Abstract P484: Defining Disease Specific Cardiac Regulatory Networks

Calnexin is a lectin-like molecular chaperone protein on the endoplasmic reticulum, mediating unfolded protein responses, the endoplasmic reticulum Ca 2+ homeostasis, and Ca 2+ signals conduction. In recent years, studies have found that calnexin plays a key role in the heart diseases. This study aims to explore the role of calnexin in the activation of cardiac fibroblasts. A transverse aortic constriction (TAC) mouse model was established to observe the activation of cardiac fibroblasts in vivo, and the in vitro cardiac fibroblasts activation model was established by transforming growth factor β1 (TGFβ1) stimulation. The adenovirus was respectively used to gene overexpression and silencing calnexin in cardiac fibroblasts to elucidate the relationship between calnexin and cardiac fibroblasts activation, as well as the possible underlying mechanism. We confirmed the establishment of TAC model by echocardiography, hematoxylin-eosin, Masson, and Sirius red staining, and detecting the expression of cardiac fibrosis markers in cardiac tissues. After TGFβ1 stimulation, markers of the activation of cardiac fibroblast, and proliferation and migration of cardiac fibroblast were detected by quantitative PCR, Western blot, EdU assay, and wound healing assay respectively. The results showed that the calnexin expression was reduced in both the TAC mice model and the activated cardiac fibroblasts. The overexpression of calnexin relieved cardiac fibroblasts activation, in contrast, the silencing of calnexin promoted cardiac fibroblasts activation. Furthermore, we found that the endoplasmic reticulum stress was activated during cardiac fibroblasts activation, and endoplasmic reticulum stress was relieved after overexpression of calnexin. Conversely, after the silencing of calnexin, endoplasmic reticulum stress was further aggravated, accompanying with the activation of cardiac fibroblasts. Our data suggest that the overexpression of calnexin may prevent cardiac fibroblasts against activation by alleviating endoplasmic reticulum stress.

Left-ventricular hypertrophy, characterized by cardiomyocyte hypertrophy, interstitial cell proliferation, and immune cell infiltration, is a high risk factor for heart failure and death. Chemokines interacting with G protein-coupled chemokine receptors probably play a role in left-ventricular hypertrophy development by promoting recruitment of activated leukocytes and modulating left-ventricular remodeling. Using the minimally invasive model of transverse aortic constriction in mice, we demonstrated that a variety of chemokine and chemokine receptor messenger Ribonucleic Acid are overexpressed in the early and late phase of hypertrophy progression. Among the chemokine receptors, Cx3cr1 and Ccr2 were most strongly overexpressed and were significantly upregulated at 3, 7, and 14 days after transverse aortic constriction. Ligands of CX3CR1 (Cx3cl1) and CCR2 (Ccl2, Ccl7, Ccl12) were significantly overexpressed in the left ventricle at the early stages after mechanical pressure overload. Pharmacological inhibition of CX3CR1 signaling using the antagonist AZD8797 led to a significant reduction of hypertrophy, whereas inhibition of CCR2 with the RS504393 antagonist did not show any effect. Furthermore, AZD8797 treatment reduced the expression of the hypertrophic marker genes Nppa and Nppb as well as the profibrotic genes Tgfb1 and Col1a1 at 14 days after transverse aortic constriction. These findings strongly suggest the involvement of the CX3CR1/CX3CL1 pathway in the pathogenesis of left-ventricular hypertrophy.

Chlorogenic acid targets STING to alleviate pressure overload-induced heart failure and myocardial fibrosis.

Tetracycline is a powerful tool for controlling the expression of specific transgenes (TGs) in various tissues, including heart. In these mouse systems, TG expression is repressed/enhanced by adding doxycycline (Dox) to the diet. However, Dox has been shown to attenuate matrix metalloproteinase (MMP) expression and activity in various tissues, and MMP inactivation mitigates left ventricular (LV) remodeling in animal models of heart failure. Therefore, we examined the influence of Dox on LV remodeling and MMP expression in mice after transverse aortic constriction (TAC). One month after TAC, cardiac hypertrophy (99% vs. 67%) and the proportion of mice exhibiting congestive heart failure (CHF, 74% vs. 32%) were higher in the TAC + Dox group than in the TAC group (P < 0.05). These differences were no longer seen 2 mo after TAC, although LV was more severely dilated in TAC + Dox mice than in TAC mice (P < 0.05). One month after TAC, the increase in brain natriuretic peptide and beta-myosin heavy chain mRNA levels was 1.6 and 1.7 times higher, respectively, in TAC + Dox mice than in TAC mice (P < 0.01). MMP-2 gelatin zymographic activity increased 1.9- and 2.4-fold in TAC and TAC + Dox mice, respectively (P < 0.01 and P < 0.05 relative to respective sham-operated animals), but the difference between TAC + Dox and TAC mice did not reach statistical significance. Dox did not significantly alter TAC-associated perivascular and interstitial myocardial fibrosis. These findings demonstrate that Dox accelerates the onset of cardiac hypertrophy and the progression to CHF following TAC in mice. Accordingly, care should be taken when designing and interpreting studies based on TG mouse models of LV hypertrophy using the tetracycline-regulated (tet)-on/tet-off system.

Mechanisms promoting the transition from hypertensive heart disease to heart failure with preserved ejection fraction are poorly understood. When inappropriate for salt status, mineralocorticoid (deoxycorticosterone acetate) excess causes hypertrophy, fibrosis, and diastolic dysfunction. Because cardiac mineralocorticoid receptors are protected from mineralocorticoid binding by the absence of 11-beta hydroxysteroid dehydrogenase, salt-mineralocorticoid-induced inflammation is postulated to cause oxidative stress and to mediate cardiac effects. Although previous studies have focused on salt/nephrectomy in accelerating mineralocorticoid-induced cardiac effects, we hypothesized that hypertensive heart disease is associated with oxidative stress and sensitizes the heart to mineralocorticoid, accelerating hypertrophy, fibrosis, and diastolic dysfunction. Cardiac structure and function, oxidative stress, and mineralocorticoid receptor-dependent gene transcription were measured in sham-operated and transverse aortic constriction (studied 2 weeks later) mice without and with deoxycorticosterone acetate administration, all in the setting of normal-salt diet. Compared with sham mice, sham plus deoxycorticosterone acetate mice had mild hypertrophy without fibrosis or diastolic dysfunction. Transverse aortic constriction mice displayed compensated hypertensive heart disease with hypertrophy, increased oxidative stress (osteopontin and NOX4 gene expression), and normal systolic function, filling pressures, and diastolic stiffness. Compared with transverse aortic constriction mice, transverse aortic constriction plus deoxycorticosterone acetate mice had similar left ventricular systolic pressure and fractional shortening but more hypertrophy, fibrosis, and diastolic dysfunction with increased lung weights, consistent with heart failure with preserved ejection fraction. There was progressive activation of markers of oxidative stress across the groups but no evidence of classic mineralocorticoid receptor-dependent gene transcription. Pressure-overload hypertrophy sensitizes the heart to mineralocorticoid excess, which promotes the transition to heart failure with preserved ejection fraction independently of classic mineralocorticoid receptor-dependent gene transcription.

Background: We have been reported that dantrolene (DAN) binds to N-terminal domain of the cardiac ryanodine receptor (RyR2) and corrects the aberrant Ca 2+ leak. We have been also reported that DAN enhances binding affinity of Calmodulin (CaM) to RyR2 in failing cardiomyocytes. In this study, we investigated whether CaM is essential for the anti-arrhythmogenic effect of DAN using pressure-overload heart failure induced by transverse aortic constriction (TAC). Methods and results: Pressure overload to 12 weeks old mice was induced by TAC using a 27-gauge needle. Cardiomyocytes isolated from mice 8 weeks after TAC operation were used in the present study. The survival rate of our TAC mice at 8 weeks after TAC operation was 32 %. LV end-diastolic dimension was significantly enlarged and LV ejection fraction was significantly decreased. Lethal arrhythmias were frequently observed after 2 mg epinephrine infusion in TAC mice, but not in Sham mice. All following experiments were done under 1 μM of KN93 to avoid the effect of CaMKII. The Ca 2+ spark frequency (SpF: s -1 ·100 μm -1 ; by fluo3) was much higher in TAC than in Sham cardiomyocytes {TAC (n=10): 7.6±4.7; Sham (n=5): 5.7±1.9; p&lt;0.01}. Addition of DAN to saponin-permeabilized TAC cells normalize Ca 2+ spark frequency {(n=5): 5.6±1.4}. This effect of DAN disappeared after suramin, CaM remover from RyR2, treatment {(n=4): 7.2±3.2}. Spontaneous Ca 2+ transients (SCaT) were measured during sequential pacing of intact cardiomyocytes from 1 Hz to 5 Hz. SCaT was rarely observed in Sham cells even at 5 Hz pacing, whereas it was observed in 20 % of TAC cells. Pre-incubation with DAN to intact TAC cells normalize inceidence of SCaT (11 %). After suramin incorporation, effect of DAN was abolished (19 %). Conclusions: In conclusion, CaM is essential for the channel stabilization effect of DAN. DAN corrects the aberrant domain-domain interaction, and as the result, CaM binding affinity to the RyR2 improves. These are the possible mechanism underlying anti-arrhythmogenic effect of DAN.

Background Phosphorylation of L-type calcium channels (LTCC) by cAMP-dependent protein kinase A (PKA) increases calcium current (ICa). However, it is unclear if PKA-dependent regulation of ICa is impaired in heart failure (HF) despite evidence for impaired β-adrenergic signaling. Recently, a novel PKA activation pathway by oxidation of regulatory subunit I (RI) has been identified. Objective We investigated the impact of redox-activated PKA for regulation of ICa, intracellular calcium (Ca) handling and contractile function in a pressure overload heart failure mouse model. Methods Knock-in mice (KI) that lack redox-dependent PKA activation (exchange of cysteine 17 of RI with serine) were compared to wild-type (WT) at baseline, 7 days and 6 weeks after transverse aortic constriction (TAC). Mouse echocardiography was performed to evaluate in-vivo cardiac function. ICa was measured by whole-cell patch clamp, PKA activity, cAMP levels, and protein levels of central Ca handling proteins were assessed at different time points in vitro. Results At baseline, no alterations of left ventricular (LV) function (echocardiography) were observed between WT and KI. TAC induced a significant RI oxidation in WT but not KI mice. Despite this difference, at 7 days after TAC, development of LV hypertrophy and impairment of systolic LV function in vivo were similar between WT and KI. Compared to baseline, 7 days after TAC a significant stimulation of peak ICa was observed in WT. In contrast to WT, the stimulation of peak ICa was absent in KI mice at 7 days after TAC. This impairment in peak ICa occurred despite a comparable increase in global PKA activity (ELISA), which was most likely due to increased cAMP levels in our TAC model (assessed by FLIM-FRET). Notably, cAMP levels were comparably increased in between groups (ELISA). Importantly, at 6 weeks after TAC, WT mice showed a mild additional deterioration of systolic LV function in vivo. In contrast, LV function was significantly more impaired in KI mice 6 weeks after TAC, which was accompanied by a significant increase in KI mice mortality. Comparing 6 weeks to 7 days after TAC, there was no stimulation of peak ICa in WT and even a significant decrease in peak ICa in KI mice. In accordance, PKA-dependent LTCC phosphorylation was absent in KI mice 6 weeks after TAC (western blotting). Conclusion Redox-activated PKA seems to exert a protective role by stimulation of ICa during pressure overload. Funding Acknowledgement Type of funding source: Public grant(s) – National budget only. Main funding source(s): Deutsche Forschungsgemeinschaft

Regulation of gene expression is a fundamental mechanism allowing the existence of complex living systems. This regulation takes shape in the form of hierarchical gene regulatory networks (GRNs), culminating in the binding of transcription factors (TFs) to regulatory elements (REs). Uncovering the structures of these GRNs has shed light on the mechanistic basis of complex spatiotemporal processes like embryonic development. The reconstruction of such GRN, for any biological process, is therefore the ultimate goal for its understanding. The planarian Schmidtea mediterranea is an important model organism for the study of adult stem cell systems, cell differentiation and regeneration. However, very little is known about the structure of GRNs in this organism due to the historical lack of adequate tools to study regulatory elements. At the beginning of my thesis, I developed a robust Start-seq protocol to study RE activity in S. mediterranea by probing for transcription initiation events. Following this, I characterized the identified putative REs on the basis of their distribution, chromatin context as well as motif content. Putative REs identified using Start-seq possess characteristic epigenomic signatures as they are situated within regions of open chromatin and are enriched for active epigenetic marks such as H3K27Ac, H3K4me3 and H3k4me1. I next showed that both planarian putative enhancers and promoter are enriched for core promoter motifs that are mostly situated at the expected position. Interestingly, the DPE motif was enriched at the transcription initiation site in both types of REs instead of its described position at ± +30 nt. Following this, I showed that most of the identified REs showed sign of bidirectional transcription initiation which is considered as a characteristic feature of REs. After characterizing the REs, I leveraged the existence of two naturally occurring biotypes within S. mediterranea (sexual and asexual) to investigate gene regulatory networks (GRNs) responsible for the development and maintenance of the planarian reproductive system. These biotypes are genetically similar, but one fails to develop sexual organs. Comparing their transcription initiation landscapes allowed me to identify multiple transcription factors potentially part of in these GRNs. Notably, the majority of the selected TF candidates exhibited expression in sexual organs, with half of the candidates being specifically expressed in these tissues. Knockdown of three candidates (tead 1, thap and cebp 4 ) showed dysregulation of many reproductive genes. In situ hybridizations on sexual markers after RNAi of the three candidates confirmed the abrogation of sexual tissues in these conditions. The results indicated that tead 1 produced a severe phenotype, where most sexual organs were absent, except for the ovaries. The thap candidate exhibited a defect in shell gland formation and yolk tissue patterning, while the cebp 4 candidate displayed an impairment in sperm differentiation. Overall, this study was not able to recreate a GRN governing the development and maintenance of the reproductive tissue in S. mediterranea. However, it lays a foundation for further research on GRNs in planaria as it shows that identification of differentially active REs using the established protocol was successful in obtaining important regulators the planarian reproductive system.

Stimulation of σ1-receptor restores abnormal mitochondrial Ca2 + mobilization and ATP production following cardiac hypertrophy

Insulin resistance (IR) is an early hallmark of pressure overload-induced myocardial injury and heart failure. Although CD36, a fatty acid transporter, regulates systemic insulin sensitivity, its role in myocardial insulin resistance under pressure overload remains unclear. This study aimed to elucidate CD36's cardioprotective mechanisms in this context. Myocardial tissues from dilated cardiomyopathy patients, transverse aortic constriction (TAC) mice, and cultured hypertrophic cardiomyocytes were analyzed for CD36 expression and insulin sensitivity. CD36 overexpression was induced via rAAV9-tnt-CD36. InsR-interacting proteins in CD36-overexpressing cells were profiled using tandem mass spectrometry, identifying HSP90α as the key partner. HSP90α agonists were used in rescue experiments. Glucose uptake (2-NBDG assay) and InsR/Akt phosphorylation were measured. Mechanistic studies included immunofluorescence, subcellular fractionation, and HSF1 transcriptional regulation analysis. CD36 levels and insulin sensitivity were reduced in dilated cardiomyopathy patients, TAC mice, and hypertrophic cardiomyocytes. CD36 overexpression enhanced glucose uptake and insulin signaling. Mass spectrometry identified HSP90α (not HSP90β) as the critical InsR partner modulated by CD36. HSP90α agonists reversed CD36-mediated improvements in glucose uptake and InsR/Akt phosphorylation. Mechanistically, CD36 disrupted HSP90α-InsR binding via (1) HSF1-dependent transcriptional suppression of HSP90α and (2) competitive displacement of HSP90α from InsR. Pathological conditions increased cytosolic InsR-HSP90α trapping, while CD36 redistributed InsR to the plasma membrane. CD36 mitigates pressure overload-induced insulin resistance by dual mechanisms: suppressing HSP90α expression via HSF1 inhibition and competitively displacing HSP90α to promote InsR membrane localization. This stabilizes insulin signaling and restores metabolic homeostasis, highlighting CD36 as a therapeutic target for heart failure involving metabolic-cardiac crosstalk.

Objectives Hypertrophic cardiomyopathy (HCM) is the most common hereditary cardiomyopathy and immune infiltration is considered an indispensable factor involved in its pathogenesis. In this study, we attempted to combine bulk sequencing and single-cell sequencing to map the immune infiltration-related genes in hypertrophic cardiomyopathy. Methods The GSE36961, GSE160997, and GSE122930 datasets were obtained from the Gene Expression Omnibus database. The compositional patterns of the 18 types of immune cell fraction and pathway enrichment score in control and HCM patients were estimated based on the GSE36961 cohort using xCell algorithm. The Weighted Gene Coexpression Network Analysis (WGCNA) was performed to identify genes associated with immune infiltration for hypertrophic cardiomyopathy. The area under the curve (AUC) value was obtained and used to evaluate the discriminatory ability of common immune-related DEGs. “NetworkAnalyst” platform was used to identify TF-gene and TF-miRNA interaction with identified common genes. Heat map was used to determine the association between common DEGs and various immune cells. Results Immune infiltration analysis by the xCell algorithm showed a higher level of CD8+ naive T cells, CD8+ T cells, as well as a lower level of activated dendritic cells (aDC), dendritic cells (DC), immature dendritic cells (iDC), conventional dendritic cells (cDC), macrophages, M1 macrophages, monocytes, and NKT cell in HCM compared with the control group in GSE36961 dataset. aDC, macrophages, and M1 macrophages were the top three discriminators between HCM and control groups with the area under the curve (AUC) of 0.907, 0.867, and 0.941. WGCNA analysis showed that 1258 immune-related genes were included in four different modules. Of these modules, the turquoise module showed a pivotal correlation with HCM. 13 common immune-related DEGs were found by intersecting common DEGs in GSE36961 and GSE160997 datasets with genes from the genes in turquoise module. 5 hub immune-related genes (S100A9, TYROBP, FCER1G, CD14, and S100A8) were identified by protein interaction network. Through analysis of single-cell sequencing data, S100a9, TYROBP, FCER1G, and S100a8 were mainly expressed by infiltrated M1 proinflammatory cells, especially Ccr2-M1 proinflammatory macrophage cells in the heart immune microenvironment while Cd14 was expressed by infiltrated M1 proinflammatory macrophage cells and M2 macrophages in transverse aortic constriction (TAC) mice at 1 week. Higher M2 macrophage and M1 proinflammatory macrophage infiltration as well as lower Ccr2-M1 proinflammatory macrophage and dendritic cells were shown in TAC 1week mice compared with sham mice. Conclusions There was a difference in immune infiltration between HCM patients and normal groups. aDC, macrophages, and M1 macrophages were the top three discriminator immune cell subsets between HCM and control groups. S100A9, TYROBP, FCER1G, CD14, and S100A8 were identified as potential biomarkers to discriminate HCM from the control group. S100a9, TYROBP, FCER1G, and S100a8 were mainly expressed by infiltrated M1 proinflammatory cells, especially Ccr2-M1 proinflammatory cells in the heart immune microenvironment while Cd14 was expressed by M2 macrophages in transverse aortic constriction (TAC) mice at 1 week.

BackgroundHeart failure (HF) is the leading cause of death in western countries. Cardiac dysfunction is accompanied by skeletal alterations resulting in muscle weakness and fatigue. Exercise is an accepted interventional approach correcting cardiac and skeletal dysfunction, thereby improving mortality, re-hospitalization and quality of life. Animal models are used to characterize underpinning mechanisms. Transverse aortic constriction (TAC) results in cardiac pressure overload and finally HF. Whether exercise training improves cardiac remodeling and peripheral cachexia in the TAC mouse model was not analyzed yet. In this study, 2 weeks post TAC animals were randomized into two groups either performing a moderate exercise program (five times per week at 60% VO2 max for 40 min for a total of 8 weeks) or staying sedentary.ResultsIn both TAC groups HF characteristics reduced ejection fraction (− 15% compared to sham, p < 0.001), cardiac remodeling (+ 22.5% cardiomyocyte cross sectional area compared to sham; p < 0.001) and coronary artery congestion (+ 34% diameter compared to sham; p = 0.008) were observed. Unexpectedly, peripheral cachexia was not detected. Furthermore, compared to sedentary group animals from the exercise group showed aggravated HF symptoms [heart area + 9% (p = 0.026), heart circumference + 7% (p = 0.002), right ventricular wall thickness − 30% (p = 0.003)] while muscle parameters were unchanged [Musculus soleus fiber diameter (p = 0.55), Musculus extensor digitorum longus contraction force (p = 0.90)].ConclusionThe severe TAC model is inappropriate to study moderate exercise effects in HF with respect to cardiac and skeletal muscle improvements. Further, the phenotype induced by different TAC procedures should be well documented and taken into account when planning experiments.

Recent data suggest adiponectin, an adipocyte-derived hormone, affects development of heart failure in response to hypertension. Severe short-term pressure overload [1-3 wk of transverse aortic constriction (TAC)] in adiponectin(-/-) mice causes greater left ventricle (LV) hypertrophy than in wild-type (WT) mice, but conflicting results are reported regarding LV remodeling, with either increased or decreased LV end diastolic volume compared with WT mice. Here we assessed the effects of prolonged TAC on LV hypertrophy and remodeling. WT and adiponectin(-/-) mice were subjected to TAC and maintained for 6 wk. Regardless of strain, TAC induced similar LV hypertrophy ( approximately 70%) and upregulation of mRNA for heart failure marker genes. However, LV chamber size was dramatically different, with classic LV dilation in WT TAC mice but concentric LV hypertrophy in adiponectin(-/-) mice. LV end diastolic and systolic volumes were lower and ejection fraction higher in adiponectin(-/-) TAC mice compared with WT, indicating that adiponectin deletion prevented LV remodeling and deterioration in systolic function. The activities of marker enzymes of mitochondrial oxidative capacity were reduced in WT TAC mice by approximately 35%, whereas enzyme activities were maintained at sham levels in adiponectin(-/-) TAC mice. In conclusion, in WT mice, long-term pressure overload caused dilated LV hypertrophy accompanied by decreased activity of mitochondrial oxidative enzymes. Although adiponectin deletion did not affect LV hypertrophy, it prevented LV chamber remodeling and preserved mitochondrial oxidative capacity, suggesting that adiponectin plays a permissive role in mediating changes in cardiac structure and metabolism in response to pressure overload.

Mechanical overload can be classified into pressure overload and volume overload, causing concentric and eccentric cardiac hypertrophy, respectively. Here, we aimed to differentiate the load-mediated signaling pathways involved in pressure versus volume overload cardiac hypertrophy. Pressure or volume overload was imposed on C57BL/6J mice by transverse aortic constriction (TAC) or aortic regurgitation (AR), respectively. After surgery (2 wk), left ventricular structure and function were evaluated by echocardiographic, hemodynamic, and histological analyses. Signaling pathways related to hypertrophy, fibrosis, angiogenesis, and apoptosis were studied by histological analysis, RT-PCR, and Western blot analysis. Although mean wall stress was similar in both TAC and AR mice, systolic wall stress was significantly increased in TAC and diastolic wall stress was mainly elevated in AR. TAC or AR induced concentric or eccentric compensated hypertrophy, respectively. TAC was associated with more significant fibrosis and apoptosis, whereas AR was associated with more significant angiogenesis. MAPK kinase family, β-arrestin-2, Akt, and Ca2+-related signaling pathways were markedly activated in TAC but mildly upregulated or unchanged in AR. Pressure overload and volume overload induce different phenotypic and molecular adaptations in cardiac hypertrophy. Most load-related signaling pathways assessed in this study predominate in pressure but not volume overload. The stimulus-specific heterogeneity in the signaling pathways requires distinct manipulations for further mechanistic and pharmacological studies. NEW & NOTEWORTHY Using the transverse aortic constriction mouse model and the newly developed aortic regurgitation mouse model, we delineated the prominent differences between concentric and eccentric cardiac hypertrophy on morphological, functional, and molecular levels. Our findings are important for the precise diagnosis and treatment of these two types of cardiac hypertrophy. Listen to this article's corresponding podcast at http://ajpheart.podbean.com/e/chinese-english-language-podcast-on-differential-cardiac-remodeling-in-tac-vs-ar/ .

Expression and role of SerpinE2 in cardiac fibrosis