Ying-Ju Lai

BACKGROUND: Arrhythmogenic cardiomyopathy is an inherited disorder characterized by fibro-fatty myocardial replacement and ventricular arrhythmias. Although desmosomal mutations such as desmoglein-2 ( DSG2 ) are well-established causes, the pathogenic mechanisms of specific missense variants remain incompletely defined. METHODS: We generated a physiologically relevant Dsg2 ^F536C/F536C knock-in mouse model using CRISPR/Cas9 to mimic the human DSG2 p.Phe531Cys mutation. Comprehensive phenotyping included histopathology, immunostaining, transcriptomic profiling, in vitro cardiomyocyte and fibroblast assays, in vivo imaging and ECG analysis, and ex vivo optical mapping. Therapeutic potential was assessed using the PPAR-γ (peroxisome proliferator-activated receptor gamma) antagonist GW9662. RESULTS: Dsg2 ^F536C/F536C mice developed progressive cardiac hypertrophy, interstitial fibrosis, lipid accumulation, and inducible ventricular arrhythmias following isoproterenol infusion and programmed electrical stimulation. These changes led to severe cardiac dysfunction and reduced survival. Mechanistically, the mutation caused reduced DSG2 and nuclear accumulation of β-catenin and PPAR-γ, promoting triacylglycerol biosynthesis, oxidative stress, cardiomyocyte death, and calcium-handling abnormalities. We also identified activation of epicardial epithelial-to-mesenchymal transition and paracrine fibroblast activation via IL-6 (interleukin-6) and PDGF-BB (platelet-derived growth factor-BB) as key contributors to fibrotic remodeling. Optical mapping revealed prolonged and heterogeneous action potential duration, with both reentrant and focal ectopic mechanisms of ventricular tachycardia. Treatment with GW9662 attenuated lipid accumulation, fibrosis, reactive oxygen species production, and arrhythmogenic susceptibility. CONCLUSIONS: The Dsg2 ^F536C/F536C knock-in mouse is a genotype-specific arrhythmogenic cardiomyopathy model that links desmosomal dysfunction to metabolic remodeling, epicardial epithelial-to-mesenchymal transition, and electrophysiological instability. PPAR-γ inhibition ameliorated structural and arrhythmogenic remodeling, supporting PPAR-γ as a potential therapeutic target and advancing precision strategies for desmosome-related cardiomyopathies.

Chronic thromboembolic pulmonary hypertension (CTEPH) is a progressive vascular disorder characterized by persistent thromboembolic obstruction and fibrotic remodeling of the pulmonary arteries. Although autophagy has been implicated in various forms of pulmonary hypertension, its specific role in CTEPH pathogenesis remains unclear. In this study, histological, immunohistochemical, and molecular analyses of pulmonary artery lesions from CTEPH patients revealed increased collagen deposition and elevated expression of autophagy markers beclin-1 and microtubule-associated protein 1A/1B-light chain 3 (LC3) compared to non-CTEPH controls. Primary pulmonary artery endothelial cells (PAECs) derived from CTEPH patients exhibited dysregulated autophagy-related protein expression. Mechanistically, TGF-β stimulation induced autophagy via the Smad3 signaling pathway. Notably, riociguat, a soluble guanylate cyclase stimulator approved for CTEPH, modulated autophagy-related proteins in vitro. In hypoxia- and TGF-β-induced pulmonary hypertension mouse models, riociguat attenuated LC3 but not beclin-1 expression and significantly reduced right ventricular systolic pressure. These findings suggest that LC3 may be a key autophagy marker in CTEPH. These findings identify the TGF-β/Smad3-LC3 axis as a critical mechanism driving CTEPH and suggest that pharmacological modulation of autophagy, particularly via riociguat, offers a promising therapeutic opportunity, especially for patients ineligible for surgical intervention. KEY MESSAGES: Beclin-1 and LC3, key autophagy markers, are upregulated in thrombofibrotic pulmonary arteries from patients with CTEPH. TGF-β activates autophagy in pulmonary artery endothelial cells through the Smad3 signaling pathway. Riociguat treatment downregulates LC3 expression, improves pulmonary hemodynamics, and reduces vascular remodeling in experimental PH models. LC3 as a potential biomarker and therapeutic target in the progression of CTEPH driven by autophagy.

Right heart failure is a major cause of mortality in patients with pulmonary arterial hypertension (PAH). This study aimed to evaluate the preventive anti‐remodeling effect of a natural phenolic compound, caffeic acid phenethyl ester (CAPE), on pressure‐overloaded right hearts. PAH was induced in Sprague–Dawley rats by intraperitoneal injection of monocrotaline (MCT; 60 mg/kg). The rats were randomly treated with CAPE (30 mg/kg/day, i.p.) or vehicle for 28 days. Right ventricular (RV) function was assessed, and remodeling was examined using both ex vivo and in vitro analyses. Chronic CAPE treatment in MCT rats attenuated right heart hypertrophy, fibrosis, and oxidative stress. CAPE improved RV function and normalized the elevated RV pressure and QTc interval in anesthetized animals. It also restored the prolonged QT interval and ventricular refractory period and reduced arrhythmia vulnerability in perfused hearts. CAPE normalized the prolonged action potential duration in right heart tissues. In RV myocytes, the delayed kinetics of Ca ²⁺ transients and cell contraction were also corrected. Furthermore, CAPE reversed the PAH‐induced downregulation of sarco(endo)plasmic reticulum Ca ²⁺ ‐ATPase 2a (SERCA2a) and restored the densities of transient outward, steady‐state outward, and inward rectifier K ⁺ currents, along with the expression of their corresponding channel proteins. CAPE also significantly mitigated RV remodeling induced by PA banding, a model of fixed PA stenosis. These results indicate that CAPE ameliorates structural and electromechanical abnormalities in pressure‐overloaded right hearts, likely through inhibition of oxidative stress. CAPE may represent a potential therapeutic candidate in PAH‐related cardiac remodeling and dysfunction. Trial Registration: NCT03049046

Chronic thromboembolic pulmonary hypertension (CTEPH) is a progressive vascular disorder characterized by persistent thromboembolic obstruction and fibrotic remodeling of the pulmonary arteries. Although autophagy has been implicated in various forms of pulmonary hypertension, its specific role in CTEPH pathogenesis remains unclear. In this study, histological, immunohistochemical, and molecular analyses of pulmonary artery lesions from CTEPH patients revealed increased collagen deposition and elevated expression of autophagy markers beclin-1 and microtubule-associated protein 1A/1B-light chain 3 (LC3) compared to non-CTEPH controls. Primary pulmonary artery endothelial cells (PAECs) derived from CTEPH patients exhibited dysregulated autophagy-related protein expression. Mechanistically, TGF-β stimulation induced autophagy via the Smad3 signaling pathway. Notably, riociguat, a soluble guanylate cyclase stimulator approved for CTEPH, modulated autophagy-related proteins in vitro. In hypoxia- and TGF-β-induced pulmonary hypertension mouse models, riociguat attenuated LC3 but not beclin-1 expression and significantly reduced right ventricular systolic pressure. These findings suggest that LC3 may be a key autophagy marker in CTEPH. These findings identify the TGF-β/Smad3-LC3 axis as a critical mechanism driving CTEPH and suggest that pharmacological modulation of autophagy, particularly via riociguat, offers a promising therapeutic opportunity, especially for patients ineligible for surgical intervention.

The loss of peroxisome proliferator-activated receptor gamma (PPARγ) exacerbates pulmonary arterial hypertension (PAH), while its upregulation reduces cell proliferation and vascular remodeling, thereby decreasing PAH severity. SGLT2 inhibitors, developed for type 2 diabetes, might also affect signal transduction in addition to modulating sodium-glucose cotransporters. Pulmonary arterial smooth muscle cells (PASMCs) isolated from patients with idiopathic pulmonary arterial hypertension (IPAH) were treated with three SGLT2 inhibitors, canagliflozin (Cana), dapagliflozin (Dapa), and empagliflozin (Empa), to investigate their antiproliferative effects. To assess the impact of Empa on PPARγ, luciferase reporter assays and siRNA-mediated PPARγ knockdown were employed to examine regulation of the γ-secretase complex and its downstream target Notch3. Therapy involving daily administration of Empa was initiated 21 days after inducing hypoxia-induced PAH in mice. Empa exhibited significant antiproliferative effects on fast-growing IPAH PASMCs. Empa activated PPARγ to prevent formation of the γ-secretase complex, with specific impacts on presenilin enhancer 2 (PEN2), which plays a crucial role in maintaining γ-secretase complex stability, thereby inhibiting Notch3. Similar results were obtained in lung tissue of chronically hypoxic mice. Empa attenuated pulmonary arterial remodeling and right ventricle hypertrophy in a hypoxic PAH mouse model. Moreover, PPARγ expression was significantly decreased and PEN2, and Notch3 levels were increased in lung tissue from PAH patients compared with non-PAH lung tissue. Empa reverses vascular remodeling by activating PPARγ to suppress the γ-secretase-Notch3 axis. We propose Empa as a PPARγ activator and potential therapeutic for PAH.

The loss of semaphorin 3A (Sema3A), which is related to endothelial‐to‐mesenchymal transition (EndMT) in atrial fibrosis, is implicated in the pathogenesis of atrial fibrillation (AF). To explore the mechanisms by which EndMT affects atrial fibrosis and assess the potential of a Sema3A activator (naringin) to prevent atrial fibrosis by targeting transforming growth factor‐beta (TGF‐β)‐induced EndMT, we used human atria, isolated human atrial endocardial endothelial cells (AEECs), and used transgenic mice expressing TGF‐β specifically in cardiac tissues (TGF‐β transgenic mice). We evaluated an EndMT marker (Twist), a proliferation marker (proliferating cell nuclear antigen; PCNA), and an endothelial cell (EC) marker (CD31) through triple immunohistochemistry and confirmed that both EndMT and EC proliferation contribute to atrial endocardial fibrosis during AF in TGF‐β transgenic mice and AF patient tissue sections. Additionally, we investigated the impact of naringin on EndMT and EC proliferation in AEECs and atrial fibroblasts. Naringin exhibited an antiproliferative effect, to which AEECs were more responsive. Subsequently, we downregulated Sema3A in AEECs using small interfering RNA to clarify a correlation between the reduction in Sema3A and the elevation of EndMT markers. Naringin treatment induced the expression of Sema3A and a concurrent decrease in EndMT markers. Furthermore, naringin administration ameliorated AF and endocardial fibrosis in TGF‐β transgenic mice by stimulating Sema3A expression, inhibiting EndMT markers, reducing atrial fibrosis, and lowering AF vulnerability. This suggests therapeutic potential for naringin in AF treatment.

Patients with systemic lupus erythematosus (SLE) associated with pulmonary arterial hypnertension (PAH) receive targeted therapy for PAH to decrease pulmonary arterial systolic pressure and significantly prolong their survival. Cysteine cathepsin proteases play critical roles in the progression of cardiovascular disease. Inhibition of cathepsin S (Cat S) has been shown to improve SLE and lupus nephritis. However, the effect of Cat S inhibitors on SLE-associated PAH (SLE-PAH) remains unclear, and there is no animal model for translational research on SLE-PAH. We hypothesized that the inhibition of Cat S may affect PAH development and arterial remodeling associated with SLE. A female animal model of SLE-PAH, female MRL/lpr (Lupus), was used to evaluate the role of pulmonary arterial remodeling in SLE. The key finding of the research work is the establishment of an animal model of SLE associated with PAH in female MRL/lpr mice that is able to evaluate pulmonary arterial remodeling starting from the age of 11 weeks to 15 weeks. Cat S protein level was identified as a marker of experimental SLE. Pulmonary hypertension in female MRL/lpr (Lupus) mice was treated by administering the selective Cat S inhibitor Millipore-219393, which stimulated peroxisome proliferator-activated receptor-gamma (PPARγ) in the lungs to inhibit Cat S expression and pulmonary arterial remodeling. Studies provide an animal model of female MRL/lpr (Lupus) associated with PAH and a deeper understanding of the pathogenesis of SLE-PAH. The results may define the role of cathepsin S in preventing progressive and fatal SLE-PAH and provide approaches for therapeutic interventions in SLE-PAH.

Atrial fibrosis is an essential contributor to atrial fibrillation (AF). It remains unclear whether atrial endocardial endothelial cells (AEECs) that undergo endothelial-mesenchymal transition (EndMT) are among the sources of atrial fibroblasts. We studied human atria, TGF-β–treated human AEECs, cardiac-specific TGF-β–transgenic mice, and heart failure rabbits to identify the underlying mechanism of EndMT in atrial fibrosis. Using isolated AEECs, we found that miR-181b was induced in TGF-β–treated AEECs, which decreased semaphorin 3A (Sema3A) and increased EndMT markers, and these effects could be reversed by a miR-181b antagomir. Experiments in which Sema3A was increased by a peptide or decreased by a siRNA in AEECs revealed a mechanistic link between Sema3A and LIM-kinase 1/phosphorylated cofilin (LIMK/p-cofilin) signaling and suggested that Sema3A is upstream of LIMK in regulating actin remodeling through p-cofilin. Administration of the miR-181b antagomir or recombinant Sema3A to TGF-β–transgenic mice evoked increased Sema3A, reduced EndMT markers, and significantly decreased atrial fibrosis and AF vulnerability. Our study provides a mechanistic link between the induction of EndMT by TGF-β via miR-181b/Sema3A/LIMK/p-cofilin signaling to atrial fibrosis. Blocking miR-181b and increasing Sema3A are potential strategies for AF therapeutic intervention.