Abstract MP224: IL-10 Protects The Heart From Late Phage Cardiac Inflammation Associated With Intestinal Permeability Following Transaortic Constriction

Introduction: Impaired intestinal permeability is known to augment cardiac dysfunction. Though the cardioprotective role of IL-10 is known in early phase cardiac inflammation, following transaortic constriction (TAC), its role in alleviating late phase cardiac inflammation associated with intestinal permeability is not known. Hypothesis: We hypothesized that IL-10 reduces late-phase cardiac inflammation by minimizing intestinal permeability and systemic reduction of pathogen-associated molecular pattern (PAMP), which ultimately improve cardiac function. Methods: WT and IL-10 KO mice (n=6 in each group) were subjected to TAC or sham surgery, and echocardiography was performed to evaluate cardiac function for up to 56 days. Heart and intestine were collected to evaluate inflammation (RT-qPCR), immune cell infiltration (flow cytometry), and intestinal permeability (WB and immunohistochemistry). Blood was collected to quantify the level of peptidoglycan (PGN, a PAMP) and other bacterial metabolites. The effect of PGN on mitochondrial function was evaluated in neonatal rat ventricular myocyte (NRVM). Results: Cardiac functions were significantly reduced in IL10-KO mice, following TAC till 56 days. Furthermore, exacerbated inflammation was observed both at early phase (within 14 days) and late phase (around 56 days) in IL-10 KO TAC mice as compare to the WT counterpart. Interestingly, early cardiac inflammation coincided with immune cell infiltration into the intestine of IL-10 KO mice following TAC. Enhanced inflammatory immune cells homing to the intestine following TAC led to gut dysbiosis (16S RNA sequencing) which ultimately caused impaired gut wall permeability in the IL-10 KO mice as compared to the WT mice. Further, plasma PGN level was significantly increased in IL-10 KO mice following TAC. Finally, our cardiotoxicity array in neonatal rat cardiomyocytes following PGN treatment showed altered expression of proteins which are involved in the regulation of mitochondrial function and cardiac contractility. Conclusion: IL-10 plays an important role in limiting late phase cardiac inflammation by reducing intestinal permeability and systemic increase in PGN which induce inflammation and mitochondrial dysfunction in cardiac muscle.

(-)-Epicatechin Inhibits Cardiac Fibrosis via SIRT1/AKT/GSK3β Pathway

Background: (-)-Epicatechin (EPI) is an important substance involved in protective effects of flavanol-rich foods. Many studies indicate EPI has cardioprotective effect, but the effect of EPI in inhibition of cardiac fibrosis is unclear. Thus, we aimed to evaluate the effect of EPI in preventing cardiac fibrosis and unveil the molecular mechanisms. Methods: Cardiac fibrosis model was established by transaortic constriction (TAC). The acutely isolated cardiac fibroblasts were induced to myofibroblasts with angiotensin II (AngII). Results: EPI markedly attenuated TAC-induced cardiac dysfunction and fibrosis in mice. In cultured CFs, EPI blocked AngII-induced myofibroblast transformation and collagen production. Furthermore, EPI conducted anti-fibrotic effects by activating the the SIRT1/AKT/GSK3β pathway. Conclusions: These findings will supply new agent and mechanism of action for treating cardiac fibrosis in the future.

Pressure Overload–Mediated Sustained PKR2 (Prokineticin-2 Receptor) Signaling in Cardiomyocytes Contributes to Cardiac Hypertrophy and Endotheliopathies

Chronic cardiac pressure overload, caused by conditions, such as hypertension, induces pathological hypertrophic growth of myocardium and vascular rarefaction, with largely unknown mechanisms. Here, we described that expression of the PKR2 (prokineticin-2 receptor) is increased in the cardiomyocytes of mice following transaortic constriction pressure overload–mediated pathological hypertrophy. To identify PKR2-induced pathways, we performed microarray analysis on TG-PKR2 (transgenic mice overexpressing cardiomyocyte-restricted human PKR2) hearts and cytokine analyses in hPKR2 overexpressing H9c2-lines (PKR2-cardiomyocytes). An enrichment of activin pathway gene sets was found in both TG-PKR2 and transaortic constriction-operated hearts. Elevated levels of 2 cytokines activin A and its coreceptor, sENG (soluble Endoglin), were found in both PKR2-cardiomyocytes and in PKR2-cardiomyocytes conditioned medium. ELISA analyses of the cardiomyocytes derived from both TG-PKR2 and transaortic constriction hearts revealed high levels of these cardiokines that were repressed with antibodies blocking PKR2, indicating a PKR2-dependent event. The conditioned medium of PKR2-cardiomyocytes induced fenestration of endothelial cells and inhibited tube-like formations. These endotheliopathies were blocked by either depleting activin A or sENG from conditioned medium or by using 2 pharmacological inhibitors, follistatin, and TRC105. In addition, similar endotheliopathies were produced by exogenous administration of activin A and ENG. Prolonged exposure to prokineticin-2 in PKR2-cardiomyocytes increased cell volume by the PKR2/Gα 12/13 /ERK5-pathway. Activation of the PKR2/Gα 12/13 /matrix metalloprotease-pathway promoted both activin A and sENG release. This study reveals that pressure overload–mediated PKR2 signaling in cardiomyocytes contributes to cardiac hypertrophy through autocrine signaling, and vascular rarefaction via cardiac cytokine-mediated cardiomyocyte–endothelial cell communications. Our results may contribute to the development of potential therapeutic targets for heart failure.

Exchange protein directly activated by cAMP (Epac) mediates cardiac repolarization and arrhythmogenesis during chronic heart failure

Most sudden cardiac death in chronic heart failure (CHF) is caused by malignant ventricular arrhythmia (VA). However, the molecular mechanism remains unclear. This study aims to explore the effect of exchange proteins directly activated by cAMP (Epac) on VA in CHF and the potential molecular mechanism. Transaortic constriction was performed to prepare CHF guinea pigs. Epac activation model was obtained with 8-pCPT administration. Programmed electrical stimulation (PES) was performed to detect effective refractory period (ERP) or induce VA. Isolated adult cardiomyocytes were treated with 8-pCPT and/or the Epac inhibitor. Cellular electrophysiology was examined by whole-cell patch clamp. With Epac activation, corrected QT duration (QTc) was lengthened by 12.6%. 8-pCPT increased action potential duration (APD) (APD50: 236.9±18.07ms vs. 328.8±11.27ms, p<0.05; APD90: 264.6±18.22ms vs. 388.6±6.47ms, p<0.05) and decreased IKr current (tail current density: 1.1±0.08pA/pF vs. 0.7±0.03pA/pF, p<0.05). PES induced more malignant arrhythmias in the 8-pCPT group than in the control group (3/4 vs. 0/8, p<0.05). The selective Epac1 inhibitor CE3F4 rescued the drop in IKr after 8-pCPT stimulation (tail current density: 0.5 ± 0.02pA/pF vs. 0.6 ± 0.03pA/pF, p<0.05). In conclusion, Epac1 regulates IKr, APD and ERP in guinea pigs, which could contribute to the proarrhythmic effect of Epac1 in CHF.

Extreme Acetylation of the Cardiac Mitochondrial Proteome Does Not Promote Heart Failure

Rationale: Circumstantial evidence links the development of heart failure to posttranslational modifications of mitochondrial proteins, including lysine acetylation (Kac). Nonetheless, direct evidence that Kac compromises mitochondrial performance remains sparse. Objective: This study sought to explore the premise that mitochondrial Kac contributes to heart failure by disrupting oxidative metabolism. Methods and Results: A DKO (dual knockout) mouse line with deficiencies in CrAT (carnitine acetyltransferase) and Sirt3 (sirtuin 3)—enzymes that oppose Kac by buffering the acetyl group pool and catalyzing lysine deacetylation, respectively—was developed to model extreme mitochondrial Kac in cardiac muscle, as confirmed by quantitative acetyl-proteomics. The resulting impact on mitochondrial bioenergetics was evaluated using a respiratory diagnostics platform that permits comprehensive assessment of mitochondrial function and energy transduction. Susceptibility of DKO mice to heart failure was investigated using transaortic constriction as a model of cardiac pressure overload. The mitochondrial acetyl-lysine landscape of DKO hearts was elevated well beyond that observed in response to pressure overload or Sirt3 deficiency alone. Relative changes in the abundance of specific acetylated lysine peptides measured in DKO versus Sirt3 KO hearts were strongly correlated. A proteomics comparison across multiple settings of hyperacetylation revealed ≈86% overlap between the populations of Kac peptides affected by the DKO manipulation as compared with experimental heart failure. Despite the severity of cardiac Kac in DKO mice relative to other conditions, deep phenotyping of mitochondrial function revealed a surprisingly normal bioenergetics profile. Thus, of the >120 mitochondrial energy fluxes evaluated, including substrate-specific dehydrogenase activities, respiratory responses, redox charge, mitochondrial membrane potential, and electron leak, we found minimal evidence of oxidative insufficiencies. Similarly, DKO hearts were not more vulnerable to dysfunction caused by transaortic constriction–induced pressure overload. Conclusions: The findings challenge the premise that hyperacetylation per se threatens metabolic resilience in the myocardium by causing broad-ranging disruption to mitochondrial oxidative machinery.

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.

How does pressure overload cause cardiac hypertrophy and dysfunction? High-ouabain affinity cardiac Na+ pumps are crucial

Left ventricular hypertrophy is frequently observed in hypertensive patients and is believed to be due to the pressure overload and cardiomyocyte stretch. Three recent reports on mice with genetically engineered Na+ pumps, however, have demonstrated that cardiac ouabain-sensitive α2-Na+ pumps play a key role in the pathogenesis of transaortic constriction-induced hypertrophy. Hypertrophy was delayed/attenuated in mice with mutant, ouabain-resistant α2-Na+ pumps and in mice with cardiac-selective knockout or transgenic overexpression of α2-Na+ pumps. The latter, seemingly paradoxical, findings can be explained by comparing the numbers of available (ouabain-free) high-affinity (α2) ouabain-binding sites in wild-type, knockout, and transgenic hearts. Conversely, hypertrophy was accelerated in α2-ouabain-resistant (R) mice in which the normally ouabain-resistant α1-Na+ pumps were mutated to an ouabain-sensitive (S) form (α1S/Sα2R/R or “SWAP” vs. wild-type or α1R/R α2S/S mice). Furthermore, transaortic constriction-induced hypertrophy in SWAP mice was prevented/reversed by immunoneutralizing circulating endogenous ouabain (EO). These findings show that EO and its receptor, ouabain-sensitive α2, are critical factors in pressure overload-induced cardiac hypertrophy. This complements reports linking elevated plasma EO to hypertension, cardiac hypertrophy, and failure in humans and elucidates the underappreciated role of the EO-Na+ pump pathway in cardiovascular disease.

Abstract 154: CCDC80 Functions as a Protein Kinase GI Substrate and is Secreted by Cardiac Myocytes

Background: Protein kinase G I alpha (PKGIa) inhibits cardiac hypertrophy, remodeling, and dysfunction. Downstream PKGI substrates remain incompletely understood and represent potential novel therapeutic targets for myocardial disease. We previously identified through a molecular screen that PKGIa binds and phosphorylates the protein coiled-coiled domain containing 80 (Ccdc80; also termed SSG1 and URB) in vascular smooth muscle cells. Previous work also identified that Ccdc80 is secreted from adipocytes. However, the expression and secretion of Ccdc80 from the cardiac myocyte has not been investigated. The current study tested the hypothesis that Ccdc80 is expressed in and secreted from the cardiac myocyte. Results: In cultured rat cardiac myocytes (CM), we detected Ccdc80 by western blot. Western blot for Ccdc80 also detected a band of the predicted Ccdc80 molecular weight present in media from these cells, but not in uncultured media. Ccdc80 could be detected in the human left ventricle (LV), though expression did not differ between hearts of normal controls and patients with hypertrophic cardiomyopathy. In the setting of LV pressure overload induced by transaortic constriction (TAC), we observed an increase in Ccdc80 expression in 1 week TAC LVs, compared with sham LVs (5.0 +/- 0.3 arbitrary densitometric units in sham versus 9.6 +/- 0.9 in TAC; n=4 per group). Conclusion: Taken together, our findings identify that the PKGIa substrate Ccdc80 expresses in cardiac myocytes, becomes secreted from CMs, resides in the human heart, and increases in expression in the mouse LV in response to pressure overload. Given the anti-remodeling role of PKGIa, these findings support future studies to understand the in vivo role of Ccdc80 in the cardiovascular system. Future studies will also explore the significance of Ccdc80 secretion from the CM and its potential regulation by PKG.

Abstract 77: Metabolic Reprograming Driven by Interaction AMP-activated Protein Kinase and Coactivator SRC-2 Regulate Cardiac Stress Response

Flexibility in fuel source for the heart is necessary to meet ATP demands during a high and variable workload such as transaortic constriction (TAC). During such chronic metabolic and hemodynamic stresses, shifts occur in substrate utilization in the heart based on magnitude of the demand, oxygen and nutrient availability. These shifts directly affect the amount of ATP available for other stress responsive pathways and therefore their efficacy. Communication from metabolic changes to these other pathways is crucial. As a result, it is important understand the molecular communication between metabolic and hypertrophic pathways under stress. We have found that chronic loss of cardiomyocyte SRC-2 and transient activation of AMPK both effect metabolic inputs prior to TAC and result in a blunted hypertrophic response. We hypothesized that SRC-2 and AMPK are critical upstream regulators of the cardiac stress response controlling metabolic changes translating into altered hypertrophy. Furthermore, we proposed that they could be regulating each other to control both the temporal changes to the metabolic machinery through AMPK and long-term genetic changes through SRC-2. We identified AMPK as a coregulator of SRC-2 activity and confirmed a direct interaction between them in cardiac cells. We have identified putative AMPK phosphorylation sites on SRC-2 that control SRC-2 stability and activity. Conversely, we found by ChIP that SRC-2 localized to AMPK subunit genes. Additionally, we performed co-immunoprecipitation and luciferase reporters and found that SRC-2 can interact with AMPK subunits and regulate its activity. We believe that disruption of these interactions is a main determinant in efficient hypertrophy in response to metabolic changes. Our results suggest the basis of a mechanism whereby energetic changes are sensed via AMPK and translated to transcriptional machinery of diverse targets via SRC-2 which allows AMPK to effect multiple cardiac transcription pathways. This mechanism provides insights into the role of AMPK-SRC-2 interaction in regulating myocardial function, metabolism, hypertrophy, and the progression to heart failure.