Nonfailing Donor Research Articles

The Potential of Metabolism-Related Gene OGDHL as a Biomarker for Myocardial Remodeling in Dilated Cardiomyopathy.

The development of dilated cardiomyopathy (DCM) is accompanied by a series of metabolic disorders, resulting in myocardial remodeling or exacerbation, while the mechanism remains not completely clear. This study was to find out the key metabolism-related genes involved in the onset of DCM, providing new insight into the pathogenesis of this disease. The datasets of GSE57338, GSE116250, and GSE5406 associated with hearts of patients with DCM were downloaded from the Gene Expression Omnibus database. GSE57338 was analyzed to screen out metabolism-related differentially expressed genes (DEGs), while GSE116250 and GSE5406 were utilized to verify the optimal genes through R software. Support vector machine recursive feature elimination algorithm and least absolute shrinkage and selection operator algorithm were used to determine key genes. Finally, 6 of 39 metabolism-related DEGs were screened out and identified as the optimal genes. After quantitative reverse-transcription polymerase chain reaction (qRT-PCR) validation performed on the samples drawn from the left ventricles of human hearts, it showed that only the expression of oxoglutarate dehydrogenase-like (OGDHL) increased while PLA2G2 decreased significantly in patients with DCM compared with non-failing donors, respectively. Furthermore, the higher OGDHL protein expression, except the change of PLA2G2, was also found in DCM hearts, and its mRNA expression was negatively correlated with myocardial Masson’s scores (r = –0.84, P = 0.009) and left ventricular end-diastolic diameter (LVEDd; r = –0.82, P = 0.014), which might be regulated by miR-3925-5p through further bioinformatics prediction and qRT-PCR verification. The data then suggested that the metabolism-related gene OGDHL was associated with myocardial fibrosis of DCM and probably a biomarker for myocardial remodeling in patients with DCM.

Effects of Atrial Fibrillation on the Human Ventricle.

Rationale:Atrial fibrillation (AF) and heart failure often coexist, but their interaction is poorly understood. Clinical data indicate that the arrhythmic component of AF may contribute to left ventricular (LV) dysfunction.Objective:This study investigates the effects and molecular mechanisms of AF on the human LV.Methods and Results:Ventricular myocardium from patients with aortic stenosis and preserved LV function with sinus rhythm or rate-controlled AF was studied. LV myocardium from patients with sinus rhythm and patients with AF showed no differences in fibrosis. In functional studies, systolic Ca2+ transient amplitude of LV cardiomyocytes was reduced in patients with AF, while diastolic Ca2+ levels and Ca2+ transient kinetics were not statistically different. These results were confirmed in LV cardiomyocytes from nonfailing donors with sinus rhythm or AF. Moreover, normofrequent AF was simulated in vitro using arrhythmic or rhythmic pacing (both at 60 bpm). After 24 hours of AF-simulation, human LV cardiomyocytes from nonfailing donors showed an impaired Ca2+ transient amplitude. For a standardized investigation of AF-simulation, human iPSC-cardiomyocytes were tested. Seven days of AF-simulation caused reduced systolic Ca2+ transient amplitude and sarcoplasmic reticulum Ca2+ load likely because of an increased diastolic sarcoplasmic reticulum Ca2+ leak. Moreover, cytosolic Na+ concentration was elevated and action potential duration was prolonged after AF-simulation. We detected an increased late Na+ current as a potential trigger for the detrimentally altered Ca2+/Na+-interplay. Mechanistically, reactive oxygen species were higher in the LV of patients with AF. CaMKII (Ca2+/calmodulin-dependent protein kinase IIδc) was found to be more oxidized at Met281/282 in the LV of patients with AF leading to an increased CaMKII activity and consequent increased RyR2 phosphorylation. CaMKII inhibition and ROS scavenging ameliorated impaired systolic Ca2+ handling after AF-simulation.Conclusions:AF causes distinct functional and molecular remodeling of the human LV. This translational study provides the first mechanistic characterization and the potential negative impact of AF in the absence of tachycardia on the human ventricle.

A Mechanical Bridge to Recovery as a Bridge to Discovery: Learning From Few and Applying to Many.

A Mechanical Bridge to Recovery as a Bridge to Discovery: Learning From Few and Applying to Many.

Identification of Hub Diagnostic Biomarkers and Candidate Therapeutic Drugs in Heart Failure.

PurposeThe objective of this study was to identify the potential regulatory mechanisms, diagnostic biomarkers, and therapeutic drugs for heart failure (HF).MethodsDifferentially expressed genes (DEGs) between HF and non-failing donors were screened from the GSE57345, GSE5406, and GSE3586 datasets. Database for Annotation Visualization and Integrated Discovery and Metascape were used for Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analyses respectively. The GSE57345 dataset was used for weighted gene co-expression network analysis (WGCNA). The intersecting hub genes from the DEGs and WGCNA were identified and verified with the GSE5406 and GSE3586 datasets. The diagnostic value of the hub genes was calculated through receiver operating characteristic analysis and net reclassification index (NRI). Gene set enrichment analysis (GSEA) was used to filter out the signaling pathways associated with the hub genes. SYBYL 2.1 was used for molecular docking of hub targets and potential HF drugs obtained from the connection map.ResultsFunctional annotation of the DEGs showed enrichment of negative regulation of angiogenesis, endoplasmic reticulum stress response, and heart development. PTN, LUM, ISLR, and ASPN were identified as the hub genes of HF. GSEA showed that the key genes were related to the transforming growth factor-β (TGF-β) and Wnt signaling pathways. Sirolimus, LY-294002, and wortmannin have been confirmed as potential drugs for HF.ConclusionWe identified new hub genes and candidate therapeutic drugs for HF, which are potential diagnostic, therapeutic and prognostic targets and warrant further investigation.

Abstract 12001: Atrial Fibrillation Induces Ventricular Dysfunction via Altered Ca 2+ Handling and Oxidative CaMKII Activation in the Human Heart

Introduction: The effects of atrial fibrillation (AF) on left ventricular (LV) function are poorly understood. Clinical data indicate that the arrhythmic component of AF may contribute to LV dysfunction. This study investigated the effects of AF on the human LV. Methods and Results: LV myocardium from patients with preserved LV function (EF>50%) with sinus rhythm (SR, n=31) or rate-controlled AF (n=24) obtained during surgery for aortic stenosis was studied. Clinical data were mostly balanced without changes in LV function and remodeling. LV myocardium from SR and AF patients showed no changes in fibrosis. In functional studies, systolic Ca 2+ transient amplitude of human LV cardiomyocytes (CM) was reduced in AF patients, while diastolic Ca 2+ levels and Ca 2+ transient kinetics were unaltered. These results were confirmed in LV CM from non-failing donors with AF (n=4) vs. SR (n=8). In addition, normofrequent AF was simulated in vitro using arrhythmic or rhythmic electrical culture pacing (both at 60 bpm). After 24h of AF-simulation, human LV CM from non-failing donors (n=9) showed also an impaired Ca 2+ transient amplitude. For a standardized chronic AF-simulation, human iPSC-CM (n=22 differentiations/5 individuals) were tested. 7 days of AF-simulation caused reduced systolic Ca 2+ transient amplitude and sarcoplasmic reticulum Ca 2+ load, which could be explained by an increased diastolic Ca 2+ leak. Mechanistically, oxidative stress markers were higher in the LV of AF patients. Ca 2+ /calmodulin-dependent protein kinase IIδ (CaMKII) was found to be more oxidized at Met281/282 in the LV of AF patients leading to an increased CaMKII activity. Thus, RyR2 phosphorylation was elevated, which likely underlies the depression of LV Ca 2+ handling in AF. In translational contractility experiments of human ventricular trabecula from patients with heart failure (n=8), in vitro AF-simulation for 8h worsened systolic and diastolic contractile function. Conclusions: Here we could firstly show that AF impairs human LV function via increased oxidative stress and CaMKII activity in different patient populations and models. Thus, this translational study demonstrates the detrimental effects and mechanisms of AF in the absence of tachycardia on the human LV.

Abstract 10254: HDAC Inhibition Regulates Cardiac Function via Myofilament Acetylation

Introduction: Heart failure (HF) is a global issue and approximately 50% of patients are classified as having HF with preserved Ejection Fraction (HFpEF), with no FDA approved therapies available. We previously characterized a large animal model that recapitulates key features of HFpEF and tested the effects of suberanilohydroxamic acid (SAHA, pan-HDAC inhibitor), which reversed and prevented the development of cardiopulmonary dysfunction. Parallel studies in cardiac tissue from humans and large mammals were designed to evaluate the effects of SAHA at the cellular level. Hypothesis: SAHA treatment will improve cardiomyocyte function. Methods: Adult feline ventricular cardiomyocytes (AFVM) were isolated from male domestic short hair cats and treated with 2.5μM SAHA or vehicle (DMSO) for 90 minutes, then incubated with a calcium (Ca 2+ ) indicator (Fluo-4AM) and electrically stimulated (0.5Hz) to record Ca 2+ transients and contractions. Human left ventricle (LV) trabeculae isolated from non-failing donor hearts were treated with 10μM SAHA or vehicle for 120 minutes while being electrically stimulated (1Hz) to record developed force and relaxation parameters. Skinned myocytes were isolated from treated AFVM and human trabeculae to assess myofilament Ca 2+ sensitivity and passive stiffness. Mass spectrometry (MS) was performed on AFVM skinned myocytes to identify novel acetylation sites. Results: There was no difference in AFVM peak Ca 2+ transients with SAHA treatment, but calcium removal was increased (tau, time to 30% baseline). There was a significant increase in contractility (fractional shortening) and relaxation kinetics (time to 50% baseline, return velocity). Trabeculae treated with SAHA had decreased diastolic tension and an increase in developed force. Skinned myocytes were isolated from treated AFVMs and trabeculae and both had a significant improvement in myofilament Ca 2+ sensitivity and significant decrease in passive stiffness with SAHA. MS of AFVM skinned myocytes revealed increased acetylation of the myosin regulatory light chain, driven by lysine 115. Conclusions: These findings suggest that SAHA may have an important role in regulating cardiac function via the cardiomyocyte and myofilament in feline and human myocardium.

Abstract 10240: Key Pathomechanisms of Cardiomyopathy Due to TTN Truncation

Introduction: Heterozygous truncating variants in TTN (TTNtv) coding for titin cause dilated cardiomyopathy (DCM) but the underlying pathomechanisms are unclear and disease management remains uncertain. Here, we aim to elucidate the key pathomechanisms of TTNtv-DCM. Hypothesis: Stable expression of truncated titin proteins and titin haploinsufficiency characterize TTNtv-DCM and represent potential targets for therapy. Methods and Results: We studied left ventricular tissues from 14 nonfailing donor hearts and 113 endstage failing DCM patients and identified a TTNtv in 22 DCM patients (19.5%) by next generation sequencing. Using titin protein gel electrophoresis and western blot we demonstrate, for the first time, titin haploinsufficiency in TTNtv-DCM hearts. Strikingly, all 21 adult TTNtv-DCM hearts of our cohort showed stable expression of truncated titin proteins. Expression was variable, up to one-half of the total titin protein pool, and negatively correlated to patient age at heart transplantation. Truncated titin proteins were not detected in sarcomeres but were present in intracellular aggregates. Deregulated ubiquitin-dependent protein quality control was apparent. Next, we produced human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs), comparing wildtype controls to cells with a patient-derived, prototypical A-band TTNtv or a CRISPR/Cas9-edited M-band TTNtv. TTNtv hiPSC-CMs showed reduced wildtype titin expression and contained truncated titin proteins, whose proportion increased upon inhibition of proteasomal activity. In engineered heart muscle generated from hiPSC-CMs, depressed contractility caused by TTNtv could be reversed by correction of the mutation using CRISPR/Cas9, thereby eliminating truncated titin proteins and raising wildtype titin content. Functional improvement also occurred when titin protein content was increased by proteasome inhibition. Conclusions: Our study reveals the major pathomechanisms of TTNtv-DCM, which include titin haploinsufficiency as a life-long condition and truncated titin protein enrichment with aggregate formation, along with aberrant protein quality control. Results can be exploited for new therapies of TTNtv cardiomyopathies.

Effects of atrial fibrillation on ventricular remodeling in the human heart

Abstract Atrial fibrillation (AF) is often found in patients with heart failure (HF). Clinical data indicated that the arrhythmic component of AF alone could contribute to left-ventricular (LV) dysfunction. However, the effects of non-tachycardic AF with arrhythmic excitation of the human LV, are unknown. We investigated human LV myocardium from patients with sinus rhythm (SR) or normofrequent AF (mean EF>50%, matched clinical data, derived from septal resections during AVR). In histological analysis we detected no difference between SR (n=17 patients) and AF patients (n=18) regarding the amount and distribution of fibrosis. We isolated human LV cardiomyocytes (CM) and studied cellular Ca-handling (Fura-2). Systolic Ca-transient amplitude of LV CM was reduced in patients suffering from AF (n=8 AF patients vs. 11 SR), while diastolic Ca-levels and Ca-transient kinetics were not significantly changed. These results were confirmed in LV CM from non-failing donors (NF) with AF (n=4 AF patients vs. 8 SR). For the standardized investigation of a normofrequent arrhythmia, we simulated AF in vitro by using arrhythmic (60 bpm, 40% beat-to-beat variability) or rhythmic (60 bpm) field stimulation. Human LV CM from NF SR patients (n=8) showed an impaired Ca-transient amplitude after 24h arrhythmic culture pacing without changes in diastolic Ca and Ca-transient kinetics. For studying a model suitable for more standardized chronic pacing, we utilized human iPSC cardiomyocytes (iPSC-CM) from healthy donors (n=6). After 7 days, arrhythmically paced iPSC-CM exhibited a reduced systolic Ca-transient amplitude, a trend towards a prolonged Ca-elimination time and a reduced sarcoplasmic reticulum Ca-load. Confocal line-scans of arrhythmically paced cells (Fluo-4 AM) showed an increased diastolic Ca-leak from the sarcoplasmic reticulum, possibly underlying the reduced Ca-load. Coupled with the Ca changes, cytosolic Na was elevated after arrhythmia. We found an increased late INa, which could explain the detrimentally altered Ca/Na-interplay. Accordingly, Patch-clamp experiments revealed a prolonged action potential duration after arrhythmia. We further elucidated the underlying mechanisms of this electrophysiological remodeling by showing that oxidative stress (H2O2, LPO) is increased in the LV of patients suffering from AF (n=6 AF patients vs. 6 SR), which was associated with an enhanced NOX2/-4 activity. Consecutively, Ca2+/calmodulin-dependent protein kinase IIδ (CaMKII) was found to be more oxidized (CaMKII-Met281/282) in the LV of AF patients (n=7 AF patients vs. 7 SR) leading to an increased CaMKII activity, which adversely regulated EC-coupling protein phosphorylation including RyR2 hyperphosphorylation. Normofrequent arrhythmia/AF impairs human ventricular EC-coupling via increased oxidative stress and enhanced CaMKII. Thus, this translational study provides the first mechanistic characterization and the potential negative impact of isolated AF on the human LV. Funding Acknowledgement Type of funding sources: Public Institution(s). Main funding source(s): Else Kröner-Fresenius-Stiftung (EKFS) and Deutsche Gesellschaft für Innere Medizin

Altered microRNA and mRNA profiles during heart failure in the human sinoatrial node

Heart failure (HF) is frequently accompanied with the sinoatrial node (SAN) dysfunction, which causes tachy-brady arrhythmias and increased mortality. MicroRNA (miR) alterations are associated with HF progression. However, the transcriptome of HF human SAN, and its role in HF-associated remodeling of ion channels, transporters, and receptors responsible for SAN automaticity and conduction impairments is unknown. We conducted comprehensive high-throughput transcriptomic analysis of pure human SAN primary pacemaker tissue and neighboring right atrial tissue from human transplanted HF hearts (n = 10) and non-failing (nHF) donor hearts (n = 9), using next-generation sequencing. Overall, 47 miRs and 832 mRNAs related to multiple signaling pathways, including cardiac diseases, tachy-brady arrhythmias and fibrosis, were significantly altered in HF SAN. Of the altered miRs, 27 are predicted to regulate mRNAs of major ion channels and neurotransmitter receptors which are involved in SAN automaticity (e.g. HCN1, HCN4, SLC8A1) and intranodal conduction (e.g. SCN5A, SCN8A) or both (e.g. KCNJ3, KCNJ5). Luciferase reporter assays were used to validate interactions of miRs with predicted mRNA targets. In conclusion, our study provides a profile of altered miRs in HF human SAN, and a novel transcriptome blueprint to identify molecular targets for SAN dysfunction and arrhythmia treatments in HF.

Identification of the distinctive role of DPT in dilated cardiomyopathy: a study based on bulk and single-cell transcriptomic analysis.

BackgroundDilated cardiomyopathy (DCM) is a common cause of heart failure. Cardiac remodeling is the main pathological change in DCM, yet the molecular mechanism is still unclear. Therefore, the present study aims to find potential crucial genes and regulators through bulk and single-cell transcriptomic analysis.MethodsThree microarray datasets of DCM (GSE57338, GSE42955, GSE79962) were chosen from gene expression omnibus (GEO) to analyze the differentially expressed genes (DEGs). LASSO regression, SVM-RFE, and PPI network methods were then carried out to identify key genes. Another dataset (GSE116250) was used to validate these findings. To further identify DCM-associated specific cell types, transcription factors, and cell-cell interaction networks, GSEA, SCENIC, and CellPhoneDB were conducted on public datasets for single-cell RNA sequencing analysis of DCM (GSE109816 and GSE121893). Finally, reverse transcription-polymerase chain reaction (RT-PCR), western blot, and immunohistochemical were performed to validate DPT expression in fibroblasts and DCM.ResultsThere were 281 DEGs between DCM and non-failing donors. CCL5 and DPT were identified to be key genes and both genes had a 0.844 area under the receiver operating curve (AUC) in the validation dataset. Further single-cell sequencing analysis revealed three main findings: (I) DPT was mainly expressed in fibroblasts and was significantly upregulated in DCM fibroblasts; (II) DPT+ fibroblasts were involved in the organization of the extracellular matrix (ECM) and collagen fibrils and were regulated by the transcription factor STAT3; and (III) DPT+ fibroblasts had high interactions with endothelial cells through including Ephrin-Eph, ACKR-CXCL, and JAG-NOTCH signal pathways. RT-PCR, western blot, and immunohistochemical confirmed that DPT was highly expressed and co-localized with Vimentin and p-STAT3 in DCM patients. STAT3 inhibitor S3I-201 reduced the expression of DPT in mouse cardiac fibroblasts.ConclusionsDPT could be used as a diagnostic marker and therapeutic target of DCM. DPT+ fibroblasts could be a novel regulator of the cardiac remodeling process in DCM.

Noncanonical WNT Activation in Human Right Ventricular Heart Failure.

Background: No medical therapies exist to treat right ventricular (RV) remodeling and RV failure (RVF), in large part because molecular pathways that are specifically activated in pathologic human RV remodeling remain poorly defined. Murine models have suggested involvement of Wnt signaling, but this has not been well-defined in human RVF.Methods: Using a candidate gene approach, we sought to identify genes specifically expressed in human pathologic RV remodeling by assessing the expression of 28 WNT-related genes in the RVs of three groups: explanted nonfailing donors (NF, n = 29), explanted dilated and ischemic cardiomyopathy, obtained at the time of cardiac transplantation, either with preserved RV function (pRV, n = 78) or with RVF (n = 35).Results: We identified the noncanonical WNT receptor ROR2 as transcriptionally strongly upregulated in RVF compared to pRV and NF (Benjamini-Hochberg adjusted P < 0.05). ROR2 protein expression correlated linearly to mRNA expression (R2 = 0.41, P = 8.1 × 10−18) among all RVs, and to higher right atrial to pulmonary capillary wedge ratio in RVF (R2 = 0.40, P = 3.0 × 10−5). Utilizing Masson's trichrome and ROR2 immunohistochemistry, we identified preferential ROR2 protein expression in fibrotic regions by both cardiomyocytes and noncardiomyocytes. We compared RVF with high and low ROR2 expression, and found that high ROR2 expression was associated with increased expression of the WNT5A/ROR2/Ca2+ responsive protease calpain-μ, cleavage of its target FLNA, and FLNA phosphorylation, another marker of activation downstream of ROR2. ROR2 protein expression as a continuous variable, correlated strongly to expression of calpain-μ (R2 = 0.25), total FLNA (R2 = 0.67), calpain cleaved FLNA (R2 = 0.32) and FLNA phosphorylation (R2 = 0.62, P < 0.05 for all).Conclusion: We demonstrate robust reactivation of a fetal WNT gene program, specifically its noncanonical arm, in human RVF characterized by activation of ROR2/calpain mediated cytoskeleton protein cleavage.

Distinct hypertrophic cardiomyopathy genotypes result in convergent sarcomeric proteoform profiles revealed by top-down proteomics

Hypertrophic cardiomyopathy (HCM) is the most common heritable heart disease. Although the genetic cause of HCM has been linked to mutations in genes encoding sarcomeric proteins, the ability to predict clinical outcomes based on specific mutations in HCM patients is limited. Moreover, how mutations in different sarcomeric proteins can result in highly similar clinical phenotypes remains unknown. Posttranslational modifications (PTMs) and alternative splicing regulate the function of sarcomeric proteins; hence, it is critical to study HCM at the level of proteoforms to gain insights into the mechanisms underlying HCM. Herein, we employed high-resolution mass spectrometry-based top-down proteomics to comprehensively characterize sarcomeric proteoforms in septal myectomy tissues from HCM patients exhibiting severe outflow track obstruction (n = 16) compared to nonfailing donor hearts (n = 16). We observed a complex landscape of sarcomeric proteoforms arising from combinatorial PTMs, alternative splicing, and genetic variation in HCM. A coordinated decrease of phosphorylation in important myofilament and Z-disk proteins with a linear correlation suggests PTM cross-talk in the sarcomere and dysregulation of protein kinase A pathways in HCM. Strikingly, we discovered that the sarcomeric proteoform alterations in the myocardium of HCM patients undergoing septal myectomy were remarkably consistent, regardless of the underlying HCM-causing mutations. This study suggests that the manifestation of severe HCM coalesces at the proteoform level despite distinct genotype, which underscores the importance of molecular characterization of HCM phenotype and presents an opportunity to identify broad-spectrum treatments to mitigate the most severe manifestations of this genetically heterogenous disease.

Abstract 384: Predicting Drugs and Drug Targets Associated With Dilated Cardiomyopathy Using a Gene Expression-based Systems Medicine Platform

Among the genetic and environmental contributors to dilated cardiomyopathy (DCM), the pathogenesis of drug-induced DCM is heterogeneous, poorly understood, and difficult to predict. In this study, transcriptomic data from two recently published datasets were used to identify genes which are differentially expressed in DCM compared to cardiac tissue from non-failing donors. DCM-associated genes were then mapped using gene set enrichment analysis to associated biochemical pathways, gene ontology functions, and protein-protein interactions, creating a DCM-associated biological perturbation network. Corresponding multi-scale pharmacologic activity networks for over 9000 drugs were produced using public repositories of known drug-protein interactions. Drugs were then rank-ordered based on multi-scale statistical significance of concordance with the DCM perturbation network while accounting for direction of effect using a causal analysis algorithm. A predictive algorithm using overrepresentation analysis was developed using the SIDER database of adverse drug reactions (ADRs) to formulate ADR-protein and ADR-pathway associations, which were then compared to the drug-DCM association networks produced by our model. In addition to a demonstrated concordance between the rank lists for each dataset, this platform prioritized multiple drugs and metabolites which are known to be associated with DCM, such as doxorubicin, risperidone, and 4-hydroxynonenal, as well as associated mechanisms such as bile acid excess and inhibition of DNA topoisomerase II and mitochondrial complex III. Multi-scale concordance of drug and DCM networks highlighted the involvement of heat shock proteins and associated angiotensin II activity, supporting current evidence that its inhibition may be cardioprotective to drug-induced DCM. Our approach also recapitulated the roles of drugs, genes, and pathways that hold prognostic and therapeutic significance for DCM, such as the abilities of sulforaphane and fenofibrate to prevent DCM in animal models of type 2 diabetes. This platform can be used to predict a wide array of adverse drug reactions and their underlying mechanisms based on bioactivity data.

Abstract 376: Functional Significance of Anatomically Heterogeneous Distribution of Transversal-axial Tubule System in Mouse and Human Atrial Myocardium

Background: In cardiac myocytes, efficient and synchronous excitation-contraction coupling (ECC) occurs through a specialized network of membrane invaginations called transverse-axial tubule system (TATS). In contrast to an extensive TATS in ventricular myocytes, atrial cells show a significantly heterogeneous organization of TATS, varying from a ventricular-like organized TATS to absent/irregular TATS. However, anatomical distribution of atrial TATS and its functional significance remains unknown. We hypothesized that atrial myocytes with a different TATS organization have a specific anatomical location within the atria that determines their impact on atrial ECC and contraction. Methods and Results: In order to avoid the potential bias associated with cell isolation, we applied a mosaic imaging protocol at the whole-mount isolated mouse atrial preparation stained with a membrane dye RH-237. We developed an algorithm that can automatically remove the outer membrane and analyze TATS organization. This analysis showed that myocytes located within the inter-caval region (ICR, i.e., between the superior and inferior vena cava and between the crista terminalis in atrioventricular junction) have a lower TATS organization than cells located within the right atrial appendage (RAA): 0.21±0.01 A.U. vs. 0.50±0.02 A.U. in ICR vs. RAA, respectively, P&lt;0.01. Fluorescent optical mapping of Ca 2+ transients from isolated mouse atrial preparations revealed that ICR region has a longer Ca 2+ transient rise up time compared to RAA (7.8±0.3 ms vs. 6.3±0.3 ms, P&lt;0.01). Moreover, a similar region-specific TATS organization was found in human non-failing (donor) atrial tissue samples. The analysis of TATS density in specimens selected from ICR and RAA regions of the human hearts (n=4) showed a significantly lower density of TATS in ICR compared to RAA region (5.8±0.2% vs. 10.6±0.4%, P&lt;0.01). Conclusion: Our findings show a region-specific structural and functional differences of myocytes from RAA and ICR regions of mouse and human heart that could represent a general organization of the mammalian atria. It may have important implications into establishment of different functional roles of cells with a specific TATS structure in atrial physiology and pathology.

Abstract 237: Noncanonical WNT Activation in Human Right Ventricular Heart Failure

Background: Drivers of adaptive and pathologic human right ventricular (RV) remodeling are poorly defined, but there is some evidence that WNT signaling contributes to these processes. Methods: Using a candidate gene approach, we sought to identify genes specifically expressed in RV failure by assessing the expression of 28 WNT-related genes in the RVs of three groups: human explanted nonfailing donor hearts (NF, n = 29), pre-transplant cardiomyopathy hearts with preserved RV function (pRV, n = 78), and pre-transplant cardiomyopathy hearts with RV failure (RVF, n = 35). Results: We identified the noncanonical WNT receptor ROR2 as transcriptionally strongly upregulated in RVF compared to pRV and NF (p &lt; 0.05, adjusted for multiple hypothesis testing using Benjamin-Hochberg). By western blot, ROR2 protein expression correlated to mRNA expression (R 2 = 0.41, p = 8.1 x 10-18) and increased linearly with higher right atrial to pulmonary capillary wedge ratio in RVF (R 2 =0.40, p = 3.0 x 10-5). Using trichrome, immunohistochemistry, and RNAscope, we identified preferential ROR2 mRNA and protein expression in fibrotic regions by both cardiomyocytes and noncardiomyocytes. High ROR2 expression correlated with increased expression of a known ROR2 target, the WNT/Ca2+ responsive protease calpain and its target FLNA (R 2 0.25 and 0.67, respectively, p &lt; 0.05), as well as with increased relative phosphorylation of FLNA (R 2 0.62, p &lt; 0.05), a marker of its activation by ROR2. Finally, mice with a range of RVF severity, generated by pulmonary artery constriction, demonstrated a robust increase in ROR2 and FLNA expression compared to sham-treated animals, and ROR2 and FLNA expression correlated strongly with each other (R 2 0.89, p = 6.9 x 10-5) and with increasing RV weight normalized to body weight (R 2 ~0.8, p &lt; 0.001, both). Conclusion: ROR2 expression in the RV is dramatically increased in humans and mice with severe RVF, and ROR2 expression correlates with worsening RV hemodynamics, calpain expression, FLNA cleavage, and FLNA phosphorylation. Taken together, the data reveal the robust activation of noncanonical WNT signaling in human RVF, and identify ROR2-mediated cytoskeleton protein cleavage as a potential novel target of human pathologic RV remodeling.

Differential Expression of the Angiotensin-(1-12)/Chymase Axis in Human Atrial Tissue

BackgroundHeart chymase rather than angiotensin (Ang)-converting enzyme has higher specificity for Ang I conversion into Ang II in humans. A new pathway for direct cardiac Ang II generation has been revealed through the demonstration that Ang-(1-12) is cleaved by chymase to generate Ang II directly. Herein, we address whether Ang-(1-12), chymase messenger RNA (mRNA), and activity levels can be differentiated in human atrial tissue from normal and diseased hearts and if these measures associate with various pathologic heart conditions. Materials and methodsAtrial appendages were collected from 11 nonfailing donor hearts and 111 patients undergoing heart surgery for the correction of valvular heart disease, resistant atrial fibrillation, or ischemic heart disease. Chymase mRNA was analyzed by real-time polymerase chain reaction and enzymatic activity by high-performance liquid chromatography using Ang-(1-12) as the substrate. Ang-(1-12) levels were determined by immunohistochemical staining. ResultsChymase gene transcripts, chymase activity, and immunoreactive Ang-(1-12) expression levels were higher in left atrial tissue compared with right atrial tissue, irrespective of cardiac disease. In addition, left atrial chymase mRNA expression was significantly higher in stroke versus nonstroke patients and in cardiac surgery patients who had a history of postoperative atrial fibrillation versus nonatrial fibrillation. Correlation analysis showed that left atrial chymase mRNA was positively related to left atrial enlargement, as determined by echocardiography. ConclusionsAs Ang-(1-12) expression and chymase gene transcripts and enzymatic activity levels were positively linked to left atrial size in patients with left ventricular heart disease, an important alternate Ang II forming pathway, via Ang-(1-12) and chymase, in maladaptive atrial and ventricular remodeling in humans is uncovered.

The Role of Nonglycolytic Glucose Metabolism in Myocardial Recovery Upon Mechanical Unloading and Circulatory Support in Chronic Heart Failure.

Significant improvements in myocardial structure and function have been reported in some patients with advanced heart failure (termed responders [R]) following left ventricular assist device (LVAD)-induced mechanical unloading. This therapeutic strategy may alter myocardial energy metabolism in a manner that reverses the deleterious metabolic adaptations of the failing heart. Specifically, our previous work demonstrated a post-LVAD dissociation of glycolysis and oxidative-phosphorylation characterized by induction of glycolysis without subsequent increase in pyruvate oxidation through the tricarboxylic acid cycle. The underlying mechanisms responsible for this dissociation are not well understood. We hypothesized that the accumulated glycolytic intermediates are channeled into cardioprotective and repair pathways, such as the pentose-phosphate pathway and 1-carbon metabolism, which may mediate myocardial recovery in R. We prospectively obtained paired left ventricular apical myocardial tissue from nonfailing donor hearts as well as R and nonresponders at LVAD implantation (pre-LVAD) and transplantation (post-LVAD). We conducted protein expression and metabolite profiling and evaluated mitochondrial structure using electron microscopy. Western blot analysis shows significant increase in rate-limiting enzymes of pentose-phosphate pathway and 1-carbon metabolism in post-LVAD R (post-R) as compared with post-LVAD nonresponders (post-NR). The metabolite levels of these enzyme substrates, such as sedoheptulose-6-phosphate (pentose phosphate pathway) and serine and glycine (1-carbon metabolism) were also decreased in Post-R. Furthermore, post-R had significantly higher reduced nicotinamide adenine dinucleotide phosphate levels, reduced reactive oxygen species levels, improved mitochondrial density, and enhanced glycosylation of the extracellular matrix protein, α-dystroglycan, all consistent with enhanced pentose-phosphate pathway and 1-carbon metabolism that correlated with the observed myocardial recovery. The recovering heart appears to direct glycolytic metabolites into pentose-phosphate pathway and 1-carbon metabolism, which could contribute to cardioprotection by generating reduced nicotinamide adenine dinucleotide phosphate to enhance biosynthesis and by reducing oxidative stress. These findings provide further insights into mechanisms responsible for the beneficial effect of glycolysis induction during the recovery of failing human hearts after mechanical unloading.

The Role of Non-Glycolytic Glucose Metabolism in Myocardial Recovery Following Mechanical Unloading and Circulatory Support in Chronic Heart Failure

Following left ventricular assist device (LVAD) mechanical unloading significant improvements in myocardial structure and function have been reported in some advanced heart failure (HF) patients, termed as Responders (R). Our prior work demonstrated a post-LVAD mismatch of glycolysis and oxidative phosphorylation characterized by upregulation of glycolysis without subsequent increase in pyruvate oxidation via the Krebs cycle. We hypothesized that the accumulated glycolytic intermediates are channeled into cardioprotective and repair pathways, such as the pentose phosphate pathway (PPP) and one-carbon metabolism (OCM), which may mediate myocardial structural and functional improvement in Responders. We prospectively obtained myocardial tissue from Non-Failing Donor hearts (n=15) and from Responders (n=11) and Non-Responders (n=30) at LVAD implant (Pre-LVAD) and later at transplantation (Post-LVAD). Responders and Non-Responders were defined based on prior published criteria. We conducted protein expression and metabolite profiling and evaluated mitochondrial structure using electron microscopy. Western blot analysis of key PPP and OCM rate-limiting enzymes, glucose-6-phosphate dehydrogenase and phosphoglycerate dehydrogenase were significantly increased in Post-LVAD Responders (Post-R). In agreement with these findings the metabolite levels of these enzyme substrates such as sedoheptulose-6-phosphate (PPP) and serine and glycine (OCM) were decreased in Post-R. Furthermore, Post-R had significantly higher NADPH levels, reduced ROS levels, improved mitochondrial dynamics and enhanced glycosylation of the extracellular matrix (ECM) protein, α-dystroglycan, indicating increased flux of glucose into the PPP and OCM pathways and all together these findings correlated with the observed myocardial structural and functional improvement. The recovering human heart appears to direct glycolytic metabolites into the PPP and OCM pathways which could contribute to cardioprotection and improved myocardial function by generating NADPH to enhance biosynthesis for repair and by reducing oxidative stress.

Defining decreased protein succinylation of failing human cardiac myofibrils in ischemic cardiomyopathy

Succinylation is a post-translational modification of protein lysine residues with succinyl groups derived from succinyl CoA. Succinylation is considered a significant post-translational modification with the potential to impact protein function which is highly conserved across numerous species. The role of succinylation in the heart, especially in heart failure and myofibril mechanics, remains largely unexplored. Mechanical parameters were measured in myofibrils isolated from failing hearts of ischemic cardiomyopathy patients and non-failing donor controls. We employed mass spectrometry to quantify differential protein expression in myofibrils from failing ischemic cardiomyopathy hearts compared to non-failing hearts. In addition, we combined peptide enrichment by immunoprecipitation with liquid chromatography tandem mass spectrometry to quantitatively analyze succinylated lysine residues in these myofibrils. Several key parameters of sarcomeric mechanical interactions were altered in myofibrils isolated from failing ischemic cardiomyopathy hearts, including lower resting tension and a faster rate of activation. Of the 100 differentially expressed proteins, 46 showed increased expression in ischemic heart failure, while 54 demonstrated decreased expression in ischemic heart failure. Our quantitative succinylome analysis identified a total of 572 unique succinylated lysine sites located on 181 proteins, with 307 significantly changed succinylation events. We found that 297 succinyl-Lys demonstrated decreased succinylation on 104 proteins, while 10 residues demonstrated increased succinylation on 4 proteins. Investigating succinyl CoA generation, enzyme activity assays demonstrated that α-ketoglutarate dehydrogenase and succinate dehydrogenase activities were significantly decreased in ischemic heart failure. An activity assay for succinyl CoA synthetase demonstrated a significant increase in ischemic heart failure. Taken together, our findings support the hypothesis that succinyl CoA production is decreased and succinyl CoA turnover is increased in ischemic heart failure, potentially resulting in an overall decrease in the mitochondrial succinyl CoA pool, which may contribute to decreased myofibril protein succinylation in heart failure.

P3480Tissue-specific regulation of the mitochondrial proteome by adiponectin receptor 1

Abstract Lack of the adiponectin target receptor adiponectin receptor 1 (AdipoR1) impairs gene expression of mitochondrial OXPHOS proteins due to impaired AMPK-SIRT1-PGC-1α signaling. Since decreased adiponectin serum levels in diabetes mellitus should thus compromise AdipoR1 signaling, we hypothesized that impaired AdipoR1 signaling may causally contribute to typically observed mitochondrial defects in diabetes complications. Thus, we performed comparative proteomics in cardiac, renal and hepatic tissue of AdipoR1−/− mice using LC-MS/MS. Using principal component analysis, heatmapping and hierarchical clustering, a significant separation of genotypes was observed across tissues. Enrichment analysis of differentially expressed proteins revealed disproportionate representation of proteins involved in oxidative phosphorylation, TCA cycle and fatty acid oxidation in all tissues. While 121 or 98 or 78 proteins were differentially regulated in cardiac or renal or hepatic tissue, respectively, only 15 proteins were regulated in the same direction across all tissues. Pathway analysis identified HNF4, NRF1, LONP, RICTOR, SURF1, insulin receptor and PGC-1α as most likely upstream regulators. Importantly, we found a dramatic downregulation of AdipoR1 expression in heart (−70%), liver (−90%) and kidney (−80%; all p&lt;0.05) of high fat-fed and prediabetic non-transgenic mice compared to low fat-fed mice. In addition and beyond diabetes, AdipoR1 expression was also decreased in endstage failing hearts of non-diabetic human subjects compared to non-failing donor hearts. Thus, we conclude that AdipoR1 signaling regulates mitochondrial protein composition across all tissues in a functionally conserved, yet molecularly distinct, manner. Impaired AdipoR1 signaling may causally contribute to mitochondrial defects in diabetic complications and even human heart failure.