End-stage Human Heart Failure Research Articles

YB-1 Is a Novel Target for the Inhibition of α-Adrenergic-Induced Hypertrophy.

Cardiac hypertrophy resulting from sympathetic nervous system activation triggers the development of heart failure. The transcription factor Y-box binding protein 1 (YB-1) can interact with transcription factors involved in cardiac hypertrophy and may thereby interfere with the hypertrophy growth process. Therefore, the question arises as to whether YB-1 influences cardiomyocyte hypertrophy and might thereby influence the development of heart failure. YB-1 expression is downregulated in human heart biopsies of patients with ischemic cardiomyopathy (n = 8), leading to heart failure. To study the impact of reduced YB-1 in cardiac cells, we performed small interfering RNA (siRNA) experiments in H9C2 cells as well as in adult cardiomyocytes (CMs) of rats. The specificity of YB-1 siRNA was analyzed by a miRNA-like off-target prediction assay identifying potential genes. Testing three high-scoring genes by transfecting cardiac cells with YB-1 siRNA did not result in downregulation of these genes in contrast to YB-1, whose downregulation increased hypertrophic growth. Hypertrophic growth was mediated by PI3K under PE stimulation, as well by downregulation with YB-1 siRNA. On the other hand, overexpression of YB-1 in CMs, caused by infection with an adenovirus encoding YB-1 (AdYB-1), prevented hypertrophic growth under α-adrenergic stimulation with phenylephrine (PE), but not under stimulation with growth differentiation factor 15 (GDF15; n = 10-16). An adenovirus encoding the green fluorescent protein (AdGFP) served as the control. YB-1 overexpression enhanced the mRNA expression of the Gq inhibitor regulator of G-protein signaling 2 (RGS2) under PE stimulation (n = 6), potentially explaining its inhibitory effect on PE-induced hypertrophic growth. This study shows that YB-1 protects cardiomyocytes against PE-induced hypertrophic growth. Like in human end-stage heart failure, YB-1 downregulation may cause the heart to lose its protection against hypertrophic stimuli and progress to heart failure. Therefore, the transcription factor YB-1 is a pivotal signaling molecule, providing perspectives for therapeutic approaches.

Essential role of obscurin kinase-1 in cardiomyocyte coupling via N-cadherin phosphorylation.

Obscurins are giant cytoskeletal proteins with structural and regulatory roles. Obscurin-B (~870 kDa), the largest known isoform, contains 2 enzymatically active Ser/Thr kinase (kin) domains, kin1 and kin2, which belong to the myosin light chain kinase family. Kin1 binds to and phosphorylates N-cadherin, a major component of the intercalated disc, the unique sarcolemmal microdomain that mediates the mechanochemical coupling of adjacent cardiomyocytes. Obscurin-B containing kin1 and N-cadherin colocalize at cell junctions in embryonic rat ventricular myocytes (ERVMs), and their codistribution is regulated by Ca2+. Phosphoproteomics analysis revealed that obscurin-kin1 phosphorylates N-cadherin at Ser-788 located within the juxtamembrane region of its cytoplasmic domain, with an apparent Kcat of approximately 5.05 min-1. Overexpression of obscurin-kin1 or phosphomimic-Ser-788-Glu N-cadherin in ERVMs markedly increases cell adhesion and chemical coupling. Importantly, phosphomimic Ser-788-Glu N-cadherin exhibits significantly reduced binding to p120-catenin, while overexpression of phosphoablated Ser-788-Ala N-cadherin increases RhoA activity. Consistent with an essential role of the obscurin-kin1/N-cadherin axis in cardiomyocyte coupling, it is deregulated in end-stage human heart failure. Given the nearly ubiquitous expression of obscurin and N-cadherin, our findings may have broad applicability in deciphering the obscurin-kin1/N-cadherin axis that likely mediates cell coupling in diverse tissues and organs.

InsP3R-RyR channel crosstalk augments sarcoplasmic reticulum Ca2+ release and arrhythmogenic activity in post-MI pig cardiomyocytes

Ca2+ transients (CaT) underlying cardiomyocyte (CM) contraction require efficient Ca2+ coupling between sarcolemmal Ca2+ channels and sarcoplasmic reticulum (SR) ryanodine receptor Ca2+ channels (RyR) for their generation; reduced coupling in disease contributes to diminished CaT and arrhythmogenic Ca2+ events. SR Ca2+ release also occurs via inositol 1,4,5-trisphosphate receptors (InsP3R) in CM. While this pathway contributes negligeably to Ca2+ handling in healthy CM, rodent studies support a role in altered Ca2+ dynamics and arrhythmogenic Ca2+ release involving InsP3R crosstalk with RyRs in disease. Whether this mechanism persists in larger mammals with lower T-tubular density and coupling of RyRs is not fully resolved. We have recently shown an arrhythmogenic action of InsP3-induced Ca2+ release (IICR) in end stage human heart failure (HF), often associated with underlying ischemic heart disease (IHD). How IICR contributes to early stages of disease is however not determined but highly relevant. To access this stage, we chose a porcine model of IHD, which shows substantial remodelling of the area adjacent to the infarct. In cells from this region, IICR preferentially augmented Ca2+ release from non-coupled RyR clusters that otherwise showed delayed activation during the CaT. IICR in turn synchronised Ca2+ release during the CaT but also induced arrhythmogenic delayed afterdepolarizations and action potentials. Nanoscale imaging identified co-clustering of InsP3Rs and RyRs, thereby allowing Ca2+-mediated channel crosstalk. Mathematical modelling supported and further delineated this mechanism of enhanced InsP3R-RyRs coupling in MI. Our findings highlight the role of InsP3R-RyR channel crosstalk in Ca2+ release and arrhythmia during post-MI remodelling.

Changes in transcriptomic landscape in human end-stage heart failure with distinct etiology

SummaryHeart failure (HF) is the ultimate outcome of a variety of heart diseases, including restrictive cardiomyopathy (RCM), ischemic heart disease (IHD), and valvular heart disease (VHD). To date, accumulating evidence has suggested an important role of noncoding RNAs (ncRNAs) in HF. We performed RNA-sequencing studies with myocardial mRNAs/lncRNAs/circRNAs/miRNAs from non-failing hearts (donor heart tissue from heart transplantation) and three groups of patients with HF (RCM, IHD, and VHD). HF-related gene regulatory networks and gene co-expression networks were constructed based on the interaction relationship and expression profiles of differentially expressed mRNAs/ncRNAs. Our results indicated that HF with different etiologies is regulated by complex lncRNA/circRNA/miRNA/mRNA regulatory networks, comprising common pathways that are shared by all HF types as well as distinct pathways that are enriched in specific HF types. In addition, the HF biomarkers identified in our study have an important clinical application value in HF staging and HF type diagnosis.

Abstract 100: Integrated Cardiac Metabolism In End-Stage Human Heart Failure

Heart failure affects millions of people worldwide with mortality near 50% within five years. This disease is characterized by widespread cardiac and systemic metabolic changes, but a comprehensive evaluation of metabolism in failing human hearts is lacking. Here, we provide a comprehensive depiction of cardiac and systemic metabolic changes in 89 explanted failing and non-failing human hearts through integration of plasma and cardiac tissue metabolomics, genome-wide RNAseq, and proteomic data. The data confirm a profound bioenergetic defect in end-stage human heart failure and demonstrate extensive changes in metabolic homeostasis. The data indicate a substantial defect in fatty acid (FA) use in failing hearts, in particular unsaturated FAs. Reduction of FAs and acyl-carnitines in failing tissue in contrast to concomitant elevations in plasma suggest a defect in import of FAs into the cell, rather than a defect in FA oxidation. Intermediates of glycolysis, the pentose phosphate pathway, and glycogen synthesis are all similarly reduced, as is expression of GLUT1, indicating diminished glucose uptake. However, there was no significant change in tissue pyruvate content, suggesting an increase in lactate utilization. The data suggest increased flux of pyruvate into mitochondria, likely promoting pyruvate oxidation but not pyruvate carboxylation. Blunted anabolic pyruvate flux, in turn, likely leads to insufficient TCA cycle intermediates. Ketone levels were increased in both failing tissue and plasma, as previously reported. The phospholipid content of failing human hearts is greatly increased in both failing tissue and plasma. Nucleotide synthesis pathways also appear to be reprogrammed, with a notable decrease in adenosine metabolism, specifically. Together, these data indicate widespread change in the local cardiac and greater systemic metabolic landscape in severe human heart failure.

Myocardial ketone body utilization in patients with heart failure: The impact of oral ketone ester

AimsUpregulation of ketone body (β-hydroxybutyrate, βHB) utilization has been documented in human end-stage heart failure (HF), but is unclear if this is due to intrinsic cardiac metabolic remodeling or a HF-related catabolic state. This study sought to evaluate the maximal ketone body utilization capacity and its determinants in controls and in patients with moderate HF and reduced ejection fraction (HFrEF). Methods and results19 HFrEF patients and 9 controls underwent sampling from the arterial circulation (A) and coronary sinus (CS) to measure transmyocardial extraction of energy-providing substrates and oxygen. In a separate experiment, measurements were performed 80-min after oral administration of 25 g of ketone ester (KE, (R)-3-hydroxybutyl(R)-3-hydroxybutyrate) drink in 11 HFrEF and 6 control subjects. There were no statistically significant differences in fasting substrate levels and fractional extractions between HF and controls. Administration of KE increased βHB by 12.9-fold, revealing an increased ability to utilize ketones in HFrEF as compared to controls (fractional extraction, FE%: 52 vs 39%, p = 0.035). βHB FE% correlated directly with βHB myocardial delivery (r = 0.90), LV mass (r = 0.56), LV diameter (r = 0.65) and inversely with LV EF (−0.59) (all p < 0.05). βHB FE% positively correlated with lactate FE% (p < 0.01), but not with FFA or glucose FE%, arguing against substrate competition. ConclusionsAcute nutritional ketosis enhances βHB extraction in patients with HFrEF compared to controls, and this enhancement correlates with degree of cardiac dysfunction and remodeling. Data suggest that subclinical metabolic remodeling occurs early in HF progression. Further studies are needed to determine whether exogenous ketones may have a potential therapeutic role.

Atrial fibrillation is not associated with altered left atrial microRNA expression profile in ischemic end-stage human heart failure

Abstract Introduction In patients with chronic heart failure (CHF) left ventricular dysfunction results in elevated left atrial (LA) pressure, triggering pathological atrial remodelling and atrial fibrillation (AF). Nevertheless, it has been reported that some patients with CHF remain in sinus rhythm (SR) despite of the pathological structural alterations (e.g. dilation and fibrosis) of the LA. Of particular interest, data is scarce regarding the molecular explanation for the observed variability in AF development among CHF patients. Recent studies have indicated that alterations in microRNA (miRNA) expression might contribute to the pathogenesis of AF. However, the majority of previous studies focusing on miRNA expression compared healthy LA with SR to pathologically remodelled, dilated LA with AF. Consequently, whether dysregulation of miRNA expression directly contribute to AF and not only to pathological LA remodelling has not been tested before. Purpose The present study aimed to investigate miRNA expression in comparably remodelled LA from end-stage CHF patients with permanent AF (CHF-AF) or SR (CHF-SR). Methods LA samples were collected from male, non-diabetic, ischemic end-stage CHF patients undergoing heart transplantation (n=24). Patients were carefully selected to avoid any differences in age (55±2 vs. 54±2 years, CHF-AF vs. CHF-SR, n.s.), ejection fraction ([EF]: 22.5±1.8 vs. 23.3±2.5%, CHF-AF vs. CHF-SR, n.s.) LA diameters (longitudinal LA diameter: 56±4 vs. 48±5mm.; CHF-AF vs. CHF-SR, n.s.; horizontal LA diameter: 61±2 vs. 54±3, CHF-AF vs. CHF-SR, n.s.) and NYHA stage. As a molecular marker of atrial load, the mRNA expression of atrial natriuretic peptide (ANP) was measured with qRT-PCR. The extent of left atrial fibrosis was assessed on picrosirius red stained histological sections. Global LA miRNA expression profiling (including the measurement of 800 human miRNA) was carried out using a commercially available kit. Results LA mRNA expression of ANP was comparable between the AF-CHF and the SR-CHF groups, suggesting that atrial load occurred to the same level in the two experimental groups. Furthermore, no differences could be observed in the extent of atrial collagen content between the AF-CHF and the SR-CHF groups (collagen area: 20.3±1.3% vs. 23.9±3.1%, n.s.), providing evidence that fibrotic remodelling had occurred to a similar magnitude. The high-throughput miRNA measurement revealed no differences in atrial miRNA expression between the two study groups. Conclusion The present study provides evidence for the first time that AF is not associated with different LA miRNA expression in end-stage CHF patients with comparable level of LA dilatation, ANP expression (atrial load) and interstitial fibrosis. Based on these findings, the potential of miRNA-based therapeutic interventions might be limited in AF patients with ischemic end-stage CHF. Funding Acknowledgement Type of funding source: Public grant(s) – National budget only. Main funding source(s): NVKP_16-1-2016-0017

Alterations of the renin angiotensin system in human end-stage heart failure before and after mechanical cardiac unloading by LVAD support

Heart transplantation is often an unrealizable therapeutic option for end-stage heart failure, which is why mechanical left ventricular assist devices (LVADs) become an increasingly important therapeutic alternative. Currently, there is a lack of information about molecular mechanisms which are influenced by LVADs, particularly regarding the pathophysiologically critical renin angiotensin system (RAS). We, therefore, determined regulation patterns of key components of the RAS and the β-arrestin signaling pathways in left ventricular (LV) tissue specimens from 8 patients with end-stage ischemic cardiomyopathy (ICM) and 12 patients with terminal dilated cardiomyopathy (DCM) before and after LVAD implantation and compared them with non-failing (NF) left ventricular tissue samples: AT1R, AT2R, ACE, ACE2, MasR, and ADAM17 were analyzed by polymerase chain reaction. ERK, phosphorylated ERK, p38, phosphorylated p38, JNK, phosphorylated JNK, GRK2, β-arrestin 2, PI3K, Akt, and phosphorylated Akt were determined by Western blot analysis. Angiotensin I and Angiotensin II were quantified by mass spectrometry. Patients were predominantly middle-aged (53 ± 10 years) men with severely impaired LV function (LVEF 19 ± 8%), when receiving LVAD therapy for a mean duration of 331 ± 317 days. Baseline characteristics did not differ significantly between ICM and DCM patients. By comparing failing with non-failing left ventricles, i.e., before LVAD implantation, a downregulation of AT1R, AT2R, and MasR and an upregulation of ACE, ACE2, GRK, β-arrestin, ERK, PI3K, and Akt were seen. Following LVAD support, then angiotensin I, ACE2, GRK, and β-arrestin were downregulated and AT2R, JNK, and p38 were upregulated. ACE, angiotensin II, AT1R, ADAM17, MasR, ERK, PI3K, and Akt remained unchanged. Some regulation patterns were influenced by the underlying etiology of heart failure, the severity of LV dysfunction at baseline, and the duration of LVAD therapy. Key components of the RAS and β-arrestin signaling pathways were divergently altered in failing left ventricles both before and after LVAD implantation, whereas a remarkable fraction remained unchanged. This indicates a rather incomplete molecular reverse remodeling, whose functional relevance has to be further evaluated.

ERBB2 Targeting Increases Cardiomyocyte β1-Adrenergic Receptor Density

Objective: To determine the underlying pathways that promote cardiotoxicity following anti-ERBB2 cancer therapeutics. Background: Epidermal growth factor receptor 2 (EGFR2/ERBB2) is a member of EGFR (ERBB) family represented by ERBB 1 through 4 that either homo- or hetero-dimerize to signal. ERBB2 has constitutively active confirmation but explicitly needs to hetero-dimerize with either ERBB1 or ERBB4 to mediate signaling and cardiac hypertrophy. Less is known about the mechanisms through which ERBB2 mediates the adaptive cardiac hypertrophy given that anti-ERBB2 therapeutics results in cardiac dilation of the otherwise healthy heart. Methods: We generated transgenic (Tg) mice with cardiomyocyte-specific overexpression of microRNA-7 (miRNA-7) that targets ERBB2. Echocardiography, immunohistochemistry and β-adrenergic receptor (βAR) density were assessed upon aging and transverse aortic constriction (TAC). To determine whether inhibition of ERBB2 has similar outcomes, C57Bl/6 mice were administered ERBB2-specific inhibitor AG825. Also, βAR density was assessed in end-stage human heart failure patient samples with Adriamycin cardiotoxicity. Results: Loss in ERBB2 expression due to miRNA-7 targeting or ERRB2 inhibition by AG 825 results in cardiac dilation. Loss of ERBB2 in miRNA-7 Tg mice leads to increased fibrosis post-TAC. Selective significant increase in β1AR density is observed in miRNA-7 Tg mice or in mice following ERBB2 inhibition by AG825 and in the human heart failure patient samples due to Adriamycin cardiotoxicity. Conclusions: Cardiomyocyte ERBB2 expression and function is required for maintaining the normal cardiac function. Targeting ERBB2 leads to selective and significant increase in β1ARs that may promote cardiac dilation and heart failure.

Activation of endogenous protein phosphatase 1 enhances the calcium sensitivity of the ryanodine receptor type 2 in murine ventricular cardiomyocytes.

Increased protein phosphatase 1 (PP-1) activity has been found in end stage human heart failure. Although PP-1 has been extensively studied, a detailed understanding of its role in the excitation-contraction coupling mechanism, in normal and diseased hearts, remains elusive. The present study investigates the functional effect of the PP-1 activity on local Ca2+ release events in ventricular cardiomyocytes, by using an activating peptide (PDP3) for the stimulation of the endogenous PP-1 protein. We report that acute de-phosphorylation may increase the sensitivity of RyR2 channels to Ca2+ in situ, and that the RyR2-serine2808 phosphorylation site may mediate such a process. Our approach unmasks the functional importance of PP-1 in the regulation of RyR2 activity, suggesting a potential role in the generation of a pathophysiological sarcoplasmic reticulum Ca2+ leak in the diseased heart. Changes in cardiac ryanodine receptor (RyR2) phosphorylation are considered to be important regulatory and disease related post-translational protein modifications. The extent of RyR2 phosphorylation is mainly determined by the balance of the activities of protein kinases and phosphatases, respectively. Increased protein phosphatase-1 (PP-1) activity has been observed in heart failure, although the regulatory role of this enzyme on intracellular Ca2+ handling remains poorly understood. To determine the physiological and pathophysiological significance of increased PP-1 activity, we investigated how the PP-1 catalytic subunit (PP-1c) alters Ca2+ sparks in permeabilized cardiomyocytes and we also applied a PP-1-disrupting peptide (PDP3) to specifically activate endogenous PP-1, including the one anchored on the RyR2 macromolecular complex. We compared wild-type and transgenic mice in which the usually highly phosphorylated site RyR2-S2808 has been ablated to investigate its involvement in RyR2 modulation (S2808A+/+ ). In wild-type myocytes, PP-1 increased Ca2+ spark frequency by two-fold, followed by depletion of the sarcoplasmic reticulum Ca2+ store. Similarly, PDP3 transiently increased spark frequency and decreased sarcoplasmic reticulum Ca2+ load. RyR2 Ca2+ sensitivity, which was assessed by Ca2+ spark recovery analysis, was increased in the presence of PDP3 compared to a negative control peptide. S2808A+/+ cardiomyocytes did not respond to both PP-1c and PDP3 treatment. Our results suggest an increased Ca2+ sensitivity of RyR2 upon de-phosphorylation by PP-1. Furthermore, we have confirmed the S2808 site as a target for PP-1 and as a potential link between RyR2s modulation and the cellular response.

TRP Channels Expression Profile in Human End-Stage Heart Failure.

Objectives: Many studies indicate the involvement of transient receptor potential (TRP) channels in the development of heart hypertrophy. However, the data is often conflicted and has originated in animal models. Here, we provide systematic analysis of TRP channels expression in human failing myocardium. Methods and results: Left-ventricular tissue samples were isolated from explanted hearts of NYHA III-IV patients undergoing heart transplants (n = 43). Quantitative real-time PCR was performed to assess the mRNA levels of TRPC, TRPM and TRPV channels. Analysis of functional, clinical and biochemical data was used to confirm an end-stage heart failure diagnosis. Compared to myocardium samples from healthy donor hearts (n = 5), we detected a distinct increase in the expression of TRPC1, TRPC5, TRPM4 and TRPM7, and decreased expression of TRPC4 and TRPV2. These changes were not dependent on gender, clinical or biochemical parameters, nor functional parameters of the heart. We detected, however, a significant correlation of TRPC1 and MEF2c expression. Conclusions: The end-stage heart failure displays distinct expressional changes of TRP channels. Our findings provide a systematic description of TRP channel expression in human heart failure. The results highlight the complex interplay between TRP channels and the need for deeper analysis of early stages of hypertrophy and heart failure development.

AdipoRon, an adiponectin receptor agonist, attenuates cardiac remodeling induced by pressure overload.

AdipoRon, a small-molecule adiponectin receptor (AdipoR) agonist, has been reported to be implicated in cardiovascular diseases. However, its role in pressure-overload-induced cardiac remodeling is still elusive. To elucidate the role of AdipoRon in the pathogenesis of cardiac remodeling in vivo and vitro, in the left ventricle of human end-stage heart failure, the expression of AdipoR2 is upregulated. Meanwhile, increased expression of AdipoR2 was also observed in mice failing hearts. Oral administration of AdipoRon alleviated cardiac hypertrophy and fibrosis induced by pressure overload, as evidenced by the beneficial change of cross-sectional area of cardiomyocytes, heart weight-to-body weight ratio, gene expression of hypertrophic markers, ventricle collagen ratio, and cardiac function. The AMPKα activation mediated by AdipoRon significantly inhibited AngII-induced TGF-β1 expression and cardiac fibroblast differentiation, and these inhibitory effects were abrogated by treatment with the AMPK inhibitor Compound C. Consistent with the above results, AdipoRon abolished the ability to retard AngII-induced TGF-β1 expression in AMPKα2-/- cardiac fibroblasts. In AMPKα2-/- mice subjected to aortic banding, AdipoRon abolished the protective effect, as indicated by increased cross-sectional area, cardiac collagen ratio, and cardiac dysfunction. Our results demonstrated that AdipoR2 expression was markedly increased in the failing hearts. AdipoRon inhibited TGF-β1 expression and myofibroblast differentiation in AMPKα-dependent manner in vitro. In line with the vitro results, AMPKα2-/- mice markedly abrogated the inhibitory effects of AdipoRon in cardiac remodeling. These results indicated AdipoRon may hold promise of an effective therapy against pressure-overload-induced cardiac remodeling. KEY MESSAGES: • The increased expression of AdipoR2 is observed in human and mice failing hearts, the changeable expression of AdipoR suggests the possible role of AdipoR in cardiac remodeling. • Oral administration of AdipoRon alleviates cardiac hypertrophy and fibrosis induced by pressure overload, and AMPKα activation mediated by AdipoRon significantly inhibited AngII-induced TGF-β1 expression and cardiac fibroblast differentiation. • These findings provide new mechanistic insight and open new therapeutic pathways for heart failure.

Mapping the in vitro interactome of cardiac sodium (Na+)‐calcium (Ca2+) exchanger 1 (NCX1)

The sodium (Na+ )-calcium (Ca2+ ) exchanger 1 (NCX1) is an antiporter membrane protein encoded by the SLC8A1 gene. In the heart, it maintains cytosolic Ca2+ homeostasis, serving as the primary mechanism for Ca2+ extrusion during relaxation. Dysregulation of NCX1 is observed in end-stage human heart failure. In this study, we used affinity purification coupled with MS in rat left ventricle lysates to identify novel NCX1 interacting proteins in the heart. Two screens were conducted using: (1) anti-NCX1 against endogenous NCX1 and (2) anti-His (where His is histidine) with His-trigger factor-NCX1cyt recombinant protein as bait. The respective methods identified 112 and 350 protein partners, of which several were known NCX1 partners from the literature, and 29 occurred in both screens. Ten novel protein partners (DYRK1A, PPP2R2A, SNTB1, DMD, RABGGTA, DNAJB4, BAG3, PDE3A, POPDC2, STK39) were validated for binding to NCX1, and two partners (DYRK1A, SNTB1) increased NCX1 activity when expressed in HEK293 cells. A cardiac NCX1 protein-protein interaction map was constructed. The map was highly connected, containing distinct clusters of proteins with different biological functions, where "cell communication" and "signal transduction" formed the largest clusters. The NCX1 interactome was also significantly enriched with proteins/genes involved in "cardiovascular disease" which can be explored as novel drug targets in future research.

Abstract 380: Mapping the i n vitro Interactome of the Cardiac Sodium-Calcium Exchanger 1

The sodium (Na + )-calcium (Ca 2+ ) exchanger 1 (NCX1) is an antiporter membrane protein encoded by the SLC8A1 gene. In the heart, it maintains cytosolic Ca 2+ homeostasis, serving as the primary mechanism for Ca 2+ extrusion during relaxation. Dysregulation of NCX1 is observed in end-stage human heart failure. In this study we used affinity purification coupled with mass spectrometry in rat left ventricle lysates to identify novel NCX1 interacting proteins in the heart. Two screens were conducted using: 1) anti-NCX1 against endogenous NCX1 and 2) anti-His with His-TF-NCX1 cyt recombinant protein as bait. The respective methods identified 112 and 350 protein partners, of which several were known NCX1 partners from the literature and 29 occurred in both screens. Selected protein partners were validated for binding to NCX1 expressed in HEK293 cells. A cardiac NCX1 protein-protein interaction map was constructed. The map was highly connected, containing distinct clusters of proteins with different biological functions, where cell communication and signal transduction formed the largest clusters. The NCX1 interactome was also significantly enriched with proteins/genes involved in cardiovascular disease. Exploring the molecular mechanisms of these protein partners in future studies may aid in elucidation of NCX1 regulation and facilitate selective therapeutic targeting of NCX1.

A unique microRNA profile in end-stage heart failure indicates alterations in specific cardiovascular signaling networks.

It is well established that the gene expression patterns are substantially altered in cardiac hypertrophy and heart failure, however, less is known about the reasons behind such global differences. MicroRNAs (miRNAs) are short non-coding RNAs that can target multiple molecules to regulate wide array of proteins in diverse pathways. The goal of the study was to profile alterations in miRNA expression using end-stage human heart failure samples with an aim to build signaling network pathways using predicted targets for the altered miRNA and to determine nodal molecules regulating individual networks. Profiling of miRNAs using custom designed microarray and validation with an independent set of samples identified eight miRNAs that are altered in human heart failure including one novel miRNA yet to be implicated in cardiac pathology. To gain an unbiased perspective on global regulation by top eight altered miRNAs, functional relationship of predicted targets for these eight miRNAs were examined by network analysis. Ingenuity Pathways Analysis network algorithm was used to build global signaling networks based on the targets of altered miRNAs which allowed us to identify participating networks and nodal molecules that could contribute to cardiac pathophysiology. Majority of the nodal molecules identified in our analysis are targets of altered miRNAs and known regulators of cardiovascular signaling. Cardio-genomics heart failure gene expression public data base was used to analyze trends in expression pattern for target nodal molecules and indeed changes in expression of nodal molecules inversely correlated to miRNA alterations. We have used NF kappa B network as an example to show that targeting other molecules in the network could alter the nodal NF kappa B despite not being a miRNA target suggesting an integrated network response. Thus, using network analysis we show that altering key functional target proteins may regulate expression of the myriad signaling pathways underlying the cardiac pathology.

Sheet-Like Remodeling of the Transverse Tubular System in Human Heart Failure Impairs Excitation-Contraction Coupling and Functional Recovery by Mechanical Unloading.

Cardiac recovery in response to mechanical unloading by left ventricular assist devices (LVADs) has been demonstrated in subgroups of patients with chronic heart failure (HF). Hallmarks of HF are depletion and disorganization of the transverse tubular system (t-system) in cardiomyocytes. Here, we investigated remodeling of the t-system in human end-stage HF and its role in cardiac recovery. Left ventricular biopsies were obtained from 5 donors and 26 patients with chronic HF undergoing implantation of LVADs. Three-dimensional confocal microscopy and computational image analysis were applied to assess t-system structure, density, and distance of ryanodine receptor clusters to the sarcolemma, including the t-system. Recovery of cardiac function in response to mechanical unloading was assessed by echocardiography during turndown of the LVAD. The majority of HF myocytes showed remarkable t-system remodeling, particularly sheet-like invaginations of the sarcolemma. Circularity of t-system components was decreased in HF versus controls (0.37±0.01 versus 0.46±0.02; P<0.01), and the volume/length ratio was increased in HF (0.36±0.01 versus 0.25±0.02 µm2; P<0.0001). T-system density was reduced in HF, leading to increased ryanodine receptor-sarcolemma distances (0.96±0.05 versus 0.64±0.1 µm; P<0.01). Low ryanodine receptor-sarcolemma distances at the time of LVAD implantation predicted high post-LVAD left ventricular ejection fractions (P<0.01) and ejection fraction increases during unloading (P<0.01). Ejection fraction in patients with pre-LVAD ryanodine receptor-sarcolemma distances >1 µm did not improve after mechanical unloading. In addition, calcium transients were recorded in field-stimulated isolated human cardiomyocytes and analyzed with respect to local t-system density. Calcium release in HF myocytes was restricted to regions proximal to the sarcolemma. Local calcium upstroke was delayed (23.9±4.9 versus 10.3±1.7 milliseconds; P<0.05) and more asynchronous (18.1±1.5 versus 8.9±2.2 milliseconds; P<0.01) in HF cells with low t-system density versus cells with high t-system density. The t-system in end-stage human HF presents a characteristic novel phenotype consisting of sheet-like invaginations of the sarcolemma. Our results suggest that the remodeled t-system impairs excitation-contraction coupling and functional recovery during chronic LVAD unloading. An intact t-system at the time of LVAD implantation may constitute a precondition and predictor for functional cardiac recovery after mechanical unloading.

Nucleoside Diphosphate Kinase-C Suppresses cAMP Formation in Human Heart Failure.

Chronic heart failure (HF) is associated with altered signal transduction via β-adrenoceptors and G proteins and with reduced cAMP formation. Nucleoside diphosphate kinases (NDPKs) are enriched at the plasma membrane of patients with end-stage HF, but the functional consequences of this are largely unknown, particularly for NDPK-C. Here, we investigated the potential role of NDPK-C in cardiac cAMP formation and contractility. Real-time polymerase chain reaction, (far) Western blot, immunoprecipitation, and immunocytochemistry were used to study the expression, interaction with G proteins, and localization of NDPKs. cAMP levels were determined with immunoassays or fluorescent resonance energy transfer, and contractility was determined in cardiomyocytes (cell shortening) and in vivo (fractional shortening). NDPK-C was essential for the formation of an NDPK-B/G protein complex. Protein and mRNA levels of NDPK-C were upregulated in end-stage human HF, in rats after long-term isoprenaline stimulation through osmotic minipumps, and after incubation of rat neonatal cardiomyocytes with isoprenaline. Isoprenaline also promoted translocation of NDPK-C to the plasma membrane. Overexpression of NDPK-C in cardiomyocytes increased cAMP levels and sensitized cardiomyocytes to isoprenaline-induced augmentation of contractility, whereas NDPK-C knockdown decreased cAMP levels. In vivo, depletion of NDPK-C in zebrafish embryos caused cardiac edema and ventricular dysfunction. NDPK-B knockout mice had unaltered NDPK-C expression but showed contractile dysfunction and exacerbated cardiac remodeling during long-term isoprenaline stimulation. In human end-stage HF, the complex formation between NDPK-C and Gαi2 was increased whereas the NDPK-C/Gαs interaction was decreased, producing a switch that may contribute to an NDPK-C-dependent cAMP reduction in HF. Our findings identify NDPK-C as an essential requirement for both the interaction between NDPK isoforms and between NDPK isoforms and G proteins. NDPK-C is a novel critical regulator of β-adrenoceptor/cAMP signaling and cardiac contractility. By switching from Gαs to Gαi2 activation, NDPK-C may contribute to lower cAMP levels and the related contractile dysfunction in HF.

Abstract 128: Impaired Beta Adrenergic Receptor Resensitization Contributes to Human Heart Failture

Beta adrenergic receptors (βARs) are one of the most powerful regulators of cardiac function, and βAR downregulation and desensitization are hallmarks of heart failure. Agonist occupied βARs undergo desensitization by G-protein coupled receptor kinase phosphorylation leading to βAR internalization. Desensitized βARs are resensitized by dephosphorylation in the endosomes by PP2A; however, less is known about role of resensitization in heart failure. Although we have previously shown that resensitization is regulated by inhibition of PP2A by I2PP2A via PI3Kγ (Vasudevan et. al., 2011), the underlying mechanisms of I2PP2A binding to PP2A are not well understood. We used PyMOL software to find the binding interaction between PP2A and I2PP2A. Based on in silico predictions, we generated a mutant PP2A that when expressed would compete out I2PP2A and inhibit I2PP2A from binding to endogenous PP2A. Expression of PP2A mutant in β2AR expressing cells showed preservation of β2AR function following stimulation as measured by reduced β2AR phosphorylation, increased cAMP generation and increased phosphatase function. We also tested whether resensitization as a mechanism is altered in endstage human heart failure samples. Since resensitization occurs in endosomes, plasma membranes and endosomes were isolated from human heart samples and assessed for PI3K activity, PP2A activity, β2AR phosphorylation and adenylyl cyclase (AC) activity as a measure of βAR function. Significant PI3Kγ activity was observed to increase in heart failure samples while AC activity was reduced in plasma membranes. Consistent with the reduced recovery in βAR function in heart failure samples, significant β2AR phosphorylation was observed in the endosomes and plasma membranes of human heart failure samples compared to non-failing. Correspondingly, we observed significant reduction in endosomal PP2A activity in heart failure samples versus controls suggesting inhibited resensitization occurs in human heart failure. Together our studies suggest that resensitization is inhibited in human heart failure and targeting I2PP2A may provide preservation of receptor function and beneficial cardiac remodeling.

Abstract 207: Left Ventricular Assist Device (LVAD) Reverses Inhibition on Beta-adrenergic Receptor Resensitization

Beta-adrenergic receptor (βAR) down-regulation and desensitization are hallmarks of heart failure. Agonist occupied βAR undergo desensitization through phosphorylation by G-protein coupled receptor kinases leading to βAR internalization. Phosphorylated βAR becomes resensitized following dephosphorylation by protein phosphatase 2A (PP2A) in the endosomes and we have shown previously shown that PP2A activity is regulated by phosphoinositide3-kinaseγ (PI3Kγ). Traditionally, it has been considered that increased desensitization mechanisms underlie βAR dysfunction in heart failure but it is not known whether resensitization of βARs is altered and an integral contributor to heart failure. To test whether resensitization mechanisms are altered in heart failure and could play a role in recovery of βAR function post-LVAD, we used paired pre- and post-LVAD human heart samples along with non-failing hearts. Since resensitization occurs in endosomes, plasma membranes and endosomes were isolated from these human heart samples and assessed for PI3K activity, PP2A activity, β2AR phosphorylation and adenylyl cyclase (AC) activity as a measure of recovery in βAR function by G-protein coupling. Significant PI3Kγ activity was observed in the endosomes of pre-LVAD compared post-LVAD and non-failing human heart samples while plasma membrane PI3K activity remained similar across all samples. Similarly, AC activity was markedly reduced in plasma membranes and endosomes of pre-LVAD samples compared to post-LVAD and non-failing showing reduced ability of receptors to couple to G-proteins indicating reduced recovery of receptor function in post-LVAD heart samples. Consistent with the reduced recovery in βAR function in pre-LVAD samples, significant β2AR phosphorylation was observed in the endosomes and plasma membranes of pre-LVAD compared to post-LVAD and non-failing. Correspondingly, we observed significant reduction in endosomal PP2A activity in the pre-LVAD samples which was remarkably reversed in post-LVAD samples similar to the activity in non-failing. These studies suggest that resensitization is inhibited in end-stage human heart failure and may critically contribute to cardiac remodeling and hypertrophic response upon cardiac stress.

Abstract 390: End-stage Human Heart Failure is Characterized by a Deficit in Energetic Lipids in Both Diabetic and Non-diabetic Patients

Introduction: Animal models and human studies have identified increased intramyocardial lipid accumulation and a lipotoxicity hypothesis has been emerging as a mechanism of myocardial dysfunction in diabetes. We have identified a significant decrease in energetic lipids in chronic advanced non-diabetic heart failure. We hypothesized that intramyocardial lipid species would be increased in diabetic as compared to non-diabetic end-stage heart failure patients. Methods: Left ventricular samples procured at the time of orthotopic heart transplantation from non-diabetic (IDCM n=8) and diabetic (DCMDM n=8) patients as well as organ donors with (NFDM) and without a history of diabetes (Donor) were quantitated for lipids with high-resolution mass spectrometry . Stable isotope labeled essential nutrient in cell culture internal standards for acyl-CoAs were generated using [ 13 C 3 15 N 1 ]-pantotheonate in Hepa1c1c7 cells. Results: The lipidomic signature of end-stage failing myocardium marked by a significant decrease in energetic lipids, a decreased myocardial Succinyl CoA (p-value 0.0079 for failing versus non-failing diabetic subjects) and a decreased ratio of [Succinyl CoA]/[Acetyl Coa] consistent with deficient TCA cycling, is indistinguishable in diabetic and non-diabetic patients (Figure 1). Discussion: Despite the known bioenergetic deficits in insulin-resistant diabetes, we have not identified any significant differences between diabetic and non-diabetic subjects in the lipidomic signature of end-stae failing myocardium. Future studies are needed with focused metabolomics to elucidate differences in the diabetic phenotype of human heart failure.