Human Heart Failure Patients Research Articles

Abstract 15535: SGK1 is a Key Mediator of Pathological Cardiac Fibrosis

Background: Cardiac fibroblasts initially respond to injury adaptively, proliferating and differentiating into myofibroblasts to maintain cardiac structure and function. However, prolonged cardiac stress leads to pathological cardiac fibrosis, ultimately resulting in heart failure (HF). Thus, there is a major interest in developing therapeutic strategies to constrain fibroblast differentiation and sustained activation. We recently identified the cytosolic protein kinase, serum/glucocorticoid regulated kinase 1 (SGK1), as being largely involved with pathological remodeling in the cardiac myocyte. While there have been reports that SGK1 may also play a role in fibrosis, the mechanistic role for SGK1 in cardiac fibroblasts in response to injury remains unknown. Hypothesis: SGK1 promotes pathological cardiac fibrosis and exacerbates progression to HF. Methods: Transforming growth factor beta, (TGFβ) was used in primary cardiac fibroblasts (CFBs), in vitro , and transverse aortic constriction (TAC) was used as a HF model in mice, in vivo . CFBs were transfected with siRNA to knockdown SGK1, in vitro , and SGK1 floxed mice were injected with a novel AAV9 utilizing a myofibroblast-specific promoter to knockout SGK1 specifically in myofibroblasts (SGK1 mfbKO), in vivo . Results: SGK1 signaling was elevated in cardiac explants obtained from ischemic human HF patients which led us to further evaluate SGK1 activity in CFBs. SGK1 knockdown in CFBs attenuated TGFβ-mediated differentiation and fibrotic signaling. SGK1 mfbKO mice subjected to TAC showed preserved systolic and diastolic cardiac function, measured by echocardiography. At the biochemical level, pathological remodeling and gene markers of fibrosis were attenuated in SGK1 mfbKO mice as determined by qRT-PCR of cardiac extracts. Finally, treatment of CFBs with a small molecule SGK1 inhibitor attenuated differentiation and fibrotic signaling in response to TGFβ treatment. Conclusion: SGK1 knockdown is protective against cardiac fibrosis, attenuating differentiation and fibrotic signaling in CFBs. Further, SGK1 mfbKO protected against pathological cardiac remodeling and functional decline in a mouse model of HF, highlighting the specific pathophysiological contributions of CFBs.

FRI118 3D Reconstruction Of Mitochondria From Human Heart Failure Structures Is Associated With Alterations In CHCHD6 And CHCHD3 Expression

Abstract Disclosure: A.G. Marshall: None. Z. Vue, PhD: None. E. Garza-Lopez: None. H. Beasley, PhD: None. V. Exil: None. K. Actkins: None. T. Miller-Fleming: None. A.O. Hinton: None. Heart failure (HF) is accompanied by symptoms and signs caused by cardiac dysfunction, resulting in reduced longevity. Critically, HF has been associated with mitochondria dysfunction, causing pathophysiology that might contribute to age-dependent decrease in cardiac function. Thus, we hypothesize that reduced cardiac function in response to HF, affects mitochondrial function and morphology. To test this, we investigated mitochondrial morphological changes using 3D reconstruction from 4 control human hearts and 4 human heart failure patients. Mitochondrial samples were 3D rendered and quantified using serial block facing-scanning electron microscopy and the Amira software. From our HF reconstructions, there are many phenotypes, including fragmentation, nanotunnels, and mitochondrial clustering, compared to controls, which may be associated to mitochondrial dysfunction. Surprisingly, we saw altered expression of CHCHD6 and CHCHD3 in human heart failure samples, based on immunofluorescence. Thus, we knocked out CHCHD6 and CHCHD3 in mouse fibroblasts, myotubes and human-induced pluripotent stem cell-derived cardiomyocytes and saw fragmentation in all three models. Presently, HF represents an unmet need with no approved clinical therapies for damaged myocardium replacement. Here, we suggest that CHCHD3 and CHCHD6 may be a molecular target that functions in the development of HF and possibly develop novel therapeutics for the treatment of HF. Presentation: Friday, June 16, 2023

Biotransformation of the Novel Myeloperoxidase Inhibitor AZD4831 in Preclinical Species and Humans.

We report herein an in-depth analysis of the metabolism of the novel myeloperoxidase inhibitor AZD4831 ((R)-1-(2-(1-aminoethyl)-4-chlorobenzyl)-2-thioxo-2,3-dihydro-1H-pyrrolo[3,2-d]pyrimidin-4(5H)-one) in animals and human. Quantitative and qualitative metabolite profiling were performed on samples collected from mass balance studies in rats and humans. Exposure of circulating human metabolites with comparable levels in animal species used in safety assessment were also included. Structural characterization of 20 metabolites was performed by liquid chromatography high-resolution mass spectrometry, and quantification was performed by either 14C analysis using solid phase scintillation counting or accelerator mass spectrometry and, where available, authentication with synthesized metabolite standards. A complete mass balance study in rats is presented, while data from dogs and human are limited to metabolite profiling and characterization. The metabolism of AZD4831 is mainly comprised of reactions at the primary amine nitrogen and the thiourea sulfur, resulting in several conjugated metabolites with or without desulfurization. A carbamoyl glucuronide metabolite of AZD4831 (M7) was the most abundant plasma metabolite in both human healthy volunteers and heart failure patients after single and repeated dose administration of AZD4831, accounting for 75%-80% of the total drug-related exposure. Exposures to M7 and other human circulating metabolites were covered in rats and/or dogs, the two models most frequently used in the toxicology studies, and were also highly abundant in the mouse, the second model other than rat used in carcinogenicity studies. The carbamoyl glucuronide M7 was the main metabolite in rat bile, while a desulfurized and cyclized metabolite (M5) was abundant in rat plasma and excreta. SIGNIFICANCE STATEMENT: The biotransformation of AZD4831, a novel myeloperoxidase inhibitor inhibiting xanthine derivative bearing thiourea and primary aliphatic amine functions, is described. Twenty characterized metabolites demonstrate the involvement of carbamoylation with glucuronidation, desulfurization, and cyclization as main biotransformation reactions. The carbamoyl glucuronide was the main metabolite in human plasma, likely governed by a significant species difference in plasma protein binding for this metabolite, but this and other human plasma metabolites were covered in animals used in the toxicity studies.

Abstract P2048: Matricellular Protein Cilp1 Promotes Myocardial Fibrosis In Response To Myocardial Infarction

Rationale: Cartilage intermediate layer protein 1 (Cilp1) is a secreted extracellular matrix (ECM) protein normally associated with bone and cartilage development. Its function and mechanism of action in adult heart disease remain elusive. Objective: To establish the function and mechanism of action of Cilp1 in post-myocardial infarction (MI) cardiac remodeling. Methods and Results: We investigated the expression of Cilp1 in mouse models of pathological cardiac remodeling and human heart failure patients. Cilp1 was expressed predominantly in cardiac fibroblasts and upregulated in response to cardiac injury and in the heart and blood of heart failure patients. We generated Cilp1 knock out (KO) and transgenic (Tg) mice with N-terminal half of the protein (NCilp1) overexpressed in myofibroblasts. Cilp1 KO mice had better cardiac function, reduced number of immune cells and myofibroblasts, and enhanced microvascular survival after MI compared to wild-type (WT) littermates. Conversely, NCilp1-Tg mice had augmented loss of cardiac function, increased number of myofibroblasts and infarct size after the MI injury. RNA-seq and gene ontology analysis indicated that cell proliferation and mTORC1 signaling were downregulated in KO hearts compared to WT hearts. In vivo BrdU labeling and immunofluorescence staining showed that myofibroblast proliferation in the Cilp1 KO heart was downregulated. Biaxial mechanical testing and ECM gene expression analysis indicated that while MI caused significant stiffness in WT hearts it had little effect on KO hearts. Upregulation of collagen expression after MI injury was attenuated in KO hearts. Recombinant CILP1 protein or NCilp1-conditioned medium promoted proliferation of neonatal rat ventricular cardiac fibroblasts via the mTORC1 signaling pathway. Conclusions: Our studies established a pathological role of Cilp1 in promoting post-MI remodeling, identified a novel function of Cilp1 in promoting myofibroblast proliferation, and suggested that Cilp1 may serve as a potential biomarker for pathological cardiac remodeling and target for fibrotic heart disease.

Abstract P3068: TNFR1 Regulates Cellular Proliferation And Not Pathogenicity Of Cd4 + T-lymphocytes During Ischemic Heart Failure

Ischemic heart failure (HF) is associated with elevated inflammatory responses including pro-inflammatory cytokines, such as Tumor necrosis factor (TNF) α [and its cognate receptors, TNF receptor (TNFR) 1 and TNFR2] and pathological switching of CD4 + T-cells. TNF-TNFR2 signaling is known to promote CD4 + T-cell activation and proliferation. However, it is unknown if CD4 + T-cells also express TNFR1, and if elevated TNFα-TNFR1 signaling during HF mediates pathological effects of CD4 + T-cells. Using flow cytometry, we show that TNFR1 + CD4 + T-cells are amplified in the hearts at 3 days (d) and 8 weeks (wks) post-MI (HF) in mice. As compared to sham, while TNFα expression (and %) is blunted in CD4 + T-cells at 3 d post-MI, it is increased during HF with significantly higher expression in TNFR1 + T-cells as compared to TNFR1 - T-cells. Splenic CD4 + T-cells activated using anti-CD3/CD28 antibodies and treated with neutralizing anti-TNFR1 antibody showed increased T-cell proliferation, and Bcl-xL (pro-survival factor) and TNFα expression without affecting activation marker (CD69) suggesting a role of TNFR1 in mediating pro-apoptotic signaling without affecting activation. To confirm these results, we adoptively transferred (AT) HF-activated splenic WT and TNFR1 -/- T-cells (CD45.2) into CD45.1 naïve mice, and measured changes in cardiac function of recipient mice by echocardiography (as an indicator of pathological activity), and donor T-cells (as a measure of cell survival) in the blood, heart, spleen and mediastinal lymph nodes (mLNs) at 14 wks post-AT. Compared to WT, we observed amplified levels of donor TNFR1 -/- T-cells in the blood, spleen, and mLNs which were not different in the hearts. Consistently, recipient mice injected either with WT and TNFR1 -/- T-cells showed similar degrees of cardiac dysfunction. Interestingly, at 14 wks post-AT the expression of ki67, a proliferation marker, was magnified in HF-activated TNFR1 -/- T-cells (as compared to WT) infiltrated into the myocardium suggesting that TNF-TNFR1 mediates pro-survival and proliferative signaling in T-cells during HF, and not their pathogenicity. Our results also provide deeper insights into the paradoxical outcomes observed in the clinical trials antagonizing TNFα in human HF patients.

Sphingomyelinase activity promotes atrophy and attenuates force in human muscle fibres and is elevated in heart failure patients.

BackgroundActivation of sphingomyelinase (SMase) as a result of a general inflammatory response has been implicated as a mechanism underlying disease‐related loss of skeletal muscle mass and function in several clinical conditions including heart failure. Here, for the first time, we characterize the effects of SMase activity on human muscle fibre contractile function and assess skeletal muscle SMase activity in heart failure patients.MethodsThe effects of SMase on force production and intracellular Ca2+ handling were investigated in single intact human muscle fibres. Additional mechanistic studies were performed in single mouse toe muscle fibres. RNA sequencing was performed in human muscle bundles exposed to SMase. Intramuscular SMase activity was measured from heart failure patients (n = 61, age 69 ± 0.8 years, NYHA III‐IV, ejection fraction 25 ± 1.0%, peak VO2 14.4 ± 0.6 mL × kg × min) and healthy age‐matched control subjects (n = 10, age 71 ± 2.2 years, ejection fraction 60 ± 1.2%, peak VO2 25.8 ± 1.1 mL × kg × min). SMase activity was related to circulatory factors known to be associated with progression and disease severity in heart failure.ResultsSphingomyelinase reduced muscle fibre force production (−30%, P < 0.05) by impairing sarcoplasmic reticulum (SR) Ca2+ release (P < 0.05) and reducing myofibrillar Ca2+ sensitivity. In human muscle bundles exposed to SMase, RNA sequencing analysis revealed 180 and 291 genes as up‐regulated and down‐regulated, respectively, at a FDR of 1%. Gene‐set enrichment analysis identified ‘proteasome degradation’ as an up‐regulated pathway (average fold‐change 1.1, P = 0.008), while the pathway ‘cytoplasmic ribosomal proteins’ (average fold‐change 0.8, P < 0.0001) and factors involving proliferation of muscle cells (average fold‐change 0.8, P = 0.0002) where identified as down‐regulated. Intramuscular SMase activity was ~20% higher (P < 0.05) in human heart failure patients than in age‐matched healthy controls and was positively correlated with markers of disease severity and progression, and with several circulating inflammatory proteins, including TNF‐receptor 1 and 2. In a longitudinal cohort of heart failure patients (n = 6, mean follow‐up time 2.5 ± 0.2 years), SMase activity was demonstrated to increase by 30% (P < 0.05) with duration of disease.ConclusionsThe present findings implicate activation of skeletal muscle SMase as a mechanism underlying human heart failure‐related loss of muscle mass and function. Moreover, our findings strengthen the idea that SMase activation may underpin disease‐related loss of muscle mass and function in other clinical conditions, acting as a common patophysiological mechanism for the myopathy often reported in diseases associated with a systemic inflammatory response.

Abstract 11592: Glycogen Synthase Kinase 3β Localizes to the Z-Disc to Maintain Length Dependent Activation

Glycogen Synthase Kinase 3β (GSK-3β) can modulate myofilament function in vitro . However, it’s in vivo role, mechanism, and translational relevance are unknown, which we investigated here using inducible cardiomyocyte specific GSK-3β KO mice. Compared to tamoxifen-treated GSK-3β fl/fl Cre- mice (Con), skinned myocytes from KO mice had reduced calcium sensitivity at long sarcomere lengths (SL = 2.2 μm), but there were no differences at short SL (~1.9 μm). Thus, myocytes from KO mice did not sensitize to calcium with stretch, a mechanism called length dependent activation (LDA) that underlies the organ-level Frank-Starling law. LDA has been attributed to (1) phosphorylation of myofilament proteins, (2) altered lattice-spacing, and (3) changes to titin’s elastic properties. Using mass spectrometry and small energy x-ray diffraction we ruled out the first two mechanisms, however we did find that GSK-3β KO myocytes had decreased passive tension – indicating the loss of LDA was due to loss of titin as a length sensor. Interestingly, immunofluorescence showed that GSK-3β localized to the z-disc when phosphorylated at Y216 and, via mass spectrometry, phosphorylated primarily z-disc proteins including several sites on the structural protein Ablim-1, which we showed also localizes to the z-disc. These data suggested that GSK-3β’s effect on titin is likely through altering its ability to anchor to the z-disc through targeting of these z-disc proteins. To provide further evidence that GSK-3β is specifically altering LDA, we genetically removed a downstream effecter of LDA, cardiac myosin binding protein-C (cMyBP-C). In vitro treatment with exogenous GSK-3β was able to increase calcium sensitivity at long SLs in both KO and WT mice but had no effect on cMyBPC KO mice or mice lacking the c-terminal domains of cMyBPC that are known to be important for LDA. Lastly, we found that human heart failure patients had less myofilament GSK-3β compared to non-failing patients, and that these same samples had a depressed LDA. This work has identified a novel mechanism by which GSK-3β localizes to the myofilament to modulate LDA and indicates that z-disc localized GSK-3β may be a possible therapeutic target to restore the Frank-Starling mechanism in heart failure patients.

Implication of sphingolipid metabolism gene dysregulation and cardiac sphingosine-1-phosphate accumulation in heart failure

Abstract Introduction and purpose Abnormal levels of bioactive sphingolipids have been linked to disturbances in a variety of biological functions of several diseases, including cardiovascular diseases. However, changes in sphingolipid metabolism associated with progressive human heart failure (HF) have not been described, and S1P levels in HF tissues have not been evaluated. Methods We investigated changes in the transcriptome of human heart tissues using RNA sequencing technologies. We collected 26 left ventricular (LV) tissue samples from HF patients undergoing heart transplantation and 10 samples from healthy controls (CNT), and subjected these to mRNA-seq; and an additional 42 HF tissues and 8 CNT for the ncRNA-seq. In addition, we determined the bioactive sphingolipid levels in these tissues (HF, n=36; CNT, n=5) using ELISA. Results Most major changes were observed in expression of genes belonging to de novo (SPTSSA, −1.46±0.25; SPTSSB, −2.46±0.21; SPTLC1, −1.52±0.37; SPTLC3, −1.60±0.36; PPARA, −1.19±0.17; CERS1, 1.98±1.05; p&amp;lt;0.05), and salvage pathways (ACER1, −1.70±0.28; ACER3, 1.19±0.26; SGPP1, −1.49±0.24; p&amp;lt;0.05; SGPP2, −2.83±0.28; S1PR3, −1.78±0.28, p&amp;lt;0.01). Furthermore, we observed that the tight regulation of these genes by their miRNA regulatory partners is largely dysregulated in HF. In addition, we verified that two key bioactive sphingolipids, ceramide (307±218 vs. 81±79 ng/mL, p&amp;lt;0.01) and S1P (1908±625 vs. 963±489 ng/mL; p&amp;lt;0.05) accumulate in cardiac tissue during HF, with the ceramide/S1P ratio increasing by as much as 57% in HF patients. Interestingly, CERS1 expression was linked to changes in LV mass (r=0.797, p&amp;lt;0.0001), LV end-systolic (LVESD, r=0.561, p&amp;lt;0.05) and end-diastolic diameters (LVEDD, r=0.601, p&amp;lt;0.01), while S1P levels were inversely correlated with these parameters (LVESD, r=−0.552; LVEDD, r=−0.541; p&amp;lt;0.05). Conclusions In this study, we outline the changes in the key components of the sphingolipid metabolism, mainly in the de novo and the salvage pathways, which lead to cardiac ceramide and S1P accumulation and the increase in the ceramide/S1P ratios in HF patients. Gene modulation targeting ceramide and/or S1P levels may be beneficial in the treatment of these patients. Specifically, CERS1 is linked to cardiac remodeling, making it a good candidate for the treatment of patients with HF. Funding Acknowledgement Type of funding sources: Public grant(s) – National budget only. Main funding source(s): National Institute of Health Carlos IIIEuropean Regional Development Fund (ERDF)

Inflammation in Human Heart Failure: Major Mediators and Therapeutic Targets.

Inflammation has been recognized as a major pathophysiological contributor to the entire spectrum of human heart failure (HF), including HF with reduced ejection fraction, HF with preserved ejection fraction, acute HF and cardiogenic shock. Nevertheless, the results of several trials attempting anti-inflammatory strategies in HF patients have not been consistent or motivating and the clinical implementation of anti-inflammatory treatments for HF still requires larger and longer trials, as well as novel and/or more specific drugs. The present work reviews the different inflammatory mechanisms contributing to each type of HF, the major inflammatory mediators involved, namely tumor necrosis factor alpha, the interleukins 1, 6, 8, 10, 18, and 33, C-reactive protein and the enzymes myeloperoxidase and inducible nitric oxide synthase, and their effects on heart function. Furthermore, several trials targeting these mediators or involving other anti-inflammatory treatments in human HF are also described and analyzed. Future therapeutic advances will likely involve tailored anti-inflammatory treatments according to the patient’s inflammatory profile, as well as the development of resolution pharmacology aimed at stimulating resolution of inflammation pathways in HF.

Matricellular Protein Cilp1 Promotes Myocardial Fibrosis in Response to Myocardial Infarction.

[Figure: see text].

Abstract P499: Histone H4K20 Trimethylation Is Differentially Regulated In Heart Disease

It has been well established that many cardiac pathologies result from dynamic changes in gene expression and conversely that modulating key epigenetic factors in murine models is capable of preventing or abrogating ischemic injury and pathological remodeling. One epigenetic mechanism is the post-translational modification of histones, which are reversibly methylated on lysine (K) residues and can accept up to three methyl groups (Me1, Me2 and Me3). In the heart, significant changes in global levels of histone H3K4Me3 and H3K9Me3 have been previously reported to be upregulated and downregulated, respectively, during hypertrophy and failure in mice. However, the majority of post-translational modifications on histones have never been examined to quantify global abundance in the heart during disease. In particular, histone H4K20Me3 is important in heterochromatin formation and gene repression in non-cardiac cells but has never been evaluated in the heart. Therefore, we utilized cardiac tissue from three animal models of cardiac stress and employed western blotting and mass spectrometry to quantify the global abundance of total histone H4 and H4K20 methylation. We specifically evaluated tissue from mice subjected to LAD ligation, transverse aortic banding and isoproterenol infusion (via mini-osmotic pump). In addition, we also utilized primary neonatal cardiomyocytes treated with the hypertrophic agonist phenylephrine to quantify H4K20 methylation. Our data show that global levels of histone H4K20Me3 are differentially regulated in some models of cardiac dysfunction, but not all (i.e. isoproterenol infusion). In addition, we measured the abundance of histone methyltransferases and demethylases (via western blotting and qPCR) which are responsible for adding or removing this methyl mark in mouse cardiac tissue, and compared this to published data from human heart failure patients. These analyses allowed us to identify two enzymes, the methyltransferase Smyd5 and demethylase KDM7B, which are also differentially expressed in cardiac tissue during disease. Together these results are the first analysis of histone H4K20 methylation in the heart and suggest a novel role for this methylation site in the pathophysiology of cardiovascular disease.

Exaggerated exercise pressor reflex in a mouse model of femoral artery ligation

The exercise pressor reflex (EPR) is a reflexive mechanism in which blood pressure and heart rate increase in response to skeletal muscle contraction. These reflexive increases are caused by both mechanical and metabolic stimulation of group III and IV afferent neurons innervating in contracting skeletal muscle. Patients with peripheral artery disease (PAD) have an exaggerated EPR which contributes to exercise intolerance and morbidity and mortality. Previous studies in our laboratory have demonstrated that the EPR is overactive in a rat model of heart failure similar to it is in human heart failure patients. We determined that this abnormality was initiated by a blunting of the metabolic component but maintained by the mechanosensitive component of the reflex. Recently, we developed a mouse model of EPR, and have validated that the mouse model is similar to our rat model. In this model, studied the EPR in a model of peripheral artery disease (PAD). Our current results demonstrated that the blood pressure increase induced by passive stretch was exaggerated in mice 72 hours following femoral artery ligation. This exaggerated rise in blood pressure was not affected by SB-705498 (a TRPv1 receptor inhibitor) but it was nearly abolished by Gadolinium (a mechanical sensitive group III afferent neurons inhibitor) and it was partially reduced by GsMTx-4 (a selective inhibitor of the Piezo2 receptor). These results indicate that the exaggerated EPR in a murine model of PAD is mediated by mechanically sensitive group III neurons, which is mediated, in part, by Piezo2 receptors.

Abstract 16406: Epigenetic Regulation of Myofibroblast Differentiation and Persistence is Mediated by Enhanced Glutaminolysis

Cardiac fibrosis is mediated by the activation and differentiation of resident cardiac fibroblasts. While initially reparative, chronic activation leads to progressive cardiac dysfunction. Associated with myofibroblast formation are changes in metabolites directly linked to the activity of epigenetic-modifying enzymes. To define the signaling linking fibroblast activation with changes to the epigenetic landscape, we treated primary adult murine cardiac fibroblasts (CFs) with the pro-fibrotic agonist, TGFβ, and subjected them to next-gen sequencing techniques (RNA-seq, ATAC-seq). Following activation, we observed alterations in chromatin accessibility corresponding to the transcriptional activation and suppression of key fibrotic genes. Intriguingly, increased chromatin accessibility was strongly associated with both transcriptional activation and suppression, while decreased accessibility correlated with neither transcriptional repression or activation. Regions of increased accessibility were enriched for canonical fibrotic TF binding motifs (e.g. SMADs, NFAT). We recently reported that αKG-dependent lysine demethylases may be required for myofibroblast formation, so we made correlative comparisons between chromatin remodeling and the synthesis and utilization of metabolites critical for (de)methylation by applying unbiased, stable isotope metabolomics. These studies indicated enhanced glutaminolysis as the driver of increased αKG abundance. In the presence of TGFβ, pharmacologic inhibition (CB-839) or genetic deletion ( Gls1 -/- ) of glutaminolysis prevented myofibroblast formation as indicated by αSMA positivity, fibrotic gene program activation, and collagen contraction assays. Following myofibroblast formation, inhibition of glutaminolysis was sufficient to revert activated myofibroblasts to a non-fibrotic phenotype, during sustained stress. Remarkably, these results were recapitulated in CFs from human heart failure patients. Collectively, these findings suggest that metabolic remodeling is necessary for the epigenetic modifications that underlie myofibroblast formation and persistence, providing substantial rationale to evaluate several new therapeutic targets to treat fibrosis.

Transcriptional regulation by methyltransferases and their role in the heart: highlighting novel emerging functionality.

Methyltransferases are a superfamily of enzymes that transfer methyl groups to proteins, nucleic acids, and small molecules. Traditionally, these enzymes have been shown to carry out a specific modification (mono-, di-, or trimethylation) on a single, or limited number of, amino acid(s). The largest subgroup of this family, protein methyltransferases, target arginine and lysine side chains of histone molecules to regulate gene expression. Although there is a large number of functional studies that have been performed on individual methyltransferases describing their methylation targets and effects on biological processes, no analyses exist describing the spatial distribution across tissues or their differential expression in the diseased heart. For this review, we performed tissue profiling in protein databases of 199 confirmed or putative methyltransferases to demonstrate the unique tissue-specific expression of these individual proteins. In addition, we examined transcript data sets from human heart failure patients and murine models of heart disease to identify 40 methyltransferases in humans and 15 in mice, which are differentially regulated in the heart, although many have never been functionally interrogated. Lastly, we focused our analysis on the largest subgroup, that of protein methyltransferases, and present a newly emerging phenomenon in which 16 of these enzymes have been shown to play dual roles in regulating transcription by maintaining the ability to both activate and repress transcription through methyltransferase-dependent or -independent mechanisms. Overall, this review highlights a novel paradigm shift in our understanding of the function of histone methyltransferases and correlates their expression in heart disease.

Abstract 343: Glycogen Synthase Kinase 3β Localizes to the Cardiac Myofilament via Py216 and Dynamically Modulates Calcium Sensitivity

Localization of kinases to sub-cellular compartments allows them dynamic control over specific subsets of their targets. We previously found GSK-3β could modulate myofilament Ca 2+ sensitivity. However, whether GSK-3β modulates Ca 2+ sensitivity in vivo , if it localizes to the myofilament, and the consequences, are unknown. In myofilament enriched LV tissue from human non-failing and heart failure (HF) patients (n = 66) we found GSK-3β does localize to the myofilament and is altered by sex, age, and HF. To determine its in vivo functional role, we used myocyte specific inducible GSK-3β knock-out mice for skinned myocyte force-calcium experiments and found that GSK-3β reduction reduced calcium sensitivity. Further, we measured function in human samples and found myofilament GSK-3β levels directly correlated to Ca 2+ sensitivity. To establish how GSK-3β binds to the myofilament, we performed co-IP and IHC with phosphorylated forms of GSK-3β. GSK-3β phosphorylated at Y216 had a high affinity for the myofilament and localized to the z-disc. Mutating Y216 to a phospho-mimetic increased binding to the myofilament while mutating it to a phospho-null ablated binding.To identify GSK-3β’s myofilament targets, we performed mass spectrometry on myofilament phospho-enriched samples from GSK-3β KO and WT mice. As GSK-3β modulated Ca 2+ sensitivity, we expected to detect thin filament proteins. However, in agreement with its localization to the z-disc, GSK-3β primarily phosphorylated z-disc proteins.In the GSK-3β KO mice, strain analysis revealed the posterior wall contracted significantly earlier than the anterior wall, indicating baseline mechanical dyssynchrony. The WT mice had synchronous contraction, and interestingly there was significantly higher myofilament GSK-3β in the anterior wall compared to the posterior wall, a difference we hypothesize maintains synchrony. Thus, losing this fine control over Ca 2+ sensitivity as in the GSK-3β KO mice would induce mechanical dyssynchrony. Overall, these findings reveal that GSK-3β dynamically localizes to the myofilament to modulate Ca 2+ sensitivity through Y216 phosphorylation. The consequence of this “fine tuning” maintains chamber level mechanical synchrony.

Enhanced Cardiomyocyte Function in Hypertensive Rats With Diastolic Dysfunction and Human Heart Failure Patients After Acute Treatment With Soluble Guanylyl Cyclase (sGC) Activator.

AimsOur aim was to investigate the effect of nitric oxide (NO)-independent activation of soluble guanylyl cyclase (sGC) on cardiomyocyte function in a hypertensive animal model with diastolic dysfunction and in biopsies from human heart failure with preserved ejection fraction (HFpEF).MethodsDahl salt-sensitive (DSS) rats and control rats were fed a high-salt diet for 10 weeks and then acutely treated in vivo with the sGC activator BAY 58-2667 (cinaciguat) for 30 min. Single skinned cardiomyocyte passive stiffness (Fpassive) was determined in rats and human myocardium biopsies before and after acute treatment. Titin phosphorylation, activation of the NO/sGC/cyclic guanosine monophosphate (cGMP)/protein kinase G (PKG) cascade, as well as hypertrophic pathways including NO/sGC/cGMP/PKG, PKA, calcium–calmodulin kinase II (CaMKII), extracellular signal-regulated kinase 2 (ERK2), and PKC were assessed. In addition, we explored the contribution of pro-inflammatory cytokines and oxidative stress levels to the modulation of cardiomyocyte function. Immunohistochemistry and electron microscopy were used to assess the translocation of sGC and connexin 43 proteins in the rat model before and after treatment.ResultsHigh cardiomyocyte Fpassive was found in rats and human myocardial biopsies compared to control groups, which was attributed to hypophosphorylation of total titin and to deranged site-specific phosphorylation of elastic titin regions. This was accompanied by lower levels of PKG and PKA activity, along with dysregulation of hypertrophic pathway markers such as CaMKII, PKC, and ERK2. Furthermore, DSS rats and human myocardium biopsies showed higher pro-inflammatory cytokines and oxidative stress compared to controls. DSS animals benefited from treatment with the sGC activator, as Fpassive, titin phosphorylation, PKG and the hypertrophic pathway kinases, pro-inflammatory cytokines, and oxidative stress markers all significantly improved to the level observed in controls. Immunohistochemistry and electron microscopy revealed a translocation of sGC protein toward the intercalated disc and t-tubuli following treatment in both control and DSS samples. This translocation was confirmed by staining for the gap junction protein connexin 43 at the intercalated disk. DSS rats showed a disrupted connexin 43 pattern, and sGC activator was able to partially reduce disruption and increase expression of connexin 43. In human HFpEF biopsies, the high Fpassive, reduced titin phosphorylation, dysregulation of the NO–sGC–cGMP–PKG pathway and PKA activity level, and activity of kinases involved in hypertrophic pathways CaMKII, PKC, and ERK2 were all significantly improved by sGC treatment and accompanied by a reduction in pro-inflammatory cytokines and oxidative stress markers.ConclusionOur data show that sGC activator improves cardiomyocyte function, reduces inflammation and oxidative stress, improves sGC–PKG signaling, and normalizes hypertrophic kinases, indicating that it is a potential treatment option for HFpEF patients and perhaps also for cases with increased hypertrophic signaling.

Cerebral venous congestion promotes blood‐brain barrier disruption and neuroinflammation, impairing cognitive function in mice.

Cognitive impairment is one of the most common co‐occurring chronic conditions among elderly heart failure patients (incidence: up to ~ 80%); however, the underlying mechanisms are not completely understood. It is hypothesized that in addition to decreased cardiac output, increases in central‐and consequentially, cerebral‐venous pressure (backward failure) also contribute significantly to the genesis of cognitive impairment. To test this hypothesis and elucidate the specific pathogenic role of venous congestion in the brain, we have established a novel model of increased cerebral venous pressure: mice with jugular vein ligation (JVL). To test the hypothesis that increased venous pressure in the brain contributes to the development of cognitive deficits by causing blood‐brain barrier disruption, dysregulation of blood flow, and/or promoting neuroinflammation, in C57BL/6 mice, the internal and external jugular veins were ligated. Cognitive function (radial arm water maze), gait function (CatWalk), and motor coordination (rotarod) were tested post‐JVL. Neurovascular coupling responses were assessed by measuring changes in cerebral blood flow in the whisker barrel cortex in response to contralateral whisker stimulation by laser speckle contrast imaging through a closed cranial window. Blood‐brain barrier integrity (IgG extravasation) and microglia activation (Iba1 staining) were assessed in brain slices by immunohistochemistry. Neuroinflammation‐related gene expression profile was assessed by a targeted qPCR array. After jugular vein ligation, mice exhibited impaired spatial learning and memory, altered motor coordination, and impaired gait function, mimicking important aspects of altered brain function observed in human heart failure patients. JVL did not alter neurovascular coupling responses. In the brains of mice with JVL, significant extravasation of IgG was detected, indicating blood‐brain barrier disruption, which was associated with histological markers of neuroinflammation (increased presence of activated microglia) and a proinflammatory shift in gene expression profile. Thus, cerebral venous congestion per se can cause blood‐brain barrier disruption and neuroinflammation, which likely contribute to the genesis of cognitive impairment. These findings have relevance to the pathogenesis of cognitive decline associated with heart failure as well as increased cerebal venous pressure due to increased jugular venous reflux in elderly human patients.Support or Funding InformationThis work was supported by grants the Oklahoma Center for the Advancement of Science and Technology, the Oklahoma Shared Clinical and Translational Resources (OSCTR) program funded by the National Institute of General Medical Sciences (GM104938), the NIA‐supported Geroscience Training Program in Oklahoma (T32AG052363), the Oklahoma Nathan Shock Center (P30AG050911), the Cellular and Molecular GeroScience CoBRE (1P20GM125528, sub#5337), and the NIGMS supported Center of Biomedical Research Excellence (CoBRE)..

Sex-Specific Programming of Cardiac DNA Methylation by Developmental Phthalate Exposure.

Phthalate plasticizers are ubiquitous chemicals linked to several cardiovascular diseases in animal models and humans. Despite this, the mechanisms by which phthalate exposures cause adverse cardiac health outcomes are unclear. In particular, whether phthalate exposures during pregnancy interfere with normal developmental programming of the cardiovascular system, and the resulting implications this may have for long-term disease risk, are unknown. Recent studies suggest that the effects of phthalates on metabolic and neurobehavioral outcomes are sex-specific. However, the influence of sex on cardiac susceptibility to phthalate exposures has not been investigated. One mechanism by which developmental exposures may influence long-term health is through altered programming of DNA methylation. In this work, we utilized an established mouse model of human-relevant perinatal exposure and enhanced reduced representation bisulfite sequencing to investigate the long-term effects of diethylhexyl phthalate (DEHP) exposure on DNA methylation in the hearts of adult male and female offspring at 5 months of age (n = 5-7 mice per sex and exposure). Perinatal DEHP exposure led to hundreds of sex-specific, differentially methylated cytosines (DMCs) and differentially methylated regions (DMRs) in the heart. Pathway analysis of DMCs revealed enrichment for several pathways in females, including insulin signaling, regulation of histone methylation, and tyrosine phosphatase activity. In males, DMCs were enriched for glucose transport, energy generation, and developmental programs. Notably, many sex-specific genes differentially methylated with DEHP exposure in our mouse model were also differentially methylated in published data of heart tissues collected from human heart failure patients. Together, these data highlight the potential role for DNA methylation in DEHP-induced cardiac effects and emphasize the importance of sex as a biological variable in environmental health studies.

Cerebral venous congestion promotes blood-brain barrier disruption and neuroinflammation, impairing cognitive function in mice.

Cognitive impairment is one of the most common co-occurring chronic conditions among elderly heart failure patients (incidence: up to ~ 80%); however, the underlying mechanisms are not completely understood. It is hypothesized that in addition to decreased cardiac output, increases in central-and consequentially, cerebral-venous pressure (backward failure) also contribute significantly to the genesis of cognitive impairment. To test this hypothesis and elucidate the specific pathogenic role of venous congestion in the brain, we have established a novel model of increased cerebral venous pressure: mice with jugular vein ligation (JVL). To test the hypothesis that increased venous pressure in the brain contributes to the development of cognitive deficits by causing blood-brain barrier disruption, dysregulation of blood flow, and/or promoting neuroinflammation, in C57BL/6 mice, the internal and external jugular veins were ligated. Cognitive function (radial arm water maze), gait function (CatWalk), and motor coordination (rotarod) were tested post-JVL. Neurovascular coupling responses were assessed by measuring changes in cerebral blood flow in the whisker barrel cortex in response to contralateral whisker stimulation by laser speckle contrast imaging through a closed cranial window. Blood-brain barrier integrity (IgG extravasation) and microglia activation (Iba1 staining) were assessed in brain slices by immunohistochemistry. Neuroinflammation-related gene expression profile was assessed by a targeted qPCR array. After jugular vein ligation, mice exhibited impaired spatial learning and memory, altered motor coordination, and impaired gait function, mimicking important aspects of altered brain function observed in human heart failure patients. JVL did not alter neurovascular coupling responses. In the brains of mice with JVL, significant extravasation of IgG was detected, indicating blood-brain barrier disruption, which was associated with histological markers of neuroinflammation (increased presence of activated microglia) and a pro-inflammatory shift in gene expression profile. Thus, cerebral venous congestion per se can cause blood-brain barrier disruption and neuroinflammation, which likely contribute to the genesis of cognitive impairment. These findings have relevance to the pathogenesis of cognitive decline associated with heart failure as well as increased cerebal venous pressure due to increased jugular venous reflux in elderly human patients.

Abstract 945: A Novel Role of Cardiac DNA Methylation as a Regulator of Fibrosis in Human Diabetic Heart Failure

Human heart failure (HF) is accompanied by changes in cardiac gene expression; however, the impact of diabetes mellitus on this transcriptional regulation remains unclear. Our laboratory studies the role of epigenetics in HF and focuses on DNA methylation, commonly accepted as a negative regulator of gene expression when present in a gene’s promoter. In the current study, we hypothesized that diabetes alters cardiac DNA methylation and the transcriptome. We performed an integrated analysis of RNA-sequencing and DNA methylation using human left ventricle biopsies from HF patients to determine the effect of diabetes on this reprogramming. Our analysis identified global changes in cardiac DNA methylation as well as mRNA expression sufficient to distinguish diabetic from non-diabetic patients. Specifically, we identified significant regulation of 1,133 genes (|1.5| fold-change, P&lt;0.05) and 9,302 methylation sites (|5%| methylation, P&lt;0.05). Within the 391 co-regulated genes, only 174 genes had altered promoter-associated methylation. Consistent with the accepted dogma that promoter methylation inversely regulates gene expression, we found that 119 of those 174 genes displayed this inverse relationship; of these, an overwhelming majority (102) were hypomethylated in diabetic relative to non-diabetic hearts. These findings contrast the cardiac hypermethylation we have reported for ischemic HF. Gene set enrichment of the 119 inversely-regulated genes identified numerous pathways involved in fibrosis, including extracellular matrix organization (FDR&lt;0.05, 3.9% enriched) and collagen biosynthesis (FDR&lt;0.05, 6.3% enriched), which suggest that DNA methylation contributes to the adverse cardiac remodeling associated with diabetic heart failure. To identify putative regulators of these transcriptional and epigenetic differences, a candidate gene approach was used to reveal induction (2.5-fold, Q&lt;0.05) and demethylation (20.5%, P&lt;0.05) of EGR2, a known regulator of fibrosis. Furthermore, we found induction of GADD45beta (2.6-fold, Q &lt; 0.05) a known regulator of DNA demethylation. Taken together, these observations suggest that epigenetic mechanisms underlie an etiology-specific reprogramming of cardiac fibrosis in diabetic HF.