Small molecule-mediated rapid maturation of human induced pluripotent stem cell-derived cardiomyocytes.

BACKGROUND: Human induced pluripotent stem cell (iPSC)-derived cardiomyocytes (iPSC-CMs) do not display all hallmarks of mature primary cardiomyocytes, especially the ability to use fatty acids (FA) as an energy source, containing high mitochondrial mass, presenting binucleation and increased DNA content per nuclei (polyploidism), and synchronized electrical conduction. This immaturity represents a bottleneck to their application in (1) disease modelling-as most cardiac (genetic) diseases have a middle-age onset-and (2) clinically relevant models, where integration and functional coupling are key. So far, several methods have been reported to enhance iPSC-CM maturation; however, these protocols are laborious, costly, and not easily scalable. Therefore, we developed a simple, low-cost, and rapid protocol to promote cardiomyocyte maturation using two small molecule activators of the peroxisome proliferator-activated receptor ß/δ and gamma coactivator 1-alpha (PPAR/PGC-1α) pathway: asiatic acid (AA) and GW501516 (GW). METHODS AND RESULTS: Monolayers of iPSC-CMs were incubated with AA or GW every other day for ten days resulting in increased expression of FA metabolism-related genes and markers for mitochondrial activity. AA-treated iPSC-CMs responsiveness to the mitochondrial respiratory chain inhibitors increased and exhibited higher flexibility in substrate utilization. Additionally, structural maturity improved after treatment as demonstrated by an increase in mRNA expression of sarcomeric-related genes and higher nuclear polyploidy in AA-treated samples. Furthermore, treatment led to increased ion channel gene expression and protein levels. CONCLUSIONS: Collectively, we developed a fast, easy, and economical method to induce iPSC-CMs maturation via PPAR/PGC-1α activation. Treatment with AA or GW led to increased metabolic, structural, functional, and electrophysiological maturation, evaluated using a multiparametric quality assessment.

Clinical Phenotypes of Heart Failure With Preserved Ejection Fraction to Select Preclinical Animal Models.

At least one-half of the growing heart failure population consists of heart failure with preserved ejection fraction (HFpEF). The limited therapeutic options, the complexity of the syndrome, and many related comorbidities emphasize the need for adequate experimental animal models to study the etiology of HFpEF, as well as its comorbidities and pathophysiological changes. The strengths and weaknesses of available animal models have been reviewed extensively with the general consensus that a "1-size-fits-all" model does not exist, because no uniform HFpEF patient exists. In fact, HFpEF patients have been categorized into HFpEF phenogroups based on comorbidities and symptoms. In this review, we therefore study which animal model is best suited to study the different phenogroups-to improve model selection and refinement of animal research. Based on the published data, we extrapolated human HFpEF phenogroups into 3 animal phenogroups (containing small and large animals) based on reports and definitions of the authors: animal models with high (cardiac) age (phenogroup aging); animal models focusing on hypertension and kidney dysfunction (phenogroup hypertension/kidney failure); and models with hypertension, obesity, and type 2 diabetes mellitus (phenogroup cardiometabolic syndrome). We subsequently evaluated characteristics of HFpEF, such as left ventricular diastolic dysfunction parameters, systemic inflammation, cardiac fibrosis, and sex-specificity in the different models. Finally, we scored these parameters concluded how to best apply these models. Based on our findings, we propose an easy-to-use classification for future animal research based on clinical phenogroups of interest.

Ventricular TLR4 Levels Abrogate TLR2-Mediated Adverse Cardiac Remodeling upon Pressure Overload in Mice.

Involvement of the Toll-like receptor 4 (TLR4) in maladaptive cardiac remodeling and heart failure (HF) upon pressure overload has been studied extensively, but less is known about the role of TLR2. Interplay and redundancy of TLR4 with TLR2 have been reported in other organs but were not investigated during cardiac dysfunction. We explored whether TLR2 deficiency leads to less adverse cardiac remodeling upon chronic pressure overload and whether TLR2 and TLR4 additively contribute to this. We subjected 35 male C57BL/6J mice (wildtype (WT) or TLR2 knockout (KO)) to sham or transverse aortic constriction (TAC) surgery. After 12 weeks, echocardiography and electrocardiography were performed, and hearts were extracted for molecular and histological analysis. TLR2 deficiency (n = 14) was confirmed in all KO mice by PCR and resulted in less hypertrophy (heart weight to tibia length ratio (HW/TL), smaller cross-sectional cardiomyocyte area and decreased brain natriuretic peptide (BNP) mRNA expression, p < 0.05), increased contractility (QRS and QTc, p < 0.05), and less inflammation (e.g., interleukins 6 and 1ß, p < 0.05) after TAC compared to WT animals (n = 11). Even though TLR2 KO TAC animals presented with lower levels of ventricular TLR4 mRNA than WT TAC animals (13.2 ± 0.8 vs. 16.6 ± 0.7 mg/mm, p < 0.01), TLR4 mRNA expression was increased in animals with the largest ventricular mass, highest hypertrophy, and lowest ejection fraction, leading to two distinct groups of TLR2 KO TAC animals with variations in cardiac remodeling. This variation, however, was not seen in WT TAC animals even though heart weight/tibia length correlated with expression of TLR4 in these animals (r = 0.078, p = 0.005). Our data suggest that TLR2 deficiency ameliorates adverse cardiac remodeling and that ventricular TLR2 and TLR4 additively contribute to adverse cardiac remodeling during chronic pressure overload. Therefore, both TLRs may be therapeutic targets to prevent or interfere in the underlying molecular processes.

Immunomodulation in Heart Failure with Preserved Ejection Fraction: Current State and Future Perspectives.

The heart failure (HF) epidemic is growing and approximately half of the HF patients have heart failure with preserved ejection fraction (HFpEF). HFpEF is a heterogeneous syndrome, characterized by a preserved left ventricular ejection fraction (LVEF ≥ 50%) with diastolic dysfunction, and is associated with high morbidity and mortality. Underlying comorbidities of HFpEF, i.e., hypertension, type 2 diabetes mellitus, obesity, and renal failure, lead to a systemic pro-inflammatory state, thereby affecting normal cardiac function. Increased inflammatory biomarkers predict incident HFpEF and are higher in patients with HFpEF as compared with heart failure with reduced ejection fraction (HFrEF). Randomized trials in HFpEF patients using traditional HF medication failed to demonstrate a clear benefit on hard endpoints (mortality and/or HF hospitalization). Therefore, therapies targeting underlying comorbidities and systemic inflammation in early HFpEF may provide better opportunities. Here, we provide an overview of the current state and future perspectives of immunomodulatory therapies for HFpEF.

Sex-Specific Aspects in the Pathophysiology and Imaging of Coronary Macro- and Microvascular Disease.

Sex differences in coronary artery disease (CAD) are well established, with women presenting with non-obstructive CAD more often than men do. However, recent evidence has identified coronary microvascular dysfunction as the underlying cause for cardiac complaints, yet sex-specific prevalence numbers are inconclusive. This review summarises known sex-specific aspects in the pathophysiology of both macro- and microvascular dysfunction and identifies currently existing knowledge gaps. In addition, this review describes current diagnostic approaches and whether these should take underlying sex differences into account by, for example, using different techniques or cut-off values for women and men. Future research into both innovation of imaging techniques and perfusion-related sex differences is needed to fill evidence gaps and enable the implementation of the available knowledge in daily clinical practice.

Estrogen Contributions to Microvascular Dysfunction Evolving to Heart Failure With Preserved Ejection Fraction.

Heart failure with preserved ejection fraction (HFpEF) is a syndrome involving microvascular dysfunction. No treatment is available yet and as the HFpEF patient group is expanding due to the aging population, more knowledge on dysfunction of the cardiac microvasculature is required. Endothelial dysfunction, impaired angiogenesis, (perivascular) fibrosis and the pruning of capillaries (rarefaction) may all contribute to microvascular dysfunction in the heart and other organs, e.g., the kidneys. The HFpEF patient group consists mainly of post-menopausal women and female sex itself is a risk factor for this syndrome. This may point toward a role of estrogen depletion after menopause in the development of HFpEF. Estrogens favor the ratio of vasodilating over vasoconstricting factors, which results in an overall lower blood pressure in women than in men. Furthermore, estrogens improve angiogenic capacity and attenuate (perivascular) fibrosis formation. Therefore, we hypothesize that the drop of estrogen levels after menopause contributes to myocardial microvascular dysfunction and renders post-menopausal women more vulnerable for heart diseases that involve the microvasculature. This review provides a detailed summary of molecular targets of estrogen, which might guide future research and treatment options.

Sex-specific influence on cardiac structural remodeling and therapy in cardiovascular disease.

BACKGROUND: Cardiovascular diseases (CVDs) culminating into heart failure (HF) are major causes of death in men and women. Prevalence and manifestation, however, differ between sexes, since men mainly present with coronary artery disease (CAD) and myocardial infarction (MI), and post-menopausal women predominantly present with hypertension. These discrepancies are probably influenced by underlying genetic and molecular differences in structural remodeling pathways involved in hypertrophy, inflammation, fibrosis, and apoptosis. In general, men mainly develop eccentric forms, while women develop concentric forms of hypertrophy. Besides that, women show less inflammation, fibrosis, and apoptosis upon HF. This seems to emerge, at least partially, from the fact that the underlying pathways might be modulated by estrogen, which changes after menopause due to declining of the estrogen levels. CONCLUSION: In this review, sex-dependent alterations in adverse cardiac remodeling are discussed for various CVDs. Moreover, potential therapeutic options, like estrogen treatment, are reviewed.

Flotillins in the intercalated disc are potential modulators of cardiac excitability.

BACKGROUND: The intercalated disc (ID) is important for cardiac remodeling and has become a subject of intensive research efforts. However, as yet the composition of the ID has still not been conclusively resolved and the role of many proteins identified in the ID, like Flotillin-2, is often unknown. The Flotillin proteins are known to be involved in the stabilization of cadherins and desmosomes in the epidermis and upon cancer development. However, their role in the heart has so far not been investigated. Therefore, in this study, we aimed at identifying the role of Flotillin-1 and Flotillin-2 in the cardiac ID. METHODS: Location of Flotillins in human and murine cardiac tissue was evaluated by fluorescent immunolabeling and co-immunoprecipitation. In addition, the effect of Flotillin knockout (KO) on proteins of the ID and in electrical excitation and conduction was investigated in cardiac samples of wildtype (WT), Flotillin-1 KO, Flotilin-2 KO and Flotilin-1/2 double KO mice. Consequences of Flotillin knockdown (KD) on cardiac function were studied (patch clamp and Multi Electrode Array (MEA)) in neonatal rat cardiomyocytes (NRCMs) transfected with siRNAs against Flotillin-1 and/or Flotillin-2. RESULTS: First, we confirmed presence in the ID and mutual binding of Flotillin-1 and Flotillin-2 in murine and human cardiac tissue. Flotillin KO mice did not show cardiac fibrosis, nor hypertrophy or changes in expression of the desmosomal ID proteins. However, protein expression of the cardiac sodium channel NaV1.5 was significantly decreased in Flotillin-1 and Flotillin-1/2 KO mice compared to WT mice. In addition, sodium current density showed a significant decrease upon Flotillin-1/2 KD in NRCMs as compared to scrambled siRNA-transfected NRCMs. MEA recordings of Flotillin-2 KD NRCM cultures showed a significantly decreased spike amplitude and a tendency of a reduced spike slope when compared to control and scrambled siRNA-transfected cultures. CONCLUSIONS: In this study, we demonstrate the presence of Flotillin-1, in addition to Flotillin-2 in the cardiac ID. Our findings indicate a modulatory role of Flotillins on NaV1.5 expression at the ID, with potential consequences for cardiac excitation.

Disturbed Desmoglein-2 in the intercalated disc of pediatric patients with dilated cardiomyopathy.

Dilated cardiomyopathy (DCM) leads to disturbed contraction and force transduction, and is associated with substantial mortality in all age groups. Involvement of a disrupted composition of the intercalated disc (ID) has been reported. However, in children, little is established about such subcellular changes during disease, because of the pathological mix-up with the ongoing cardiac maturation. This leaves maladaptive remodeling often undetected. We aimed at illustrating subcellular alterations in children diagnosed with DCM compared to age-matched controls, focusing on ID proteins known to be crucially stable under healthy conditions and destabilized during cardiac injury in adults. Left ventricular or septal pediatric specimens were collected from 7 individuals diagnosed with DCM (age: 23 weeks in utero to 8 weeks postnatal) and age-matched controls that died of non-cardiovascular cause. We determined the amount of fibrosis and localization of ID proteins by immunohistochemistry. In pediatric DCM, most ID proteins follow similar spatiotemporal changes in localization as in controls. However, although no mutations were found, the signal of the desmosomal protein Desmoglein-2 was reduced in all pediatric DCM specimens, but not in controls or adult DCM patients. Endocardial and transmural fibrosis was increased in all pediatric DCM patients compared to age-matched controls. Composition of the ID in pediatric DCM patients is similar to controls, except for the localization of Desmoglein-2 and presence of severe fibrosis. This suggests that the architecture of desmosomes is already disturbed in the early stages of DCM. These findings contribute to the understanding of pediatric DCM.

Leukocytic Toll-Like Receptor 2 Deficiency Preserves Cardiac Function And Reduces Fibrosis In Sustained Pressure Overload.

An involement of Toll-like receptor 2 (TLR2) has been established in cardiac dysfunction after acute myocardial infarction; however, its role in chronic pressure overload is unclear. We sought to evaluate the role of TLR2 in cardiac hypertrophy, fibrosis and dysfunction in sustained pressure overload. We induced pressure overload via transverse aortic constriction (TAC) in TLR2-/- and wild type (WT) mice, and followed temporal changes over 8 weeks. Despite similar increases in heart weight, left ventricular (LV) ejection fraction (EF) and diastolic function (mitral E/A ratio) were preserved in TLR2-/- mice but impaired in WT mice following TAC. TAC produced less LV fibrosis in TLR2-/- mice associated with lower mRNA levels of collagen genes (Col1a1 and Col3a1) and lower protein level of TGFbeta1, compared to WT mice. Following TAC, the influx of macrophages and CD3 T cells into LV was similar between TLR2-/- and WT mice, whereas levels of cyto/chemokines were lower in the heart and plasma in TLR2-/- mice. TLR2-/- bone marrow-derived cells protected against LVEF decline and fibrosis following TAC. Our findings show that leukocytic TLR2 deficiency protects against LV dysfunction and fibrosis probably via a reduction in inflammatory signaling in sustained pressure overload.

CTGF knockout does not affect cardiac hypertrophy and fibrosis formation upon chronic pressure overload.

BACKGROUND: One of the main contributors to maladaptive cardiac remodeling is fibrosis. Connective tissue growth factor (CTGF), a matricellular protein that is secreted into the cardiac extracellular matrix by both cardiomyocytes and fibroblasts, is often associated with development of fibrosis. However, recent studies have questioned the role of CTGF as a pro-fibrotic factor. Therefore, we aimed to investigate the effect of CTGF on cardiac fibrosis, and on functional, structural, and electrophysiological parameters in a mouse model of CTGF knockout (KO) and chronic pressure overload. METHODS AND RESULTS: A new mouse model of global conditional CTGF KO induced by tamoxifen-driven deletion of CTGF, was subjected to 16weeks of chronic pressure overload via transverse aortic constriction (TAC, control was sham surgery). CTGF KO TAC mice presented with hypertrophic hearts, and echocardiography revealed a decrease in contractility on a similar level as control TAC mice. Ex vivo epicardial mapping showed a low incidence of pacing-induced ventricular arrhythmias (2/12 in control TAC vs. 0/10 in CTGF KO TAC, n.s.) and a tendency towards recovery of the longitudinal conduction velocity of CTGF KO TAC hearts. Picrosirius Red staining on these hearts unveiled increased fibrosis at a similar level as control TAC hearts. Furthermore, genes related to fibrogenesis were also similarly upregulated in both TAC groups. Histological analysis revealed an increase in fibronectin and vimentin protein expression, a significant reduction in connexin43 (Cx43) protein expression, and no difference in NaV1.5 expression of CTGF KO ventricles as compared with sham treated animals. CONCLUSION: Conditional CTGF inhibition failed to prevent TAC-induced cardiac fibrosis and hypertrophy. Additionally, no large differences were found in other parameters between CTGF KO and control TAC mice. With no profound effect of CTGF on fibrosis formation, other factors or pathways are likely responsible for fibrosis development.

A fishing trip to cure arrhythmogenic cardiomyopathy?

The paper entitled "Identification of a New Modulator of the Intercalated Disc in a Zebrafish Model of Arrhythmogenic Cardiomyopathy", as published in 2014 in Science Translational Medicine, examined the effects of the newly discovered drug SB216763 (SB21) on arrhythmogenic cardiomyopathy (ACM). In this paper, the authors focused on mechanisms underlying ACM and the accompanying molecular and cellular alterations. Most importantly they showed that SB21 was able to rescue and partly reverse the ACM phenotype in three different experimental models: (I) a zebrafish model of Naxos disease induced by the overexpression of the 2057del2 mutation in plakoglobin (PKG); (II) neonatal rat cardiomyocytes overexpressing the same mutation in PKG; (III) cardiomyocytes derived from induced pluripotent stem cells expressing two different forms of mutations in plakophilin-2. This editorial will focus on the potency and possible restrictions concerning SB21 treatment as a potential intervention for ACM and the usefulness of the applied zebrafish models in general.

Passive ventricular remodeling in cardiac disease: focus on heterogeneity.

Passive ventricular remodeling is defined by the process of molecular ventricular adaptation to different forms of cardiac pathophysiology. It includes changes in tissue architecture, such as hypertrophy, fiber disarray, alterations in cell size and fibrosis. Besides that, it also includes molecular remodeling of gap junctions, especially those composed by Connexin43 proteins (Cx43) in the ventricles that affect cell-to-cell propagation of the electrical impulse, and changes in the sodium channels that modify excitability. All those alterations appear mainly in a heterogeneous manner, creating irregular and inhomogeneous electrical and mechanical coupling throughout the heart. This can predispose to reentry arrhythmias and adds to a further deterioration into heart failure. In this review, passive ventricular remodeling is described in Hypertrophic Cardiomyopathy (HCM), Dilated Cardiomyopathy (DCM), Ischemic Cardiomyopathy (ICM), and Arrhythmogenic Cardiomyopathy (ACM), with a main focus on the heterogeneity of those alterations mentioned above.