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Aims Potential loss-of-function variants of ATP13A3 , the gene encoding a P5B-type transport ATPase of undefined function, were recently identified in patients with pulmonary arterial hypertension (PAH). ATP13A3 is implicated in polyamine transport but its function has not been fully elucidated. In this study, we sought to determine the biological function of ATP13A3 in vascular endothelial cells (ECs) and how PAH-associated variants may contribute to disease pathogenesis. Methods and results We studied the impact of ATP13A3 deficiency and overexpression in EC models [human pulmonary ECs, blood outgrowth ECs (BOECs), and human microvascular EC 1], including a PAH patient–derived BOEC line harbouring an ATP13A3 variant (LK726X). We also generated mice harbouring an Atp13a3 variant analogous to a human disease–associated variant to establish whether these mice develop PAH. ATP13A3 localized to the recycling endosomes of human ECs. Knockdown of ATP13A3 in ECs generally reduced the basal polyamine content and altered the expression of enzymes involved in polyamine metabolism. Conversely, overexpression of wild-type ATP13A3 increased polyamine uptake. Functionally, loss of ATP13A3 was associated with reduced EC proliferation, increased apoptosis in serum starvation, and increased monolayer permeability to thrombin. The assessment of five PAH-associated missense ATP13A3 variants (L675V, M850I, V855M, R858H, and L956P) confirmed loss-of-function phenotypes represented by impaired polyamine transport and dysregulated EC function. Furthermore, mice carrying a heterozygous germline Atp13a3 frameshift variant representing a human variant spontaneously developed a PAH phenotype, with increased pulmonary pressures, right ventricular remodelling, and muscularization of pulmonary vessels. Conclusion We identify ATP13A3 as a polyamine transporter controlling polyamine homeostasis in ECs, a deficiency of which leads to EC dysfunction and predisposes to PAH. This suggests a need for targeted therapies to alleviate the imbalances in polyamine homeostasis and EC dysfunction in PAH. [ABSTRACT FROM AUTHOR]

The article discusses the role of the epicardium, a layer of cells that covers the outer surface of the heart, in cardiac development and regeneration. The authors highlight recent research on epicardioids, which are cardiac organoids that mimic the self-organization and functionality of the ventricular myocardium with an external epicardial layer. These epicardioids have provided insights into the functions of the epicardium, including its role as a source of progenitor cells and a facilitator of myocardial compaction and maturation. The article also emphasizes the potential of epicardioids for studying developmental processes and investigating therapeutic strategies for cardiac regeneration. [Extracted from the article]

Aims Ventricular tachyarrhythmias (VTs) are common in the pathologically remodelled heart. These arrhythmias can be lethal, necessitating acute treatment like electrical cardioversion to restore normal rhythm. Recently, it has been proposed that cardioversion may also be realized via optically controlled generation of bioelectricity by the arrhythmic heart itself through optogenetics and therefore without the need of traumatizing high-voltage shocks. However, crucial mechanistic and translational aspects of this strategy have remained largely unaddressed. Therefore, we investigated optogenetic termination of VTs (i) in the pathologically remodelled heart using an (ii) implantable multi-LED device for (iii) in vivo closed-chest, local illumination. Methods and results In order to mimic a clinically relevant sequence of events, transverse aortic constriction (TAC) was applied to adult male Wistar rats before optogenetic modification. This modification took place 3 weeks later by intravenous delivery of adeno-associated virus vectors encoding red-activatable channelrhodopsin or Citrine for control experiments. At 8–10 weeks after TAC, VTs were induced ex vivo and in vivo , followed by programmed local illumination of the ventricular apex by a custom-made implanted multi-LED device. This resulted in effective and repetitive VT termination in the remodelled adult rat heart after optogenetic modification, leading to sustained restoration of sinus rhythm in the intact animal. Mechanistically, studies on the single cell and tissue level revealed collectively that, despite the cardiac remodelling, there were no significant differences in bioelectricity generation and subsequent transmembrane voltage responses between diseased and control animals, thereby providing insight into the observed robustness of optogenetic VT termination. Conclusion Our results show that implant-based optical cardioversion of VTs is feasible in the pathologically remodelled heart in vivo after local optogenetic targeting because of preserved optical control over bioelectricity generation. These findings add novel mechanistic and translational insight into optical ventricular cardioversion. [ABSTRACT FROM AUTHOR]

Aims Inflammation plays a role in atrial fibrillation (AF), but classical anti-inflammatory molecules are ineffective. Recent evidence suggests that failure of inflammation-resolution causes persistent inflammatory signalling and that a novel drug-family called resolvins promotes inflammation-resolution. Right heart disease (RHD) is associated with AF; experimental RHD shows signs of atrial inflammatory-pathway activation. Here, we evaluated resolvin-therapy effects on atrial arrhythmogenic remodelling in experimental RHD. Methods and results Pulmonary hypertension and RHD were induced in rats with an intraperitoneal injection of 60 mg/kg monocrotaline (MCT). An intervention group received daily resolvin-D1 (RvD1), starting 1 day before MCT administration. Right atrial (RA) conduction and gene-expression were analysed respectively by optical mapping and qPCR/gene-microarray. RvD1 had no or minimal effects on MCT-induced pulmonary artery or right ventricular remodelling. Nevertheless, in vivo transoesophageal pacing induced atrial tachyarrhythmias in no CTRL rats vs. 100% MCT-only rats, and only 33% RvD1-treated MCT rats (P  < 0.001 vs. MCT-only). Conduction velocity was significantly decreased by MCT, an effect prevented by RvD1. RHD caused RA dilation and fibrosis. RvD1 strongly attenuated RA fibrosis but had no effect on RA dilation. MCT increased RA expression of inflammation- and fibrosis-related gene-expression pathways on gene-microarray transcriptomic analysis, effects significantly attenuated by RvD1 (334 pathways enriched in MCT-rats vs. control; only 177 dysregulated by MCT with RvD1 treatment). MCT significantly increased RA content of type 1 (proinflammatory) CD68-positive M1 macrophages without affecting type 2 (anti-inflammatory) M2 macrophages. RvD1-treated MCT-rat RA showed significant reductions in proinflammatory M1 macrophages and increases in anti-inflammatory M2 macrophages vs. MCT-only. MCT caused statistically significant increases in protein-expression (western blot) of COL3A1, ASC, CASP1, CASP8, IL1β, TGFβ3, CXCL1, and CXCL2, and decreases in MMP2, vs. control. RvD1-treatment suppressed all these MCT-induced protein-expression changes. Conclusion The inflammation-resolution enhancing molecule RvD1 prevents AF-promoting RA remodelling, while suppressing inflammatory changes and fibrotic/electrical remodelling, in RHD. Resolvins show potential promise in combating atrial arrhythmogenic remodelling by suppressing ongoing inflammatory signalling. [ABSTRACT FROM AUTHOR]

Aims Cardiovascular disease remains the greatest cause of mortality in Americans over 65. The stretch-activated transient receptor potential vanilloid-4 (TRPV4) ion channel is expressed in cardiomyocytes of the aged heart. This investigation tests the hypothesis that TRPV4 alters Ca2+ handling and cardiac function in response to increased ventricular preload and cardiomyocyte stretch. Methods and results Left ventricular maximal pressure (P Max) was monitored in isolated working hearts of Aged (24–27 months) mice following preload elevation from 5 to 20mmHg, with and without TRPV4 antagonist HC067047 (HC, 1 µmol/L). In preload responsive hearts, P Max prior to and immediately following preload elevation (i.e. Frank–Starling response) was similar between Aged and Aged +HC. Within 1 min following preload elevation, Aged hearts demonstrated secondary P Max augmentation (Aged > Aged +HC) suggesting a role for stretch-activated TRPV4 in cardiac hypercontractility. However, after 20 min at 20 mmHg Aged exhibited depressed P Max (Aged < Aged +HC) suggestive of TRPV4-dependent contractile dysfunction with sustained stretch. To examine stretch-induced Ca2+ homeostasis at the single-cell level, isolated cardiomyocytes were stretched 10–15% of slack length while measuring intracellular Ca2+ with fura-2. Uniaxial longitudinal stretch increased intracellular Ca2+ levels and triggered Ca2+ overload and terminal cellular contracture in Aged , but not Aged +HC. Preload elevation in hearts of young/middle-age (3–12 months) mice produced an initial P Max increase (Frank–Starling response) without secondary P Max augmentation, and cardiomyocyte stretch did not affect intracellular Ca2+ levels. Hearts of transgenic mice with cardiac-specific TRPV4 expression exhibited P Max similar to 3- to 12-month control mice prior to and immediately following preload elevation but displayed secondary P Max augmentation. Cardiomyocytes of mice with transgenic TRPV4 expression were highly sensitive to mechanical stimulation and exhibited elevated Ca2+ levels, Ca2+ overload, and terminal contracture upon cellular attachment and stretch. Conclusion TRPV4 contributes to a stretch-induced increase in cardiomyocyte Ca2+ and cardiac hypercontractility, yet sustained stretch leads to cardiomyocyte Ca2+ overload and contractile dysfunction in the aged heart. [ABSTRACT FROM AUTHOR]

Improvements in early interventions after acute myocardial infarction (AMI), notably, the increased use of timely reperfusion therapy, have increased survival dramatically in recent decades. Despite this, maladaptive ventricular remodelling and subsequent heart failure (HF) following AMI remain a significant clinical challenge, particularly because several pre-clinical strategies to attenuate remodelling have failed to translate into clinical practice. Monocytes and macrophages, pleiotropic cells of the innate immune system, are integral in both the initial inflammatory response to injury and subsequent wound healing in many tissues, including the heart. However, maladaptive immune cell behaviour contributes to ventricular remodelling in mouse models, prompting experimental efforts to modulate the immune response to prevent the development of HF. Seminal work in macrophage biology defined macrophages as monocyte-derived cells that are comprised of two populations, pro-inflammatory M1 macrophages and reparative M2 macrophages, and initial investigations into cardiac macrophage populations following AMI suggested they aligned well to this model. However, more recent data, in the heart and other tissues, demonstrate remarkable heterogeneity and plasticity in macrophage development, phenotype, and function. These recent insights into macrophage biology may explain the failure of non-specific immunosuppressive strategies and offer novel opportunities for therapeutic targeting to prevent HF following AMI. Here, we summarize the traditional monocyte-macrophage paradigm, experimental evidence for the significance of these cells in HF after AMI, and the potential relevance of emerging evidence that refutes canonical models of monocyte and macrophage biology. [ABSTRACT FROM AUTHOR]

Aims Heart failure with reduced ejection fraction (HFrEF) causes lung remodelling with myofibroblasts proliferation and fibrosis leading to a restrictive lung syndrome with pulmonary hypertension (PH) and right ventricular (RV) dysfunction. PBI-4050 is a first-in-class anti-fibrotic, anti-inflammatory, and anti-proliferative compound. The present study evaluated the therapeutic impact of PBI-4050 on PH in an HFrEF model. Methods and results HFrEF was induced after myocardial infarction (MI) in rats. Two weeks later, sham-operated and MI groups received PBI-4050 (200 mg/kg/day by gavage) or saline for 3 weeks. Animals were analysed according to infarct size as large (≥30% left ventricle) or medium MI (<30%). Large MI caused PH and RV hypertrophy (RVH) with a restrictive lung syndrome. PBI-4050 did not adversely affect left ventricular (LV) function but markedly reduced PH and RVH and improved RV dysfunction. PBI-4050 reduced lung remodelling and improved respiratory compliance with decreased lung fibrosis, alveolar wall cellular proliferation and α-smooth muscle actin expression. The increased expression of endothelin-1 (ET-1), transforming growth factor beta (TGF-β), interleukin-6 (IL-6) and of tissue inhibitor of metalloprotease-1 in the lungs from HFrEF were reduced with PBI-4050 therapy. Activation of isolated human lung fibroblasts (HLFs) to a myofibroblastic pro-fibrogenic phenotype was markedly reduced by PBI-4050. The fatty acid receptor GPR84 was increased in HFrEF lungs and in activated HLFs, and reduced by PBI-4050. GPR84 agonists activated fibrogenesis in HLFs and finally, PBI-4050 reduced ERK1/2 phosphorylation. Conclusions PBI-4050 reduces PH and RVH in HFrEF by decreasing lung fibrosis and remodelling. This novel agent decreases the associated restrictive lung syndrome and recovers RV function. A contributing mechanism involves reducing the activation of lung fibroblasts by IL-6, TGF-β, and ET-1 by antagonism of GPR84 and reduced ERK1/2 phosphorylation. PBI-4050 is a novel promising therapy for targeting lung remodelling in group II PH. [ABSTRACT FROM AUTHOR]

Aims The Toll-like receptor 7 (TLR7) is an intracellular innate immune receptor activated by nucleic acids shed from dying cells leading to activation of the innate immune system. Since innate immune system activation is involved in the response to myocardial infarction (MI), this study aims to identify if TLR7 is involved in post-MI ischaemic injury and adverse remodelling after MI. Methods and results TLR7 involvement in MI was investigated in human tissue from patients with ischaemic heart failure, as well as in a mouse model of permanent left anterior descending artery occlusion in C57BL/6J wild type and TLR7 deficient (TLR7−/−) mice. TLR7 expression was up-regulated in human and mouse ischaemic myocardium after MI. Compared to wild type mice, TLR7−/− mice had less acute cardiac rupture associated with blunted activation of matrix metalloproteinase 2, increased expression of tissue inhibitor of metalloproteinase 1, recruitment of more myofibroblasts, and the formation of a myocardial scar with higher collagen fibre density. Furthermore, inflammatory cell influx and inflammatory cytokine expression post-MI were reduced in the TLR7−/− heart. During a 28-day follow-up after MI, TLR7 deficiency resulted in less chronic adverse left ventricular remodelling and better cardiac function. Bone marrow (BM) transplantation experiments showed that TLR7 deficiency in BM-derived cells preserved cardiac function after MI. Conclusions In acute MI, TLR7 mediates the response to acute cardiac injury and chronic remodelling probably via modulation of post-MI scar formation and BM-derived inflammatory infiltration of the myocardium. [ABSTRACT FROM AUTHOR]

Aims The ability of the right ventricle (RV) to adapt to increased afterload is the major determinant of survival in patients with pulmonary hypertension (PH). In this study, we explored the effect of genetic background on RV adaptation and survival in a rat model of severe pulmonary arterial hypertension (PAH). Methods and results PH was induced by a single injection of SU5416 (SU) in age-matched Sprague Dawley (SD) or Fischer rats, followed by a 3-week exposure to chronic hypoxia (SUHx). SD and Fischer rats exhibited similar elevations in RV systolic pressure, number of occlusive pulmonary vascular lesions, and RV hypertrophy (RV/LV+S) in response to SUHx. However, no Fischer rats survived beyond 7 weeks compared with complete survival for SD rats. This high early mortality of Fischer rats was associated with significantly greater RV dilatation and reduced ejection fraction, cardiac output, and exercise capacity at 4 weeks post-SU. Moreover, microarray analysis revealed that over 300 genes were uniquely regulated in the RV in the severe PAH model in the Fischer compared with SD rats, mainly related to angiogenesis and vascular homoeostasis, fatty acid metabolism, and innate immunity. A focused polymerase chain reaction array confirmed down-regulation of angiogenic genes in the Fischer compared with SD RV. Furthermore, Fischer rats demonstrated significantly lower RV capillary density compared with SD rats in response to SUHx. Conclusion Fischer rats are prone to develop RV failure in response to increased afterload. Moreover, the high mortality in the SUHx model of severe PAH was caused by a failure of RV adaptation associated with lack of adequate microvascular angiogenesis, together with metabolic and immunological responses in the hypertrophied RV. [ABSTRACT FROM AUTHOR]

Aims Tenascin-C (TN-C) is an extracellular matrix protein undetected in the normal adult heart, but expressed in several heart diseases associated with inflammation. We previously reported that serum TN-C levels of myocardial infarction (MI) patients were elevated during the acute stage, and that patients with high peak TN-C levels were at high risk of left ventricular (LV) remodelling and poor outcome, suggesting that TN-C could play a significant role in the progression of ventricular remodelling. However, the detailed molecular mechanisms associated with this process remain unknown. We aimed to elucidate the role and underlying mechanisms associated with TN-C in adverse remodelling after MI. Methods and results MI was induced by permanent ligation of the coronary artery of TN-C knockout (TN-C-KO) and wild type (WT) mice. In WT mice, TN-C was expressed at the borders between intact and necrotic areas, with a peak at 3 days post-MI and observed in the immediate vicinity of infiltrating macrophages. TN-C-KO mice were protected from ventricular adverse remodelling as evidenced by a higher LV ejection fraction as compared with WT mice (19.0 ± 6.3% vs. 10.6 ± 4.4%; P  <   0.001) at 3 months post-MI. During the acute phase, flow-cytometric analyses showed a decrease in F4/80+CD206lowCD45+ M1 macrophages and an increase in F4/80+CD206highCD45+ M2 macrophages in the TN-C-KO heart. To clarify the role of TN-C on macrophage polarization, we examined the direct effect of TN-C on bone marrow-derived macrophages in culture, observing that TN-C promoted macrophage shifting into an M1 phenotype via Toll-like receptor 4 (TLR4). Under M2-skewing conditions, TN-C suppressed the expression of interferon regulatory factor 4, a key transcription factor that controls M2-macrophage polarization, via TLR4, thereby inhibiting M2 polarization. Conclusion These results suggested that TN-C accelerates LV remodelling after MI, at least in part, by modulating M1/M2-macrophage polarization. [ABSTRACT FROM AUTHOR]

Aims Cardiac remodelling and heart failure are promoted by persistent sympathetic activity. We recently reported that nuclear receptor Nur77 may protect against sympathetic agonist-induced cardiac remodelling in mice. The sympathetic co-transmitter neuropeptide Y (NPY) is co-released with catecholamines and is a known cardiac modulator and predictor of heart failure mortality. Recently, transcriptome analyses revealed NPY as a putative target of Nur77. In this study, we assess whether Nur77 modulates adverse cardiac remodelling via NPY signalling. Methods and results Nur77 represses NPY expression in the PC12 adrenal chromaffin cell line. Accordingly, NPY levels are higher in adrenal gland, plasma, and heart from Nur77-KO compared to wild-type mice. Conditioned medium from Nur77-silenced chromaffin cells and serum from Nur77-KO mice induce marked hypertrophy in cultured neonatal rat cardiomyocytes, which is inhibited by the NPY type 1 receptor (NPY1R) antagonist BIBO3304. In cardiomyocytes from Nur77-KO mice, intracellular Ca2+ is increased partially via the NPY1R. This is independent from elevated circulating NPY since cardiomyocyte-specific Nur77-deficient mice (CM-KO) do not have elevated circulating NPY, but do exhibit BIBO3304-sensitive, increased cardiomyocyte intracellular Ca2+. In vivo, this translates to NPY1R antagonism attenuating cardiac calcineurin activity and isoproterenol-induced cardiomyocyte hypertrophy and fibrosis in full-body Nur77-KO mice, but not in CM-KO mice. Conclusions The cardioprotective action of Nur77 can be ascribed to both inhibition of circulating NPY levels and to cardiomyocyte-specific modulation of NPY-NPY1R signalling. These results reveal the underlying mechanism of Nur77 as a promising modifier gene in heart failure. [ABSTRACT FROM AUTHOR]

Aims Pulmonary arterial hypertension (PAH) is a devastating disease and treatment options are limited. Urocortin-2 (Ucn-2) has shown promising therapeutic effects in experimental and clinical left ventricular heart failure (HF). Our aim was to analyse the expression of Ucn-2 in human and experimental PAH, and to investigate the effects of human Ucn-2 (hUcn-2) administration in rats with monocrotaline (MCT)-induced pulmonary hypertension (PH). Methods and results Tissue samples were collected from patients with and without PAH and from rats with MCT-induced PH. hUcn-2 (5 lg/kg, bi-daily, i.p., for 10 days) or vehicle was administered to male wistar rats subjected to MCT injection or to pulmonary artery banding (PAB) to induce right ventricular (RV) overload without PAH. Expression of Ucn-2 and its receptor was increased in the RV of patients and rats with PAH. hUcn-2 treatment reduced PAH in MCT rats, resulting in decreased morbidity, improved exercise capacity and attenuated pulmonary arterial and RV remodelling and dysfunction. Additionally, RV gene expression of hypertrophy and failure signalling pathways were attenuated. hUcn-2 treatment also attenuated PAB-induced RV hypertrophy. Conclusions Ucn-2 levels are altered in human and experimental PAH. hUcn-2 treatment attenuates PAH and RV dysfunction in MCT-induced PH, has direct anti-remodelling effects on the pressure-overloaded RV, and improves pulmonary vascular function. [ABSTRACT FROM AUTHOR]

Aims: NADPH oxidase-4 (Nox4) is an important reactive oxygen species (ROS) source that is upregulated in the haemodynamically overloaded heart. Our previous studies using global Nox4 knockout (Nox4KO) mice demonstrated a protective role of Nox4 during chronic abdominal aortic banding, involving a paracrine enhancement of myocardial capillary density. However, other authors who studied cardiac-specific Nox4KO mice reported detrimental effects of Nox4 in response to transverse aortic constriction (TAC). It has been speculated that these divergent results are due to cell-specific actions of Nox4 (i.e. cardiomyocyte Nox4 detrimental but endothelial Nox4 beneficial) and/or differences in the model of pressure overload (i.e. abdominal banding vs. TAC). This study aimed to (i) investigate whether the effects of Nox4 on pressure overload-induced cardiac remodelling vary according to the pressure overload model and (ii) compare the roles of cardiomyocyte vs. endothelial cell Nox4. Methods And Results: Global Nox4KO mice subjected to TAC developed worse cardiac remodelling and contractile dysfunction than wild-type littermates, consistent with our previous results with abdominal aortic banding. Next, we generated inducible cardiomyocyte-specific Nox4 KO mice (Cardio-Nox4KO) and endothelial-specific Nox4 KO mice (Endo-Nox4KO) and studied their responses to pressure overload. Both Cardio-Nox4KO and Endo-Nox4KO developed worse pressure overload-induced cardiac remodelling and dysfunction than wild-type littermates, associated with significant decrease in protein levels of HIF1α and VEGF and impairment of myocardial capillarization. Conclusions: Cardiomyocyte as well as endothelial cell Nox4 contributes to protection against chronic hemodynamic overload-induced cardiac remodelling, at least in part through common effects on myocardial capillary density. [ABSTRACT FROM AUTHOR]

Canonical innate immune signalling involves complex cascades: multiple germline-encoded pattern recognition receptors rapidly recognize pathogen-associated or damage-associated molecular patterns to induce the production of cytokines, which bind to their corresponding receptors to orchestrate subsequent host defense phases. Inflammation is a healthy response to pathogenic signals, which are typically rapid and specific and they terminate once the threat has passed. However, excessive activation or suppression of innate immune or inflammatory responses can lead to considerable human suffering, such as cardiac remodelling. Interestingly, recent studies have revealed that innate immune molecules in the parenchymal cells of the heart influence cardiac homeostasis not only by directly regulating innate immune responses but also through reprogrammed signalling pathways, which are independent of conventional innate immune signalling. Elucidating 'innate immune signalling reprogramming' events will help us better understand the functions of innate immune molecules and, moreover, the pathogenesis of cardiac diseases. [ABSTRACT FROM AUTHOR]

Aims: Left-atrial (LA) fibrosis is an important feature of many atrial fibrillation (AF) substrates. The JAK-STAT system contributes to cardiac remodelling, but its role in AF is unknown. Here we investigated JAK-STAT changes in an AF-model and their potential contributions to LA-fibrosis. Methods and results: LA-remodelling was studied in dogs with heart failure (HF) induced by ventricular tachypacing (VTP, 240 bpm), and in mice with left-ventricular (LV) dysfunction due to myocardial infarction (MI). The selective STAT-3 inhibitor S3I-201 was administered to fibroblasts in vitro or mice in vivo (10 mg/kg/d, osmotic mini-pump). HF-dogs developed LA-selective fibrosis and AF-susceptibility at 1-week VTP. The mRNA-expression of platelet-derived growth factor (PDGF, a JAK-STAT activator) isoforms A, C and D, as well as JAK2, increased in LA fibroblasts from 1-week VTP. HF upregulated protein-expression of PDGF-receptor-β and phosphorylated (activated) signal transducer and activator of transcription 3 (STAT3) in LA. PDGF-AB stimulation of LA fibroblasts increased PDGFR-α, STAT3 and phosphorylated-STAT3 expression, as well as collagen-1 and fibronectin-1 protein secretion (by 1.6- to 20-fold), with smaller changes in LV fibroblasts. Phosphorylated-STAT3 and collagen upregulation were suppressed by the JAK2 inhibitor AG-490, PDGF receptor inhibitor AG1296 and STAT3-inhibitor SI3-201. In vivo S3I-201 treatment of MI-mice attenuated LA-fibrosis, LA-dilation and P-wave duration changes versus vehicle-control. Conclusions: HF activates the LA JAK-STAT system and enhances PDGF-signalling. JAK-STAT inhibition reduces the profibrotic effects of PDGF stimulation on canine fibroblasts in vitro while attenuating in vivo LA-fibrosis and remodelling in post-MI mice, suggesting that the JAK/STAT pathway contributes to LA-fibrogenesis and might be a potential target for LA-fibrosis prevention. [ABSTRACT FROM AUTHOR]

Aims Impaired Ca2+cycling and myocyte contractility are a hallmark of heart failure triggered by pathological stress such as hemodynamic overload. The A-Kinase anchoring protein AKAP150 has been shown to coordinate key aspects of adrenergic regulation of Ca2+cycling and excitation-contraction in cardiomyocytes. However, the role of the AKAP150 signalling complexes in the pathogenesis of heart failure has not been investigated. Methods and results Here we examined how AKAP150 signalling complexes impact Ca2+cycling, myocyte contractility, and heart failure susceptibility following pathological stress. We detected a significant reduction of AKAP150 expression in the failing mouse heart induced by pressure overload. Importantly, cardiac-specific AKAP150 knockout mice were predisposed to develop dilated cardiomyopathy with severe cardiac dysfunction and fibrosis after pressure overload. Loss of AKAP150 also promoted pathological remodelling and heart failure progression following myocardial infarction. However, ablation of AKAP150 did not affect calcineurin-nuclear factor of activated T cells signalling in cardiomyocytes or pressure overload- or agonist-induced cardiac hypertrophy. Immunoprecipitation studies showed that AKAP150 was associated with SERCA2, phospholamban, and ryanodine receptor-2, providing a targeted control of sarcoplasmic reticulum Ca2+regulatory proteins. Mechanistically, loss of AKAP150 led to impaired Ca2+cycling and reduced myocyte contractility reserve following adrenergic stimulation or pressure overload. Conclusions These findings define a critical role for AKAP150 in regulating Ca2+cycling and myocardial ionotropy following pathological stress, suggesting the AKAP150 signalling pathway may serve as a novel therapeutic target for heart failure. [ABSTRACT FROM AUTHOR]

Aims Obesity and type 2 diabetes (T2D) trigger a harmful stress-induced cardiac remodeling process known as cardiomyopathy. These diseases represent a serious and widespread health problem in the Western world; however the underlying molecular basis is not clear. ATF3 is an 'immediate early' gene whose expression is highly and transiently induced in response to multiple stressors such as metabolic, oxidative, endoplasmic reticulum and inflammation, stressors that are involved in T2D cardiomyopathy. The role of ATF3 in diabetic cardiomyopathy is currently unknown. Our research has aimed to study the effect of ATF3 expression on cardiomyocytes, heart function and glucose homeostasis in an obesity-induced T2D mouse model. Methods and results We used wild type mice (WT) as well as mutant mice with a cardiac-specific ATF3 deficiency (ATF3-cKO). Mice were fed a high-fat diet (HFD) for 15 weeks. HFD induced high ATF3 expression in cardiomyocytes. Mice were examined for cardiac remodeling processes and the diabetic state was assessed. HFD-fed ATF3-cKO mice exhibited severe cardiac fibrosis, higher levels of heart hypertrophic markers, increased inflammation and worse cardiac function, as compared to WT mice. Interestingly, HFD-fed ATF3-cKO mice display increased hyperglycemia and reduced glucose tolerance, despite higher blood insulin levels, as compared to HFD-fed WT mice. Elevated levels of the cardiac inflammatory cytokines IL-6 and TNFα leading to impaired insulin signalling may partially explain the peripheral glucose intolerance. Conclusions Cardiac ATF3 has a protective role in dampening the HFD-induced cardiac remodeling processes. ATF3 exerts both local and systemic effects related to T2D-induced cardiomyopathy. This study provides a strong relationship between heart remodeling processes and blood glucose homeostasis. [ABSTRACT FROM AUTHOR]

Aims: Although increased Na+/Ca2+ exchanger 1 (NCX1) expression is observed during heart failure (HF), the pathological role of NCX1 during the progression of HF remains unclear. We examined alterations of NCX1 expression and activity in hearts after transverse aortic constriction (TAC) surgery and explored whether NCX1 influences pressure overloadinduced pathological cardiac remodelling. Methods and results: We generated novel transgenic mice in which NCX1 expression is controlled by a cardiac-specific, doxycycline (DOX)- dependent promoter. In the absence of DOX, TAC surgery caused substantial chamber dilation with a gradual decrease in contractility by 16 weeks. Cardiomyocytes showed a decline in contractility with abnormal Ca2+ handling during excitation- contraction (E-C) coupling. Reduced NCX1 activity was observed 8 weeks after TAC and was still apparent at 17 weeks. Induced NCX1 overexpression by DOX treatment starting 8 weeks after TAC returned NCX1 activity to pre-TAC levels and prevented chamber dilation with cardiac dysfunction. DOX treatment not only upregulated NCX1 expression in TAC-operated hearts but also returned L-type Ca2+ channel and sarcoplasmic reticulum (SR) Ca2+ ATPase expression levels to those in sham-operated hearts. In DOX-treated myocytes, contractility, T-tubule integrity, synchrony of Ca2+ release from the SR, and Ca2+ handling during E-C coupling was preserved 16 weeks after TAC surgery. In addition, DOX treatment attenuated the down-regulation of survival signalling and up-regulation of apoptosis signalling 16 weeks after TAC surgery. Conclusion: Induced overexpression of NCX1 attenuated pressure overload-induced pathological cardiac remodelling. Thus, maintaining NCX1 activity may be a potential therapeutic strategy for preventing the progression of HF. [ABSTRACT FROM AUTHOR]

Aims Leucine-rich α2-glycoprotein (LRG) is considered as a biomarker of the clinical activities of chronic inflammatory diseases, including heart failure. However, its pathophysiological roles in cardiac remodelling after myocardial infarction (MI) remain to be clarified. In this study, we have addressed functional roles of LRG in cardiac remodelling after MI. Methods and results MI was generated by ligating the left coronary artery in mice. Real-time reverse transcription (RT)-PCR and immunoblot analyses revealed that the expressions of LRG transcript and protein were up-regulated in post-infarct myocardium. LRG protein was produced by heart-infiltrating myeloid cells, such as macrophages and neutrophils. To elucidate functional roles of LRG in cardiac remodelling, we generated MI in wild-type (WT) and LRG-deficient (LRG-1-) mice and found that LRG gene ablation aggravated myocardial fibrosis with cardiac dysfunction after MI. Immunohistochemical analyses with anti-CD31 antibody revealed that capillary density decreased at border zone in LRG-1- mice compared with WT mice. Consistently, the expression of apelin receptor was reduced in LRG-1- mice, implying that the impaired angiogenic activity is associated with adverse cardiac remodelling in LRG-1- mice. Moreover, LRG gene ablation suppressed the activation of smad1/5/8, a pro-angiogenic signalling pathway. Finally, the transplantation of WT bone marrow cells into LRG-1- mice attenuated cardiac fibrosis with functional improvement after MI, accompanied by restoration of capillary density compared with the bone marrow transplantation from LRG-1- mice. Conclusion LRG, produced by heart-infiltrating myeloid cells, suppresses adverse cardiac remodelling after MI as a novel cardioprotective factor. LRG signalling could be a therapeutic target against cardiovascular diseases. [ABSTRACT FROM AUTHOR]

Aims: Despite its known cardiovascular benefits, the intracellular signalling mechanisms underlying physiological cardiac growth remain poorly understood. Therefore, the purpose of this study was to investigate a novel role of p21-activated kinase-1 (Pak1) in the regulation of exercise-induced cardiac hypertrophy. Methods and results: Wild-type (WT) and Pak1 KO mice were subjected to 6 weeks of treadmill endurance exercise training (ex-training). Cardiac function was assessed via echocardiography, in situ haemodynamics, and the pCa-force relations in skinned fibre preparations at baseline and at the end of the training regimen. Post-translational modifications to the sarcomeric proteins and expression levels of calcium-regulating proteins were also assessed following ex-training. Heart weight/ tibia length and echocardiography data revealed that there was marked hypertrophy following ex-training in the WT mice, which was not evident in the KO mice. Additionally, following ex-training, WT mice demonstrated an increase in cardiac contractility, myofilament calcium sensitivity, and phosphorylation of cardiac myosin-binding protein C, cardiac TnT, and tropomyosin compared with KO mice. With ex-training in WT mice, there were also increased protein levels of calcineurin and increased phosphorylation of phospholamban. Conclusions: Our data suggest that Pak1 is essential for adaptive physiological cardiac remodelling and support previous evidence that demonstrates Pak1 signalling is important for cardiac growth and survival. [ABSTRACT FROM AUTHOR]

Aims: Postinfarction remodelling is accompanied and influenced by perturbations in MAPK signalling. The Growth arrest and DNA-damage-inducible 45 (Gadd45) proteins are small acidic proteins involved in DNA repair and modulation of MAPK activity. Little is known about the role of Gadd45 in the heart. Here, we explored the potential contribution of Gadd45 gamma (γ) isoform to the acute and late phase of heart failure (HF) after myocardial infarction (MI) and determine the mechanisms underlying Gadd45γ actions. Methods and results: The Gadd45γ isoform is upregulated in murine cardiomyocytes subject to simulated ischemia and in the mouse heart during MI. In order to mimic the situation observed during MI, we enhanced Gadd45γ content in cardiomyocytes with a single injection of an adeno-associated viral (AAV9) vector encoding Gadd45γ under the cTNT promoter. Gadd45γ overexpression induces cardiomyocyte apoptosis, fibrosis, left ventricular dysfunction and HF. On the other hand, genetic deletion of Gadd45γ in KO mice confers resistance to ischemic injury, at least in part by limiting cardiomyocyte apoptosis. Mechanistically, Gadd45γ activates RIP1 and caspase-8 in a p38 MAPK-dependent manner to promote cardiomyocyte death. Conclusions: This work is the first to demonstrate that Gadd45? accumulation during MI promotes the development and persistence of HF by inducing cardiomyocyte apoptosis in a p38 MAPK-dependent manner. We clearly identify Gadd45γ as a therapeutic target in the development of HF. [ABSTRACT FROM AUTHOR]

Aims Post-infarction remodelled failing hearts have reduced metabolic efficiency. Paradoxically, they have increased tolerance to further ischaemic injury. This study was designed to investigate the metabolic mechanisms that may contribute to this phenomenon and to examine the relationship between ischaemic tolerance and metabolic efficiency during post-ischaemic reperfusion. Methods and results Male C57BL/6 mice were subjected to coronary artery ligation (CAL) or SHAM surgery. After 4 weeks, in vivo mechanical function was assessed by echocardiography, and then isolated working hearts were perfused in this sequence: 45 min aerobic, 15 min global no-flow ischaemia, and 30 min aerobic reperfusion. Left ventricular (LV) function, metabolic rates, and metabolic efficiency were measured. Relative to SHAM, both in vivo and in vitro CAL hearts had depressed cardiac function under aerobic conditions (45 and 36%, respectively), but they had a greater recovery of LV function during post-ischaemic reperfusion (67 vs. 49%, P< 0.05). While metabolic efficiency (LV work per ATP produced) was 50% lower during reperfusion of SHAM hearts, metabolic efficiency in CAL hearts did not decrease. During ischaemia, glycogenolysis was 28% lower in CAL hearts, indicative of lower ischaemic proton production. There were no differences in mitochondrial abundance, calcium handling proteins, or key metabolic enzymes. Conclusion Compared with SHAM, remodelled CAL hearts are more tolerant to ischaemic injury and undergo no further deterioration of metabolic efficiency during reperfusion. Less glycogen utilization in CAL hearts during ischaemia may contribute to increased ischaemic tolerance by limiting ischaemic proton production that may improve ion homeostasis during early reperfusion. [ABSTRACT FROM AUTHOR]

Funding Acknowledgements Type of funding sources: Public grant(s) – National budget only. Main funding source(s): This work was supported by grants from Spain´s Ministerio de Economía y Competitividad [(PI18/00543, CIBERCV (CB16/11/00264), co-funded by Fondo Europeo de Desarrollo Regional (FEDER)]; Instituto de Investigación Sanitaria Marqués de Valdecilla, IDIVAL (INNVAL18/20; PREVAL18/03); Grants4Targets from BAYER AG (ID 2017-03-2088). Background Aortic valve stenosis (AS) confronts the left ventricle (LV) with a progressive pressure overload that leads to myocardial remodelling (hypertrophy and fibrosis) and ultimately to heart failure. BMP7, through BMPR1A-mediated pSMAD1/5/(8)9 signalling, protects the LV against maladaptive remodelling. Small BMP-based molecules for therapeutic applications are gaining momentum in different clinical fields. A synthetic peptide agonist of BMPR1A, THR184, has been reported to prevent and reverse experimental renal fibrotic processes. Purpose To assess the potential of THR184 to halt or reverse the LV pathological remodelling induced by pressure overload in mice and evaluate the mechanisms involved in cultured cells. Methods and Results Mice subjected to a 4-week transverse aortic constriction (TAC) period received daily i.p injections of THR184 (n=9) (5µg/g) or saline (n=10) during the 3rd and 4th weeks after TAC, when LV remodelling was already ongoing. LV morphology and function were assessed by echocardiography. LV samples were analysed by qPCR and Western blot. THR184 halted the LV morpho-functional deterioration developed four weeks after TAC as compared to the saline group: [LV mass: THR184, 3.74±0.27 mg vs saline, 4.44±0.71 mg*; long-axis systolic function (MAPSE): THR184, 0.60±0.04 mm vs saline, 0.49±0.06 mm*; LV filling pressures (E/e'): THR184, 36.92±5.41 vs saline, 58.41±19.34**]. Moreover, THR184 partially reverted LV dysfunction, both systolic (MAPSE: 4 wk, 0.60±0.04 mm vs 2 wk, 0.49±0.03 mm***) and diastolic (E/e': 4 wk, 36.92±5.41 vs 2 wk, 46.87±10.04*). The myocardial overexpression of the remodelling-related genes induced by TAC was attenuated by THR184 compared to saline: [Collagen I (Col I): THR184, 6.77±1.72 vs saline, 14.61±1.94 ***); β myosin heavy chain (β-MHC): THR184, 3.08±1.52 vs saline, 5.67±1.14*]. The expression of pSMAD1/5/(8)9 was reduced in saline-treated TAC mice (TAC, 0.67±0.09 vs sham, 1.04±0.05*), while it was rescued in THR184-treated TAC mice (THR184, 1.11±0.14 vs saline, 0.67±0.09**). In cultured cells, THR184 reduced TGF-β1-mediated transcriptional activation of Col I in 3T3 fibroblasts (THR184, 2.40±0.45 vs TGF-β1, 4.34±0.55***) and β-MHC in H9C2 cardiomyocytes (THR184, 1.02±0.38 vs TGF-β1, 4.10±1.56*). The involvement of BMPR1A in the effects of THR184 was supported by the antagonism with LDN or DMH2. Conclusion THR184 activated BMPR1A signalling through pSMAD1/5/(8)9 to halt the maladaptive remodelling of the LV by counteracting TGF-β1-mediated pro-fibrotic and pro-hypertrophic actions. We provide a preclinical proof-of-concept on THR184, a BMP7-based BMPR1A agonist, to treat the deleterious consequences of LV pressure overload. [ABSTRACT FROM AUTHOR]

Funding Acknowledgements Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Institute of Carlos III the Spanish Ministry of Economy and Competitiveness Background Primary percutaneous coronary intervention (PPCI) in patients with ST-segment elevation myocardial infarction (STEMI) improves the survival of patients. Nevertheless, some patients develop left ventricular adverse remodeling (LVAR). The main objective of this study is to find reliable prognostic biomarkers for patients developing LVAR, focusing in the role of pro-inflammatory mediators, including cell populations and secreted molecules. Methods We assessed the level of pro-inflammatory cell subsets, before and after the revascularization, 1 and 6 months after the PPCI, using flow cytometry. We also performed miRNAs microarray in isolated peripheral blood mononuclear cells (PBMCs), and examined the levels of 27 cytokines in patients' serum by multiplex ELISA. Results We observed that levels of pro-inflammatory monocytes were increased soon after PPCI, in patients who afterward developed LVAR. Furthermore, we detected significant increase in the concentration of secreted cytokines such as IL-1b, IL-17, IFN-g and VEGF in LVAR patients post-PPCI. Moreover, specific miRNAs levels regulating these cytokines secretion, also correlated with LVAR. Multivariate regression analysis and ROC curves indicated that these inflammatory mediators were good biomarkers to correlate with the appearance of LVAR in patients. In addition, we found that the combined analysis of these biomarkers increased the specificity and sensibility to identify patients with LVAR. Conclusion Our data suggest that the combined analysis of these inflammatory axes and related molecules, could reliably predict LVAR in STEMI patients. [ABSTRACT FROM AUTHOR]

Funding Acknowledgements Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Ministerio de Ciencia e Innovación (Spain) Diputación General de Aragón Introduction Endothelin-1 (ET-1) is a vasoconstrictor peptide secreted by endothelial cells and cardiac myocytes and fibroblasts. It is involved in oxidative stress, apoptosis regulation and ventricular remodeling processes associated with heart failure and ischemic cardiomyopathy, including myocardial hypertrophy, fibrosis and impaired conduction. ET-1 has been shown to influence cardiac electrophysiology by modulation of calcium and potassium currents and to contribute to arrhythmogenesis and sudden cardiac death. Purpose We aim to characterize the functional role of ET-1 in the electrophysiology of healed myocardial infarction (MI) by analysis of porcine ventricular slices as a highly representative model of ventricular tissue with preserved cellular cross-talks and architecture. Methods Domestic pigs (60–80 kg, n = 3) were infarcted by temporal occlusion of the left anterior descending coronary artery. 8-12 weeks after infarct induction, animals were cardioplegically arrested under deep anesthesia and sacrificed. All animal procedures conformed to the guidelines from Directive 2010/63/EU and were approved by local authorities. 350 µm-thick ventricular slices were produced from transmural tissue blocks of healed MI ventricles. Tissue blocks were taken from remote, adjacent and border zones of the infarct area. Slices were optically mapped within 8 hours after tissue collection to record transmembrane potential and intracellular calcium. Action Potential Duration (APD) and Calcium Transient Duration (CaTD) were measured at 80% repolarization for 0.5, 1 and 2 Hz pacing frequencies in the presence and absence of 100 nM ET-1. The notation n/N is used to denote n tissue slices from N pigs. Results ET-1 prolonged the APD at all frequencies in remote zones, with mean prolongation percentages of 30.5%, 32%, 26.2% at 0.5, 1 and 2 Hz, respectively, n/N=7/3. However, only minor effects were observed in adjacent (mean APD prolongation of 3.3%, 4.9% and 10.7%, n/N=5/3) and border zones (7%, 4% and 3.6%, n/N=5/3). ET-1 caused an increase in CaTD at 1 Hz in the three zones, with no significant regional differences in the amount of CaTD increase: mean prolongation of 14.1% (n/N=7/3) in the remote zone, 12.4 % (n/N=5/3) in the adjacent zone and 20.9% (n/N=4/3) in the border zone. Conclusions In chronic MI pigs, ET-1 induces strong APD and moderate CaTD prolongation of remote normal myocardium at low (0.5 Hz) to high (2 Hz) frequencies. The ET-1-induced effects on the AP of normal tissue, but not on CaT, are disrupted in the border zones of the infarct area and in its proximity. Our results point to ET-1 acting to enhance ventricular repolarization dispersion in chronic MI pigs, which might contribute to increased arrhythmia vulnerability. Open in new tab Download slide [ABSTRACT FROM AUTHOR]

Aims Cardiac remodelling is one of the key pathological changes that occur with cardiovascular disease. Previous studies have demonstrated the beneficial effects of CYP2J2 expression on cardiac injury. In the present study, we investigated the effects of cardiomyocyte-specific CYP2J2 expression and EET treatment on angiotensin II-induced cardiac remodelling and sought to determine the underlying molecular mechanisms involved in this process. Methods and results Eight-week-old mice with cardiomyocyte-specific CYP2J2 expression (αMHC-CYP2J2-Tr) and wild-type (WT) control mice were treated with Ang-II. Ang-II treatment of WT mice induced changes in heart morphology, cardiac hypertrophy and dysfunction, as well as collagen accumulation; however, cardiomyocyte-specific expression of CYP2J2 attenuated these effects. The cardioprotective effects observed in α-MHC-CYP2J2-Tr mice were associated with peroxisome proliferator-activated receptor (PPAR)-γ activation, reduced oxidative stress, reduced NF-κB p65 nuclear translocation, and inhibition of TGF-β1/smad pathway. The effects seen with cardiomyocyte-specific expression of CYP2J2 were partially blocked by treatment with PPAR-γ antagonist GW9662. In in vitro studies, 11,12-EET(1 μmol/L) treatment attenuated cardiomyocyte hypertrophy and remodelling-related protein (collagen I, TGF-β1, TIMP1) expression by inhibiting the oxidative stress-mediated NF-κB pathway via PPAR-γ activation. Furthermore, conditioned media from neonatal cardiomyocytes treated with 11,12-EET inhibited activation of cardiac fibroblasts and TGF-β1/smad pathway. Conclusion Cardiomyocyte-specific expression of CYP2J2 or treatment with EETs protects against cardiac remodelling by attenuating oxidative stress-mediated NF-κBp65 nuclear translocation via PPAR-γ activation. [ABSTRACT FROM PUBLISHER]

Aims Cardiomyocyte swelling occurs in multiple pathological situations and has been associated with contractile dysfunction, cell death, and enhanced propensity to arrhythmias. We investigate whether hypotonic swelling promotes nitric oxide (NO) release in cardiomyocytes, and whether it impacts on swelling-induced contractile dysfunction. Methods and results Superfusing rat cardiomyocytes with a hypotonic solution (HS; 217 mOsm), increased cell volume, reduced myocyte contraction and Ca2+ transient, and increased NO-sensitive 4-amino-5-methylamino-2′,7′-difluorofluorescein diacetate (DAF-FM) fluorescence. When cells were exposed to HS + 2.5 mM of the NO synthase inhibitor l-NAME, cell swelling occurred in the absence of NO release. Swelling-induced NO release was also prevented by the nitric oxide synthase 1 (NOS1) inhibitor, nitroguanidine, and significantly reduced in NOS1 knockout mice. Additionally, colchicine (inhibitor of microtubule polymerization) prevented the increase in DAF-FM fluorescence induced by HS, indicating that microtubule integrity is necessary for swelling-induced NO release. The swelling-induced negative inotropic effect was exacerbated in the presence of either l-NAME, nitroguandine, the guanylate cyclase inhibitor, ODQ, or the PKG inhibitor, KT5823, suggesting that NOS1-derived NO provides contractile support via a cGMP/PKG-dependent mechanism. Indeed, ODQ reduced Ca2+ wave velocity and both ODQ and KT5823 reduced the HS-induced increment in ryanodine receptor (RyR2, Ser2808) phosphorylation, suggesting that in this context, cGMP/PKG may contribute to preserve contractile function by enhancing sarcoplasmic reticulum Ca2+ release. Conclusions Our findings suggest a novel mechanism for NO release in cardiomyocytes with putative pathophysiological relevance determined, at least in part, by its capability to reduce the extent of contractile dysfunction associated with hypotonic swelling. [ABSTRACT FROM AUTHOR]

Aims Pulmonary arterial hypertension (PAH) reflects abnormal pulmonary vascular resistance and causes right ventricular (RV) hypertrophy. Enhancement of the late sodium current (INaL) may result from hypertrophic remodelling. The study tests whether: (i) constitutive INaL enhancement may occur as part of PAH-induced myocardial remodelling; (ii) ranolazine (RAN), a clinically available INaL blocker, may prevent constitutive INaL enhancement and PAH-induced myocardial remodelling. Methods and results PAH was induced in rats by a single monocrotaline (MCT) injection [60 mg/kg intraperitoneally (i.p.)]; studies were performed 3 weeks later. RAN (30 mg/kg bid i.p.) was administered 48 h after MCT and washed-out 15 h before studies. MCT increased RV systolic pressure and caused RV hypertrophy and loss of left ventricular (LV) mass. In the RV, collagen was increased; myocytes were enlarged with T-tubule disarray and displayed myosin heavy chain isoform switch. INaL was markedly enhanced; diastolic Ca2+ was increased and Ca2+ release was facilitated. K+ currents were down-regulated and APD was prolonged. In the LV, INaL was enhanced to a lesser extent and cell Ca2+ content was strongly depressed. Electrical remodelling was less prominent than in the RV. RAN completely prevented INaL enhancement and limited most aspects of PAH-induced remodelling, but failed to affect in vivo contractile performance. RAN blunted the MCT-induced increase in RV pressure and medial thickening in pulmonary arterioles. Conclusion PAH induced remodelling with chamber-specific aspects. RAN prevented constitutive INaL enhancement and blunted myocardial remodelling. Partial mechanical unloading, resulting from an unexpected effect of RAN on pulmonary vasculature, might contribute to this effect. [ABSTRACT FROM PUBLISHER]

Aims The overloaded heart remodels by cardiomyocyte hypertrophy and interstitial fibrosis, which contributes to the development of heart failure. Signalling via the TGFβ-pathway is crucial for this remodelling. Here we tested the hypothesis that microRNAs in the overloaded heart regulate this remodelling process via inhibition of the TGFβ-pathway. Methods and results We show that the miRNA-15 family, which we found to be up-regulated in the overloaded heart in multiple species, inhibits the TGFβ-pathway by targeting of TGFBR1 and several other genes within this pathway directly or indirectly, including p38, SMAD3, SMAD7, and endoglin. Inhibition of miR-15b by subcutaneous injections of LNA-based antimiRs in C57BL/6 mice subjected to transverse aorta constriction aggravated fibrosis and to a lesser extent also hypertrophy. Conclusion We identified the miR-15 family as a novel regulator of cardiac hypertrophy and fibrosis acting by inhibition of the TGFβ-pathway. [ABSTRACT FROM PUBLISHER]

Aims We previously demonstrated that pharmacological activation of mitochondrial aldehyde dehydrogenase 2 (ALDH2) protects the heart against acute ischaemia/reperfusion injury. Here, we determined the benefits of chronic activation of ALDH2 on the progression of heart failure (HF) using a post-myocardial infarction model. Methods and results We showed that a 6-week treatment of myocardial infarction-induced HF rats with a selective ALDH2 activator (Alda-1), starting 4 weeks after myocardial infarction at a time when ventricular remodelling and cardiac dysfunction were present, improved cardiomyocyte shortening, cardiac function, left ventricular compliance and diastolic function under basal conditions, and after isoproterenol stimulation. Importantly, sustained Alda-1 treatment showed no toxicity and promoted a cardiac anti-remodelling effect by suppressing myocardial hypertrophy and fibrosis. Moreover, accumulation of 4-hydroxynonenal (4-HNE)-protein adducts and protein carbonyls seen in HF was not observed in Alda-1-treated rats, suggesting that increasing the activity of ALDH2 contributes to the reduction of aldehydic load in failing hearts. ALDH2 activation was associated with improved mitochondrial function, including elevated mitochondrial respiratory control ratios and reduced H2O2 release. Importantly, selective ALDH2 activation decreased mitochondrial Ca2+-induced permeability transition and cytochrome c release in failing hearts. Further supporting a mitochondrial mechanism for ALDH2, Alda-1 treatment preserved mitochondrial function upon in vitro aldehydic load. Conclusions Selective activation of mitochondrial ALDH2 is sufficient to improve the HF outcome by reducing the toxic effects of aldehydic overload on mitochondrial bioenergetics and reactive oxygen species generation, suggesting that ALDH2 activators, such as Alda-1, have a potential therapeutic value for treating HF patients. [ABSTRACT FROM AUTHOR]

Aims Tissue inhibitor of metalloproteinases (TIMPs) can mediate myocardial remodelling, hypertrophy, and fibrosis in heart disease. We investigated the impact of TIMP2 vs. TIMP3 deficiency in angiotensin II (Ang II)-induced myocardial remodelling and cardiac dysfunction. Methods and results TIMP2−/−, TIMP3−/−, and wild-type (WT) mice received Ang II/saline (Alzet pump) for 2 weeks. Ang II infusion resulted in enhanced myocardial hypertrophy and lack of fibrosis in TIMP2−/−, and conversely, excess fibrosis without hypertrophy in TIMP3−/− mice. Echocardiographic imaging revealed preserved ejection fraction in all groups; however, exacerbated left ventricular (LV) diastolic dysfunction was detected in Ang II-infused TIMP2−/− and TIMP3−/− mice, despite the suppressed Ang II-induced hypertension in TIMP3−/− mice. Enhanced hypertrophy in TIMP2−/− mice impaired active relaxation, while excess fibrosis in TIMP3−/− mice increased LV passive stiffness. Adult WT cardiomyocytes, only when co-cultured with cardiac fibroblasts, exhibited Ang II-induced hypertrophy which was suppressed in TIMP3−/− cardiomyocytes. In vitro studies on adult cardiofibroblasts (quiescent and cyclically stretched), and in vivo analyses, revealed that the increased fibrosis in TIMP3−/−-Ang II hearts is due to post-translational stabilization and deposition of collagen by matricellular proteins [osteopontin and Secreted Protein Acidic and Rich in Cysteine (SPARC)], which correlated with increased inflammation, rather than increased de novo synthesis. Reduced cross-linking enzymes, LOX and PLOD1, could underlie suppressed collagen deposition in TIMP2−/−-Ang II hearts. Conclusion TIMP2 and TIMP3 play fundamental and differential roles in mediating pathological remodelling, independent from their MMP-inhibitory function. TIMP2−/− and TIMP3−/− mice provide a unique opportunity to study myocardial hypertrophy and fibrosis independently, and their impact on cardiac dysfunction. [ABSTRACT FROM AUTHOR]

Signalling and transcriptional control involve precise programmes of gene activation and suppression necessary for cardiovascular physiology. Deep sequencing of DNA-bound transcription factors reveals a remarkable complexity of co-activators or co-repressors that serve to alter chromatin modification and regulate gene expression. The regulated complexes characterized by genome-wide mapping implicate the recruitment and exchange of proteins with specific enzymatic activities that include roles for histone acetylation and methylation in key developmental programmes of the heart. As for transcriptional changes in response to pathological stress, co-regulatory complexes are also differentially utilized to regulate genes in cardiac disease. Members of the histone deacetylase (HDAC) family catalyse the removal of acetyl groups from proteins whose pharmacological inhibition has profound effects preventing heart failure. HDACs interact with a complex co-regulatory network of transcription factors, chromatin-remodelling complexes, and specific histone modifiers to regulate gene expression in the heart. For example, the histone methyltransferase (HMT), enhancer of zeste homolog 2 (Ezh2), is regulated by HDAC inhibition and associated with pathological cardiac hypertrophy. The challenge now is to target the activity of enzymes involved in protein modification to prevent or reverse the expression of genes implicated with cardiac hypertrophy. In this review, we discuss the role of HDACs and HMTs with a focus on chromatin modification and gene function as well as the clinical treatment of heart failure. [ABSTRACT FROM PUBLISHER]

Aims Pellino1 is an evolutionally conserved immune regulator and participates in the regulation of Toll-like receptor/interleukin-1 receptor (TLR/IL-1R)-mediated signalling. Recent studies have shown that TLR/IL-1R contributes to the left ventricular (LV) remodelling after myocardial infarction (MI). However, the role of Pellino1 in LV remodelling following MI has not been investigated. This study examined the effect of Pellino1 silencing on cardiac function and LV remodelling after MI. Methods and results Male C57BL/6 mice were subjected to permanent ligation of left anterior descending coronary artery (LAD) to induce MI. The levels of Pellino1 were significantly increased in the myocardium 3 days and sustained for 4 weeks after MI, when compared with the sham control. Hypoxia increased Pellino1 expression in cultured cardiomyocytes and fibroblasts. To examine whether Pellino1 plays a role in MI-induced cardiac dysfunction and the LV remodelling, we suppressed the expression of Pellino1 either by intramyocardial delivery of adenovirus expressing siRNA for Pellino1 (AdsiPeli1) or by Cre-LoxP-mediated conditional deletion of Pellino1 from the myocardium. In both models, silencing of Pellino1 significantly attenuated MI-induced cardiac dysfunction, decreased scar size, and reduced collagen deposition, when compared with the control groups. Pellino1 silencing in mice also attenuated MI-induced Pellino1 E3 ligase activity, receptor-interacting protein 1 and tumor necrosis factor receptor associated factor 6 (TRAF6) ubiquitination, nuclear factor Kappa B (NF-κB) activity, cytokine production, and inflammatory cell infiltration into the myocardium when compared with the MI group. Conclusions Our data demonstrate that Pellino1 plays an important role in the pathogenesis of MI. Targeting Pellino1 may ameliorate cardiac dysfunction and remodelling following MI. [ABSTRACT FROM PUBLISHER]

Aims Melusin is a muscle-specific chaperone protein whose expression is required for a compensatory hypertrophy response to pressure overload. Here, we evaluated the consequences of melusin overexpression in the setting of myocardial infarction (MI) using a comprehensive multicentre approach. Methods and results Mice overexpressing melusin in the heart (TG) and wild-type controls (WT) were subjected to permanent LAD ligation and both the acute response (Day 3) and subsequent remodelling (2 weeks) were examined. Mortality in wild-type mice was significant between Days 3 and 7, primarily due to cardiac rupture, but melusin's overexpression strongly reduced mortality (43.2% in wild-type vs. 27.3% in melusin-TG, P = 0.005). At Day 3 after MI, a time point preceding the mortality peak, TG hearts had increased heat shock protein 70 expression, increased ERK1/2 signalling, reduced cardiomyocyte hyper-contractility and inflammatory cell infiltrates, and increased matricellular protein expression in the infarcted area. Conclusions Consistent with its function as a chaperone protein, melusin overexpression exerts a dual protective action following MI reducing an array of maladaptive processes. In the early phase after MI, reduced inflammation and myocyte remodelling protect against cardiac rupture. Chronically, reduced myocyte loss and matrix remodelling, with preserved myocyte contractility, confer adaptive LV remodelling. [ABSTRACT FROM AUTHOR]

Aims G protein-coupled receptor 35 (GPR35) has been characterized to be one of the genes that are up-regulated in human heart failure. Since mechanisms controlling GPR35 expression are not known, we investigated the regulation of GPR35 gene and protein expression in cardiac myocytes and in the mouse models of cardiac failure. Methods and results In cardiac myocytes, GPR35 gene expression was found to be exceptionally sensitive to hypoxia and induced by hypoxia-inducible factor-1 (HIF-1) activation. HIF-1-dependent regulation was established by genetic (HIF-1/VP16, Inhibitory Per/Arnt/Sim domain protein) and chemical [desferrioxamine (DFO)] modulation of the HIF-1 pathway and further confirmed by mutation analysis of the GPR35 promoter and by demonstrating direct binding of endogenous HIF-1 to the gene promoter. Hypoxia increased the number and density of GPR35 receptors on the cardiomyocyte cell membranes. Chemical GPR35 agonist Zaprinast caused GPR35 activation and receptor internalization in cardiac myocytes. In addition, overexpressed GPR35 disrupted actin cytoskeleton arrangement and caused morphological changes in cultured cardiomyocytes. GPR35 gene and protein expressions were also induced in mouse models of cardiac failure; the acute phase of myocardial infarction and during the compensatory and decompensatory phase of pressure-load induced cardiac hypertrophy. Conclusions Cardiac expression of GPR35 is regulated by hypoxia through activation of HIF-1. The expression of GPR35 in mouse models of cardiac infarction and pressure load suggests that GPR35 could be used as an early marker of progressive cardiac failure. [ABSTRACT FROM AUTHOR]

Aims Toll-interacting protein (Tollip) is a critical regulator of the Toll-like receptor-mediated signalling pathway. However, the role of Tollip in chronic pressure overload-induced cardiac hypertrophy remains unclear. This study aimed to determine the functional significance of Tollip in the regulation of aortic banding-induced cardiac remodelling and its underlying mechanisms. Methods and results First, we observed that Tollip was down-regulated in human failing hearts and murine hypertrophic hearts, as determined by western blotting and RT–PCR. Using cultured neonatal rat cardiomyocytes, we found that adenovirus vector-mediated overexpression of Tollip limited angiotensin II-induced cell hypertrophy; whereas knockdown of Tollip by shRNA exhibited the opposite effects. We then generated a transgenic (TG) mouse model with cardiac specific-overexpression of Tollip and subjected them to aortic banding (AB) for 8 weeks. When compared with AB-treated wild-type mouse hearts, Tollip-TGs showed a significant attenuation of cardiac hypertrophy, fibrosis, and dysfunction, as measured by echocardiography, immune-staining, and molecular/biochemical analysis. Conversely, a global Tollip-knockout mouse model revealed an aggravated cardiac hypertrophy and accelerated maladaptation to chronic pressure overloading. Mechanistically, we discovered that Tollip interacted with AKT and suppressed its downstream signalling pathway. Pre-activation of AKT in cardiomyocytes largely offset the Tollip-elicited anti-hypertrophic effects. Conclusion Our results provide the first evidence that Tollip serves as a negative regulator of pathological cardiac hypertrophy by blocking the AKT signalling pathway. [ABSTRACT FROM AUTHOR]

Aims Calcium fluctuations and cardiac hypertrophy occur during pregnancy, but the role of the well-studied calcium-activated phosphatase, calcineurin, has not been studied in this setting. The purpose of this study was to determine whether calcineurin signalling is required for cardiac remodelling during pregnancy in mice. Methods and results We first examined calcineurin expression in the heart of mice during pregnancy. We found both calcineurin levels and activity were significantly increased in early-pregnancy and decreased in late-pregnancy. Since progesterone levels start to rise in early-pregnancy, we investigated whether progesterone alone was sufficient to modulate calcineurin levels in vivo. After implantation of progesterone pellets in non-pregnant female mice, cardiac mass increased, whereas cardiac function was maintained. In addition, calcineurin levels increased, which is also consistent with early-pregnancy. To determine whether these effects were occurring in the cardiac myocytes, we treated neonatal rat ventricular myocytes (NRVMs) with pregnancy-associated sex hormones. We found that progesterone treatment, but not oestradiol, increased calcineurin levels. To obtain a functional read-out of increased calcineurin activity, we measured the activity of the transcription factor NFAT, a downstream target of calcineurin. Progesterone treatment significantly increased NFAT activity in NRVMs, and this was blocked by the calcineurin inhibitor cyclosporine A (CsA), showing that the progesterone-mediated increase in NFAT activity requires calcineurin activity. Importantly, CsA treatment of mice completely blocked pregnancy-induced cardiac hypertrophy. Conclusion Our results show that calcineurin is required for pregnancy-induced cardiac hypertrophy, and that calcineurin activity in early-pregnancy is due at least in part to increased progesterone. [ABSTRACT FROM PUBLISHER]

Aims The endothelial nitric oxide synthase (eNOS) contributes to cardiac remodelling. We studied the role of eNOS in the development of myocardial fibrosis during cardiac overload. Methods and results Ten-week-old male C57/Bl6 wildtype (WT) and eNOS mice (eNOS−/−) were subjected to transverse aortic constriction (TAC, 360 μm) and WT were treated with l-NG-nitroarginine methyl ester (l-NAME, 100 mg/kg/day) for 35 days. Inhibition of eNOS by l-NAME induced interstitial fibrosis, augmented replacement fibrosis, and induced apoptosis of cardiac fibroblasts and cardiomyocytes. l-NAME and eNOS−/− markedly increased the fibrosis induced by TAC and enhanced the myocardial prevalence of CXCR4pos fibroblasts. Myocardial stromal-derived factor-1 (SDF-1) expression was up-regulated by l-NAME and down-regulated after TAC. Blood pressure lowering by co-treatment with hydralazine (250 mg/L/day) did not reverse the l-NAME effects. Conclusion eNOS inhibition induces and enhances cardiac fibrosis independently of blood pressure by activating SDF-1/CXCR4, extracellular matrix production in cardiac fibroblasts and by increasing recruitment of fibrocytes from the bone marrow. [ABSTRACT FROM AUTHOR]

Aims HSPA12B is a newly discovered and endothelial-cell-specifically expressed heat shock protein. We have reported recently that overexpression of HSPA12B increased endothelial nitric oxide synthase (eNOS) expression in mouse cardiac tissues during endotoxemia. Endothelial NOS has been shown to protect heart from ischaemic injury. We hypothesized that overexpression of HSPA12B will attenuate cardiac dysfunction and remodelling after myocardial infarction (MI) through an eNOS-dependant mechanism. Methods and results MI was induced by permanent ligation of the left anterior descending coronary artery in the transgenic mice (Tg) overexpressing hspa12b gene and its wild-type (WT) littermates. Echocardiographic analysis revealed that Tg mice exhibited improvements in cardiac dysfunction and remodelling at 1 and 4 weeks after MI. These improvements were accompanied by a significant decrease in cardiomyocyte apoptosis and increase in capillary and arteriolar densities. Significant up-regulation of eNOS, VEGF, Ang-1, and Bcl-2 was also observed in Tg hearts compared with WT hearts after MI. However, pharmacological inhibition of eNOS abolished the HSPA12B-induced decrease in cardiomyocyte apoptosis and increase in capillary formation after MI. Most importantly, inhibition of eNOS abrogated the protection of HSPA12B against cardiac dysfunction and remodelling after MI. Conclusions These data demonstrate for the first time that the overexpression of HSPA12B attenuates cardiac dysfunction and remodelling after MI. This action of HSPA12B was mediated, at least in part, by prevention of cardiomyocyte apoptosis and promotion of myocardial angiogenesis via an eNOS-dependent mechanism. HSPA12B could be a novel target for the management of patients with post-MI cardiac dysfunction and remodelling. [ABSTRACT FROM AUTHOR]

Aims Smad7 plays a negative regulatory role in many inflammatory diseases, but its effect on hypertensive disease remains unknown. The present study tested the hypothesis that overexpression of Smad7 may have therapeutic potential for angiotensin II (Ang II)-mediated hypertensive cardiac remodelling. Methods and results Hypertensive heart disease was induced in mice by subcutaneous infusion of Ang II for 28 days and treated with Smad7 by a non-invasive ultrasound-microbubble-mediated inducible Smad7 gene transfer. We found that cardiac Smad7 was largely reduced in the hypertensive heart and overexpression of cardiac Smad7 protected against the fall in the left ventricular (LV) ejection fraction (EF), an increase in LV mass, and cardiac inflammation and fibrosis such as up-regulation of pro-inflammatory cytokines (IL-1β, TNF-α) and fibrotic markers (collagen I, α-SMA), and infiltration of CD3+ T cells and F4/80+ macrophages. Further studies revealed that inactivation of the Sp1-TGF-β/Smad3-NF-κB (NF-κB, nuclear factor κB) pathways and prevention of cardiac miR-29 loss were mechanisms by which overexpression of Smad7 inhibited Ang II-mediated cardiac remodelling. Importantly, we also found that treatment with Smad7 when hypertensive cardiopathy established at day 14 halted the progression of cardiac injury by blunting the fall of EF and an increase in LV mass, and blocking TGF-β/Smad3-mediated cardiac fibrosis and NF-κB-driven inflammation. Conclusion Smad7 plays a protective role in Ang II-induced cardiac remodelling via mechanisms involving the Sp1-TGF-β/Smad-NF-κB-miR-29 regulatory network. Thus, Smad7 may be a novel therapeutic agent for hypertensive cardiovascular diseases. [ABSTRACT FROM AUTHOR]

Aims While chronic alterations in cardiac triacylglycerol (TAG) metabolism and accumulation are associated with cardiomyopathy, it is unclear whether TAG catabolizing enzymes such as adipose triglyceride lipase (ATGL) play a role in acquired cardiomyopathies. Importantly, germline deletion of ATGL leads to marked cardiac steatosis and heart failure in part through reducing peroxisome proliferator-activated receptor α (PPARα) activity and subsequent fatty acid oxidation (FAO). However, whether ATGL deficiency specifically in adult cardiomyocytes contributes to impaired PPARα activity, cardiac function, and metabolism is not known. Methods and results To study the effects of acquired cardiac ATGL deficiency on cardiac PPARα activity, function, and metabolism, we generated adult mice with tamoxifen-inducible cardiomyocyte-specific ATGL deficiency (icAtglKO). Within 4–6 weeks following ATGL ablation, icAtglKO mice had markedly increased myocardial TAG accumulation, fibrotic remodelling, and pathological hypertrophy. Echocardiographic analysis of hearts in vivo revealed that contractile function was moderately reduced in icAtglKO mice. Analysis of energy metabolism in ex vivo perfused working hearts showed diminished FAO rates which was not paralleled by markedly impaired PPARα target gene expression. Conclusions This study shows that acquired cardiomyocyte-specific ATGL deficiency in adult mice is sufficient to promote fibrotic and hypertrophic cardiomyopathy and impair myocardial FAO in the absence of markedly reduced PPARα signalling. [ABSTRACT FROM PUBLISHER]

Aims Ageing is an important risk factor of cardiovascular diseases including heart failure. Senescence marker protein 30 (SMP30), which was originally identified as an important ageing marker protein, is assumed to act as a novel anti-ageing factor in various organs. However, the role of SMP30 in the heart has not been previously explored. In this study, our aim was to elucidate the functional role of SMP30 on cardiac remodelling. Methods and results SMP30 knockout (KO) mice and wild-type (WT) mice were subjected to continuous angiotensin II (Ang II) infusion. After 14 days, the extent of cardiac hypertrophy and myocardial fibrosis was significantly higher in SMP30-KO mice than in WT mice. Echocardiography revealed that SMP30-KO mice had more severely depressed systolic and diastolic function with left ventricular dilatation compared with WT mice. Generation of reactive oxygen species related with activation of nicotinamide adenine dinucleotide phosphate-oxidase was greater in SMP30-KO mice than in WT mice. The number of deoxynucleotidyl transferase-mediated dUTP nick end-labelling positive nuclei was markedly increased in SMP30-KO mice with activation of caspase-3, increases in the Bax to Bcl-2 ratio and phosphorylation of c-Jun N-terminal kinase compared with WT mice. Furthermore, the number of senescence-associated β-galactosidase-positive cells was significantly increased via up-regulation of p21 gene expression in SMP30-KO mice compared with WT mice. Conclusion This study demonstrated the first evidence that deficiency of SMP30 exacerbates Ang II-induced cardiac hypertrophy, dysfunction, and remodelling, suggesting that SMP30 has a cardio-protective role in cardiac remodelling with anti-oxidative and anti-apoptotic effects in response to Ang II. [ABSTRACT FROM PUBLISHER]

Aims Infarct-remodelled hearts are less amenable to protection against ischaemia/reperfusion. Understanding preservation of energy metabolism in diseased vs. healthy hearts may help to develop anti-ischaemic strategies effective also in jeopardized myocardium. Methods and results Isolated infarct-remodelled/sham Sprague–Dawley rat hearts were perfused in the working mode and subjected to 15 min of ischaemia and 30 min of reperfusion. Protection of post-ischaemic ventricular work was achieved by pharmacological conditioning with sevoflurane. Oxidative metabolism was measured by substrate flux in fatty acid and glucose oxidation using [3H]palmitate and [14C]glucose. Mitochondrial oxygen consumption was measured in saponin-permeabilized left ventricular muscle fibres. Activity assays of citric acid synthase, hydroxyacyl-CoA dehydrogenase, and pyruvate dehydrogenase and mass spectrometry for acylcarnitine profiling were also performed. Six weeks after coronary artery ligation, the hearts exhibited macroscopic and molecular signs of hypertrophy consistent with remodelling and limited respiratory chain and citric acid cycle capacity. Unprotected remodelled hearts showed a marked decline in palmitate oxidation and acetyl-CoA energy production after ischaemia/reperfusion, which normalized in sevoflurane-protected remodelled hearts. Protected remodelled hearts also showed higher β-oxidation flux as determined by increased oxygen consumption with palmitoylcarnitine/malate in isolated fibres and a lower ratio of C16:1+C16OH/C14 carnitine species, indicative of a higher long-chain hydroxyacyl-CoA dehydrogenase activity. Remodelled hearts exhibited higher PPARα-PGC-1α but defective HIF-1α signalling, and conditioning enabled them to mobilize fatty acids from endogenous triglyceride stores, which closely correlated with improved recovery. Conclusions Protected infarct-remodelled hearts secure post-ischaemic energy production by activation of β-oxidation and mobilization of fatty acids from endogenous triglyceride stores. [ABSTRACT FROM AUTHOR]