Transaortic Constriction Research Articles

P748Afterload-induced TRIF-dependent signaling promotes fibrosis but not cardiomyocyte hypertrophy

Abstract Background Pressure overload dependent cardiac remodeling is characterized by a continuous functional loss of cardiomyocytes, cardiomyocyte hypertrophy, cell death and fibrosis. The relevance of the MyD88- and NFκB-independent inflammatory TRIF (TIR-domain-containing adapter protein inducing IFN-β) signaling pathway in myocardial hypertrophy is incompletely understood. Methods and results The murine model of afterload induced myocardial hypertrophy (trans-aortic constriction, TAC) was used to investigate the time dependent chemokine expression in C57Bl/6J Wild type (WT) mice after intervention. Myd88- as well as TRIF-dependent chemokines reached their expression maximum at d7 post intervention. This time point was determined for further investigations on TRIF knockout-mice (TRIF−/−) compared to WT mice. While left ventricular (LV) mRNA-expression of TNFalpha and IL6 was upregulated in TRIF−/− mice, CXCL10, CXCL11, RANTES and pathway molecule IRF3 remained unchanged. In the stage of established hypertrophy (35 days after TAC) cytokine levels returned to baseline in WT as well as in TRIF−/− mice. Cellular hypertrophy (increase in cardiomyocyte size) as well as echocardiographic determined increase of LV mass was similar in TRIF−/− and WT mice at d7 as well as at d35 after TAC. Additionally at d7 and d35 the fractional shortening function didn't show any differences between the groups after TAC. On the other hand LV interstitial fibrosis (determined by collagen content) was significantly less distinct in TRIF−/− mice (1.4±0.2%) than in WT mice (2.3±0.3%, (p<0.01 TRIF−/− vs. WT) at d7 following TAC. According to that TGFbeta protein expression was more pronounced in WT (318±85% of sham-operated controls) than in TRIF−/− mice (111±15% of sham-operated controls; p<0.05 TRIF−/− vs. WT). These differences in fibrosis and TGFbeta remained at d35. Even the hydroxyproline concentration was higher in LV tissue of the WT mice than in the TRIF−/− mice d35 after TAC (WT: 93.4±7.7μM, TRIF−/− 54.8±9.0μM, p<0.5 TRIF−/− vs. WT). The expression of TGFbeta signaling pathway associated SMAD proteins (SMAD 3, pSMAD3, SMAD4) was up to two fold higher in WT mice than in TRIF−/− mice. Additionally expression of antifibrotic miR29b was more pronounced in TRIF−/− mice (2.34±0.71) than in WT mice (0.98±0.08) after TAC d7. Conclusion In afterload induced hypertrophy TRIF-dependent signaling doesn't influence cardiomyocyte hypertrophy. But TRIF plays an important role in the regulation of fibrosis in pressure overload dependent remodeling. Through the modulation of the TGF-beta signaling pathway and antifibrotic microRNAs TRIF signaling is involved in the development of interstitial fibrosis. Especially in the early stage of the cardiac remodeling. Acknowledgement/Funding KFO196

3072Myocardial delivery of therapeutic miR-133 via inhalable nanoparticles prevents the pathologic development in a model of ventricular pressure overload

Abstract Background MicroRNAs (miRs) are regulators involved in several biological processes and have been recognized as potential novel therapeutic targets for the treatment and prevention of CDs. We previously demonstrated that the cardiac-enriched miR-133, which is inversely related to failing heart conditions, is involved in several aspects of pathological cardiac remodeling via mitigation of cardiac hypertrophy and fibrosis, fine-tuning of the β1-adrenergic receptor signaling, and protection against oxidative stress-mediated apoptosis. However, effective and clinically oriented interventions aiming to delivery exogenous miR-133 for preventing the stress-induced downregulation of miR-133 levels are still missing. Here, we applied an unconventional and effective nanotechnology-based inhalation approach enabling the delivery to diseased heart of exogenous miR-133 loaded into biocompatible and biodegradable calcium phosphate-based nanoparticles (CaPs). Methods Male C57Bl/6J mice (8 weeks) were subjected to a trans-aortic constriction (TAC), to induce a ventricular pressure overload. 4 experimental groups of TAC animals were randomly assigned to different intratracheal administrations of: 1. Saline (TAC Control); 2. CaP-miR133 (TAC CaP-miR133); 3. Pristine miR-133 (TAC miR133); 4. Unloaded CaPs (TAC CaP). Not TAC operated mice served as SHAM group. Intratracheal nebulizations were performed immediately after TAC surgery once-a-day in alternative days for 4 consecutive weeks. Echocardiography (ECO) were conducted before TAC, at 2 and 4 weeks after surgery. ECO as well as molecular and histological analyses were performed at sacrifices. Results ECO analyses showed an effective CaP-miR133-associated reversal of the failure progression, preserving left ventricular internal diameter (LVID) during cardiac cycle, ejection fraction (EF) and fraction shortening (FS), both at 2 and 4 weeks after TAC. This improvement was associated with the restoration of physiological levels of miR-133 in TAC-stressed isolated cardiomyocytes as well as its contractile activity. TAC mice receiving CaP-mir133 showed reduced indexes of fibrosis. In contrast, no beneficial effects were observed when unloaded CaPs or pristine miR-133 were administered. No major alterations of the immunological status of mice were observed after CaP-miR133 administration. Conclusions Intratracheal nebulization of miR-133-loaded nanoparticles is an effective approach for the beneficial restoration of cardiac performance, cell contractility and remodeling in a mouse model of left ventricular pressure overload. Providing the evidence for a potential innovative application of the emerging nanotechnologies, our approach might represent an important step forward for the treatment of cardiovascular diseases. Acknowledgement/Funding Fondazione Umberto Veronesi and CUPIDO project (EU's Horizon 2020, Grant Agreement 720834)

Abstract 843: Non-oxidized Protein Kinase G 1 Alpha Antagonizes mTORC1 Signaling in a Tuberin-dependent Manner to Ameliorate Cardiac Disease

Rationale: Protein kinase G 1α (PKG1α) confers anti-hypertrophic effects in hearts subjected to mechanical and neurohumoral stress. Human heart failure with a reduced ejection fraction (HFrEF) and mouse pressure overloaded hearts present with increased mechanistic target of rapamycin complex 1 (mTORC1) activity, protein aggregation, oxidative stress, and, as we previously described, increased PKG1α disulfide dimer formation indicative of PKG1α oxidation. Recently we demonstrated that stimulation of PKG1α phosphorylates tuberin (TSC2) at one specific serine, S1365, to inhibit mTORC1 signaling and attenuate pathological hypertrophy. Objective: To determine if the redox state of PKG1α impacts its ability to target TSC2 signaling in a chronic pressure-overload mouse model exhibiting pathologic hypertrophy. We hypothesize non-oxidized PKG1α will increase TSC2 S1365 phosphorylation to antagonize mTORC1 signaling, thereby enhancing autophagic flux to clear protein aggregates, culminating in ameliorated cardiac disease. Methods and Results: Mice expressing a non-oxidizable (redox-dead) PKG1α (cysteine 42 serine, CS) knock-in mutation and wild type (WT) littermate controls were subjected cardiac pressure overload stress via transaortic constriction (TAC) or sham surgeries. Following TAC, PKG1α CS mice exhibited reduced mTORC1 activation leading to increased autophagic flux and preventing protein aggregation, compared to WT mice. PKG1α CS TAC mice had decreased expression of the hypertrophic genes, attenuated cardiac hypertrophy (p<0.0001), and improve fractional shortening compared to WT TAC mice (28.14%±10.82 in WT vs. 47.42%±15.62 in CS; p<0.01). Treating WT TAC mice with an mTORC1 inhibitor (everolimus) abrogated mTORC1 hyperactivation, which lead to enhanced autophagic flux, attenuated hypertrophy, and improved cardiac function. Crossing PKG1α CS mice with TSC2 S1365 phospho-null mice resulted in increased cardiac hypertrophy and reduced lifespan (p<0.05). Conclusions: Preventing PKG1α oxidation attenuates mTORC1 activation to enhance autophagic flux, prevent protein aggregation, and ameliorate pathological hypertrophy in following cardiac pressure overload, dependent onTSC2 S1365 phosphorylation.

Abstract 844: β IV -spectrin Regulates Cardiac Fibroblast Phenotype, Fibrosis, and Cardiac Function

Increased fibrosis is associated with cardiac dysfunction and arrhythmias. Activation of quiescent cardiac fibroblasts (CFs) occurs in response to injury although the underlying mechanistic pathways remain unclear. The cytoskeletal protein β IV -spectrin has been shown to control targeting and activity of the multifunctional transcription factor Signal Transducer and Activator of Transcription 3 (STAT3) for maladaptive remodeling. The mechanism linking spectrin dysfunction to altered STAT3 signaling, fibrosis, and function remains unknown. The objective of this study was to investigate the role of stress-induced disruption of β IV -spectrin/STAT3 complex in modulating CF phenotype, fibrosis, and cardiac function. Cardiac function (echocardiography) and fibrosis (Masson’s trichrome) were evaluated in adult wildtype (WT) and qv 4J mice expressing truncated β IV -spectrin lacking STAT3 interaction. A subset of WT mice were subjected to 6 wks of transaortic constriction (TAC) to induce heart failure. From these groups, CFs were isolated from ventricles and analyzed for STAT3 localization, gene expression, and activity. CFs were treated with selective STAT3 inhibitor S3I-201 (100μM) or transfected for 72 hrs with β IV -spectrin fragment from repeat 10 to C-terminus (β IV,10-C , contains STAT3 binding motif). Ejection fraction was decreased in qv 4J and WT TAC relative to WT baseline (49.5±0.85% vs 34.9±2.2% vs 63.2±0.60% respectively, p<0.05, N=5. Ventricular fibrosis was augmented in qv 4J and WT TAC compared to WT baseline (2.4±0.09% vs 3.2±0.25% vs 0.48±0.01% respectively, p<0.05, N=5). qv 4J CFs showed STAT3 mislocalization (49.7±10.7% nuclear accumulation vs 5.3±.1.8% in WT, p<0.05, N=5) with higher STAT3 transcriptional activity (2.6±0.17-fold increase over WT, p<0.05, N=9). In parallel, qv 4J CFs displayed elevated expression of fibrotic genes associated with CF activation. At the functional level, qv 4J CFs showed increased proliferation (1.9±0.19-fold increase over WT at 24 hrs, p<0.05, N=4). STAT3 inhibition with S31-201 or transfection with β IV,10-C normalized gene expression and proliferation in qv 4J CFs compared to WT. These studies identify a requirement of β IV -spectrin for normal STAT3 signaling and quiescent phenotype in CFs.

Abstract 229: TWEAK-Fn14 Axis: A Potential Therapeutic Target for Treating Heart Failure

Heart failure (HF) is a complex disease characterized by compromised cardiac structure and function. Previous work has identified a link between upregulation of pro-inflammatory cytokines, and HF. There is a critical need to gain better understanding of the inflammatory pathways in heart and identify novel therapeutic targets to regulate cardiac inflammation and HF Recent studies have shown that under stress macrophages are recruited to heart. These macrophages are mainly implicated in the release of inflammatory cytokines that promote cardiac fibrosis, eventually resulting in heart failure. Tumor necrosis factor (TNF)-like weak inducer of apoptosis (TWEAK) is a multifunctional member of the TNF superfamily, which binds to fibroblast growth factor inducible 14 (Fn14), a ubiquitously expressed cell-surface receptor. The TWEAK-Fn14 axis has been previously implicated in promoting inflammation in multiple autoimmune disorders, such as rheumatoid arthritis, however, its role in heart in not well known. The objective of this study is to test the hypothesis that TWEAK-Fn14 pathway promotes cardiac inflammation under stress, and blocking this pathway could be a potential therapeutic strategy to reduce stress induced cardiac inflammation and HF. Wild type (WT) and Fn14 knock out (Fn14 -/- ) mice were subjected to pressure overload induced stress (Trans aortic constriction or TAC) for 1 or 6 weeks. A subset of WT TAC animals were treated with the Fn14 inhibitor [L524-0366] (9mg/kg, IP daily) or vehicle beginning at day 3 following TAC. Macrophage infiltration and cardiac fibrosis were quantified in ventricular sections using F4/80 and Masson Trichrome staining respectively, and cardiac function was measured by echocardiography. Levels of monocyte chemoattractant protein-1 ( mcp-1 ) were measured by quantitative PCR. Genetic or chemical inhibition of Fn14 reduced fibrosis [40% lower compared to WT TAC] and improved cardiac function [Δ EF of 21.7%, n=6 for WT TAC; Δ EF of 14.2%, n=6 for Fn14 -/- TAC; Δ EF of 48.5%, n=3 for vehicle; Δ EF of 27.9%, n=5 for drug] compared to WT. Fn14 inhibition also reduced mcp-1 expression and macrophage infiltration [53.5% lower compared to WT TAC] .These results support TWEAK/Fn14 axis as a potential therapeutic target for treating HF

Remodeling of substrate consumption in the murine sTAC model of heart failure

BackgroundEnergy metabolism and substrate selection are key aspects of correct myocardial mechanical function. Myocardial preference for oxidizable substrates changes in both hypertrophy and in overt failure. Previous work has shown that glucose oxidation is upregulated in overpressure hypertrophy, but its fate in overt failure is less clear. Anaplerotic flux of pyruvate into the tricarboxylic acid cycle (TCA) has been posited as a secondary fate of glycolysis, aside from pyruvate oxidation or lactate production. Methods and resultsA model of heart failure that emulates both valvular and hypertensive heart disease, the severe transaortic constriction (sTAC) mouse, was assayed for changes in substrate preference using metabolomic and carbon-13 flux measurements. Quantitative measures of O2 consumption in the Langendorff perfused mouse heart were paired with 13C isotopomer analysis to assess TCA cycle turnover. Since the heart accommodates oxidation of all physiological energy sources, the utilization of carbohydrates, fatty acids, and ketones were measured simultaneously using a triple-tracer NMR method. The fractional contribution of glucose to acetyl-CoA production was upregulated in heart failure, while other sources were not significantly different. A model that includes both pyruvate carboxylation and anaplerosis through succinyl-CoA produced superior fits to the data compared to a model using only pyruvate carboxylation. In the sTAC heart, anaplerosis through succinyl-CoA is elevated, while pyruvate carboxylation was not. Metabolomic data showed depleted TCA cycle intermediate pool sizes versus the control, in agreement with previous results. ConclusionIn the sTAC heart failure model, the glucose contribution to acetyl-CoA production was significantly higher, with compensatory changes in fatty acid and ketone oxidation not reaching a significant level. Anaplerosis through succinyl-CoA is also upregulated, and is likely used to preserve TCA cycle intermediate pool sizes. The triple tracer method used here is new, and can be used to assess sources of acetyl-CoA production in any oxidative tissue.

MAS AGONISTS FOR THE TREATMENT OF NEURODEGENERATIVE DISEASE

Alzheimer's disease (AD) is associated with loss of memory and executive function. Several studies have shown a direct correlation between hypertension and an increased incidence of AD like symptoms, including cognitive deficits and increased Aβ deposition. Most interestingly, individuals who are on anti-hypertensive medications (angiotensin receptor blockers and angiotensin converting enzyme inhibitors) targeting the Renin-Angiotensin System (RAS), have a reduced risk of developing AD. Recently, the protective arm of RAS, angiotensin (1-7)[A(1-7)]/Mas receptor/angiotensin converting enzyme 2, is reduced in AD patients. Mas activation increases cognition and is cardioprotective. Thus, we hypothesized that Mas agonists could help ameliorate AD-like pathology. Mice underwent transaortic constriction (TAC), a mouse model of heart failure and hypertension. One week after induction of TAC, mice started treatment with saline or one of three Mas agonists. Seven weeks after surgery, the cardiac function was assessed by echocardiography followed by assessment of their cognitive abilities (8 weeks) using novel object recognition. Upon necropsy, brain, heart, bone marrow and blood were collected. TAC surgery decreased cognition and increased mitochondrial oxidative stress (OS) in neurons and microglia. Treatment with Mas agonists increased cognition and reduced brain OS. Further, treatment with Mas agonists reduced inflammatory cytokines in the hippocampus. These data support the development of Mas agonist for the treatment of neurodegenerative diseases.

Therapeutic Effect of Targeting Branched-Chain Amino Acid Catabolic Flux in Pressure-Overload Induced Heart Failure.

BackgroundBranched‐chain amino acid (BCAA) catabolic defect is an emerging metabolic hallmark in failing hearts in human and animal models. The therapeutic impact of targeting BCAA catabolic flux under pathological conditions remains understudied.Methods and ResultsBT2 (3,6‐dichlorobenzo[b]thiophene‐2‐carboxylic acid), a small‐molecule inhibitor of branched‐chain ketoacid dehydrogenase kinase, was used to enhance BCAA catabolism. After 2 weeks of transaortic constriction, mice with significant cardiac dysfunctions were treated with vehicle or BT2. Serial echocardiograms showed continuing pathological deterioration in left ventricle of the vehicle‐treated mice, whereas the BT2‐treated mice showed significantly preserved cardiac function and structure. Moreover, BT2 treatment improved systolic contractility and diastolic mechanics. These therapeutic benefits appeared to be independent of impacts on left ventricle hypertrophy but associated with increased gene expression involved in fatty acid utilization. The BT2 administration showed no signs of apparent toxicity.ConclusionsOur data provide the first proof‐of‐concept evidence for the therapeutic efficacy of restoring BCAA catabolic flux in hearts with preexisting dysfunctions. The BCAA catabolic pathway represents a novel and potentially efficacious target for treatment of heart failure.

Emodin alleviates cardiac fibrosis by suppressing activation of cardiac fibroblasts via upregulating metastasis associated protein 3

Excess activation of cardiac fibroblasts inevitably induces cardiac fibrosis. Emodin has been used as a natural medicine against several chronic diseases. The objective of this study is to determine the effects of emodin on cardiac fibrosis and the underlying molecular mechanisms. Intragastric administration of emodin markedly decreased left ventricular wall thickness in a mouse model of pathological cardiac hypertrophy with excess fibrosis induced by transaortic constriction (TAC) and suppressed activation of cardiac fibroblasts induced by angiotensin II (AngII). Emodin upregulated expression of metastasis associated protein 3 (MTA3) and restored the MTA3 expression in the setting of cardiac fibrosis. Moreover, overexpression of MTA3 promoted cardiac fibrosis; in contrast, silence of MTA3 abrogated the inhibitory effect of emodin on fibroblast activation. Our findings unraveled the potential of emodin to alleviate cardiac fibrosis via upregulating MTA3 and highlight the regulatory role of MTA3 in the development of cardiac fibrosis.

Cardiomyocyte-GSK-3α promotes mPTP opening and heart failure in mice with chronic pressure overload

Chronic pressure-overload (PO)- induced cardiomyopathy is one of the leading causes of left ventricular (LV) remodeling and heart failure. The role of the α isoform of glycogen synthase kinase-3 (GSK-3α) in PO-induced cardiac remodeling is unclear and its downstream molecular targets are largely unknown. To investigate the potential roles of GSK-3α, cardiomyocyte-specific GSK-3α conditional knockout (cKO) and control mice underwent trans-aortic constriction (TAC) or sham surgeries. Cardiac function in the cKOs and littermate controls declined equally up to 2 weeks of TAC. At 4 week, cKO animals retained concentric LV remodeling and showed significantly less decline in contractile function both at systole and diastole, vs. controls which remained same until the end of the study (6 wk). Histological analysis confirmed preservation of LV chamber and protection against TAC-induced cellular hypertrophy in the cKO. Consistent with attenuated hypertrophy, significantly lower level of cardiomyocyte apoptosis was observed in the cKO. Mechanistically, GSK-3α was found to regulate mitochondrial permeability transition pore (mPTP) opening and GSK-3α-deficient mitochondria showed delayed mPTP opening in response to Ca2+ overload. Consistently, overexpression of GSK-3α in cardiomyocytes resulted in elevated Bax expression, increased apoptosis, as well as a reduction of maximum respiration capacity and cell viability.Taken together, we show for the first time that GSK-3α regulates mPTP opening under pathological conditions, likely through Bax overexpression. Genetic ablation of cardiomyocyte GSK-3α protects against chronic PO-induced cardiomyopathy and adverse LV remodeling, and preserves contractile function. Selective inhibition of GSK-3α using isoform-specific inhibitors could be a viable therapeutic strategy to limit PO-induced heart failure.

Dysregulation of proangiogeneic factors in pressure-overload left-ventricular hypertrophy results in inadequate capillary growth.

Background:Pressure-overload left-ventricular hypertrophy (LVH) is an increasingly prevalent pathological condition of the myocardial muscle and an independent risk factor for a variety of cardiac diseases. We investigated changes in expression levels of proangiogeneic genes in a small animal model of LVH.Methods:Myocardial hypertrophy was induced by transaortic constriction (TAC) in C57BL/6 mice and compared with sham-operated controls. The myocardial expression levels of vascular endothelial growth factor (VEGF), its receptors (KDR and FLT-1), stromal-cell-derived factor 1 (SDF1) and the transcription factors hypoxia-inducible factor-1 and 2 (HIF1 and HIF2) were analyzed by quantitative polymerase chain reaction over the course of 25 weeks. Histological sections were stained for caveolin-1 to visualize endothelial cells and determine the capillary density. The left-ventricular morphology and function were assessed weekly by electrocardiogram-gated magnetic resonance imaging.Results:The heart weight of TAC animals increased significantly from week 4 to 25 (p = 0.005) compared with sham-treated animals. At 1 day after TAC, the expression of VEGF and SDF1 also increased, but was downregulated again after 1 week. The expression of HIF2 was significantly downregulated after 1 week and remained at a lower level in the subsequent weeks. The expression level of FLT-1 was also significantly decreased 1 week after TAC. HIF-1 and KDR showed similar changes compared with sham-operated animals. However, the expression levels of HIF1 after 4 and 8 weeks were significantly decreased compared with day 1. KDR changes were significantly decreased after 1, 2, 4, 8 and 25 weeks compared with week 3. After 4 weeks post-TAC, the size of the capillary vessels increased (p = 0.005) while the capillary density itself decreased (TAC: 2143 ± 293 /mm2 versus sham: 2531 ± 321 /mm2; p = 0.021). Starting from week 4, the left-ventricular ejection fraction decreased compared with controls (p = 0.049).Conclusions:The decrease in capillary density in the hypertrophic myocardium appears to be linked to the dysregulation in the expression of proangiogeneic factors. The results suggest that overcoming this dysregulation may lead to reconstitution of capillary density in the hypertrophic heart, and thus be beneficial for cardiac function and survival.

βIV-Spectrin regulates STAT3 targeting to tune cardiac response to pressure overload.

Heart failure (HF) remains a major source of morbidity and mortality in the US. The multifunctional Ca2+/calmodulin-dependent kinase II (CaMKII) has emerged as a critical regulator of cardiac hypertrophy and failure, although the mechanisms remain unclear. Previous studies have established that the cytoskeletal protein βIV-spectrin coordinates local CaMKII signaling. Here, we sought to determine the role of a spectrin-CaMKII complex in maladaptive remodeling in HF. Chronic pressure overload (6 weeks of transaortic constriction [TAC]) induced a decrease in cardiac function in WT mice but not in animals expressing truncated βIV-spectrin lacking spectrin-CaMKII interaction (qv3J mice). Underlying the observed differences in function was an unexpected differential regulation of STAT3-related genes in qv3J TAC hearts. In vitro experiments demonstrated that βIV-spectrin serves as a target for CaMKII phosphorylation, which regulates its stability. Cardiac-specific βIV-spectrin-KO (βIV-cKO) mice showed STAT3 dysregulation, fibrosis, and decreased cardiac function at baseline, similar to what was observed with TAC in WT mice. STAT3 inhibition restored normal cardiac structure and function in βIV-cKO and WT TAC hearts. Our studies identify a spectrin-based complex essential for regulation of the cardiac response to chronic pressure overload. We anticipate that strategies targeting the new spectrin-based "statosome" will be effective at suppressing maladaptive remodeling in response to chronic stress.

Abstract 284: Loss of Cardiomyocyte General Control of Amino-Acid Synthesis 5-like 1 Expression Impairs Mitochondrial Function and Exacerbates Heart Failure Progression

Background: Energy starvation resulting from impaired mitochondrial function is one of the key mechanisms of heart failure development. Emerging evidence shows that lysine acetylation is one of the critical post-translational modifications that modulates mitochondrial bioenergetic output. We have identified General Control of Amino-Acid Synthesis 5-like 1 (GCN5L1) as a key acetyltransferase protein responsible for dynamic mitochondrial protein acetylation, and that this mechanism plays an essential role in regulating fatty acid oxidation. However, the role of GCN5L1 regulation on mitochondrial function in heart failure development is unknown. Methods and results: We examined GCN5L1 expression in cultured neonatal cardiac myocytes (RNCM) challenged with hypertrophy inducer phenylepherine (PE), and in failing hearts. We found that GCN5L1 mRNA and protein expression is markedly decreased in RNCM treated with PE, and in heart failure induced by mouse transaortic constriction (TAC). In addition, total mitochondrial protein acetylation in failing hearts was significantly decreased, corresponding to the decreased levels of GCN5L1. Next, we investigated the impact of decreased level of GCN5L1 on mitochondrial function and cardiac function in response to pathological stress. We found that GCN5L1 knockdown by shRNA in RNCM results in a decrease in basal oxygen consumption rate and ATP-linked respiration upon PE stimulation. More importantly, knocking down GCN5L1 in the presence of PE exacerbates the RNCM hypertrophic response assessed by increased cellular area and elevated ANP and BNP. Consistent with these in vitro studies, cardiac specific GCN5L1 knockout mice subjected to TAC display accelerated cardiac hypertrophy and heart failure, compared to WT littermates (fractional shortening 15.7% vs 26.4%, n=6-7 in each group, P < 0.05). Conclusion: GCN5L1 plays a critical role in mitochondrial function and cardiac bioenergetics in response to stress. Impaired GCN5L1 function might be one of the key mechanisms of metabolic derangement in heart failure development, and could be a promising therapeutic target.

Abstract 103: Cytochrome b5 Reductase 3 Regulates Myoglobin Redox State and Controls Cardiac Function

Oxidative stress contributes to the adverse pathophysiology of cardiac remodeling in heart failure. During oxidative stress, hemoprotein heme-iron can be oxidized, often altering the function of these proteins. We previously reported that cytochrome b5 reductase 3 (Cyb5R3) regulates heme iron redox in the vascular wall. While conducting vasoreactivity studies with a small-molecule Cyb5R3 inhibitor, we unexpectedly observed inhibition resulted in dilated cardiomyopathy (DCM) with reduced ejection fraction. Based on this study, we generated the first cardiomyocyte Cyb5R3 knockout mouse. Heart specific Cyb5R3 knockout resulted in DCM with >50% lethality in 15 days. H&E staining, wheat germ agglutinin immunofluorescence staining, transmission electron microscopy and diffusion tensor MRI shows that loss of Cyb5R3 causes myocardial atrophy and fibrosis. Due to the known heme iron reducing function of Cyb5R3, electron paramagnetic resonance (EPR) was used to assess the abundance of reduced and oxidized myoglobin ex vivo. Cyb5R3 knockout hearts had approximately 30% more oxidation relative to controls. Hypoxyprobe staining, which senses tissue pO2 below 5mmHg, showed Cyb5R3 expression is required for maintaining intra-cardiomyocyte pO 2 , likely through myoglobin heme-iron reduction. Decreased pO 2 was supported by altered mitochondrial dynamics. Complex IV activity was significantly reduced, along with decreased mitochondrial size and total ATP. Heme degradation protein, heme oxygenase-1 (HO-1) was significantly upregulated, suggesting that Cyb5R3 knockout may result in increased free-heme toxicity leading to intracellular damage. To translate these findings to humans, we conducted a retrospective study using a high-frequency T117S variant in African Americans. T117S carriers had significantly accelerated mortality post-first acute cardiac event. A T117S mouse was generated and received trans-aortic constriction (TAC) for 2 weeks. Preliminary results suggest T117S TAC mice have accelerated cardiac remodeling and time to death after pressure overload relative to C57B6/J TAC controls. Together, these data support Cyb5R3 expression may have an important role as a heme-iron reductase in cardiomyocyte function.

Truncations of the titin Z-disc predispose to a heart failure with preserved ejection phenotype in the context of pressure overload.

Titin (TTN) Truncating variants (TTNtv) in the A-band of TTN predispose the mouse heart to systolic dysfunction when subjected to pressure-loading. However, the effects of TTNtv of the Z-disc are largely unexplored. A rat model of pressure-loaded heart is developed by trans-aortic constriction (TAC). Rats with TTNtv of the Z-disc were randomly assigned to TAC (Z-TAC) or sham-surgery (Z-Sham) and wildtype (WT) littermates served as controls (WT-TAC or WT-Sham). Left ventricular (LV) function was assessed by echocardiography. Pressure volume (PV) loops, histology and molecular profiling were performed eight months after surgery. Pressure-load by TAC increased LV mass in all cases when compared with Sham animals. Notably, systolic function was preserved in TAC animals throughout the study period, which was confirmed by terminal PV loops. Diastolic function was impaired in Z-disc TTNtv rats at baseline as compared to WT and became impaired further after TAC (dp/dtmin, mmHg/s): Z-TAC = -3435±763, WT-TAC = -6497±1299 (p<0.01). Z-TAC animals had greater cardiac fibrosis, with elevated collagen content and decreased vascular density as compared to WT-TAC animals associated with enhanced apoptosis of myocyte and non-myocyte populations. In the context of pressure overload, Z-disc TTNtv is associated with cardiac fibrosis, diastolic dysfunction, and capillary rarefaction in the absence of overt systolic dysfunction.

MicroRNA-101a suppresses fibrotic programming in isolated cardiac fibroblasts and in vivo fibrosis following trans-aortic constriction

AimsMiR-101a is reported to reduce post-infarction myocardial fibrosis through targeting c-FOS and TGFbr1. However the actions of miR-101a within the TGF-β signaling pathway are largely unknown. We demonstrate the mechanisms underlying mutual inhibition between miR-101a and TGF-β signaling and explore the therapeutic potential of miR-101a in suppressing pressure overload-induced cardiac fibrosis. Methods and resultsThe effects of miR-101a on fibroblast proliferation, myofibroblast transdifferentiation, collagen synthesis, apoptosis, and autophagy were assessed in isolated rat cardiac fibroblasts (cFB). MiR-101a effects upon cFB TGF-β signaling were assessed by qPCR, Western blotting, 3′UTR luciferase reporter assay and promoter activity assessments. Rats subjected to transverse aortic constriction (TAC) were treated with PBS or 1 mg/kg of a miR-101a mimic i.v. at 1, 3, and 7-day post-surgery. Left ventricular (LV) function (echocardiography; LV catheterization) and LV fibrosis (picro-sirius red staining, qPCR and WB) were assessed at 2-day and 2 & 10-week post-surgery. MiR-101a inhibited cFB proliferation by inducing cell apoptosis rather than through cell cycle inhibition; and reduced collagen synthesis by downregulation of collagen gene expression and increased autophagy. MiR-101a inhibited TGF-β signaling pathways by directly targeting TGFbr1, reducing Smad3 phosphorylation and inhibiting Tab3 promoter activity. Conversely TGF-β inhibited promoter activity for both miR-101a and b. In vivo endogenous miR-101a expression was downregulated 2-day post-TAC returning to baseline by 14-day. MiR-101a mimic treatment inhibited myocardial TGF-β signaling and collagen gene up-regulation at 2-day post-TAC. MiR-101a reduced fibrosis, improved +dP/dt and lowered end diastolic pressure (EDP) at 2-week post-TAC. Treatment also attenuated adverse LV remodeling and preserved cardiac ejection fraction at 10-week post-TAC. ConclusionsMiR-101a and TGF-β are mutually inhibitory and co-direct the activation, proliferation, and collagen synthesis of cFBs. The predominance of TGF-β signaling over reduced miR-101a expression early post-TAC is associated with cardiac fibrosis and dysfunction. Treatment with miR-101a, introduced early after imposition of abnormal pressure loading, inhibits TGF-β signaling, reduces cardiac fibrosis and preserves LV function.

Cardiac contractility modulation improves left ventricular systolic function partially via miR-25 mediated SERCA2A expression in rabbit trans aortic constriction heart failure model.

The purpose of this study was to investigate the underlying mechanism of cardiac contractility modulation (CCM) in improving trans aortic constriction (TAC)-induced heart failure (HF) left ventricular (LV) systolic function. A total of 25 New Zealand white rabbits were randomly divided into 5 groups: sham operation group (SHM), TAC-induced HF group (HF), TAC-induced HF followed by CCM stimulation group (HF + CCM), TAC-induced HF followed by injection of anti-miR-25 group (HF + anti-miR-25), TAC-induced HF followed by CCM stimulation and AAV9-miR-25 injection group (HF + CCM + miR-25). CCM current was performed 6 hours a day for 4 weeks. The left ventricle ejection fraction (LVEF) was measured by ultrasound. Reverse transcription-polymerase chain reaction (RT-PCR) and Western blot were used for measuring RNA and protein levels. The sarcoplasmic reticulum calcium ATPase (SERCA2A) and LVEF were reduced, while the miR-25 expression was improved in HF group compared to SHM group. Conversely, the SERCA2A and LVEF were improved, and the miR-25 reduced in the HF + CCM and the HF + anti-miR-25 groups compared to the HF group. Moreover, the SERCA2A and LVEF were reduced, while the miR-25 was improved in the HF + CCM + miR-25 group compared to the HF + CCM group. CCM is a potentially effective procedure for improving LV systolic function, which might partially by inhibiting miR-25 expression, further improved SERCA2A expression in TAC HF models.

Apelin and APJ orchestrate complex tissue-specific control of cardiomyocyte hypertrophy and contractility in the hypertrophy-heart failure transition.

The G protein-coupled receptor APJ is a promising therapeutic target for heart failure. Constitutive deletion of APJ in the mouse is protective against the hypertrophy-heart failure transition via elimination of ligand-independent, β-arrestin-dependent stretch transduction. However, the cellular origin of this stretch transduction and the details of its interaction with apelin signaling remain unknown. We generated mice with conditional elimination of APJ in the endothelium (APJendo-/-) and myocardium (APJmyo-/-). No baseline difference was observed in left ventricular function in APJendo-/-, APJmyo-/-, or control (APJendo+/+, APJmyo+/+) mice. After exposure to transaortic constriction, APJendo-/- mice displayed decreased left ventricular systolic function and increased wall thickness, whereas APJmyo-/- mice were protected. At the cellular level, carbon fiber stretch of freshly isolated single cardiomyocytes demonstrated decreased contractile responses to stretch in APJ-/- cardiomyocytes compared with APJ+/+ cardiomyocytes. Ca2+ transients did not change with stretch in either APJ-/- or APJ+/+ cardiomyocytes. Application of apelin to APJ+/+ cardiomyocytes resulted in decreased Ca2+ transients. Furthermore, hearts of mice treated with apelin exhibited decreased phosphorylation in cardiac troponin I NH2-terminal residues (Ser22 and Ser23) consistent with increased Ca2+ sensitivity. These data establish that APJ stretch transduction is mediated specifically by myocardial APJ, that APJ is necessary for stretch-induced increases in contractility, and that apelin opposes APJ's stretch-mediated hypertrophy signaling by lowering Ca2+ transients while maintaining contractility through myofilament Ca2+ sensitization. These findings underscore apelin's unique potential as a therapeutic agent that can simultaneously support cardiac function and protect against the hypertrophy-heart failure transition. NEW & NOTEWORTHY These data address fundamental gaps in our understanding of apelin-APJ signaling in heart failure by localizing APJ's ligand-independent stretch sensing to the myocardium, identifying a novel mechanism of apelin-APJ inotropy via myofilament Ca2+ sensitization, and identifying potential mitigating effects of apelin in APJ stretch-induced hypertrophic signaling.

Abstract 251: miRNA Mapping of Cardiac Endothelial and Fibroblast Cells during Hypertrophy Progression

MicroRNAs (MiRNA/miRs) are known key players in cardiovascular disorders. Here we created and analyzed a global miRNA map in isolated cardiac endothelial and fibroblast cells during hypertrophy progression. Hypertrophy was induced in male C57BL/6 mice by trans-aortic constriction (TAC). Hypertrophic phenotyping was performed at 3 days (3d), 2 weeks (2w) or 4 weeks (4w) post- SHAM/TAC operation, using Millar system and echocardiography. Thereafter, pure single cell fractions from hearts were recovered by retrograde collagenase-II perfusion, followed by pre-plating of fibroblasts and magnetic sorting of endothelial cells. Transcriptomic analysis was performed on RNA isolated from different heart cell fractions at 3d, 2w or 4w post-SHAM/TAC. Additional 6w and 13w SHAM/TAC groups were included for validation of selected miRNAs. Cytokine secretome was performed using multiplex assay after transfection of a miR library to primary cardiac fibroblasts. Unsupervised hierarchical clustering revealed specific miRNA profile of each cardiac cell fraction. Principle component analysis projected strong effect of individual cellular compartments. Based on miRnome screening, miR-709, miR-30e-5p, miR-146a, miR-34a and miR-204, miR-1187 were validated by RTPCR to confirm spatial and temporal regulation (n=3-5, p&lt;0.05) of these miRNAs in non-cardiomyocyte fractions. Corresponding time-dependent downregulation of miR-146a targets, Pten and Timp-2, was also observed. Cytokine secretome analysis upon miR-precursor library transfection in cardiac fibroblasts confirmed an increase in FGF, LIF-1, MCP-1, MIP-1 secretion (1.5-5 folds) by miR-146a and miR-34a. Gradual activation of the TGF beta pathway in endothelial cells may initiate endothelial to mesenchymal transition, whereas cytokine secretion from fibroblasts may affect the cellular hemostasis. Our study represents a global miRNome of cardiac endothelial and fibroblast compartments during progressive hypertrophy to better understand the time dependent molecular changes in non-cardiomyocyte compartment during pressure-overload induced cardiac remodeling. The collective influence of miRNA deregulation may be linked to different pathways responsible for cell-proliferation, inflammation and fibrosis with the progression of hypertrophy.

Prevention of PKG-1α Oxidation Suppresses Antihypertrophic/Antifibrotic Effects From PDE5 Inhibition but not sGC Stimulation.

Stimulation of sGC (soluble guanylate cyclase) or inhibition of PDE5 (phosphodiesterase type 5) activates PKG (protein kinase G)-1α to counteract cardiac hypertrophy and failure. PKG1α acts within localized intracellular domains; however, its oxidation at cysteine 42, linking homomonomers, alters this localization, impairing suppression of pathological cardiac stress. Because PDE5 and sGC reside in separate microdomains, we speculated that PKG1α oxidation might also differentially influence the effects from their pharmacological modulation. Knock-in mice expressing a redox-dead PKG1α (PKG1αC42S) or littermate controls (PKG1αWT) were subjected to transaortic constriction to induce pressure overload and treated with a PDE5 inhibitor (sildenafil), sGC activator (BAY602770 [BAY]), or vehicle. In PKG1αWT controls, sildenafil and BAY similarly enhanced PKG activity and reduced pathological hypertrophy/fibrosis and cardiac dysfunction after transaortic constriction. However, sildenafil failed to protect the heart in PKG1αC42S, unlike BAY, which activated PKG and thereby facilitated protective effects. This corresponded with minimal PDE5 activation in PKG1αC42S exposed to transaortic constriction versus higher activity in controls and little colocalization of PDE5 with PKG1αC42S (versus colocalization with PKG1αWT) in stressed myocytes. In the stressed heart and myocytes, PKG1α C42-disulfide formation contributes to PDE5 activation. This augments the pathological role of PDE5 and so in turn enhances the therapeutic impact from its inhibition. PKG1α oxidation does not change the benefits from sGC activation. This finding favors the use of sGC activators regardless of PKG1α oxidation and may help guide precision therapy leveraging the cyclic GMP/PKG pathway to treat heart disease.