Attenuation of Adverse Postinfarction Left Ventricular Remodeling with Empagliflozin Enhances Mitochondria-Linked Cellular Energetics and Mitochondrial Biogenesis

Sodium–glucose cotransporter 2 (SGLT2) inhibitors such as empagliflozin are known to reduce the risk of hospitalizations related to heart failure irrespective of diabetic state. Meanwhile, adverse cardiac remodeling remains the leading cause of heart failure and death in the USA. Thus, understanding the mechanisms that are responsible for the beneficial effects of SGLT2 inhibitors is of the utmost relevance and importance. Our previous work illustrated a connection between adverse cardiac remodeling and the regulation of mitochondrial turnover and cellular energetics using a short-acting glucagon-like peptide-1 receptor agonist (GLP1Ra). Here, we sought to determine if the mechanism of the SGLT2 inhibitor empagliflozin (EMPA) in ameliorating adverse remodeling was similar and/or to identify what differences exist, if any. To this end, we administered permanent coronary artery ligation to induce adverse remodeling in wild-type and Parkin knockout mice and examined the progression of adverse cardiac remodeling with or without EMPA treatment over time. Like GLP1Ra, we found that EMPA affords a robust attenuation of PCAL-induced adverse remodeling. Interestingly, unlike the GLP1Ra, EMPA does not require Parkin to improve/maintain mitochondria-related cellular energetics and afford its benefits against developing adverse remodeling. These findings suggests that further investigation of EMPA is warranted as a potential path for developing therapy against adverse cardiac remodeling for patients that may have Parkin and/or mitophagy-related deficiencies.

Deep Learning Analyses to Delineate the Molecular Remodeling Process after Myocardial Infarction

Specific proteins and processes have been identified in post-myocardial infarction (MI) pathological remodeling, but a comprehensive understanding of the complete molecular evolution is lacking. We generated microarray data from swine heart biopsies at baseline and 6, 30, and 45 days after infarction to feed machine-learning algorithms. We cross-validated the results using available clinical and experimental information. MI progression was accompanied by the regulation of adipogenesis, fatty acid metabolism, and epithelial–mesenchymal transition. The infarct core region was enriched in processes related to muscle contraction and membrane depolarization. Angiogenesis was among the first morphogenic responses detected as being sustained over time, but other processes suggesting post-ischemic recapitulation of embryogenic processes were also observed. Finally, protein-triggering analysis established the key genes mediating each process at each time point, as well as the complete adverse remodeling response. We modeled the behaviors of these genes, generating a description of the integrative mechanism of action for MI progression. This mechanistic analysis overlapped at different time points; the common pathways between the source proteins and cardiac remodeling involved IGF1R, RAF1, KPCA, JUN, and PTN11 as modulators. Thus, our data delineate a structured and comprehensive picture of the molecular remodeling process, identify new potential biomarkers or therapeutic targets, and establish therapeutic windows during disease progression.

The evaluation of cTnT/CK-MB ratio is as a predictor of change in cardiac function after myocardial infarction

Objective: Cardiac enzymes that are released during acute myocardial infarction (AMI) are of prognostic importance. This study aimed to investigate the relationship between cardiac troponin T (cTnT) and creatine kinase myocardial band (CK-MB) release during AMI and 6-month post-AMI left ventricular (LV) function, as assessed by magnetic resonance imaging. Methods: This prospective cohort observational study included 131 adult patients (113 males, 18 females, mean age 53.8 (8.6) years) who had been diagnosed with a new ST-segment elevation AMI (STEMI) in the emergency department. Cardiac enzymes were assessed by serial measurements. Blood samples obtained at 12 h post-AMI were included in the analysis. The reference value for CK-MB was 2–25 U/L, while for troponin it was - 0.1 ng/mL. Values above the reference limit were accepted as positive. Patients underwent cardiovascular magnetic resonance at 2 weeks and 6 months post-AMI. LV stroke volume was quantified as LV EDV – LV ESV, and ejection fraction (EF) was determined with the following equation: EF = [(LV EDV – LV ESV)/LV EDV] × 100. Adverse remodeling was defined based on the threshold values that are commonly accepted for changes in the LV end-diastolic volume (∆LV-EDV, &qt;10%) and LV end-systolic volume (∆LV-ESV, &qt;12%). Results: All of the patients were cTnT- and CK-MB-positive at 12 h. There was no found significant difference between both groups regarding the risk factors of coronary artery disease (including diabetes mellitus, hypertension, hyperlipidemia and smoking). Adverse cardiac remodeling was observed in 32.1% (n = 42) of the patients. cTnT/CK-MB was determined to be an independent predictor of the ΔLV-EDV (β ( SE = 0.55 ( 0.08, p<0.001), ΔLV-ESV (β ( SE = 1.12 ( 0.28, p<0.001), and adverse remodeling (OR = 1.13, p<0.001). The cTnT/CK-MB ratio was able to predict adverse remodeling with 85.7% sensitivity and 74.2% specificity (area under the ROC curve (AUC) = 0.856, p<0.001). The cTnT levels were able to predict adverse remodeling with 73.8% sensitivity and 78.7% specificity (AUC = 0.796, p<0.001). CK-MB did not significantly predict adverse remodeling (AUC = 0.516, (p=0.758). Conclusion: The cTnT/CK-MB ratio was superior to its components in predicting changes in LV function after STEMI. The cTnT/CK-MB ratio can be used in clinical practice for risk stratification and treatment optimization.

Abstract MP225: Macrophage-specificα5 Integrin Signaling Protects The Infarcted Heart From Adverse Remodeling By Stimulating The Angiogenic Response

Abundant macrophages infiltrate the infarcted heart and play a critical role in repair, remodeling and fibrosis. Macrophages sense changes in the extracellular matrix (ECM) environment through Integrins, thus activating signaling pathways that regulate their function. Analysis of our RNA sequencing data identified integrin α5 (Itgα5) as one of the most upregulated integrin genes in infarct macrophages. Accordingly, we hypothesized that integrin α5 signaling in infarct macrophages transduces ECM-derived signals, regulating responses critical for repair and remodeling of the infarcted heart.In order to study the role of macrophage α5 integrin in the infarcted heart, we generated 2 lines of macrophage-specific α5 integrin KO mice: myeloid cell-specific KOs (Myα5KO, using the lysozyme-M Cre driver) and inducible macrophage-specific α5 KOs (iMaα5KO, using CX3CR1 CreER ). 28 days after infarction, Myα5KO mice had accentuated adverse remodeling, evidenced by increased LVEDV and LVESV, a trend towards reduced ejection fraction. Adverse remodeling in Mya5KO was associated with a marked reduction in microvascular density in the infarct and in the border zone. iMaα5KO mice also exhibited worse remodeling and impaired infarct angiogenesis. A PCR array in infarct macrophages showed that α5 integrin loss was associated with markedly reduced transcription of VEGF-A, and lower levels of the angiogenic CXC chemokines CXCL1 and CXCL2. RNAseq followed by bioinformatic analysis predicted that that Nrf2, Erk5, HIF1a, p38 MAPK, VEGF, RhoA, and PI3K/Akt pathways were inhibited in the absence of α5 signaling in vivo (in infarct macrophages) and in vitro (in bone marrow macrophages undergoing α5 antibody neutralization experiments).In conclusion, α5 integrin promotes an angiogenic VEGF-expressing phenotype in infarct macrophages, protecting the infarcted heart from adverse remodeling. The angiogenic effects of α5 integrin in macrophages may have important therapeutic implications for heart failure patients.

Abstract MP262: Endogenous Smad7 Restrains Myofibroblast Activation And Protects From Post-infarction Heart Failure By Suppressing Tgf-beta Signaling And By Directly Inhibiting Erbb2

Repair of the infarcted heart requires TGF-β/Smad3 signaling in cardiac myofibroblasts and formation of an organized myofibroblast-populated scar. However, TGF-β-driven myofibroblast activation needs to be tightly regulated to prevent excessive fibrosis and adverse remodeling that may precipitate heart failure. We hypothesized that induction of endogenous suppressive signals, such as the inhibitory Smad7; may restrain infarct myofibroblast activation, protecting from adverse remodeling and fibrosis, and we examined the molecular mechanisms of Smad7 actions. In a mouse model of non-reperfused infarction, Smad3 activation triggered Smad7 synthesis in α-SMA+ infarct myofibroblasts, but not in α-SMA-negative fibroblasts. Mice with myofibroblast-specific Smad7 loss had increased heart failure-related mortality, worse dysfunction, and accentuated fibrosis in the infarct border zone and papillary muscles. In isolated cardiac fibroblasts, Smad7 overexpression attenuated myofibroblast conversion and reduced synthesis of structural and extracellular matrix proteins, whereas Smad7 knockdown promoted a matrix-synthetic phenotype. Smad7 actions on TGF-β cascades involved de-activation of Smad2/3 and non-Smad Erk/Akt pathways, without affecting TGF-β receptor activity. Unbiased transcriptomic analysis identified receptor tyrosine kinase (RTK) signaling as a major target of Smad7. Proteomic arrays demonstrated that the RTK Erbb2 is a target of Smad7 in cardiac fibroblasts. Western blots and co-immunoprecipitation assays showed that Smad7 interacts with Erbb2 in a TGF-independent manner and restrains Erbb1/Erbb2 activation, suppressing expression of fibrogenic proteases, integrins and CD44. In conclusion, Smad7 induction in infarct myofibroblasts serves as an endogenous TGF-β-induced negative feedback mechanism that inhibits post-infarction fibrosis by restraining Smad- dependent and Smad-independent TGF-β responses, and by suppressing TGF-independent fibrogenic actions of Erbb2. Protective effects of Smad7 in cardiac remodeling may have important therapeutic implications for heart failure patients.

Abstract P329: Exosomes Derived From Podoplanin Positive Cells Alter The Cellular Composition Of Healthy Mouse Hearts

Fibrosis and blood hypoperfusion stimulated by paracrine signals enhances the ventricular dysfunctionafter myocardial infarction (MI). We have earlier reported that within 2 days post-MI a cohort ofpodoplanin (PDPN) positive cells populate injured heart and enhance inflammatory response by physicalinteractions with monocytes. Here we explored whether exosomes from these cells could independentlyalter healthy heart physiology and structure. PDPN+ cells were isolated 2 days after MI, culture expandedand activated with TNFα and Angiotensin II. Exosomes derived from activated PDPN+cells conditionedmedia (PDPN+exo) were used in vitro for the treatment of mouse cardiac endothelial cells (mCECs) andmouse fibroblast (3T3) and in vivo for the treatment of healthy mouse hearts. In vitro, PDPN+exoinfluenced the phenotype of mCECs, stimulating their lineage into lymphatic endothelial cells andfacilitated fibroblasts transition to myofibroblast. Characterization of the protein content of PDPN+exoshowed high concentration of Notch receptors and γ-Secretase, suggesting these cellular transitions maydepend on exosome-mediated Notch translocation and cleavage. In fact, after exosomes treatmentcleaved notch (NICD) translocated in the nuclei of mCECs and 3T3 as early as 1h of treatment and eitherHes-1 or Hey-1, major transcription factors activated by NICD were enhanced within 2d of treatment.Using DAPT, a γSecretase inhibitor, notch cleavage was inhibited, and no phenotype switching in responseto exosome treatment was observed. In vivo, PDPN+exo were injected into the left ventricle of healthymouse hearts followed by boosters delivered by retro-orbital vein injection. Treated mice developed anextended epicardial fibrosis with a subsequent impairment in the contractility and increase of the enddiastolic and systolic volumes. The fibrotic area was characterized by vessels double positive toendothelial and lymphatic endothelial markers, and infiltrating CD45+ cells. Podoplanin positive cellsrepresent 80% of the scar’s cells of a chronic infarcted myocardium and the specific exosomes cargo highlyinfluence the lineage of cardiac cells altering the biology of endothelial cells and fibroblasts which mayfacilitate adverse remodeling.

Myocardial fibrosis reversion via rhACE2-electrospun fibrous patch for ventricular remodeling prevention

Myocardial fibrosis and ventricular remodeling were the key pathology factors causing undesirable consequence after myocardial infarction. However, an efficient therapeutic method remains unclear, partly due to difficulty in continuously preventing neurohormonal overactivation and potential disadvantages of cell therapy for clinical practice. In this study, a rhACE2-electrospun fibrous patch with sustained releasing of rhACE2 to shape an induction transformation niche in situ was introduced, through micro-sol electrospinning technologies. A durable releasing pattern of rhACE2 encapsulated in hyaluronic acid (HA)—poly(L-lactic acid) (PLLA) core-shell structure was observed. By multiple in vitro studies, the rhACE2 patch demonstrated effectiveness in reducing cardiomyocytes apoptosis under hypoxia stress and inhibiting cardiac fibroblasts proliferation, which gave evidence for its in vivo efficacy. For striking mice myocardial infarction experiments, a successful prevention of adverse ventricular remodeling has been demonstrated, reflecting by improved ejection fraction, normal ventricle structure and less fibrosis. The rhACE2 patch niche showed clear superiority in long term function and structure preservation after ischemia compared with intramyocardial injection. Thus, the micro-sol electrospun rhACE2 fibrous patch niche was proved to be efficient, cost-effective and easy-to-use in preventing ventricular adverse remodeling.

Cardiac MyD88 Mediates Inflammatory Injury and Adverse Remodeling in Diabetes

Background: Hyperglycemia-associated inflammation contributes to adverse remodeling and fibrosis in diabetic heart. MyD88 is an adapter protein of many Toll-like receptors (TLRs) and is recruited to TLRs to initiate inflammatory signalling pathway in endotoxin-activated innate immunity. However, the role of MyD88 in diabetic cardiomyopathy is unknown. Methods: For genetic deficiency, cardiomyocyte-specific MyD88 knockout and littermate control mice were induced type 1 diabetes (T1D) by intraperitoneal injection of 50 mg/kg/day streptozotocin for five days consecutive and then fed for 4 moths. For pharmacological inhibition, MyD88 inhibitor LM8 were administered daily for 8 weeks by oral gavage in T1D and T2D (db/db) mice. The effect of genetic and pharmacological inhibition MyD88 to myocardial injure which were induced by 33 mM glucose in vivo.Results: In this study, we first found that MyD88 expression was increased in cardiomyocytes of diabetic mouse hearts. Cardiomyocyte-specific MyD88 knockout protected mice against hyperglycemia-induced cardiac inflammation, injury, hypertrophy, and fibrosis in T1D model. In cultured cardiomyocytes, MyD88 inhibition either by siRNA or by small-molecular inhibitor LM8 markedly blocked TLR4-MyD88 complex formation, reduced pro-inflammatory MAPKs/NF-κB cascade activation and decreased pro-inflammatory cytokine expression under high glucose condition. Moreover, pharmacologic inhibition of MyD88 by LM8 showed significantly anti-inflammatory, anti-hypertrophic and anti-fibrotic effects in the hearts of both T1D and T2D mice. These beneficial effects of MyD88 inhibition were correlated to the reduced activation of TLR4-MyD88-MAPKs/NF-κB signaling pathways in the hearts.Conclusion: Taken together, MyD88 in cardiomyocytes mediates diabetes-induced cardiac inflammatory injuries and genetic or pharmacologic inhibition of MyD88 shows significantly cardioprotective effects, indicating MyD88 as a potential therapeutic target for diabetic cardiomyopathy.