Endothelin-1 influences mechanical properties and contractility of hiPSC derived cardiomyocytes resulting in diastolic dysfunction.

A better understanding of the underlying pathomechanisms of diastolic dysfunction is crucial for the development of targeted therapeutic options with the aim to increase the patients' quality of life. In order to shed light on the processes involved, suitable models are required. Here, effects of endothelin-1 (ET-1) treatment on cardiomyocytes derived from human induced pluripotent stem cells (hiPSCs) were investigated. While it is well established, that ET-1 treatment induces hypertrophy in cardiomyocytes, resulting changes in cell mechanics and contractile behavior with focus on relaxation have not been examined before. Cardiomyocytes were treated with 10 nM of ET-1 for 24 h and 48 h, respectively. Hypertrophy was confirmed by real-time deformability cytometry (RT-DC) which was also used to assess the mechanical properties of cardiomyocytes. For investigation of the contractile behavior, 24 h phase contrast video microscopy was applied. To get a deeper insight into changes on the molecular biological level, gene expression analysis was performed using the NanoString nCounter® cardiovascular disease panel. Besides an increased cell size, ET-1 treated cardiomyocytes are stiffer and show an impaired relaxation. Gene expression patterns in ET-1 treated hiPSC derived cardiomyocytes showed that pathways associated with cardiovascular diseases, cardiac hypertrophy and extracellular matrix were upregulated while those associated with fatty acid metabolism were downregulated. We conclude that alterations in cardiomyocytes after ET-1 treatment go far beyond hypertrophy and represent a useful model for diastolic dysfunction.

Global secretome analysis of resident cardiac progenitor cells from wild-type and transgenic heart failure mice: Why ambience matters.

Resident cardiac progenitor cells (CPCs) have gained attention in cardiac regenerative medicine primarily due to their paracrine activity. In our current study we determined the role of pathological conditions such as heart failure on the autocrine-paracrine action of stem cell antigen-1 (Sca-1) expressing CPC. This comparative secretome profiling of Sca-1+ cells derived from transgenic heart failure (αMHC-cyclin-T1/Gαq overexpression [Cyc] cells) versus healthy (wild-type [Wt] cells) mice, achieved via mass-spectrometric quantification, enabled the identification of over 700 proteins. Our results demonstrate that the heart failure milieu caused a 2-fold enrichment of extracellular matrix proteins (ECM) like biglycan, versican, collagen XII, and angiogenic factors like heparan sulfate proteoglycan 2, plasminogen activator inhibitor 1 in the secretome. We further elucidated the direct influence of the secretome on the functional behavior of Sca-1 + cells via in vitro tube forming assay. Secreted factors present in the diseased milieu induced tube formation in Cyc cells (1.7-fold; p < 0.01) when compared with Wt cells after 24 hr of exposure. The presence of conditioned media moderately increased the proliferation of Cyc cells but had a more pronounced effect on Wt cells. Overall, these findings revealed global modifications in the secretory activity of adult Sca-1 + cells in the heart failure milieu. The secretion of ECM proteins and angiogenic factors, which are crucial for cardiac remodeling and recovery, was notably enriched in the supernatant of Cyc cells. Thus, during heart failure the microenvironment of Sca-1 + cells might favor angiogenesis and proliferation suggesting their potential to recover the damaged heart.

The Other Side of the RAAS: Aldosterone Improves Migration of Cardiac Progenitor Cells.

Stem cell therapy is a promising new option for patients suffering from heart failure. Though many clinical studies show encouraging results, little is known about the signals which cause stem cells to home to diseased but not to healthy hearts. We hypothesized that aldosterone as one of the main players of heart failure functions as an attractant for progenitor cells and stimulates their migration. Stem cell antigen-1 (Sca-1) positive cells were isolated from the hearts of wild type FVB mice via magnetic cell sorting. The migration rate of the cells was determined using aldosterone as an attractant in a modified Boyden chamber (n = 5). Aldosterone led to a dose dependent increase in migration rate and this effect could be prevented by adding its blocker eplerenone. The mineralocorticoid receptor could be detected on Sca-1+ cells via western blot and immunofluorescence. Therefore, aldosterone seems to play a role in stem cell migration and there the effect is most likely mediated by the mineralocorticoid receptor.

Preliminary results of the cross-sectional associations of sedentary behavior and physical activity with serum brain-derived neurotrophic factor in adults with coronary heart disease.

This is the first study to analyze the association of accelerometer-measured patterns of habitual physical activity (PA) and sedentary behavior (SB) with serum BDNF in individuals with coronary heart disease. A total of 30 individuals (M = 69.5 years; 80% men) participated in this pre-post study that aimed to test a multi-behavioral intervention. All participants underwent standardized measurement of anthropometric variables, blood collection, self-administered survey, and accelerometer-based measurement of PA and SB over seven days. Serum BDNF concentrations were measured using enzyme-linked immunosorbent assay kit. We applied separate multiple linear regression analysis to estimate the associations of baseline SB pattern measures, light and moderate-to-vigorous PA with serum BDNF (n = 29). Participants spent 508.7 ± 76.5 min/d in SB, 258.5 ± 71.2 min/d in light PA, and 21.2 ± 15.2 min/d in moderate-to-vigorous PA. Per day, individuals had 15.5 ± 3.2 numbers of 10-to-30 min bouts of SB (average length: 22.2 ± 2.1 min) and 3.4 ± 1.2 numbers of > 30 min bouts of SB (average length: 43.8 ± 2.4 min). Regression analysis revealed no significant associations between any of the accelerometer-based measures and serum BDNF. The findings of this study did not reveal an association of accelerometer-measured PA and SB pattern variables with serum BDNF in individuals with coronary heart disease. In addition, our data revealed a considerable variation of PA and SB which should be considered in future studies.

Simultaneous assessment of spontaneous cage activity and voluntary wheel running in group-housed mice.

Small animal models are frequently used to improve our understanding of the molecular and biological signaling pathways underlying the beneficial effects of physical activity and exercise. Unfortunately, when running wheels are employed, mice and rats are often kept single-housed to determine the individual running distance of each animal. However, social isolation can be stressful for rodents, and may alter an individual's propensity for or response to exercise. For example, increased stress from single housing may significantly affect the results when investigating systemic metabolic responses to exercise. We have combined two already available and well-established systems, a radiotelemetry system and a running wheel, to determine spontaneous cage activity (SCA) as well as voluntary exercise (VE) levels of the individual animal in group-housed rodents. Further, we developed a simple software tool which allows monitoring and analyzing the data. Specifically, the radiotelemetry-system utilizes radio-frequency identification via a small, implanted chip to determine the location of each animal. Since, in addition to the animals' position, also the location of the running wheel in the cage is known, the conclusion of which animal is exercising can be drawn. The developed software enables a fast and reliable assignment of the VE data to the individual animal and a simple analysis of the data collected. Hence, our combined method may be used to investigate the beneficial effects of physical activity, as well as the impact of therapeutic interventions on animal behavior in group-housed rodents.

A Transgenic Mouse Model of Eccentric Left Ventricular Hypertrophy With Preserved Ejection Fraction Exhibits Alterations in the Autophagy-Lysosomal Pathway.

The ubiquitin-proteasome system (UPS) and the autophagy-lysosomal pathway (ALP) are the main proteolytic systems involved in cellular homeostasis. Since cardiomyocytes, as terminally differentiated cells, lack the ability to share damaged proteins with their daughter cells, they are especially reliant on these protein degradation systems for their proper function. Alterations of the UPS and ALP have been reported in a wide range of cardiac diseases, including cardiomyopathies. In this study, we determined whether the UPS and ALP are altered in a mouse model of eccentric left ventricular (LV) hypertrophy expressing both cyclin T1 and Gαq under the control of the cardiac-specific α-myosin heavy chain promoter (double transgenic; DTG). Compared to wild-type (WT) littermates, DTG mice showed higher end-diastolic (ED) LV wall thicknesses and diameter with preserved ejection fraction (EF). The cardiomyopathic phenotype was further confirmed by an upregulation of the fetal gene program and genes associated with fibrosis as well as a downregulation of genes involved in Ca2+ handling. Likewise, higher NT-proBNP levels were detected in DTG mice. Investigation of the UPS showed elevated steady-state levels of (poly)ubiquitinated proteins without alterations of all proteasomal activities in DTG mice. Evaluation of ALP key marker revealed a mixed pattern with higher protein levels of microtubule-associated protein 1 light chain 3 beta (LC3)-I and lysosomal-associated membrane protein-2, lower protein levels of beclin-1 and FYVE and coiled-coil domain-containing protein 1 (FYCO1) and unchanged protein levels of p62/SQSTM1 in DTG mice when compared to WT. At transcriptional level, a > 1.2-fold expression was observed for Erbb2, Hdac6, Lamp2, Nrg1, and Sqstm1, while a < 0.8-fold expression was revealed for Fyco1 in DTG mice. The results related to the ALP suggested overall a repression of the ALP during the initiation process, but an induction of the ALP at the level of autophagosome-lysosome fusion and the delivery of ubiquitinated cargo to the ALP for degradation.

Analysis of DCM associated protein alterations of human right and left ventricles.

Dilated cardiomyopathy (DCM) is characterized by ventricular chamber enlargement and impaired myocardial function. Endomyocardial biopsies (EMB) enable immunohistochemical and molecular characterization of this disease. However, knowledge about specific molecular patterns and their relation to cardiac function in both ventricles is rare. Therefore, we performed a mass spectrometric analysis of 28 paired EMBs of left (LV) and right ventricles (RV) of patients with DCM or suspected myocarditis allowing quantitative profiling of 743 proteins. We analysed associations between protein abundance of LV and RV as well as the echocardiographic parameters LVEF, TAPSE, LVEDDI, and RVEDDI by linear regression models. Overall, more LV than RV proteins were associated with LV parameters or with RVEDDI. Most LV and RV proteins increasing in level with impairing of LVEF were annotated to structural components of cardiac tissue. Additionally, a high proportion of LV proteins with metabolic functions decreased in level with decreasing LVEF. Results were validated with LV heart sections of a genetic murine heart failure model. The study shows, that remodelling and systolic dysfunction in DCM is mirrored by distinct alterations in protein composition of both ventricles. Loss of LV systolic function is reflected predominantly by alterations in proteins assigned to metabolic functions in the LV whereas structural remodelling was more obvious in the RV. Alterations related to intermediate filaments were seen in both ventricles and highlight such proteins as early indicators of LV loss of function. SIGNIFICANCE: The present study report protein sets in the RV and the LV being associated with ventricular function and remodelling in DCM. Protein abundances in the LV and the RV emphasize and expand current knowledge on pathophysiological changes in heart failure and DCM. While RV and LV EMBs do not differ concerning diagnostic assessment of inflammatory status and virus persistence, additional information reflecting disease severity associated protein alterations can be gained by EMB protein profiling. RV and LV protein data provided complementary information. The protein pattern of the LV reflects metabolic changes and an impaired energy production, which is associated with the degree of LV systolic dysfunction and remodelling and may yield important information about the disease status in DCM. On the other hand, at this disease stage of DCM with still preserved RV function, RV alterations in structural proteins may reflect myocardial compensatory protective mechanisms for maintenance of structure and cellular function. The study highlight particular proteins being of interest as heart failure biomarkers in both ventricles which seem to reflect the severity of the disease. Further comparative studies between different HF aetiologies have to evaluate those proteins as markers specific for DCM.

[Update Myocarditis]. / Update Myokarditis.

Myocarditis is a heterogenous disease regarding aetiology, clinical presentation and course. A defined diagnostic procedure is needed to reliably detect myocarditis. While findings from medical laboratory parameters, electrocardiography and echocardiography are rather unspecific, endomyocardial biopsies supply dependable data regarding inflammatory and viral status. Analysis of cardiac MR is constantly being improved in order to increase sensitivity regarding myocarditis detection. Incidence, clinical presentation as well as disease progression and prognosis show considerable gender differences. Adamant implementation of defined diagnostic procedures is needed in order not to overlook myocarditis in women while the understanding of the pathomechanisms behind the gender differences might lead the way to new therapeutic options. Currently, treatment of myocarditis symptoms and heart failure is in the focus of clinical care. In addition, cardiac involvement in systemic inflammatory diseases should be stringently treated via immunosuppression. Avoidance of physical exercise has to be observed in order to reduce cardiac strain and consequently the number of adverse events.

Cardioprotective effect of the secretome of Sca-1+ and Sca-1- cells in heart failure: not equal, but equally important?

AIMS: Both progenitor and differentiated cells were previously shown to secrete cardioprotective substances, but so far there has been no direct comparison of the paracrine effects of the two cell types on heart failure. The study sought to compare the paracrine effect of selected progenitors and the corresponding non-progenitor mononuclear cardiac cells on the cardiac function of transgenic heart failure mice. In addition, we aimed to further enhance the paracrine effect of the cells via pretreatment with the heart failure mediator aldosterone. METHODS AND RESULTS: Transgenic heart failure mice were injected with the supernatant of murine cardiac stem cell antigen-1 positive (Sca-1+) and negative (Sca-1-) cells with or without aldosterone pretreatment. Cardiac function was determined using small animal magnetic resonance imaging. In addition, heart failure markers were determined using enzyme-linked immunosorbent assay, RT-PCR, and bead-based multiplexing assay. While only the secretome of aldosterone pretreated Sca-1+ cells led to a significant improvement in cardiac function, N-terminal pro brain natriuretic peptide plasma levels were significantly lower and galectin-1 levels significantly higher in mice that were treated with either kind of secretome compared with untreated controls. CONCLUSION: In this first direct comparison of the paracrine effects of progenitor cells and a heterogeneous population of mononuclear cardiac cells the supernatants of both cell types showed cardioprotective properties which might be of great relevance for endogenous repair. During heart failure raised aldosterone levels might further increase the paracrine effect of progenitor cells.

Sugars make the difference - Glycosylation of cardiodepressant antibodies regulates their activity in dilated cardiomyopathy.

BACKGROUND: Cardiodepressant antibodies contribute to cardiac dysfunction in dilated cardiomyopathy (DCM). Changes in immunoglobulin G (IgG) glycosylation modulate the activity of various autoimmune diseases and influence disease activity as well as severity of various autoimmune diseases. We hypothesized that alterations in IgG glycosylation are involved in the disease course of DCM. METHODS AND RESULTS: IgG glycosylation was analyzed in plasma samples of 50 DCM patients using a lectin-based ELISA. Negative inotropic (cardiodepressant) activity (NIA) of antibodies was assessed by measuring the effect of purified DCM-IgG on the shortening of isolated rat cardiomyocytes by means of a video-edge detection system. IgG obtained from plasma of healthy blood donors served as control. DCM-IgG contained significantly less sialic acid (-25%) and galactose (-16%; both P < 0.001), but showed no significant differences in core-fucosylation compared to controls. Interestingly, IgG with NIA displayed a lower percentage of sialylation (-16%, P < 0.001) core-fucosylation (-15%, P = 0.015) and galactosylation (-10%, P = 0.129) than IgG without NIA. The extent of NIA was directly associated with IgG sialylation (r = 0.68; P < 0.001) and galactosylation (r = 0.37; P = 0.001). CONCLUSION: Reduced sialylation and galactosylation of IgGs enhances their cardiodepressant activity in DCM indicating that changes in IgG glycosylation may be involved in the pathogenesis of DCM.

Brain-derived neurotrophic factor is related with adverse cardiac remodeling and high NTproBNP.

The brain-derived neurotrophic factor (BDNF) is a neuronal growth factor essential for normal cardiac contraction and relaxation. Alterations in BDNF signaling are related to the development of cardiovascular disease. Whether BDNF is related to subclinical cardiac remodeling is unclear. We related BDNF with echocardiographic parameters and NTproBNP in a large population-based cohort (n = 2,976, median age 48 years; 45% male). Transthoracic echocardiography was performed on all subjects and BDNF was measured by ELISA. Study participants with severe kidney dysfunction, previous myocardial infarction, and LV ejection fraction <40% were excluded. Linear regression models were adjusted for age, sex, lean mass, fat mass, current smoking, systolic blood pressure and depression. Low BDNF was associated with high NTproBNP. A 10,000 pg/ml lower BDNF was related with a 2.5 g higher (95%-confidence interval [CI]: 0.2 to 4.9; p = 0.036) LV mass, 0.01 cm posterior wall thickness (0.003 to 0.022; p = 0.007) and 0.02 E/A ratio (0.003 to 0.042, p = 0.026). Here we show that low BDNF levels are related with adverse cardiac remodeling and higher levels of NTproBNP. Further research is warranted to assess if BDNF may be used to monitor neuronal-cardiac damage during CVD progression.

Crataegus Extract WS®1442 Stimulates Cardiomyogenesis and Angiogenesis From Stem Cells: A Possible New Pharmacology for Hawthorn?

Extracts from the leaves and flowers of Crataegus spp. (i.e., hawthorn species) have been traditionally used with documented preclinical and clinical activities in cardiovascular medicine. Based on reported positive effects on heart muscle after ischemic injury and the overall cardioprotective profile, the present study addressed potential contributions of Crataegus extracts to cardiopoietic differentiation from stem cells. The quantified Crataegus extract WS®1442 stimulated cardiomyogenesis from murine and human embryonic stem cells (ESCs). Mechanistically, this effect was found to be induced by promoting differentiation of cardiovascular progenitor cell populations but not by proliferation. Bioassay-guided fractionation, phytochemical and analytical profiling suggested high-molecular weight ingredients as the active principle with at least part of the activity due to oligomeric procyanidines (OPCs) with a degree of polymerization between 3 and 6 (DP3-6). Transcriptome profiling in mESCs suggested two main, plausible mechanisms: These were early, stress-associated cellular events along with the modulation of distinct developmental pathways, including the upregulation of brain-derived neurotrophic factor (BDNF) and retinoic acid as well as the inhibition of transforming growth factor ß/bone morphogenetic protein (TGFß/BMP) and fibroblast growth factor (FGF) signaling. In addition, WS®1442 stimulated angiogenesis ex vivo in Sca-1+ progenitor cells from adult mice hearts. These in vitro data provide evidence for a differentiation promoting activity of WS®1442 on distinct cardiovascular stem/progenitor cells that could be valuable for therapeutic heart regeneration after myocardial infarction. However, the in vivo relevance of this new pharmacological activity of Crataegus spp. remains to be investigated and active ingredients from bioactive fractions will have to be further characterized.

Sex-Specific Associations of Brain-Derived Neurotrophic Factor and Cardiorespiratory Fitness in the General Population.

The brain-derived neurotrophic factor (BDNF) was initially considered to be neuron-specific. Meanwhile, this neurotrophin is peripherally also secreted by skeletal muscle cells and increases due to exercise. Whether BDNF is related to cardiorespiratory fitness (CRF) is currently unclear. We analyzed the association of serum BDNF levels with CRF in the general population (Study of Health in Pomerania (SHIP-TREND) from Northeast Germany; n = 1607, 51% female; median age 48 years). Sex-stratified linear regression models adjusted for age, height, smoking, body fat, lean mass, physical activity, and depression analyzed the association between BDNF and maximal oxygen consumption (VO2peak), maximal oxygen consumption normalized for body weight (VO2peak/kg), and oxygen consumption at the anaerobic threshold (VO2@AT). In women, 1 mL/min higher VO2peak, VO2peak/kg, and VO2@AT were associated with a 2.43 pg/mL (95% confidence interval [CI]: 1.16 to 3.69 pg/mL; p = 0.0002), 150.66 pg/mL (95% CI: 63.42 to 237.90 pg/mL; p = 0.0007), and 2.68 pg/mL (95% CI: 0.5 to 4.8 pg/mL; p = 0.01) higher BDNF serum concentration, respectively. No significant associations were found in men. Further research is needed to understand the sex-specific association between CRF and BDNF.

Pathophysiological aldosterone levels modify the secretory activity of cardiac progenitor cells.

Cardiac progenitor cells (CPCs) trigger regenerative processes via paracrine mechanisms in response to changes in their environment. In the present study we explored alterations in the secretory activity of CPCs induced by raised aldosterone levels symptomatic for heart failure. The cytokine profile of the supernatant of CPCs that were treated with the mineralocorticoid showed an induction of interleukin-6 secretion. Mass spectrometric analyses revealed an increase in the abundance of secreted proteins associated with regeneration and cell migration like gelsolin and galectin-1. Differential regulation of proteins associated with the extracellular matrix further points to an activation of cell migration. In response to supernatant, migration and proliferation were induced in CPCs, indicating a potential role of paracrine factors in the activation of CPCs from other regions of the heart or extra-cardiac sources. Changes in the secretory activity of CPCs might aim to compensate for the detrimental actions of aldosterone in heart failure.

Brain derived neurotrophic factor contributes to the cardiogenic potential of adult resident progenitor cells in failing murine heart.

AIMS: Resident cardiac progenitor cells show homing properties when injected into the injured but not to the healthy myocardium. The molecular background behind this difference in behavior needs to be studied to elucidate how adult progenitor cells can restore cardiac function of the damaged myocardium. Since the brain derived neurotrophic factor (BDNF) moderates cardioprotection in injured hearts, we focused on delineating its regulatory role in the damaged myocardium. METHODS AND RESULTS: Comparative gene expression profiling of freshly isolated undifferentiated Sca-1 progenitor cells derived either from heart failure transgenic αMHC-CyclinT1/Gαq overexpressing mice or wildtype littermates revealed transcriptional variations. Bdnf expression was up regulated 5-fold during heart failure which was verified by qRT-PCR and confirmed at protein level. The migratory capacity of Sca-1 cells from transgenic hearts was improved by 15% in the presence of 25 ng/ml BDNF. Furthermore, BDNF-mediated effects on Sca-1 cells were studied via pulsed Stable Isotope Labeling of Amino acids in Cell Culture (pSILAC) proteomics approach. After BDNF treatment significant differences between newly synthesized proteins in Sca-1 cells from control and transgenic hearts were observed for CDK1, SRRT, HDGF, and MAP2K3 which are known to regulate cell cycle, survival and differentiation. Moreover BDNF repressed the proliferation of Sca-1 cells from transgenic hearts. CONCLUSION: Comparative profiling of resident Sca-1 cells revealed elevated BDNF levels in the failing heart. Exogenous BDNF (i) stimulated migration, which might improve the homing ability of Sca-1 cells derived from the failing heart and (ii) repressed the cell cycle progression suggesting its potency to ameliorate heart failure.

OMICS-based exploration of the molecular phenotype of resident cardiac progenitor cells from adult murine heart.

Resident cardiac progenitor cells have emerged as a potential source of adult stem cells for regeneration of damaged myocardium. Sca-1 cells, expressing Stem cell antigen-1 as a cell surface marker, are multipotent cells that were shown to differentiate into different cell types i.e. cardiomyocytes. Previous studies have reported that Sca-1 positive cells are able to home to the injured heart. However, the mechanism of improving cardiac function is still unclear. In the current study, we have profiled the proteome and transcriptome of Sca-1 positive cells in comparison with other endogenous heart cell types to unravel the molecular phenotype of the progenitor cells. Among the 861 proteins identified with high confidence in total, 331 non-redundant proteins were overrepresented in Sca-1 positive cells. Highly abundant candidates were mostly associated with cell growth and proliferation, cell migration and cytoskeletal organization. Transcriptional profiling disclosed significant expression of surface antigens such as CD31, CD36, CD38, CD66a, CD102, and CD202B. Growth factors like KITL, JAG2, PDGFB and VEGFC showed a higher expression in Sca-1 progenitor cells than in Sca-1 negative cells. Selective candidates were validated by Western blotting. These global findings provide a basis for the study of their capability to participate in the cardiac regeneration process.

Promise of blood- and bone marrow-derived stem cell transplantation for functional cardiac repair: putting it in perspective with existing therapy.

Intracoronary transplantation of peripheral blood- or bone marrow-derived cells, as tested in several recent trials, is associated with moderate increases in left ventricular (LV) ejection fraction (EF) and a small reduction of LV end-systolic volumes. Substantial variability exists between trials, and most of them are based on a small number of patients. Meta-analyses estimated an increase in EF of 3% to 4% more in comparison with control patients. In this review, the effects are put into perspective with established treatment options for acute myocardial infarction (AMI), such as thrombolysis and acute percutaneous interventions or pharmacotherapy aimed at favorably influencing the cardiac remodeling process. Changes in functional and morphometric parameters of LV performance after cell therapy appear to be in the range of effects observed with reperfusion therapy, pharmacotherapeutic interventions influencing the renin-angiotensin-aldosterone pathway, and beta-blockers after AMI.