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Background: Autoimmunity is increasingly recognized as a key contributing factor in heart muscle diseases. The functional features of cardiac autoimmunity in humans remain undefined because of the challenge of studying immune responses in situ. We previously described a subset of c-mesenchymal epithelial transition factor (c-Met)–expressing (c-Met + ) memory T lymphocytes that preferentially migrate to cardiac tissue in mice and humans. Methods: In-depth phenotyping of peripheral blood T cells, including c-Met + T cells, was undertaken in groups of patients with inflammatory and noninflammatory cardiomyopathies, patients with noncardiac autoimmunity, and healthy controls. Validation studies were carried out using human cardiac tissue and in an experimental model of cardiac inflammation. Results: We show that c-Met + T cells are selectively increased in the circulation and in the myocardium of patients with inflammatory cardiomyopathies. The phenotype and function of c-Met + T cells are distinct from those of c-Met–negative (c-Met − ) T cells, including preferential proliferation to cardiac myosin and coproduction of multiple cytokines (interleukin-4, interleukin-17, and interleukin-22). Furthermore, circulating c-Met + T cell subpopulations in different heart muscle diseases identify distinct and overlapping mechanisms of heart inflammation. In experimental autoimmune myocarditis, elevations in autoantigen-specific c-Met + T cells in peripheral blood mark the loss of immune tolerance to the heart. Disease development can be halted by pharmacologic c-Met inhibition, indicating a causative role for c-Met + T cells. Conclusions: Our study demonstrates that the detection of circulating c-Met + T cells may have use in the diagnosis and monitoring of adaptive cardiac inflammation and definition of new targets for therapeutic intervention when cardiac autoimmunity causes or contributes to progressive cardiac injury.

Inhibition of pulmonary fibrosis (PF) by restoring sarco/endoplasmic reticulum calcium ATPase 2a isoform (SERCA2a) expression using targeted gene therapy may be a potentially powerful new treatment approach for PF. Here, we found that SERCA2a expression was significantly decreased in lung samples from patients with PF and in the bleomycin (BLM) mouse model of PF. In the BLM-induced PF model, intratracheal aerosolized adeno-associated virus serotype 1 (AAV1) encoding for human SERCA2a (AAV1.hSERCA2a) reduces lung fibrosis and associated vascular remodeling. SERCA2a gene therapy also decreases right ventricular pressure and hypertrophy in both prevention and curative protocols. In vitro, we observed that SERCA2a overexpression inhibits fibroblast proliferation, migration, and fibroblast-to-myofibroblast transition induced by transforming growth factor β (TGF-β1). Thus, pro-fibrotic gene expression is prevented by blocking nuclear factor κB (NF-κB)/interleukin-6 (IL-6)-induced signal transducer and activator of transcription 3 (STAT3) activation. This effect is signaled toward an inhibitory mechanism of small mother against decapentaplegic (SMAD)/TGF-β signaling through the repression of OTU deubiquitinase, ubiquitin aldehyde binding 1 (OTUB1) and Forkhead box M1 (FOXM1). Interestingly, this cross-inhibition leads to an increase of SKI and SnoN expression, an auto-inhibitory feedback loop of TGF-β signaling. Collectively, our results demonstrate that SERCA2a gene transfer attenuates bleomycin (BLM)-induced PF by blocking the STAT3/FOXM1 pathway and promoting the SNON/SKI Axis. Thus, SERCA2a gene therapy may be a potential therapeutic target for PF.

Background: Inflammation is a prominent feature of arrhythmogenic cardiomyopathy (ACM), but whether it contributes to the disease phenotype is not known. Methods: To define the role of inflammation in the pathogenesis of ACM, we characterized nuclear factor-κB signaling in ACM models in vitro and in vivo and in cardiac myocytes from patient induced pluripotent stem cells. Results: Activation of nuclear factor-κB signaling, indicated by increased expression and nuclear accumulation of phospho-RelA/p65, occurred in both an in vitro model of ACM (expression of JUP 2157del2 in neonatal rat ventricular myocytes) and a robust murine model of ACM (homozygous knock-in of mutant desmoglein-2 [ Dsg2 mut/mu t ]) that recapitulates the cardiac manifestations seen in patients with ACM. Bay 11-7082, a small-molecule inhibitor of nuclear factor-κB signaling, prevented the development of ACM disease features in vitro (abnormal redistribution of intercalated disk proteins, myocyte apoptosis, release of inflammatory cytokines) and in vivo (myocardial necrosis and fibrosis, left ventricular contractile dysfunction, electrocardiographic abnormalities). Hearts of Dsg2 mut/mut mice expressed markedly increased levels of inflammatory cytokines and chemotactic molecules that were attenuated by Bay 11-7082. Salutary effects of Bay 11-7082 correlated with the extent to which production of selected cytokines had been blocked. Nuclear factor-κB signaling was also activated in cardiac myocytes derived from a patient with ACM. These cells produced and secreted abundant inflammatory cytokines under basal conditions, and this was also greatly reduced by Bay 11-7082. Conclusions: Inflammatory signaling is activated in ACM and drives key features of the disease. Targeting inflammatory pathways may be an effective new mechanism-based therapy for ACM.

BACKGROUND: Electrophysiological, imaging, and pathological studies have reported the presence of subtle structural abnormalities in hearts from patients with Brugada syndrome (BrS). However, data concerning disease involvement outside of the right ventricular outflow tract are limited. OBJECTIVES: This study sought to characterize the presence and distribution of ventricular myocardial fibrosis in a cohort of decedents experiencing sudden cardiac death caused by BrS. METHODS: The authors evaluated 28 whole hearts from consecutive sudden cardiac death cases attributed to BrS and 29 hearts from a comparator group comprised of noncardiac deaths (control subjects). Cardiac tissue from 6 regions across the right and left ventricle were stained with Picrosirius red for collagen and tissue composition was determined using image analysis software. Postmortem genetic testing was performed in cases with DNA retained for analysis. RESULTS: Of 28 BrS decedents (75% men; median age of death 25 years), death occurred in sleep or at rest in 24 of 28 (86%). The highest proportion of collagen was observed in the epicardial right ventricular outflow tract of the BrS group (23.7%; 95% CI: 20.8%-26.9%). Ventricular myocardium from BrS decedents demonstrated a higher proportion of collagen compared with control subjects (ratio 1.45; 95% CI: 1.22-1.71; P < 0.001), with no significant interactions with respect to sampling location or tissue layer. There was insufficient evidence to support differences in collagen proportion in SCN5A-positive cases (n = 5) when compared with control subjects (ratio 1.23; 95% CI: 0.75-1.43; P = 0.27). CONCLUSIONS: Brugada syndrome is associated with increased collagen content throughout right and left ventricular myocardium, irrespective of sampling location or myocardial layer.

The diagnosis of arrhythmogenic cardiomyopathy (ACM) is challenging especially in children at risk of adverse events. Analysis of cardiac myocyte junctional protein distribution may have diagnostic and prognostic implications, but its utility is limited by the need for a myocardial sample. We previously reported that buccal mucosa cells show junctional protein redistribution similar to that seen in cardiac myocytes of adult patients with ACM.We aimed to determine when junctional protein distribution abnormalities first occur in children with ACM variants and whether they correlate with progression of clinically apparent disease.We analyzed buccal mucosa samples of children and adolescents with a family history of ACM (Junctional protein re-localization in buccal mucosa cells did not correlate with the presence of ACM-causing variants but instead occurred with clinical onset of disease. No changes in protein distribution were seen unless and until there was clinical evidence of disease. In addition, progressive shifts in the distribution of key proteins correlated with worsening of the disease phenotype. Finally, we observed restoration of junctional signal for Cx43 in patient with a favorable response to anti-arrhythmic therapy.Due to ethical concerns about obtaining heart biopsies in children with no apparent disease, it has not been possible to analyze molecular changes in cardiac myocytes with the onset/progression of clinical disease. Using buccal smears as a surrogate for the myocardium may facilitate future studies of mechanisms and pathophysiological consequences of junctional protein redistribution in ACM. Buccal cells may also be a safe and inexpensive tool for risk stratification and potentially monitoring response to treatment in children bearing ACM variants.

Myocyte death occurs in many inherited and acquired cardiomyopathies, including arrhythmogenic cardiomyopathy (ACM), a genetic heart disease plagued by the prevalence of sudden cardiac death. Individuals with ACM and harboring pathogenic desmosomal variants, such as desmoglein-2 (DSG2), often show myocyte necrosis with progression to exercise-associated heart failure. Here, we showed that homozygous Dsg2 mutant mice (Dsg2(mut/mut)), a model of ACM, die prematurely during swimming and display myocardial dysfunction and necrosis. We detected calcium (Ca(2+)) overload in Dsg2(mut/mut) hearts, which induced calpain-1 (CAPN1) activation, association of CAPN1 with mitochondria, and CAPN1-induced cleavage of mitochondrial-bound apoptosis-inducing factor (AIF). Cleaved AIF translocated to the myocyte nucleus triggering large-scale DNA fragmentation and cell death, an effect potentiated by mitochondrial-driven AIF oxidation. Posttranslational oxidation of AIF cysteine residues was due, in part, to a depleted mitochondrial thioredoxin-2 redox system. Hearts from exercised Dsg2(mut/mut) mice were depleted of calpastatin (CAST), an endogenous CAPN1 inhibitor, and overexpressing CAST in myocytes protected against Ca(2+) overload–induced necrosis. When cardiomyocytes differentiated from Dsg2(mut/mut) embryonic stem cells (ES-CMs) were challenged with β-adrenergic stimulation, CAPN1 inhibition attenuated CAPN1-induced AIF truncation. In addition, pretreatment of Dsg2(mut/mut) ES-CMs with an AIF-mimetic peptide, mirroring the cyclophilin-A (PPIA) binding site of AIF, blocked PPIA-mediated AIF-nuclear translocation, and reduced both apoptosis and necrosis. Thus, preventing CAPN1-induced AIF-truncation or barring binding of AIF to the nuclear chaperone, PPIA, may avert myocyte death and, ultimately, disease progression to heart failure in ACM and likely other forms of cardiomyopathies.

In the long term, 137Cs is probably the most biologically important agent released in many accidental (or malicious) radiation disasters. It can enter the food chain, and be consumed, or, if present in the environment (e.g. from fallout), can provide external irradiation over prolonged times. In either case, due to the high penetration of the energetic γ rays emitted by 137Cs, the individual will be exposed to a low dose rate, uniform, whole body, irradiation. The VADER (VAriable Dose-rate External 137Cs irradiatoR) allows modeling these exposures, bypassing many of the problems inherent in internal emitter studies. Making use of discarded 137Cs brachytherapy seeds, the VADER can provide varying low dose rate irradiations at dose rates of 0.1 to 1.2 Gy/day. The VADER includes a mouse “hotel”, designed to allow long term simultaneous residency of up to 15 mice. Two source platters containing ~ 250 mCi each of 137Cs brachytherapy seeds are mounted above and below the “hotel” and can be moved under computer control to provide constant low dose rate or a varying dose rate mimicking 137Cs biokinetics in mouse or man. We present the VADER design and characterization of its performance over 18 months of use.

In the long term, 137Cs is probably the most biologically important agent released in many accidental (or malicious) radiation disasters. It can enter the food chain, and be consumed, or, if present in the environment (e.g. from fallout), can provide external irradiation over prolonged times. In either case, due to the high penetration of the energetic γ rays emitted by 137Cs, the individual will be exposed to a low dose rate, uniform, whole body, irradiation. The VADER (VAriable Dose-rate External 137Cs irradiatoR) allows modeling these exposures, bypassing many of the problems inherent in internal emitter studies. Making use of discarded 137Cs brachytherapy seeds, the VADER can provide varying low dose rate irradiations at dose rates of 0.1 to 1.2 Gy/day. The VADER includes a mouse “hotel”, designed to allow long term simultaneous residency of up to 15 mice. Two source platters containing ~ 250 mCi each of 137Cs brachytherapy seeds are mounted above and below the “hotel” and can be moved under computer control to provide constant low dose rate or a varying dose rate mimicking 137Cs biokinetics in mouse or man. We present the VADER design and characterization of its performance over 18 months of use.

Environmental contamination and ingestion of the radionuclide Cesium-137 ((137)Cs) is a large concern in fallout from a nuclear reactor accident or improvised nuclear device, and highlights the need to develop biological assays for low-dose rate, internal emitter radiation. To mimic low-dose rates attributable to fallout, we have developed a VAriable Dose-rate External (137)Cs irradiatoR (VADER), which can provide arbitrarily varying and progressive low dose rate irradiations in the range of 0.1 to 1.2 Gy/day, while circumventing the complexities of dealing with radioactively-contaminated biomaterials. We investigated the kinetics of mouse peripheral leukocytes DNA damage response in vivo after variable, low-dose rate (137)Cs exposure. C57BL/6 mice were placed in the VADER over 7 days with total accumulated dose up to 2.7 Gy. Peripheral blood response including the leukocytes depletion, apoptosis as well as its signal protein p53 and DNA repair biomarker γ-H2AX were measured. The results illustrated that blood leukocyte numbers had significantly dropped by day 7. P53 levels peaked at day 2 (total dose= 0.91 Gy) and then declined, whereas γ-H2AX fluorescence intensity (MFI) and foci number generally increased with accumulated dose and peaked at day 5 (total dose= 2.08 Gy). ROC curve analysis for γ-H2AX provided a good discrimination of accumulated dose < 2 Gy and ≥ 2 Gy, highlighting the potential of γ-H2AX MFI as a biomarker dosimetry in a protracted, environmental exposure scenario.

Pulmonary arterial hypertension (PAH) is a devastating lung disease characterized by the progressive obstruction of the distal pulmonary arteries (PA). Structural and functional alteration of pulmonary artery smooth muscle cells (PASMC) and endothelial cells (PAEC) contributes to PA wall remodeling and vascular resistance, which may lead to maladaptive right ventricular (RV) failure and, ultimately, death. Here, we found that decreased expression of sarcoplasmic/endoplasmic reticulum Ca2+ ATPase 2a (SERCA2a) in the lung samples of PAH patients was associated with the down-regulation of bone morphogenetic protein receptor type 2 (BMPR2) and the activation of signal transducer and activator of transcription 3 (STAT3). Our results showed that the antiproliferative properties of SERCA2a are mediated through the STAT3/BMPR2 pathway. At the molecular level, transcriptome analysis of PASMCs co-overexpressing SERCA2a and BMPR2 identified STAT3 amongst the most highly regulated transcription factors. Using a specific siRNA and a potent pharmacological STAT3 inhibitor (STAT3i, HJC0152), we found that SERCA2a potentiated BMPR2 expression by repressing STAT3 activity in PASMCs and PAECs. In vivo, we used a validated and efficient model of severe PAH induced by unilateral left pneumonectomy combined with monocrotaline (PNT/MCT) to further evaluate the therapeutic potential of single and combination therapies using adeno-associated virus (AAV) technology and a STAT3i. We found that intratracheal delivery of AAV1 encoding SERCA2 or BMPR2 alone or STAT3i was sufficient to reduce the mean PA pressure and vascular remodeling while improving RV systolic pressures, RV ejection fraction, and cardiac remodeling. Interestingly, we found that combined therapy of AAV1.hSERCA2a with AAV1.hBMPR2 or STAT3i enhanced the beneficial effects of SERCA2a. Finally, we used cardiac magnetic resonance imaging to measure RV function and found that therapies using AAV1.hSERCA2a alone or combined with STAT3i significantly inhibited RV structural and functional changes in PNT/MCT-induced PAH. In conclusion, our study demonstrated that combination therapies using SERCA2a gene transfer with a STAT3 inhibitor could represent a new promising therapeutic alternative to inhibit PAH and to restore BMPR2 expression by limiting STAT3 activity.

AIMS: Cyclic adenosine monophosphate (cAMP) is the predominant intracellular second messenger that transduces signals from Gs-coupled receptors. Intriguingly, there is evidence from various cell types that an extracellular cAMP pathway is active in the extracellular space. Herein, we investigated the role of extracellular cAMP in the lung and examined whether it may act on pulmonary vascular cell proliferation and pulmonary vasculature remodeling in the pathogenesis of pulmonary hypertension (PH). METHODS AND RESULTS: The expression of cyclic AMP-metabolizing enzymes was increased in lungs from patients with PH as well as in rats treated with monocrotaline and mice exposed to Sugen/hypoxia. We report that inhibition of the endogenous extracellular cAMP pathway exacerbated Sugen/hypoxia-induced lung remodeling. We found that application of extracellular cAMP induced an increase in intracellular cAMP levels and inhibited proliferation and migration of pulmonary vascular cells in vitro. Extracellular cAMP infusion in two in vivo pulmonary hypertension models prevented and reversed pulmonary and cardiac remodeling associated with PH. Using protein expression analysis along with luciferase assays, we found that extracellular cAMP acts via the A2R/PKA/CREB/p53/Cyclin D1 pathway. CONCLUSIONS: Taken together, our data reveal the presence of an extracellular cAMP pathway in pulmonary arteries that attempts to protect the lung during PH, and suggest targeting of the extracellular cAMP signaling pathway to limit pulmonary vascular remodeling and PH. TRANSLATIONAL PERSPECTIVE: Lungs samples from patients with clinical PAH and from animals with PH display increased cyclic AMP-metabolizing enzymes expression levels. Our results indicate that an endogenous extracellular cAMP pathway is activated during PH and attempts to counteract vascular remodeling. Additionally, our study demonstrates that extracellular-cAMP inhibits chronic hypoxia-induced PH in mice and MCT-induced PH in rats by activating the A2R/PKA/CREB/p53/Cyclin D1 pathway. Importantly, PAH patients display an inactive PKA/CREB/p53/Cyclin D1 pathway that could be stimulated by extracellular-cAMP. Targeting the extracellular cAMP pathway may represent a novel therapeutic approach for the treatment of pulmonary arterial hypertension.

Environmental contamination and ingestion of the radionuclide Cesium-137 (137Cs) is a large concern in fallout from a nuclear reactor accident and improvised nuclear device and highlights the need to develop biological assays for low dose rate, internal emitter radiation. To mimic low dose rates attributable to fallout, we have developed a VAriable Dose-rate External137Cs irradiatoR (VADER), which can provide arbitrarily varying and progressive low dose rate irradiations in the range of 1.2 to 0.1 Gy/day, while circumventing the complexities of dealing with radioactively-contaminated biomaterials. We investigated the kinetics of mouse peripheral leukocytes DNA damage response in vivo after variable, low-dose rate137Cs exposure. C57BL/6 mice were placed in the VADER over 7 days with total accumulated dose up to 2.7 Gy. Peripheral blood response including the leukocytes depletion, apoptosis signal protein p53 and DNA repair biomarker γ-H2AX were measured. The results illustrated that blood leukocyte count had significantly dropped by days 7. P53 levels peaked at day 2 (total dose=0.91Gy) and then declined whereas γ-H2AX yields generally increased with accumulated dose and peaked at day 5 (total dose=2.08Gy). ROC curve analysis for γ-H2AX provided a good discrimination of accumulated dose < 2 Gy and ≥ 2 Gy, highlighting the potential of γ-H2AX as a biomarker dosimetry in a protracted, environmental exposure scenario.

Pulmonary arterial hypertension (PAH) is an uncommon, progressive, life-threatening and often fatal disease. Despite advances in PAH therapy, there is no cure for PAH, and new therapies need to be developed. Activation of G-protein-coupled receptors stimulates adenylyl cyclase, leading in vascular smooth muscle cells to an increase in intracellular cyclic adenosine monophosphate (cAMP) formation and reduced cell proliferation in vitro and in vivo . Yet, the last two decades of research have shown that there is more to the role of cAMP than ever expected from this molecule. Stimulated cells transport cAMP outside the cells, a process that is mediated by the multidrug resistance-associated proteins (MRPs). Herein, we investigated the role of extracellular cAMP in the lung and asked whether it may act on pulmonary vascular remodeling. By employing a fluorescence resonance energy transfer (FRET)-based sensor, we found that extracellular cAMP activates intracellular cAMP formation in human pulmonary artery smooth muscle cells (hPASMC) and endothelial cells (hPAEC). Extracellular cAMP, via binding of its metabolite adenosine to the type A2 receptor, reduced hPASMC and hPAEC proliferation and migration by controlling the PKA/CREB pathway. To test for a role of extracellular cAMP in the pulmonary vasculature, we used rat-monocrotaline and mouse-hypoxia as in vivo models of PAH. Rats treated with monocrotaline developed PAH with increased pulmonary artery pressures (PAP) and right ventricular (RV) hypertrophy. Extracellular cAMP infusion significantly prevented and reversed these structural changes. Extracellular-cAMP-treated rats displayed lower RV systolic pressure and Fulton index, as well as decreased PAP. In line with these observations, we found infused cAMP to potently repress RV hypertrophy, RV systolic pressure, perivascular lung fibrosis and pulmonary arteries remodeling in mice exposed to Sugen5416 and chronic hypoxia. Together, our results assign extracellular cAMP a potent regulatory role in pulmonary vascular cells, and suggest targeting the extracellular cAMP signaling pathway to limit pulmonary vascular remodeling and PAH.

BACKGROUND: Pulmonary arterial hypertension (PAH) results in right ventricular (RV) failure, electro-mechanical dysfunction and heightened risk of sudden cardiac death (SCD), although exact mechanisms and predisposing factors remain unclear. Because impaired chronotropic response to exercise is a strong predictor of early mortality in patients with PAH, we hypothesized that progressive elevation in heart rate can unmask ventricular tachyarrhythmias (VT) in a rodent model of monocrotaline (MCT)-induced PAH. We further hypothesized that intra-tracheal gene delivery of aerosolized AAV1.SERCA2a (AAV1.S2a), an approach which improves pulmonary vascular remodeling in PAH, can suppress VT in this model. OBJECTIVE: To determine the efficacy of pulmonary AAV1.S2a in reversing electrophysiological (EP) remodeling and suppressing VT in PAH. METHODS: Male rats received subcutaneous injection of MCT (60 mg/kg) leading to advanced PAH. Three weeks following MCT, rats underwent intra-tracheal delivery of aerosolized AAV1.S2a (MCT+S2a, N=8) or saline (MCT, N=9). Age-matched rats served as controls (CTRL, N=7). The EP substrate and risk of VT were determined using high-resolution optical action potential (AP) mapping ex vivo. The expression levels of key ion channel subunits, fibrosis markers and hypertrophy indices were measured by RT-PCR and histochemical analyses. RESULTS: Over 80% of MCT but none of the CTRL hearts were prone to sustained VT by rapid pacing (P

Pulmonary arterial hypertension (PAH) is a syndrome characterized by pulmonary vascular remodeling and vasoconstriction, leading to increased pulmonary vascular resistance, right ventricular pressure overload and, eventually, to right ventricular failure and premature death. Animal models have been an essential tool for understanding pulmonary hypertension pathophysiology and for the discovery and development of novel therapies.MCT-induced PAH in rats leads to a significant increase in RV pressure and pulmonary vascular remodeling, as well as greater RV hypertrophy. In this chapter, we describe protocols for inducing and assessing the monocrotaline (MCT) rat model, the most classical and widely used in vivo model of PAH. Using this protocol, rats reproducibly develop pulmonary hypertension with a mean pulmonary pressure of ~40 mmHg approximately 4 weeks after single MCT administration.

Pulmonary hypertension is a rapidly progressive, life-threatening, and often fatal disease. Despite many new developments in pulmonary arterial hypertension (PAH) therapy, there is currently no cure for PAH, and new therapies are desperately needed. PAH pathobiology involves a remodeling process in pulmonary arteries that plays a critical role in elevating pulmonary arterial and right ventricle pressures. The discovery and development of new therapies requires animal models of PAH that mimic the human disease, including vascular remodeling.Here we review and describe a detailed protocol for creating an in vivo model of Sugen/Hypoxia-induced PAH in mice that is commonly used to assess the efficiency of new therapies in PAH. Severe pulmonary hypertension can be established in 1 month using this protocol. Additional protocols to evaluate the model by invasive pressure measurements and histology are provided.

Cyclic adenosine monophosphate (cAMP) is a cellular second messenger that mediates physiological effects of G protein-coupled receptors. Yet, the last two decades of research have shown that there is more to the role of cAMP than ever expected from this small molecule. Stimulated cells transport cAMP outside the cells, a process that is mediated by the multidrug resistance-associated proteins (MRPs). The present study investigated the role of extracellular cAMP in the lung and asked whether it may act on pulmonary vasculature remodeling. By employing a fluorescence resonance energy transfer (FRET)-based sensor, we found that extracellular cAMP activates intracellular cAMP formation in human pulmonary artery smooth muscle cells (hPASMC) and endothelial cells (hPAEC). Extracellular cAMP, via binding of its metabolite adenosine to the type A2 receptor, reduced hPASMC and hPAEC growth by controlling the PKA/CREB pathway. To test for a role of extracellular cAMP in the pulmonary vasculature, we chose rat-monocrotaline and mouse-hypoxia as in vivo models for pulmonary arterial hypertension (PAH). Rats treated with monocrotaline developed PAH with increased pulmonary artery pressures (PAP) and right ventricular (RV) hypertrophy. Extracellular cAMP infusion significantly prevented and reversed these structural changes. Extracellular cAMP-treated rats displayed lower RV systolic pressure and Fulton index, as well as decreased PAP. In line with these observations, we found infused cAMP to potently repress RV hypertrophy, RV systolic pressure, perivascular lung fibrosis and pulmonary arteries remodeling in mice exposed to Sugen5416 and chronic hypoxia. Together, our results assign extracellular cAMP a potent regulatory role in pulmonary vascular cells, and suggest targeting the extracellular cAMP signaling pathway to limit pulmonary vascular remodeling and PAH.

Pulmonary arterial hypertension (PAH) is a fatal disease characterized by vascular remodeling leading to high pulmonary arterial pressure (PAP) and right ventricular (RV) heart failure. Gene therapy is a promising approach to treat PAH. The most used monocrotaline (MCT) rat model of PAH does not mimic the pathophysiology of the PAH in humans. A refined rat model, the pneumonectomy plus MCT (PNT-MCT), displays all of the features of PAH and most importantly plexiform lesions whereby therapeutics aim to reverse. In this study, we investigated whether intratracheal delivery of sarcoplasmic reticulum calcium ATPase (SERCA2a) gene reverse the severity of PAH in the PNT- MCT model. Left pneumonectomy was performed in rats. One week later, the animals received the MCT injection. At 3 weeks, the severity of PAH disease was confirmed. Thereafter, PNT-MCT rats received either intratracheal delivery of gene construct with adeno-associated virus/SERCA2a (AAV1/SERCA2a) or saline. Hemodynamic parameters were determined by magnetic resonance imaging (MRI) and RV catheterization. RV hypertrophy, heart and lung fibrosis were assessed. Molecular biology assays for gene expression and immunostaining were used to quantify SERCA2a and disease markers. Four weeks after gene delivery, RV function was improved in AAV1.SERCA2a treated-animals with an increase of stroke volume and ejection fraction compared to saline group (275 ± 22 μl vs. 192 ± 22 μl and 56 ± 3 μl vs. 44 ± 3 μl, p

Pulmonary hypertension is a rapidly progressive, life-threatening, and often fatal disease. Despite many new developments in pulmonary arterial hypertension (PAH) therapy, there is currently no cure for PAH, and new therapies are desperately needed. PAH pathobiology involves a remodeling process in pulmonary arteries that plays a critical role in elevating pulmonary arterial and right ventricle pressures. The discovery and development of new therapies requires animal models of PAH that mimic the human disease, including vascular remodeling.Here we review and describe a detailed protocol for creating an in vivo model of Sugen/Hypoxia-induced PAH in mice that is commonly used to assess the efficiency of new therapies in PAH. Severe pulmonary hypertension can be established in 1 month using this protocol. Additional protocols to evaluate the model by invasive pressure measurements and histology are provided.

Background: Pulmonary Arterial Hypertension (PAH) is a major cause of mortality due, in large part to right ventricular (RV) failure. Its electrophysiological (EP) effects however are poorly defined. Recently, a gene therapy approach targeting SERCA2a (S2A) to the lungs improved RV mechanical function in a rodent PAH model. Whether this approach improves myocardial EP properties remains unknown. Methods: Male Sprague Dawley rats received a subcutaneous injection of monocrotaline (MCT 60mg/kg) leading to PAH induced RV failure. Three wks later, rats underwent intratracheal delivery of aerosolized AAV1.S2A (1E11 gc, N=6) or no treatment (MCT, N=7). Age matched rats served as controls (CTRL, N=5). The EP substrate and risk of VT were assessed using high resolution optical action potential (AP) mapping in ex vivo perfused hearts. Results: MCT (6/7) but not CTRL (0/5) hearts were prone to pacing-induced VT (P(Fig A, B) . AP duration (APD) and heterogeneity were increased in MCT and partially reversed by AAV1.S2A. Underlying the rise in heterogeneity was selective APD prolongation on the RV side causing >90 degree clockwise shift in the orientation of the transepicardial gradient, an effect which was not reversed by AAV1.S2A (Fig C arrows) . Conclusion: S2A gene therapy to the lungs ameliorates PAH induced EP remodeling and arrhythmia propensity. Our findings highlight for the first time the utility of a non-cardiac gene therapy approach for arrhythmia suppression.