Why is early-onset atrial fibrillation uncommon in patients with Duchenne muscular dystrophy? Insights from the mdx mouse.

AIMS: A reduction in both dystrophin and neuronal nitric oxide synthase (NOS1) secondary to microRNA-31 (miR-31) up-regulation contributes to the atrial electrical remodelling that underpins human and experimental atrial fibrillation (AF). In contrast, patients with Duchenne muscular dystrophy (DMD), who lack dystrophin and NOS1 and, at least in the skeletal muscle, have raised miR-31 expression, do not have increase susceptibility to AF in the absence of left ventricular (LV) dysfunction. Here, we investigated whether dystrophin deficiency is also associated with atrial up-regulation of miR-31, loss of NOS1 protein, and increased AF susceptibility in young mdx mice. METHODS AND RESULTS: Echocardiography showed normal cardiac structure and function in 12-13 weeks mdx mice, with no indication by assay of hydroxyproline that atrial fibrosis had developed. The absence of dystrophin in mdx mice was accompanied by an overall reduction in syntrophin and a lower NOS1 protein content in the skeletal muscle and in the left atrial and ventricular myocardium, with the latter occurring alongside reduced Nos1 transcript levels (exons 1-2 by quantitative polymerase chain reaction) and an increase in NOS1 polyubiquitination [assessed using tandem polyubiquitination pulldowns; P < 0.05 vs. wild type (WT)]. Neither the up-regulation of miR-31 nor the substantial reduction in NOS activity observed in the skeletal muscle was present in the atrial tissue of mdx mice. At difference with the skeletal muscle, the mdx atrial myocardium showed a reduction in the constitutive NOS inhibitor, caveolin-1, coupled with an increase in NOS3 serine1177 phosphorylation, in the absence of differences in the protein content of other NOS isoforms or in the relative expression NOS1 splice variants. In line with these findings, transoesophageal atrial burst pacing revealed no difference in AF susceptibility between mdx mice and their WT littermates. CONCLUSION: Dystrophin depletion is not associated with atrial miR-31 up-regulation, reduced NOS activity, or increased AF susceptibility in the mdx mouse. Compared with the skeletal muscle, the milder atrial biochemical phenotype may explain why patients with DMD do not exhibit a higher prevalence of atrial arrhythmias despite a reduction in NOS1 content.

Association between heart rate variability indices and features of spontaneous ventricular tachyarrhythmias in mice.

Direct evidence is limited for the association between heart rate variability (HRV) indices and ventricular tachyarrhythmias (VTAs). While galectin-3 (Gal-3) is regarded as a causal factor for cardiac remodelling and a biomarker for arrhythmias, its regulation on VTAs and HVR is unknown. Using aged transgenic (TG) mice with cardiac overexpression of ß2 -adrenoceptors and spontaneous VTAs, we studied whether changes in HRV indices correlated with the severity of VTAs, and whether Gal-3 gene knockout (KO) in TG mice might limit VTA. Body-surface ECG was recorded (10-minute period) in 9- to 10-month-old mice of non-transgenic (nTG), TG and TG × Gal-3 knockout (TG/KO). Time-domain, frequency-domain and nonlinear-domain HRV indices were calculated using the R-R intervals extracted from ECG signals and compared with frequency of VTAs. TG and TG/KO mice developed frequent VTAs and showed significant changes in certain time-domain and nonlinear-domain HRV indices relative to nTG mice. The severity of VTAs in TG and TG/KO mice in combination, estimated by VTA counts and arrhythmia score, was significantly correlated with certain time-domain and nonlinear-domain HRV indices. In conclusion, significant changes in HRV indices were evident and correlated with the severity of spontaneous VTAs in TG mice. The frequency of VTA and HRV indices were largely comparable between TG and TG/KO mice. Deletion of Gal-3 in TG mice altered certain HRV indices implying influence by neuronally localized Gal-3 on autonomic nervous activity.

Nitric Oxide and Mechano-Electrical Transduction in Cardiomyocytes.

The ability§ of the heart to adapt to changes in the mechanical environment is critical for normal cardiac physiology. The role of nitric oxide is increasingly recognized as a mediator of mechanical signaling. Produced in the heart by nitric oxide synthases, nitric oxide affects almost all mechano-transduction pathways within the cardiomyocyte, with roles mediating mechano-sensing, mechano-electric feedback (via modulation of ion channel activity), and calcium handling. As more precise experimental techniques for applying mechanical stresses to cells are developed, the role of these forces in cardiomyocyte function can be further understood. Furthermore, specific inhibitors of different nitric oxide synthase isoforms are now available to elucidate the role of these enzymes in mediating mechano-electrical signaling. Understanding of the links between nitric oxide production and mechano-electrical signaling is incomplete, particularly whether mechanically sensitive ion channels are regulated by nitric oxide, and how this affects the cardiac action potential. This is of particular relevance to conditions such as atrial fibrillation and heart failure, in which nitric oxide production is reduced. Dysfunction of the nitric oxide/mechano-electrical signaling pathways are likely to be a feature of cardiac pathology (e.g., atrial fibrillation, cardiomyopathy, and heart failure) and a better understanding of the importance of nitric oxide signaling and its links to mechanical regulation of heart function may advance our understanding of these conditions.

Stimulation of ß-adrenoceptors up-regulates cardiac expression of galectin-3 and BIM through the Hippo signalling pathway.

BACKGROUND AND PURPOSE: Expression of the pro-fibrotic galectin-3 and the pro-apoptotic BIM is elevated in diseased heart or after ß-adrenoceptor stimulation, but the underlying mechanisms are unclear. This question was addressed in the present study. EXPERIMENTAL APPROACH: Wild-type mice and mice with cardiac transgenic expression of ß2 -adrenoceptors, mammalian sterile-20 like kinase 1 (Mst1) or dominant-negative Mst1, and non-specific galectin-3 knockout mice were used. Effects of the ß-adrenoceptor agonist isoprenaline or ß-adrenoceptor antagonists were studied. Rat cardiomyoblasts (H9c2) were used for mechanistic exploration. Biochemical assays were performed. KEY RESULTS: Isoprenaline treatment up-regulated expression of galectin-3 and BIM, and this was inhibited by non-selective or selective ß-adrenoceptor antagonists (by 60-70%). Cardiac expression of galectin-3 and BIM was increased in ß2 -adrenoceptor transgenic mice. Isoprenaline-induced up-regulation of galectin-3 and BIM was attenuated by Mst1 inactivation, but isoprenaline-induced galectin-3 expression was exaggerated by transgenic Mst1 activation. Pharmacological or genetic activation of ß-adrenoceptors induced Mst1 expression and yes-associated protein (YAP) phosphorylation. YAP hyper-phosphorylation was also evident in Mst1 transgenic hearts with up-regulated expression of galectin-3 (40-fold) and BIM as well as up-regulation of many YAP-target genes by RNA sequencing. In H9c2 cells, isoprenaline induced YAP phosphorylation and expression of galectin-3 and BIM, effects simulated by forskolin but abolished by PKA inhibitors, and YAP knockdown induced expression of galectin-3 and BIM. CONCLUSIONS AND IMPLICATIONS: Stimulation of cardiac ß-adrenoceptors activated the Mst1/Hippo pathway leading to YAP hyper-phosphorylation with enhanced expression of galectin-3 and BIM. This signalling pathway would have therapeutic potential. LINKED ARTICLES: This article is part of a themed section on Adrenoceptors-New Roles for Old Players. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v176.14/issuetoc.

ß-Adrenoceptor activation affects galectin-3 as a biomarker and therapeutic target in heart disease.

Myocardial fibrosis is a key histopathological component that drives the progression of heart disease leading to heart failure and constitutes a therapeutic target. Recent preclinical and clinical studies have implicated galectin-3 (Gal-3) as a pro-fibrotic molecule and a biomarker of heart disease and fibrosis. However, our knowledge is poor on the mechanism(s) that determine the blood level or regulate cardiac expression of Gal-3. Recent studies have demonstrated that enhanced ß-adrenoceptor activity is a determinant of both circulating concentration and cardiac expression of Gal-3. Pharmacological or transgenic activation of ß-adrenoceptors leads to increased blood levels of Gal-3 and up-regulated cardiac Gal-3 expression, effect that can be reversed with the use of ß-adrenoceptor antagonists. Conversely, Gal-3 gene deletion confers protection against isoprenaline-induced cardiotoxicity and fibrogenesis. At the transcription level, ß-adrenoceptor stimulation activates cardiac mammalian sterile-20-like kinase 1, a pivotal kinase of the Hippo signalling pathway, which is associated with Gal-3 up-regulation. Recent studies have suggested a role for the ß-adrenoceptor-Hippo signalling pathway in the regulation of cardiac Gal-3 expression thereby contributing to the onset and progression of heart disease. This implies a therapeutic potential of the suppression of Gal-3 expression. In this review, we discuss the effects of ß-adrenoceptor activity on Gal-3 as a biomarker and causative mediator in the setting of heart disease and point out pivotal knowledge gaps. LINKED ARTICLES: This article is part of a themed section on Adrenoceptors-New Roles for Old Players. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v176.14/issuetoc.

Galectin-3 deficiency ameliorates fibrosis and remodeling in dilated cardiomyopathy mice with enhanced Mst1 signaling.

Dilated cardiomyopathy (DCM) is a major cause of heart failure without effective therapy. Fibrogenesis plays a key role in the development of DCM, but little is known of the expression of the profibrotic factor galectin-3 (Gal-3) and its role in DCM pathophysiology. In a mouse DCM model with transgenic (TG) overexpression of mammalian sterile 20-like kinase 1 (Mst1), we studied Gal-3 expression and effects of the Gal-3 inhibitor modified citrus pectin (MCP) or Gal-3 gene knockout (KO). Gal-3 deletion in TG mice (TG/KO) was achieved by crossbreeding Mst1-TG mice with Gal-3 KO mice. The DCM phenotype was assessed by echocardiography and micromanometry. Cardiac expression of Gal-3 and fibrosis were determined. The cardiac transcriptome was profiled by RNA sequencing. Mst1-TG mice at 3-8 mo of age exhibited upregulated expression of Gal-3 by ~40-fold. TG mice had dilatation of cardiac chambers, suppressed left ventricular (LV) ejection fraction, poor LV contractility and relaxation, a threefold increase in LV collagen content, and upregulated fibrotic genes. Four-month treatment with MCP showed no beneficial effects. Gal-3 deletion in Mst1-TG mice attenuated chamber dilatation, organ congestion, and fibrogenesis. RNA sequencing identified profound disturbances by Mst1 overexpression in the cardiac transcriptome, which largely remained in TG/KO hearts. Gal-3 deletion in Mst1-TG mice, however, partially reversed the dysregulated transcriptional signaling involving extracellular matrix remodeling and collagen formation. We conclude that cardiac Mst1 activation leads to marked Gal-3 upregulation and transcriptome disturbances in the heart. Gal-3 deficiency attenuated cardiac remodeling and fibrotic signaling. NEW & NOTEWORTHY We found in a transgenic mouse dilated cardiomyopathy (DCM) model a pronounced upregulation of galectin-3 in cardiomyocytes. Galectin-3 gene deletion reduced cardiac fibrosis and fibrotic gene profiles and ameliorated cardiac remodeling and dysfunction. These benefits of galectin-3 deletion were in contrast to the lack of effect of treatment with the galectin-3 inhibitor modified citrus pectin. Our study suggests that suppression of galectin-3 mRNA expression could be used to treat DCM with high cardiac galectin-3 content.

Mechanisms responsible for increased circulating levels of galectin-3 in cardiomyopathy and heart failure.

Galectin-3 is a biomarker of heart disease. However, it remains unknown whether increase in galectin-3 levels is dependent on aetiology or disease-associated conditions and whether diseased heart releases galectin-3 into the circulation. We explored these questions in mouse models of heart disease and in patients with cardiomyopathy. All mouse models (dilated cardiomyopathy, DCM; fibrotic cardiomyopathy, ischemia-reperfusion, I/R; treatment with ß-adrenergic agonist isoproterenol) showed multi-fold increases in cardiac galectin-3 expression and preserved renal function. In mice with fibrotic cardiomyopathy, I/R or isoproterenol treatment, plasma galectin-3 levels and density of cardiac inflammatory cells were elevated. These models also exhibited parallel changes in cardiac and plasma galectin-3 levels and presence of trans-cardiac galectin-3 gradient, indicating cardiac release of galectin-3. DCM mice showed no change in circulating galectin-3 levels nor trans-cardiac galectin-3 gradient or myocardial inflammatory infiltration despite a 50-fold increase in cardiac galectin-3 content. In patients with hypertrophic cardiomyopathy or DCM, plasma galectin-3 increased only in those with renal dysfunction and a trans-cardiac galectin-3 gradient was not present. Collectively, this study documents the aetiology-dependency and diverse mechanisms of increment in circulating galectin-3 levels. Our findings highlight cardiac inflammation and enhanced ß-adrenoceptor activation in mediating elevated galectin-3 levels via cardiac release in the mechanism.

Upregulated galectin-3 is not a critical disease mediator of cardiomyopathy induced by ß2-adrenoceptor overexpression.

Preclinical studies have demonstrated that anti-galectin-3 (Gal-3) interventions are effective in attenuating cardiac remodeling, fibrosis, and dysfunction. We determined, in a transgenic (TG) mouse model of fibrotic cardiomyopathy, whether Gal-3 expression was elevated and whether Gal-3 played a critical role in disease development. We studied mice with fibrotic cardiomyopathy attributable to cardiac overexpression of human ß2-adrenoceptors (ß2-TG). Cardiac expression levels of Gal-3 and fibrotic or inflammatory genes were determined. The effect of Gal-3 inhibition in ß2-TG mice was studied by treatment with Gal-3 inhibitors ( N-acetyllactosamine and modified citrus pectin) or by deletion of Gal-3 through crossing ß2-TG and Gal-3 knockout mice. Changes in cardiomyopathy phenotypes were assessed by echocardiography and biochemical assays. In ß2-TG mice at 3, 6, and 9 mo of age, upregulation of Gal-3 expression was observed at mRNA (~6- to 15-fold) and protein (~4- to 8-fold) levels. Treatment of ß2-TG mice with N-acetyllactosamine (3 wk) or modified citrus pectin (3 mo) did not reverse cardiac fibrosis, inflammation, and cardiomyopathy. Similarly, Gal-3 gene deletion in ß2-TG mice aged 3 and 9 mo did not rescue the cardiomyopathy phenotype. In conclusion, the ß2-TG model of cardiomyopathy showed a robust upregulation of Gal-3 that correlated with disease severity, but Gal-3 inhibitors or Gal-3 gene deletion had no effect in halting myocardial fibrosis, remodeling, and dysfunction. Gal-3 may not be critical for cardiac fibrogenesis and remodeling in this cardiomyopathy model. NEW & NOTEWORTHY We showed a robust upregulation of cardiac galectin-3 (Gal-3) expression in a mouse model of cardiomyopathy attributable to cardiomyocyte-restricted transgenic activation of ß2-adrenoceptors. However, pharmacological and genetic inhibition of Gal-3 did not confer benefit in this model, implying that Gal-3 may not be a critical disease mediator of cardiac remodeling in this model.

Cardiac Fibrosis and Arrhythmogenesis.

Myocardial injury, mechanical stress, neurohormonal activation, inflammation, and/or aging all lead to cardiac remodeling, which is responsible for cardiac dysfunction and arrhythmogenesis. Of the key histological components of cardiac remodeling, fibrosis either in the form of interstitial, patchy, or dense scars, constitutes a key histological substrate of arrhythmias. Here we discuss current research findings focusing on the role of fibrosis, in arrhythmogenesis. Numerous studies have convincingly shown that patchy or interstitial fibrosis interferes with myocardial electrophysiology by slowing down action potential propagation, initiating reentry, promoting after-depolarizations, and increasing ectopic automaticity. Meanwhile, there has been increasing appreciation of direct involvement of myofibroblasts, the activated form of fibroblasts, in arrhythmogenesis. Myofibroblasts undergo phenotypic changes with expression of gap-junctions and ion channels thereby forming direct electrical coupling with cardiomyocytes, which potentially results in profound disturbances of electrophysiology. There is strong evidence that systemic and regional inflammatory processes contribute to fibrogenesis (i.e., structural remodeling) and dysfunction of ion channels and Ca2+ homeostasis (i.e., electrical remodeling). Recognizing the pivotal role of fibrosis in the arrhythmogenesis has promoted clinical research on characterizing fibrosis by means of cardiac imaging or fibrosis biomarkers for clinical stratification of patients at higher risk of lethal arrhythmia, as well as preclinical research on the development of antifibrotic therapies. At the end of this review, we discuss remaining key questions in this area and propose new research approaches. © 2017 American Physiological Society. Compr Physiol 7:1009-1049, 2017.

Spontaneous ventricular tachyarrhythmias in ß2-adrenoceptor transgenic mice in relation to cardiac interstitial fibrosis.

Myocardial fibrosis is regarded as a pivotal proarrhythmic substrate, but there have been no comprehensive studies showing a correlation between the severity of fibrosis and ventricular tachyarrhythmias (VTAs). Our purpose was to document this relationship in a transgenic (TG) strain of mice with fibrotic cardiomyopathy. TG mice with cardiac overexpression of ß2-adrenoceptors (ß2-AR mice) and non-TG (NTG) littermates were studied at 4-12 mo of age. VTA was quantified by ECG telemetry. The effect of pharmacological blockade of ß2-ARs on VTA was examined. Myocardial collagen content was determined by hydroxyproline assay. NTG and TG mice displayed circadian variation in heart rate, which was higher in TG mice than in NTG mice (P <0.05). Frequent spontaneous ventricular ectopic beats (VEBs) and ventricular tachycardia (VT) were prominent in TG mice but not present in NTG mice. The frequency of VEB and VT episodes in TG mice increased with age (P < 0.01). Ventricular collagen content was greater in TG mice than in NTG mice (P <0.001) and correlated with age (r = 0.71, P < 0.01). The number of VEBs or VT episodes correlated with age (r = 0.83 and r = 0.73) and the content of total or cross-linked collagen (r = 0.62∼0.66, all P <0.01). While having no effect in younger ß2-TG mice, ß2-AR blockade reduced the frequency of VTA in old ß2-TG mice with more severe fibrosis. In conclusion, ß2-TG mice exhibit interstitial fibrosis and spontaneous onset of VTA, becoming more severe with aging. The extent of cardiac fibrosis is a major determinant for both the frequency of VTA and proarrhythmic action of ß2-AR activation.

Pathological hypertrophy reverses ß2-adrenergic receptor-induced angiogenesis in mouse heart.

ß-adrenergic activation and angiogenesis are pivotal for myocardial function but the link between both events remains unclear. The aim of this study was to explore the cardiac angiogenesis profile in a mouse model with cardiomyocyte-restricted overexpression of ß2-adrenoceptors (ß2-TG), and the effect of cardiac pressure overload. ß2-TG mice had heightened cardiac angiogenesis, which was essential for maintenance of the hypercontractile phenotype seen in this model. Relative to controls, cardiomyocytes of ß2-TGs showed upregulated expression of vascular endothelial growth factor (VEGF), heightened phosphorylation of cAMP-responsive-element-binding protein (CREB), and increased recruitment of phospho-CREB, CREB-binding protein (CBP), and p300 to the VEGF promoter. However, when hearts were subjected to pressure overload by transverse aortic constriction (TAC), angiogenic signaling in ß2-TGs was inhibited within 1 week after TAC. ß2-TG hearts, but not controls, exposed to pressure overload for 1-2 weeks showed significant increases from baseline in phosphorylation of Ca(2+)/calmodulin-dependent kinase II (CaMKIIδ) and protein expression of p53, reduction in CREB phosphorylation, and reduced abundance of phospho-CREB, p300 and CBP recruited to the CREB-responsive element (CRE) site of VEGF promoter. These changes were associated with reduction in both VEGF expression and capillary density. While non-TG mice with TAC developed compensatory hypertrophy, (2-TGs exhibited exaggerated hypertrophic growth at week-1 post-TAC, followed by LV dilatation and reduced fractional shortening measured by serial echocardiography. In conclusion, angiogenesis was enhanced by the cardiomyocyte (2AR/CREB/VEGF signaling pathway. Pressure overload rapidly inhibited this signaling, likely as a consequence of activated CaMKII and p53, leading to impaired angiogenesis and functional decompensation.

Therapeutic effects of serelaxin in acute heart failure.

Over the past few decades, research on the peptide hormone, relaxin, has significantly improved our understanding of its biological actions under physiological and diseased conditions. This has facilitated the conducting of clinical trials to explore the use of serelaxin (human recombinant relaxin). Acute heart failure (AHF) is a very difficult to treat clinical entity, with limited success so far in developing new drugs to combat it. A recent phase-III RELAX-AHF trial using serelaxin therapy given during hospitalization revealed acute (ameliorated dyspnea) and chronic (improved 180-day survival) effects. Although these findings support a substantial improvement by serelaxin therapy over currently available therapies for AHF, they also raise key questions and stimulate new hypotheses. To facilitate the development of serelaxin as a new drug for heart disease, joint efforts of clinicians, research scientists and pharmacological industries are necessary to study these questions and hypotheses. In this review, after providing a brief summary of clinical findings and the pathophysiology of AHF, we present a working hypothesis of the mechanisms responsible for the observed efficacy of serelaxin in AHF patients. The existing clinical and preclinical data supporting our hypotheses are summarized and discussed. The development of serelaxin as a drug provides an excellent example of the bilateral nature of translational research.