Search

Introduction:Right ventricular dysfunction (RVD) is the leading cause of death in pulmonary arterial hypertension (PAH). Metabolic derangements are the most rigorously studied and best understood molecular signatures in RVD, but the link between metabolic dysfunction and other cellular phenotypes in cardiomyocytes are incompletely understood. We previously showed that junctophilin-2 (JPH2) misregulation in the RV causes t-tubule disruptions which promotes RVD. Recent data demonstrated JPH2 is co-regulated with mitofusin-2 (MFN2), a key regulator of mitochondrial function, in embryonic stem cell derived cardiomyocytes. However, the link between JPH2 and MFN2, mitochondrial function, and t-tubule remodeling in the RV in PAH has not been evaluated.Hypothesis:JPH2 binds and subsequently stabilizes MFN2, which links t-tubule remodeling to metabolic derangements in the RV.Methods:Western blot was used to quantify protein expression and confocal microscopy was used to examine localization patterns of JPH2 and MFN2. Co-immunoprecipitation and pulldown experiments of purified JPH2 and MFN2 were used to examine protein-protein interaction. Metabolomics were used to probe metabolic state of RV samples in monocrotaline (MCT) and pulmonary artery banded (PAB) rats.Results:In cardiomyocytes, JPH2 and MFN2 colocalize at the t-tubules, and mitochondrial enrichments showed JPH2 and MFN2 were both found in mitochondrial extracts. Co-immunoprecipitation experiments demonstrated JPH2 and MFN2 biochemically interact, and pulldown experiments of purified JPH2 and MFN2 revealed the interaction was direct. In MCT RV extracts, JPH2 and MFN2 were both reduced by over 50%, and this was associated with t-tubule remodeling and metabolic derangements. Importantly, other mitochondrial fission (Fis1 and dynamin-related protein-1) and fusion (mitofusin-1 and optic atrophy 1) proteins were not coregulated with JPH2. Conversely, in PAB rats, JPH2 and MFN2 were downregulated by only 25% which was associated with less severe t-tubule derangements and metabolic disruption.Conclusions:A direct JPH2-MFN2 interaction links t-tubule remodeling to metabolic dysfunction in RVD.

Background:Pulmonary arterial hypertension (PAH) is a disease of the cardiopulmonary unit which often results in death from failure of the right ventricle (RVF). The RV adapts to the increased afterload by hypertrophy (RVH) and increasing contractility, ultimately these compensatory mechanisms are insufficient, resulting in RVF. Increased abundance of immune cells (including macrophages) occurs in the RV of patients with PAH and preclinical models of RVH, both with PAH (monocrotaline, MCT rats) and without PAH (pulmonary artery banded rats, PAB rats). Macrophages could contribute to heart failure through activation of the nucleotide-binding domain, leucine-rich-containing family, pyrin domain-containing-3 (NLRP3) inflammasome. The role of macrophages and inflammasome in the RV dysfunction in PAH is not very well understood.Hypothesis:The inflammasome pathway is more activated in macrophages within the RV in MCT-RVH (a model with increased inflammation) compared to a noninflammatory model with similar severity of RVH (PAB).Methods:Studies were performed in adult male, Sprague Dawley rats, n= 20. The macrophages (CD68+ cells) and NLRP3 inflammasome expression in the RV were assessed using immunofluorescence confocal microscopy in control, PAB and MCT RVH. Echocardiographic assessment of RV function was performed.Results:MCT, PAB and control rats RV free wall diastolic thickness was (0.82?0.04mm, 1.22?0.25mm and 0.54? 0.10mm respectively). Macrophages prevalence was greatest in MCT (38.8? 12) vs PAB (22.1 ? 3.0) vs control (9.4 ? 1.0). However, macrophages were not the major source of NLPR3 in the heart and lungs. In the heart, NLPR3 was strongly expressed in endothelial and stromal cells (not yet identified) in MCT-rats. In the lung, expression of NLPR3 was localized to endothelial, adventitial and some CD68+ cells.Conclusion and Future Directions:MCT-RVs have a greater macrophage count and greater expression of NLRP3 than do PAB-RVs. Interestingly NLRP3 is localized to the cardiac endothelial cells. Further experiments are required to evaluate the functional significance of increased macrophage counts and NLRP3 in RVH associated with PAH.

Introduction:Pulmonary arterial hypertension (PAH) is a progressive and lethal pulmonary arterial vasculopathy that causes right ventricle dysfunction (RVD). We recently demonstrated that microtubule-mediated junctophilin-2 (MT-JPH2 pathway) downregulation promotes t-tubule disruption and RVD; however, the upstream regulator of this pathway is not defined. Interleukin-6 (IL6), an inflammatory cytokine, promotes microtubule remodeling in isolated neonatal cardiomyocytes via its downstream signaling protein signal transducer and activator of transcription 3 (STAT3). However, relationship between IL6 and the MT-JPH2 pathway in PAH related RVD is unknown.Hypothesis:IL6 signaling promotes RVD in PAH by dysregulating the MT-JPH2 pathway.Methods:Immunoblots of right ventricular (RV) and left ventricular extracts from control, monocrotaline (MCT), and pulmonary artery-banded (PAB) rats were probed for the IL6 signaling and the MT-JPH2 pathway. Ventricular cardiomyocytes were isolated and cultured in the presence of IL6 or IL6 plus a STAT3 inhibitor. Confocal microscopy was used to examine microtubule density, junctophilin-2 localization, and t-tubule organization. Echocardiography was used to quantify RV function in rats. Finally, serum levels of IL6 in PAH patients (n=72) were used to examine the link between IL6 and RVD in PAH patients.Results:MCT rats had more severe RVD than PAB rats. Western blot analysis revealed greater IL6 activation in the RV of MCT than PAB rats. This resulted in more dysregulation of the MT-JPH2 pathway in MCT RVs than PAB RVs. Importantly, IL6 signaling was blunted in MCT and PAB left ventricular extracts, which was associated with less MT stability and higher JPH2 levels. In isolated cardiomyocytes, IL6 activated the MT-JPH2 pathway which resulted in adverse t-tubule remodeling, but this effect was mitigated when a STAT3 inhibitor was used. Finally, PAH patients with higher IL6 levels had higher N-terminal pro-brain natriuretic peptide levels despite having no differences in pulmonary vascular disease severity.Conclusions:IL6 signaling dysregulates the MT-JPH2 pathway, and the degree of IL6 signaling is related to RVD severity.