Wild-type Transverse Aortic Constriction Research Articles

Role of NOD1 in atrial myopathy associated with heart failure

Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Funding: Spanish Ministry of Economy and Competitiveness and the European Regional Development Fund (ISCIII [PI17/01344 and PI20/01482]), Ministry of Education (FPU19/01973), SEC, Proyecto Traslacional 2019, FEDER, FSE, CIBER-CV, a network funded by ISCIII Introduction Heart failure (HF) is a complex syndrome that constitutes one of the leading causes of cardiovascular death worldwide. Although multifactorial mechanisms appear to be responsible of atrial and ventricular HF-myopathy, the innate immune system and the derived inflammatory response have gained much attention in the last years. Previous studies from our group have demonstrated that the nucleotide-binding oligomerization domain 1 (NOD1), a receptor of the innate immune system, plays a role in HF left ventricular (LV) dysfunction. However, its role in HF-atrial myopathy remains completely unknown. The main purpose of our study is to determine the role of NOD1 in human and experimental HF-atrial myopathy. Methods and Results The study was performed in two cohorts of individuals who underwent heart valve repair surgery, 51 non-failing individuals (NF) with LV ejection fraction (LVEF)>50% and NT-ProBNP levels<200pg/mL and 41 subjects with medium to severe reduced EF (HF)<50% and NT-ProBNP levels>200pg/mL. An informed consent was signed by all patients. Atrial myocardial tissue was excised from the atrial appendage and prepared for biochemical and histological studies. Echocardiography analysis showed that left and right atrial areas (LA and RA) were significantly increased in the HF group compared to NF, while LA and RA reservoir functions were significantly decreased in HF. Immunostaining demonstrated NOD1 location in atrial cardiomyocytes, being the protein levels of NOD1 and its specific adaptor RIP2 significantly increased in atrial tissue obtained from HF vs. NF. Although the prevalence of atrial fibrillation (AF) was significantly higher in the HF group compared to NF, the upregulation of NOD1 was not conditioned by the presence of AF. Similarly, pigs that underwent transverse aortic constriction (TAC) or proximal left circumflex coronary artery occlusion involving the LA branch (LAI), showed an upregulation of NOD1 pathway in atrial tissue, compared with the sham operated animals. Interestingly, an atrial upregulation of NOD1 axis was also observed in wild-type TAC mice, whereas Nod1-/- knockout mice showed a prevention in both structural and functional atrial remodelling induced by TAC, mainly by a prevention of intracellular calcium mishandling in isolated atrial cardiomyocytes. Finally, wild-type mice injected i.p. for 3 days with 3.3 mg/kg with a selective activator of NOD1, showed increased structural atrial remodelling and intracellular calcium mishandling compared to the vehicle-treated group. Conclusions Our results demonstrate that NOD1 axis is upregulated in human, swine and murine atrial failing myocardium. Genetic deletion of NOD1 prevented HF-atrial structural and functional remodelling, mainly by avoiding intracellular calcium mishandling, whereas its pharmacological activation induced atrial dilation and intracellular Ca2+ mishandling. All these data uncover NOD1 as a new player in atrial HF-remodelling.

CXCL10 regulates pressure overload-induced heart failure

Rationale: Heart failure involves tissue inflammation with elevated serum levels of CXCL9 and CXCL10, which are CXCR3 receptor ligands. Both chemokines are expressed by cardiac fibroblasts and myeloid cells in mice subjected to pressure overload induced by transverse aortic constriction (TAC) surgery. Although it is known that CXCR3−/− animals are protected from cardiac remodeling induced by TAC, the role of CXCL10 is unknown. The present study was undertaken to test the hypothesis that CXCL10 is crucial in the cardiac remodeling response to pressure overload induced by TAC in mice. Methods: We subjected wild-type and CXCL10−/− mice to TAC surgery, resulting in 4 experimental groups: WT Sham, WT TAC, CXCL10−/− Sham and CXCL10−/− TAC. WGA was used to analyze the cross-sectional area of cardiomyocytes. To analyze total collagen deposition, PicroSirius Red was used. Immunohistochemistry was performed to measure the number of CD64, CD3g and CD45 positive cells. Echocardiography was used to analyze cardiac function. Results are presented as the mean and standard error of the mean. Comparisons between means were made using two-way variance analysis (ANOVA), as well as Tukey's post hoc test for parametric comparisons, or Kruskal-Wallis and Dunn‘s post hoc test for non-parametric data. Differences with p<0.05 were considered significant. Results: After 1 week, the CXCL10−/− mice showed the same level of cardiac hypertrophy and similar changes in echocardiography parameters, when compared to wild-type mice. However, after 6 weeks, we observed that the CXCL10−/− mice showed an attenuation of heart weight with a reduction in the total collagen deposition compared with wild-type TAC mice. At the cellular level, the genetic deletion of CXCL10 prevented the TAC-induced increase in the cross-sectional area of cardiomyocytes. Moreover, we observed that TAC increased the number of CD3+ T-cells in the heart quantified from stained cross sections of both wild type and CXCL10−/− mice, while no differences were observed for the total number of macrophages. Importantly, echocardiographic changes, including dilation and cardiac dysfunction, induced by pressure overload in wild animals were blunted in CXCL10−/− mice. Corroborating this, the genetic deletion of CXCL10 prevented the increase in lung weight induced by TAC. Conclusion: CXCL10 regulates cardiac hypertrophy and fibrosis induced by pressure overload in mice, as well as the functional changes that characterize heart failure. We are currently unraveling the mechanism through which CXCL10 can accelerate the development of heart failure in response to cardiac pressure overload. Interaction between T cells and macrophages are likely mediating these effects. R01HL155993. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Abstract P3078: The Stimulator Of Interferon Genes (STING) Modulates Adverse Cardiac Remodeling Induced By Cardiac Pressure Overload: New Role Of Endothelial Cell Sting In Systolic Dysfunction

Introduction: Cardiac inflammation is a hallmark of heart failure (HF) defined by T cell infiltration and increased expression of pro-inflammatory cytokines. Type I Interferon (IFN-I), a cytokine induced upon activation of STING, is increased in the heart after myocardial infarction and transverse aortic constriction (TAC). We reported that absence of endothelial cell (EC) STING results in an IFN-I dependent decline in T cell extravasation to inflammatory sites. We hypothesized that STING contributes to adverse cardiac remodeling induced by TAC and EC STING regulates systolic dysfunction through modulating cardiac inflammation. Methods: Male wildtype (WT), STING -/- and inducible EC specific STING -/- mice (Cad5 ERTCre2+/- STING fl/fl treated i.p. with 85mg/kg Tamoxifen), or oil treated and Cre - littermate WT controls were subjected to TAC or Sham surgery. Systolic function by echocardiography and left ventricular (LV) hypertrophy, fibrosis, and capillary density by histology were analyzed four weeks post-surgery and T cell activation was characterized in mediastinal draining lymph nodes (MdLN) by flow cytometry. Results: In contrast to WT, STING -/- mice did not show a decline in ejection fraction (EF). LV hypertrophy was decreased in TAC STING -/- mice compared to TAC WT mice. Isolectin staining showed that capillary density was higher in STING -/- mice in response to TAC as compared to the expected capillary loss observed in WT TAC mice. The frequency of CD4+CD62 lo CD44 hi cells in the MdLN, indicative of effector T cell activation, was decreased in STING -/- mice compared to WT mice in response to TAC. Similar analyses of Cad5 ERTCre2+/- STING fl/fl mice showed that EC STING -/- mice did not show a decline in EF in response to TAC, in contrast to their littermate WT controls. However, no changes were observed in LV hypertrophy, fibrosis or capillary density between groups and activation of MdLN effector T cells was also comparable in both groups. Conclusion: STING contributes to adverse cardiac remodeling induced by TAC. While EC STING contributes to systolic dysfunction through a yet unknown mechanism, STING in other cell compartments contributes to cardiac hypertrophy, capillary density and T cell activation in response to TAC.

Genetic knockout of prestin limits cardiac hypertrophy in a murine pressure overload model.

Up until recently, Prestin (Slc26a5) was considered to be exclusively expressed in the outer hair cells of the inner ear, where it serves as a voltage-to-force convertor and mediates electromotility. Our recent studies demonstrate that prestin is expressed in the heart, where it amplifies cardiac motor function. Due to prestin's ability to modulate cardiac contractility, we hypothesize that it plays a pivotal role in pathological cardiac hypertrophy. We subjected wild-type (WT) and Slc26a5-/- mice to sham operation or chronic pressure overload induced by transverse aortic constriction (TAC) surgery. Cardiac structure and function were assessed by echocardiography. We performed single cell Ca2+ transients and cell shortening recordings on isolated cardiomyocytes. Atomic force microscopy, a high-resolution technique, was also used to quantify the Young's modulus. After 8 weeks of TAC, the expression of prestin was markedly elevated in WT TAC mice relative to WT sham mice. To test whether the increased expression is maladaptive or cardioprotective, Slc26a5-/- mice underwent the same surgeries. Slc26a5-/- sham mice had depressed cardiac function relative to WT sham mice. In contrast and unexpectedly, 4 weeks after TAC, Slc26a5-/- mice exhibited blunted cardiac structural remodeling and more robust cardiac function as compared to WT TAC mice. Moreover, we found that cardiomyocytes from Slc26a5-/- mice showed more normalized Ca2+ transients and cellular shortening relative to cardiomyocytes from WT TAC mice. Our findings demonstrate that knockout of Slc26a5 is cardioprotective in a murine model of pressure overload, highlighting its crucial role in both physiological and pathological conditions. Overexpression of prestin in cardiac hypertrophy may significantly alter the mechanical properties of cardiomyocytes, leading to worsening in hypertrophy-induced cardiac dysfunction. Our study suggests that targeting prestin may represent a viable therapeutic avenue for cardiac hypertrophy.

Abstract 12273: Crispr-Mutation of the Na + -k + Pump β1-Subunit Protects Against Cardiac Fibrosis and Heart Failure in Mice

Introduction: The Na+-K+ pump controls translocation of Na+ and K+ across the cell membrane which indirectly affects heart muscle contraction. Glutathionylation of the Na+-K+ pump’s β1 subunit (NKβ1) is a reversible post-translational oxidative modification, where a glutathione adduct forms a disulphide bond with the cysteine residue at site 45, leading to a reduction in pump activity. Using CRISPR/Cas9 technology, we created a transgenic mouse line (CRISPR-β1) that has a mutation in cysteine to serine at site 45 on NKβ1, causing insensitivity to β1-GSS. Hypothesis: We hypothesize that a mutation of the β1-GSS can inhibit transverse aortic constriction (TAC)-induced cardiac fibrosis. Methods and Results: TAC was induced in both the wild-type (WT) and CRISPR-NKβ1 mutated mice (CRISPR-β1) over a period of four weeks. Gene sequencing showed that cysteine 45 was replaced by serine, and immunoprecipitation of the free cysteine demonstrated that cysteine 45 was mutated in the β1 subunit of the CRISPR-β1 mice hearts. TAC significantly reduced cardiac function in the WT but not the CRISPR-β1 mice (ejection fraction: WT TAC: 36.24% ± 3.49%; CRISPR-β1 TAC: 57.5% ± 3.41%). Similarly, we found that the CRISPR-β1 mice were protected from cardiac hypertrophy, as evidenced by a significant reduction in cardiomyocytes size (WT TAC: 452.1 μm2 ± 30.74 μm2 ; CRISPR-β1 TAC: 271.1 μm 2 ± 11.0 μm 2 ). TAC significantly increased interstitial and perivascular fibrosis in the left ventricles of WT but not the CRISPR-β1 mice (interstitial: WT TAC: 18.4% ± 1.96%; CRISPR-β1 TAC: 5.2% ± 1.62%, perivascular: WT TAC: 42.6% ± 4.5%; CRISPR-β1 TAC: 22.01% ± 1.6%). Cardioprotective effects observed in the CRISPR-β1 mice were at least in part attributed to a significant reduction in the expression of ANP, BNP, Tgfb1, Fn1 and Ctgf genes. Conclusions: Our data indicate that CRISPR mutation of the Na+-K+ pump’s β1 subunit prevents the development of cardiac fibrosis and heart failure in a TAC-induced mouse model.

Ventricular TLR4 Levels Abrogate TLR2-Mediated Adverse Cardiac Remodeling upon Pressure Overload in Mice

Involvement of the Toll-like receptor 4 (TLR4) in maladaptive cardiac remodeling and heart failure (HF) upon pressure overload has been studied extensively, but less is known about the role of TLR2. Interplay and redundancy of TLR4 with TLR2 have been reported in other organs but were not investigated during cardiac dysfunction. We explored whether TLR2 deficiency leads to less adverse cardiac remodeling upon chronic pressure overload and whether TLR2 and TLR4 additively contribute to this. We subjected 35 male C57BL/6J mice (wildtype (WT) or TLR2 knockout (KO)) to sham or transverse aortic constriction (TAC) surgery. After 12 weeks, echocardiography and electrocardiography were performed, and hearts were extracted for molecular and histological analysis. TLR2 deficiency (n = 14) was confirmed in all KO mice by PCR and resulted in less hypertrophy (heart weight to tibia length ratio (HW/TL), smaller cross-sectional cardiomyocyte area and decreased brain natriuretic peptide (BNP) mRNA expression, p < 0.05), increased contractility (QRS and QTc, p < 0.05), and less inflammation (e.g., interleukins 6 and 1β, p < 0.05) after TAC compared to WT animals (n = 11). Even though TLR2 KO TAC animals presented with lower levels of ventricular TLR4 mRNA than WT TAC animals (13.2 ± 0.8 vs. 16.6 ± 0.7 mg/mm, p < 0.01), TLR4 mRNA expression was increased in animals with the largest ventricular mass, highest hypertrophy, and lowest ejection fraction, leading to two distinct groups of TLR2 KO TAC animals with variations in cardiac remodeling. This variation, however, was not seen in WT TAC animals even though heart weight/tibia length correlated with expression of TLR4 in these animals (r = 0.078, p = 0.005). Our data suggest that TLR2 deficiency ameliorates adverse cardiac remodeling and that ventricular TLR2 and TLR4 additively contribute to adverse cardiac remodeling during chronic pressure overload. Therefore, both TLRs may be therapeutic targets to prevent or interfere in the underlying molecular processes.

Signal transducer and transcriptional activation 1 protects against pressure overload-induced cardiac hypertrophy.

Signal transducers and transcriptional activation 1 (Stat1) is a member of the STATs family, and its role in various biological responses, including cell proliferation, differentiation, migration, apoptosis, and immune regulation has been extensively studied. We aimed to investigate its role in pathological cardiac hypertrophy, which is currently poorly understood. Experiments using H9C2 cardiomyocytes, Stat1, and IfngR cardiomyocyte-specific knockout mice revealed that Stat1 had a protective effect on cardiac hypertrophy. Using transverse aortic constriction (TAC)-induced cardiac hypertrophy in mice, we analyzed the degree of hypertrophy using echocardiography, pathology, and at the molecular level. Mice lacking Stat1 had more pronounced cardiac hypertrophy and fibrosis than wild-type TAC mice. Analysis of the molecular mechanisms suggested that Stat1 downregulated the mRNA levels of hypertrophy and fibrosis markers to inhibit cardiac hypertrophy, and promotes mitochondrial fission through the Ucp2/P-Drp1 pathway, enhancing mitochondrial function, and increasing compensatory myocardial ATP production in the compensatory phase for cardiac hypertrophy inhibition. Overall, this comprehensive analysis revealed that Stat1 inhibits cardiac hypertrophy by downregulating hypertrophic and fibrotic marker genes and enhancing the mitochondrial function to enhance cardiomyocyte function through the Ucp2/P-Drp1 signaling pathway.

Dec1 deficiency protects the heart from fibrosis, inflammation, and myocardial cell apoptosis in a mouse model of cardiac hypertrophy

Cardiac inflammation and fibrosis triggered by left ventricular pressure overload are the major causes of heart dysfunction. Differentiated embryonic chondrocyte gene 1 (Dec1) is a basic helix-loop-helix transcription factor that is comprehensively involved in inflammation and tissue fibrosis, but its role in cardiac hypertrophy remains unclear. This study explored the effects of Dec1 on cardiac fibrosis, inflammation, and apoptosis in hypertrophic conditions. Transverse aortic constriction (TAC) was performed to induce cardiac hypertrophy in wild-type (WT) mice and in Dec1 knock out (KO) mice for 4 weeks. Using the TAC mouse model, prominent differences in cardiac hypertrophy at the morphological, functional, and molecular levels were delineated by Masson’s Trichrome and TUNEL staining, immunohistochemistry, RT-PCR and Western Blot. DNA microarray and microRNA (miRNA) array analyses were carried out to identify gene and miRNA expression patterns. Dec1KO mice exhibited a more severe hypertrophic heart, whereas WT mice showed a more pronounced perivascular fibrosis after TAC at 4 weeks. The Dec1 deficiency promoted M2 phenotype macrophages. Dec1KO TAC mice showed fewer apoptotic cells than WT TAC mice. APEX1, WNT16, FGF10 and MMP-10 were differentially expressed according to DNA microarray analysis and expression levels of those genes and the corresponding miRNAs (miR-295, miR-200 b, miR-130a, miR-92a) showed the same trends. Furthermore, luciferase reporter assay confirmed that FGF10 is the direct target gene of miR-130. In conclusion, a Dec1 deficiency protects the heart from perivascular fibrosis, regulates M1/M2 macrophage polarization and reduces cell apoptosis, which may provide a novel insight for the treatment of cardiac hypertrophy.

Alpha-lipoic acid protects against pressure overload-induced heart failure via ALDH2-dependent Nrf1-FUNDC1 signaling

Alpha-lipoic acid (α-LA), a well-known antioxidant, was proved to active ALDH2 in nitrate tolerance and diabetic animal model. However, the therapeutic advantage of α-LA for heart failure and related signaling pathway have not been explored. This study was designed to examine the role of α-LA–ALDH2 in heart failure injury and mitochondrial damage. ALDH2 knockout (ALDH2−/−) mice and primary neonatal rat cardiomyocytes (NRCMs) were subjected to assessment of myocardial function and mitochondrial autophagy. Our data demonstrated α-LA significantly reduced the degree of TAC-induced LV hypertrophy and dysfunction in wild-type mice, not in ALDH2−/− mice. In molecular level, α-LA significantly restored ALDH2 activity and expression as well as increased the expression of a novel mitophagy receptor protein FUNDC1 in wild-type TAC mice. Besides, we confirmed that ALDH2 which was activated by α-LA governed the activation of Nrf1–FUNDC1 cascade. Our data suggest that α-LA played a positive role in protecting the heart against adverse effects of chronic pressure overload.

P4997Raf kinase inhibitor protein of the bone marrow contributes to cardiac fibrogenesis in pressure-overloaded myocardium

Abstract Background Raf Kinase Inhibitor Protein (RKIP) regulates myocardial remodeling under conditions of enhanced myocardial oxidative stress in pressure-overloaded left ventricle (LV) modulating myocardial production of reactive oxygen species (ROS). A second mode of action may be the mobilization of circulating fibroblasts (fibrocytes) from the bone-marrow (BM). However the underlying mechanisms are incompletely understood. Methods and results To further characterize the role of RKIP in BM cells for myocardial remodeling 10-week-old wild-type (WT) C57BL/6N mice were subjected to transplantation of bone marrow (BMT) from 10-week-old C57BL/6-RKIP-deficient (RKIP−/−) N or WT C57BL/6N mice expressing green fluorescent protein (GFP)+ ubiquitously. 28 days later, transverse aortic constriction (TAC, 360 μm) or SHAM-operation was performed. 5 weeks post TAC, LV systolic pressure (LVSP) and heart weight to tibia length ratio were significantly increased in both types of BMT, compared with corresponding SHAM. Increased afterload elicited myocardial fibrosis as assessed by picrosirius red staining (WT/WT SHAM 15±2.5%, WT/WT TAC 21.3±1.4%, p&lt;0.05; RKIP−/−/WT SHAM 17±2%, RKIP−/−/WT TAC 18±3%, p=ns) and significantly increased the number of LV fibroblasts per mm2 estimated by immunostaining for intracellular fibronectin, which were further reduced by transplantation of RKIP−/−N BM (WT/WT SHAM 5499±313, WT/WT TAC 7493±741 per mm2, p&lt;0.05; RKIP−/−/WT SHAM 5737±259, RKIP−/−/WT TAC 5282±551, per mm2, p=ns). Moreover, transplantation of RKIP−/−N BM significantly diminished the number of circulating BM-derived GFP+ fibroblasts in the peripheral blood and LV myocardium during pressure overload (WT/WT SHAM 961±129, WT/WT TAC 2326±273 per mm2, p&lt;0.05; RKIP−/−/WT SHAM 1041±209, RKIP−/−/WT TAC 1518±107, per mm2, p=ns). The myocardial redox status was assessed by the co-immunostaining for ROS production marker 8-hydroxyguanosin (8-dOHG), cardiomyocyte marker α-sarcomeric actin and fibroblast marker intracellular fibronectin. Pressure overload during 5 weeks significantly increased the percentages of 8-dOHG+cardiomyocytes (WT/WT SHAM 34±9%, WT/WT TAC 63±6%, p&lt;0.05; RKIP−/−/WT SHAM 29±6%, RKIP−/−/WT TAC 31±8%, p=ns) and 8-dOHG+fibroblasts (WT/WT SHAM 57±6%, WT/WT TAC 73±4%, p&lt;0.05; RKIP−/−/WT SHAM 58±2%, RKIP−/−/WT TAC 58±7%, p=ns) in mice transplanted with WT BM but not with RKIP−/−N BM. Conclusions In pressure-overload induced enhanced myocardial ROS production, deficiency of RKIP-expression in the bone marrow abrogates left ventricular fibrosis by reduction of myocardial ROS production and mobilization of BM-derived fibroblasts. These findings suggest that the function of RKIP in the bone marrow may be important for maladaptive myocardial remodelling. Acknowledgement/Funding Deutsche Forschungsgemeinschaft: KA4024/3-1, SFB TRR219; Saarland University HOMFOR, Dr. Marija Orlovich foundation, Corona foundation s199/10060/2014

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

Abstract 575: Loss of Function Variant in CYB5R3 Associates With Exacerbated Cardiac Hypertrophy in Mice

African Americans (AA) are 20 times more likely to be diagnosed with heart failure (HF) before the age of 50 and 2 times more likely to die from heart failure. Previous reports have shown AA with HF have diminished nitric oxide (NO) signaling, a pathway critical for cardiac contractility. NO signals, in part, via binding reduced heme iron (Fe 2+ ) in soluble guanylyl cyclase (sGC) leading to cyclic guanosine monophosphate (cGMP) generation. Recently, our lab reported that cytochrome b5 reductase 3 (Cyb5R3) reduces oxidized sGC heme-iron from the oxidized (Fe 3+ ) to the reduced (Fe 2+ ) state, thereby sensitizing sGC to NO. However, the role of Cyb5R3 in the setting of HF remains elusive. It is known that a high frequency Cyb5R3 T117S single nucleotide polymorphism (23% minor allele frequency) exists and is enriched in individuals with African ancestry. To determine the impact of T117S in HF outcomes, we completed a retrospective study from AHEFT and GRACE trials. Our data show that Cyb5R3 T117S carriers have significantly accelerated time to death/transplant. Additionally, biobank HF samples from AA samples show an enrichment of Cyb5R3 T117S carriers from 0.23 to 0.4. To assess the impact of Cyb5R3 T117S on sGC/cGMP signaling in the heart, ventricular cGMP levels in AA with HF were examined. Pooled Cyb5R3 T117S carriers have significantly decreased cGMP relative to non-carriers. Next, we determined if this variant impacts sGC heme redox state. Using purified protein activity assays, we found that Cyb5R3 T117S results in a 60% loss-of-function and an inability to reduce oxidized sGC. Lastly, to test the in vivo impact of the Cyb5R3 T117S variant in heart failure, we generated a novel Cyb5R3 T117S mouse. Transverse aortic constriction (TAC) studies in Cyb5R3 T117S mice show significantly accelerates cardiac hypertrophy relative to wild-type TAC controls. Taken together, these data suggest Cyb5R3 T117S may be a disease modifying variant that augments hypertrophic signaling through an sGC-dependent mechanism.

Hepatocyte-Specific SR-BI Gene Transfer Corrects Cardiac Dysfunction in Scarb1-Deficient Mice and Improves Pressure Overload-Induced Cardiomyopathy.

Objective- We investigated the hypothesis that HDL (high-density lipoprotein) dysfunction in Scarb1-/- mice negatively affects cardiac function both in the absence and in the presence of pressure overload. Second, we evaluated whether normalization of HDL metabolism in Scarb1-/- mice by hepatocyte-specific SR-BI (scavenger receptor class B, type I) expression after E1E3E4-deleted adenoviral AdSR-BI (E1E3E4-deleted adenoviral vector expressing SR-BI protein in hepatocytes) transfer abrogates the effects of total body SR-BI deficiency on cardiac structure and function. Approach and Results- Transverse aortic constriction (TAC) or sham operation was performed at the age of 14 weeks, 2 weeks after saline injection or after gene transfer with AdSR-BI or with the control vector Adnull. Mortality rate in Scarb1-/- TAC mice was significantly increased compared with wild-type TAC mice during 8 weeks of follow-up (hazard ratio, 2.02; 95% CI, 1.14-3.61). Hepatocyte-specific SR-BI gene transfer performed 2 weeks before induction of pressure overload by TAC potently reduced mortality in Scarb1-/- mice (hazard ratio, 0.329; 95% CI, 0.180-0.600). Hepatocyte-specific SR-BI expression abrogated increased cardiac hypertrophy and lung congestion and counteracted increased myocardial apoptosis and interstitial and perivascular fibrosis in Scarb1-/- TAC mice. Scarb1-/- sham mice were, notwithstanding the absence of detectable structural heart disease, characterized by systolic and diastolic dysfunction and hypotension, which were completely counteracted by AdSR-BI transfer. Furthermore, AdSR-BI transfer abrogated increased end-diastolic pressure and diastolic dysfunction in Scarb1-/- TAC mice. Increased oxidative stress and reduced antioxidant defense systems in Scarb1-/- mice were rescued by AdSR-BI transfer. Conclusions- The detrimental effects of SR-BI deficiency on cardiac structure and function are nullified by hepatocyte-specific SR-BI transfer, which restores HDL metabolism.

Deficiency of aldose reductase exacerbates early pressure overload-induced cardiac dysfunction and autophagy in mice

Pathological cardiac hypertrophy is associated with the accumulation of lipid peroxidation-derived aldehydes such as 4-hydroxy-trans-2-nonenal (HNE) and acrolein in the heart. These aldehydes are metabolized via several pathways, of which aldose reductase (AR) represents a broad-specificity route for their elimination. We tested the hypothesis that by preventing aldehyde removal, AR deficiency accentuates the pathological effects of transverse aortic constriction (TAC). We found that the levels of AR in the heart were increased in mice subjected to TAC for 2 weeks. In comparison with wild-type (WT), AR-null mice showed lower ejection fraction, which was exacerbated 2 weeks after TAC. Levels of atrial natriuretic peptide and myosin heavy chain were higher in AR-null than in WT TAC hearts. Deficiency of AR decreased urinary levels of the acrolein metabolite, 3-hydroxypropylmercapturic acid. Deletion of AR did not affect the levels of the other aldehyde-metabolizing enzyme - aldehyde dehydrogenase 2 in the heart, or its urinary product - (N-Acetyl-S-(2-carboxyethyl)-l-cystiene). AR-null hearts subjected to TAC showed increased accumulation of HNE- and acrolein-modified proteins, as well as increased AMPK phosphorylation and autophagy. Superfusion with HNE led to a greater increase in p62, LC3II formation, and GFP-LC3-II punctae formation in AR-null than WT cardiac myocytes. Pharmacological inactivation of JNK decreased HNE-induced autophagy in AR-null cardiac myocytes. Collectively, these results suggest that during hypertrophy the accumulation of lipid peroxidation derived aldehydes promotes pathological remodeling via excessive autophagy, and that metabolic detoxification of these aldehydes by AR may be essential for maintaining cardiac function during early stages of pressure overload.

Glucose is preferentially utilized for biomass synthesis in pressure-overloaded hearts: evidence from fatty acid-binding protein-4 and -5 knockout mice.

AimsThe metabolism of the failing heart is characterized by an increase in glucose uptake with reduced fatty acid (FA) oxidation. We previously found that the genetic deletion of FA-binding protein-4 and -5 [double knockout (DKO)] induces an increased myocardial reliance on glucose with decreased FA uptake in mice. However, whether this fuel switch confers functional benefit during the hypertrophic response remains open to debate. To address this question, we investigated the contractile function and metabolic profile of DKO hearts subjected to pressure overload.Methods and resultsTransverse aortic constriction (TAC) significantly reduced cardiac contraction in DKO mice (DKO-TAC), although an increase in cardiac mass and interstitial fibrosis was comparable with wild-type TAC (WT-TAC). DKO-TAC hearts exhibited enhanced glucose uptake by 8-fold compared with WT-TAC. Metabolic profiling and isotopomer analysis revealed that the pool size in the TCA cycle and the level of phosphocreatine were significantly reduced in DKO-TAC hearts, despite a marked increase in glycolytic flux. The ingestion of a diet enriched in medium-chain FAs restored cardiac contractile dysfunction in DKO-TAC hearts. The de novo synthesis of amino acids as well as FA from glycolytic flux was unlikely to be suppressed, despite a reduction in each precursor. The pentose phosphate pathway was also facilitated, which led to the increased production of a coenzyme for lipogenesis and a precursor for nucleotide synthesis. These findings suggest that reduced FA utilization is not sufficiently compensated by a robust increase in glucose uptake when the energy demand is elevated. Glucose utilization for sustained biomass synthesis further enhances diminishment of the pool size in the TCA cycle.ConclusionsOur data suggest that glucose is preferentially utilized for biomass synthesis rather than ATP production during pressure-overload-induced cardiac hypertrophy and that the efficient supplementation of energy substrates may restore cardiac dysfunction caused by energy insufficiency.

Abstract 423: The Valosin-containing Protein Attenuates Chronic Pressure Overload-induced Cardiac Remodeling and Heart Failure via Activating mTORC2 Pathway

Aims: The pressure overload induced cardiac hypertrophy is a key risk factor for heart failure. However, the mechanism involved remains largely unknown. We previously showed that overexpression of valosin-containing protein (VCP) promotes cardiomyocytes survival in vitro . Here we tested the hypothesis that VCP protects heart against pressure overload-induced heart failure via promoting pro-survival signaling in cardiomyocyte in vivo . Methods and Results: VCP expression were found to be notably decreased in left ventricle (LV) tissues of dilated cardiomyopathy patients compared to healthy donor at both the mRNA and protein levels. A transgenic mouse (TG) with a cardiac specific overexpression of VCP was generated and compared to its litter-matched wild-type (WT). Pressure overload was induced by transverse aortic constriction (TAC) in mice for 5 weeks and sham control was included. Cardiac structure and function were measured by echocardiography and hemodynamic analysis. After 5 week TAC, compared to sham control, WT mice developed a dilated cardiac hypertrophy and cardiac dysfunction, reflected by a significant increase in LV chamber diameter, wall thickness and the ratio of LV weight/ tibia length, and a decrease in ejection fraction (71% vs 51%) as well as an increase in ratio of lung weight /tibia length, a marker of heart failure (all, P&lt;0.01 vs WT sham). However, these pressure overload induced cardiac remodeling and dysfunction were significant suppressed in VCP TG TAC mice (P&lt;0.05 vs WT TAC). Mechanistically, compared to sham control, VCP expression in LV tissues was found to be significant decreased in WT TAC mice, but not in VCP TG mice. VCP TG TAC mice showed a significant decrease in apoptosis of cardiomyocytes vs WT TAC mice (p&lt;0.05). In addition, overexpression of VCP increased the phosphorylation of AKt at 473 and the expression of protein kinase C α in VCP TG mouse heart in vivo and in isolated cardiomyocytes in vitro which was abolished by the deletion of Rictor, indicating an activation of mammalian target of rapamycin complex 2 (mTORC2) in cardiomyocytes. Conclusion: Overexpression of VCP prevents TAC-induced cardiac remodeling and heart failure via a pro-survival mechanism involving mTORC2 signaling pathway.

Cardiomyocyte specific overexpression of a 37 amino acid domain of regulator of G protein signalling 2 inhibits cardiac hypertrophy and improves function in response to pressure overload in mice

Regulator of G protein signalling 2 (RGS2) is known to play a protective role in maladaptive cardiac hypertrophy and heart failure via its ability to inhibit Gq- and Gs- mediated GPCR signalling. We previously demonstrated that RGS2 can also inhibit protein translation and can thereby attenuate cell growth. This G protein-independent inhibitory effect has been mapped to a 37 amino acid domain (RGS2eb) within RGS2 that binds to eukaryotic initiation factor 2B (eIF2B). When expressed in neonatal rat cardiomyocytes, RGS2eb attenuates both protein synthesis and hypertrophy induced by Gq- and Gs- activating agents. In the current study, we investigated the potential cardioprotective role of RGS2eb by determining whether RGS2eb transgenic (RGS2eb TG) mice with cardiomyocyte specific overexpression of RGS2eb show resistance to the development of hypertrophy in comparison to wild-type (WT) controls. Using transverse aortic constriction (TAC) in a pressure-overload hypertrophy model, we demonstrated that cardiac hypertrophy was inhibited in RGS2eb TG mice compared to WT controls following four weeks of TAC. Expression of the hypertrophic markers atrial natriuretic peptide (ANP) and β-myosin heavy chain (MHC-β) was also reduced in RGS2eb TG compared to WT TAC animals. Furthermore, cardiac function in RGS2eb TG TAC mice was significantly improved compared to WT TAC mice. Notably, cardiomyocyte cell size was significantly decreased in TG compared to WT TAC mice. These results suggest that RGS2 may limit pathological cardiac hypertrophy at least in part via the function of its eIF2B-binding domain.

Emerging Role of the Mitochondrial Outer Membrane Translocator Protein (TSPO) in Heart Failure and Mitochondrai Quality Control

Introduction: Mitochondrial dysfunction is a hallmark of heart failure (HF), resulting in decreased energy production and impaired mitochondrial quality control (mitophagy) with resultant contractile dysfunction and cell death. The 18-kDa mitochondrial translocator protein (TSPO) has been considered a part of the mitochondrial permeability transition pore (mPTP) for a long time; however, its role in mPTP regulation is now debatable and the precise role of the TSPO in cardiac physiology and HF remains poorly understood.Objective: Determine the role of TSPO in a murine pressure-overload model of HF.Methods and Results: Conditional, cardiac-specific TSPO knockout (KO) mice were generated using the Cre-loxP system. Pressure overload by transverse aortic constriction (TAC) for 8 weeks significantly increased TSPO expression in wild type (WT) mice, but not in KO mice. While WT TAC mice showed a marked reduction in systolic function, KO TAC mice did not have a significant reduction in ejection fraction, and also exhibited fewer clinical HF signs (less cardiac dilation and fibrosis). TAC reduced mitochondrial calcium uptake and enhanced mPTP opening in permeabilized myocytes from WT animals. Calcium uptake was restored in TAC KO mice without significant effect on mPTP activity. The restoration of the mitochondrial calcium uptake coincided with reduced oxidative stress, improved oxygen consumption rate, and enhanced ATP generation and mitochondrial reserve capacity. In WT TAC, elevated mitochondrial accumulation of mCherry-Parkin1 was not coupled to the increase in LC3-autophagosomes formation indicating impaired mitophagy. In contrast, mitophagy was normalized in TSPO KO.Conclusions: These data suggest a novel mechanism to prevent HF at the cellular level via normalization of TSPO levels which leads to preservation of mitochondrial calcium uptake, oxidative phosphorylation, and mitochondrial mt-QC resulting in preservation of mitochondrial integrity and cell survival.

Abstract 16535: Conditional Knockout of the Mitochondrial 18-kDa Translocator Protein Limits Heart Failure

Introduction: Heart failure results in myocyte and mitochondrial death, and is characterized by abnormalities in mitochondrial calcium handling, energy production, and opening of the mitochondrial permeability transition pore (mPTP). The 18-kDa mitochondrial translocator protein (TSPO) has been shown to be significantly upregulated by heart failure in animals and in explanted hearts from patients, suggesting a vital role for this protein. Hypothesis: In the current experiments, we tested the hypothesis that conditional knockout of the TSPO using Cre inducible TSPO-floxed C57BL/6J mice would limit heart failure resulting from transverse aortic constriction (TAC). Methods: Mice in 4 groups_wild-type (WT) sham, WT TAC, KO sham, and KO TAC_were monitored by weekly echocardiography for 8 weeks, followed by downstream experiments. Results: TAC caused a 46±13% reduction in ejection fraction in WT mice, which was significantly lower in the KO mice (14±10%, P &lt; 0.01, Figure). Strain analysis revealed a significant improvement in radial, longitudinal, and circumferential strain in KO TAC mice, supported by tissue histology finding of significantly less collagen in KO TAC mice. KO TAC mice showed lower heart to body weight and lung to body weight ratios, with no KO TAC mice showing signs of pulmonary edema. Calcium uptake experiment using Rhod-2 AM in isolated cells revealed that KO TAC mice had higher mitochondrial calcium uptake, a crucial finding since mitochondrial calcium uptake has been shown to play a role in energetics and mPTP opening. Conclusion: Genetic modulation of the TSPO limits heart failure due to pressure overload, likely mediated by preserving mitochondrial calcium uptake and energy production, and possibly limiting mPTP opening. These data suggest that pharmacologic interventions that inhibit TSPO expression or function can limit heart failure at the sub-cellular level.

Abstract 338: Cardiac Overexpression of Creatine Kinase Improves Cardiomycytes Function in Heart Failure and During Increased Redox Stress

Aims: Several studies suggest that abnormal energy metabolism contributes to heart failure or that the failing heart is energy starved. Here we aim at testing whether an increase in intracellular CK improves myocellular contractility in experimental myocardial dysfunction and protects from increased oxidative conditions. Methods-Results: We tested the response to the β-agonist isoproterenol (2.5 nM, ISO) in field-stimulated (.5 Hz, RT) adult cardiomyocytes isolated from wild-type (WT) mice and mice overexpressing cardiac myofibrillar or mitochondrial CK (CK-M or CK-mito) from sham and failing (8 wk transverse aortic constriction (TAC)) hearts, to dissect whether overexpressing CK alters myocyte function at baseline and during increased energetic demand. There were no differences in sarcomere fractional shortening (FS) or Ca2+ transients at baseline and with ISO among sham WT, CK-M or CK-mito myocytes. However, ISO effects were significantly reduced in WT TAC myocytes, consistent with prior reports. Conversely, in CK-M or CK-mito TAC myocytes, ISO-induced inotropy was fully preserved. Interestingly, incubation with the AMPK-stimulator AICAR (1mM for at least 90’) did not have any effect on WT TAC, but increased FS in TAC CK-M (+82%) and CK-mito (+42%) myocytes significantly, supporting the important metabolic role played by enhancing CK in failing hearts. To test whether overexpressing CK-M or CK-mito confer protection against acute oxidative stress, sham myocytes were exposed to H2O2 (50μM, 10’) and the interval (seconds) between the beginning of H2O2 superfusion and the appearance of irreversible arrhythmias was measured. WT and CK-M myocytes had a similar response (416±91s vs 411±68s), whereas in CK-mito this interval was significantly prolonged (600±64s). Similarly, upon acute infusion of the anticancer TKI sunitinib (2μM), whose cardiotoxic properties have been linked also to an increase in ROS, irreversible arrhythmias appeared after 657±43s in CK-mito (p&lt;.5 vs 561±66 for WT and 467±88 for CK-M). Conclusions: Overexpressing CK-M and CK-mito under failing-TAC conditions improves myocyte function likely through better preserved Ca2+ handling, whereas only the up-regulation of CK-mito is more effective in buffering ROS effects.