic-antimycotic mix stock solution as previously described [23,50]. Cells at confluence were peeled off with Trypsin- ethylenediaminetetraacetic acid (EDTA) (Nacalai Tesque), suspended in culture medium and seeded in 6- or 12-well culture dish for experiments.…”

“…We have recently demonstrated that POSTN mRNA expression is increased in RVs of monocrotaline (MCT)-induced PAH model rats [22]. Moreover, phenotypical changes of RV fibroblasts (RVFbs) such as hyper-proliferation and migration in MCT-induced PAH model rats were observed [23]. However, the relationship between POSTN and phenotypical changes of RVFbs in the pathogenesis of PAH-induced RVF has not been elucidated.…”