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Background Empagliflozin, an SGLT2 inhibitor, has shown remarkable reductions in cardiovascular mortality and heart failure admissions (EMPA-REG OUTCOME). However, the mechanism underlying the heart failure protective effects of empagliflozin remains largely unknown. Cardiac fibroblasts play an integral role in the progression of structural cardiac remodelling and heart failure, in part, by regulating extracellular matrix (ECM) homeostasis. The objective of this study was to determine if empagliflozin has a direct effect on human cardiac myofibroblast-mediated ECM remodelling. Methods Cardiac fibroblasts were isolated via explant culture from human atrial tissue obtained at open heart surgery. Collagen gel contraction assay was used to assess myofibroblast activity. Cell morphology and cell-mediated ECM remodelling was examined with the use of confocal microscopy. Gene expression of profibrotic markers was assessed with the use of reverse-transcription quantitative polymerase chain reaction. Results Empagliflozin significantly attenuated transforming growth factor β1–induced fibroblast activation via collagen gel contraction after 72-hour exposure, with escalating concentrations (0.5 μmol/L, 1 μmol/L, and 5 μmol/L) resulting in greater attenuation. Morphologic assessment showed that myofibroblasts exposed to empagliflozin were smaller in size with shorter and fewer number of extensions, indicative of a more quiescent phenotype. Moreover, empagliflozin significantly attenuated cell-mediated ECM remodelling as measured by collagen fibre alignment index. Gene expression profiling revealed significant suppression of critical profibrotic markers by empagliflozin, including COL1A1, ACTA2, CTGF, FN1, and MMP-2. Conclusions We provide novel data showing a direct effect of empagliflozin on human cardiac myofibroblast phenotype and function by attenuation of myofibroblast activity and cell-mediated collagen remodelling. These data provide critical insights into the profound effects of empagliflozin as noted in the EMPA-REG OUTCOME study.

BackgroundInfarcted myocardium can remodel after successful reperfusion, resulting in left ventricular dilation and heart failure. Epicardial infarct repair (EIR) using a bioinductive extracellular matrix (ECM) biomaterial is a novel surgical approach to promote endogenous myocardial repair and functional recovery after myocardial infarction. Using a pre-clinical porcine model of coronary ischemia-reperfusion, we assessed the effects of EIR on regional functional recovery, safety, and possible mechanisms of benefit.MethodsAn ECM biomaterial (CorMatrix ECM) was applied to the epicardium after 75 minutes of coronary ischemia in a porcine model. Following ischemia-reperfusion injury, animals were randomly assigned in 2:1 fashion to EIR (n = 8) or sham treatment (n = 4). Serial cardiac magnetic resonance imaging was performed on normal (n = 4) and study animals at baseline (1 week) and 6 weeks after treatment. Myocardial function and tissue characteristics were assessed.ResultsFunctional myocardial recovery was significantly increased by EIR compared with sham treatment (change in regional myocardial contraction at 6 weeks, 28.6 ± 14.0% vs 4.2 ± 13.5% wall thickening, p < 0.05). Animals receiving EIR had reduced adhesions compared with animals receiving sham treatment (1.44 ± 0.51 vs 3.08 ± 0.89, p < 0.05). Myocardial fibrosis was not increased, and EIR did not cause myocardial constriction, as left ventricular compliance by passive pressure distention at matched volumes was similar between groups (13.9 ± 4.0 mm Hg in EIR group vs 16.0 ± 5.2 mm Hg in sham group, p = 0.61). Animals receiving EIR showed evidence of vasculogenesis in the region of functional recovery.ConclusionsIn addition to the beneficial effects of successful reperfusion, EIR using a bioinductive ECM enhances myocardial repair and functional recovery. Clinical translation of EIR early after myocardial infarction as an adjunct to surgical revascularization may be warranted in the future.

Background Suspected genetic causes for extracellular matrix (ECM) dysregulation in the ascending aorta in patients with bicuspid aortic valves (BAV) have influenced strategies and thresholds for surgical resection of BAV aortopathy. Using 4-dimensional (4D) flow cardiac magnetic resonance imaging (CMR), we have documented increased regional wall shear stress (WSS) in the ascending aorta of BAV patients. Objectives This study assessed the relationship between WSS and regional aortic tissue remodeling in BAV patients to determine the influence of regional WSS on the expression of ECM dysregulation. Methods BAV patients (n = 20) undergoing ascending aortic resection underwent pre-operative 4D flow CMR to regionally map WSS. Paired aortic wall samples (i.e., within-patient samples obtained from regions of elevated and normal WSS) were collected and compared for medial elastin degeneration by histology and ECM regulation by protein expression. Results Regions of increased WSS showed greater medial elastin degradation compared to adjacent areas with normal WSS: decreased total elastin (p = 0.01) with thinner fibers (p = 0.00007) that were farther apart (p = 0.001). Multiplex protein analyses of ECM regulatory molecules revealed an increase in transforming growth factor β-1 (p = 0.04), matrix metalloproteinase (MMP)-1 (p = 0.03), MMP-2 (p = 0.06), MMP-3 (p = 0.02), and tissue inhibitor of metalloproteinase-1 (p = 0.04) in elevated WSS regions, indicating ECM dysregulation in regions of high WSS. Conclusions Regions of increased WSS correspond with ECM dysregulation and elastic fiber degeneration in the ascending aorta of BAV patients, implicating valve-related hemodynamics as a contributing factor in the development of aortopathy. Further study to validate the use of 4D flow CMR as a noninvasive biomarker of disease progression and its ability to individualize resection strategies is warranted.

Objectives Fluoroquinolone (FQ) antibiotics are associated with adverse aortic clinical events. We assessed human aortic myofibroblast-mediated extracellular matrix (ECM) dysregulation as a possible cellular mechanism underlying FQ-associated aortopathy. Methods Human aortic myofibroblasts were isolated from patients with aortopathy undergoing elective ascending aortic resection (N = 9). The capacity for extracellular matrix degradation in cells exposed to FQ was assessed by multiplex analysis of secreted matrix metalloproteinases relative to tissue inhibitors of matrix metalloproteinases (TIMPs). Direct evaluation of extracellular matrix degradation was investigated in human aortic cells using a 3-dimensional gelatin-fluorescein isothiocyanate fluorescence microgel assay. Aortic cellular collagen-1 expression following FQ exposure was determined by immunoblotting and immunofluorescent staining. Cell apoptosis, necrosis, and metabolic viability was determined by annexin-V, propidium iodide staining, and water-soluble tetrazolium salt (WST1) assay. Results FQ exposure significantly decreased aortic cell TIMP-1 (P = .004) and TIMP-2 (P = .0004) protein expression compared with vehicle control. The ratio of matrix metalloproteinase-9/TIMP-2 was increased suggesting an increased capacity for extracellular matrix degradation (P = .01). In collagen gels, we show a trend toward increased aortic myofibroblast-mediated collagen fiber degradation with FQ exposure (P = .09). Similarly, FQ exposure attenuated collagen-1 expression as assessed by immunoblotting (P = .002) and immunofluorescence (P = .02). Cell apoptosis, necrosis, and metabolic viability was not significantly influenced by FQ exposure. Conclusions For the first time, we document a putative mechanism underlying FQ-associated aortopathy whereby decreased TIMP expression with impaired compensatory collagen-1 expression results in human aortic myofibroblast-mediated extracellular matrix dysregulation. These novel data may provide a cellular and molecular mechanism to explain the established clinical association between FQ exposure and acute aortic events.

We thank Dr. Torii and colleagues for their insights regarding the combination of in vivo 4-dimensional flow magnetic resonance imaging (MRI) and in silico computational fluid dynamics (CFD) to improve risk prediction associated with bicuspid aortic valve (BAV) aortopathy. In their letter, these

We thank Dr. Murakami and colleagues for their letter regarding our recently published paper [(1)][1] in the Journal examining aortic tissue from patients with bicuspid aortic valves following pre-operative 4-dimensional (4D) flow cardiac magnetic resonance imaging. Compared with tissue from regions

BACKGROUND Empagliflozin, an SGLT2 inhibitor, has shown remarkable reductions in cardiovascular mortality and heart failure admissions (EMPA-REG OUTCOME). However, the mechanism underlying the heart failure protective effects of empagliflozin remains largely unknown. Cardiac fibroblasts play an integral role in the progression of structural cardiac remodeling and heart failure, in part, by regulating extracellular matrix (ECM) homeostasis. Thus, the objective of this study was to determine if empagliflozin has a direct effect on human cardiac myofibroblast-mediated ECM remodeling. METHODS Cardiac fibroblasts were isolated via explant culture from human atrial tissue obtained at open-heart surgery. Collagen gel contraction assay was used to assess myofibroblast activity. Cell morphology and cell-mediated ECM remodeling was examined using confocal microscopy. Gene expression of pro-fibrotic markers was assessed using RT-qPCR. RESULTS Empagliflozin significantly attenuated TGFβ1-induced fibroblast activation via collagen gel contraction after 72-hour exposure with escalating concentrations (0.5μM, 1μM, and 5μM) resulting in greater attenuation. Morphological assessment showed myofibroblasts exposed to empagliflozin were smaller in size with shorter and fewer number of extensions, indicative of a more quiescent phenotype. Moreover, empagliflozin significantly attenuated cell-mediated ECM remodeling as measured by collagen fiber alignment index. Gene expression profiling revealed significant suppression of critical pro-fibrotic markers by empagliflozin including: COL1A1, ACTA2, CTGF, FN1, and MMP-2. CONCLUSIONS We provide novel data showing a direct effect of empagliflozin on human cardiac myofibroblast phenotype and function by attenuation of myofibroblast activity and cell-mediated collagen remodeling. These data provide critical insights into the profound effects of empagliflozin as noted in the EMPA-REG OUTCOME study.

s S75 phenylephrine (16-660mg/min, a1-agonist). By deep anaesthesia and Urapidil (a1-blockade), lower blood pressures were achieved. In a 3T MRI scanner, OS-images were acquired at 10-15mmHg increments in a mid left ventricular short axis slice and changes in signal intensity (SI) and myocardial flow response were expressed as %-change 70mmHg baseline. At each level, coronary blood flow was obtained and changes in O2er (D) were calculated (1⁄4100*[CaO2-CvO2/CaO2]), comparing the changes to the oxygenation response. RESULTS: With increasing MAP there was a strong correlation to the LAD blood flow response (red) and a moderate correlation to changes in myocardial oxygenation represented by signal intensity (blue). There was a weak correlation between myocardial OS-SI and flow response changes. Interestingly, there was a weak negative correlation between MAP and O2er changes (green), indicating that during the MAP increase there was a luxury perfusion that exceeded the myocardial oxygen demand, allowing OS-SI to increase, while O2er dropped. This was confirmed by the negative correlation of O2er with OS-SI and flow response. RPP showed the same trend as MAP (table) to flow and O2er, yet no relationship was found to SI. CONCLUSION: a1-mediated blood pressure changes induced by phenylephrine result in a luxury perfusion paralleled by an increase in myocardial oxygenation, despite increased myocardial workload. Oxygenation-sensitive cardiovascular magnetic resonance is a suitable tool to monitor the effects of vasopressors on myocardial oxygenation. Research in Anaesthesiology and Intensive Care Medicine, Inselspital Bern University Hospital 147 SURGICAL APPLICATION OF A NOVEL BIOMATERIAL ATTENUATES POST-MI REMODELING AND HEART FAILURE: IMPACT OF INTERVENTION TIMING RELATIVE TO INFARCT STAGE ON FUNCTIONAL RECOVERY HE Mewhort, JD Turnbull, G Teng, DD Belke, DA Svystonyuk, DG Guzzardi, HJ Duff, PW Fedak

BACKGROUND: Epicardial infarct repair (EIR) using a bioinductive extracellular matrix (ECM) biomaterial is a novel surgical approach that targets the infarcted myocardium to enhance myocardial repair and prevent ischemic HF. We previously demonstrated that EIR prevents maladaptive LV remodeling and improves post-MI functional recovery. It is unclear if these benefits are a consequence of passive biomechanical infarct restraint or secondary to active paracrine signals from within the biomaterial (bio-inductive effect). In this study we outline the mechanisms by which EIR enhances myocardial repair following ischemic injury. METHODS AND RESULTS: Epicardial infarct repair performed using a biologically active ECM-biomaterial (CorMatrix-ECM, CorMatrix Cardiovascular Inc., GA, USA) was compared to EIR performed using the same ECM-biomaterial biologically inactivated by gluteraldehyde fixation. The active (N1⁄44) or inactive (N1⁄46) ECM-biomaterial was surgically applied to the epicardial surface of the infarcted myocardium following permanent coronary artery ligation in a rat model. Using a conductance catheter, indices of cardiac performance were quantified by pressure volume loop analysis 14-weeks posttreatment. EIR with either biomaterial resulted in functional recovery above sham-treated (N1⁄44) animals (ejection fraction: 42.43 13.09% vs. 25.15 6.99%, respectively; P1⁄40.035), however we observed a trend toward greater functional improvement in active EIR-treated animals as compared to inactive EIR-treated animals (ejection fraction: 47.13 15.45% vs. 30.50 13.40%, respectively; P1⁄40.16; stroke work: 10618 1548mmHg*uL vs. 8148 2514mmHg*uL, respectively; P1⁄40.17). Increased vascularity was observed within the infarcted myocardium of active EIR-treated animals as compared to inactive EIR-treated animals (11.8 5.6 vs. 3.7 3.9 blood vessels per high power field, respectively; P1⁄40.0008) potentially resulting in increased microvascular perfusion accounting for the functional recovery observed. Increased epicardial thickness, indicative of epicardial activation, was also observed in active EIR-treated animals as compared to inactive EIR-treated animals (4.6 1.6 vs. 2.9 0.7 fold increase relative to sham-treated animals, respectively; P1⁄40.0012). An increase in the number of epicardium derived progenerator cells (EDPCs), identified by Wilms’ tumor-1 expression and nuclearization of b-catenin was also observed in active EIR-treated animals, specifically in the infarcted myocardium indicative of increased epithelial to mesenchymal transition. Vascular structures positive for both nuclear b-catenin and a-SMA identified within the infarcted myocardium of active biomaterial-treated animals indicate that EDPCs undergo EMT and differentiate into vascular smooth muscle cells (VSMCs) through vasculogenesis (Figure). CONCLUSION: These data suggest that EIR with a biologically active biomaterial enhances myocardial repair through a bio-inductive mechanism beyond passive infarct restraint. The epicardial application of ECM-biomaterial stimulates epicardial progenitor cell mobilization and increases vasculogenesis to enhance functional recovery post-MI.

BACKGROUND: Patients with congenital bicuspid aortic valves (BAV) are predisposed to progressive dilatation of the ascending aorta (“bicuspid aortopathy”). A possible inherited/genetic etiology has led to aggressive surgical resection strategies to remove fragile tissue prone to complications. We recently demonstrated patterns of altered wall shear stresses (WSS) in the BAV aorta. These observations suggest regional hemodynamics may trigger local aortic extracellular matrix (ECM) degeneration leading to aortopathy. If validated, a more patient-specific and targeted resection strategy based on preoperative regional hemodynamic assessments may be warranted. For the first time, we correlated regional aortic tissue pathology with local hemodynamics using 4D-MRI in BAVpatients undergoing ascending aortic resection. METHODS AND RESULTS: BAV patients (N1⁄411) referred for ascending aortic resection received preoperative 4D-MRI. Regional WSS differences within each patient’s aorta relative to a normal tricuspid aortic valve control population for “elevated” (>1.96 SD) and “normal” WSS were defined. Paired aortic wall samples were collected during surgical resection from the predefined “elevated” and “normal” WSS regions. Aortic tissue was examined for histologic abnormalities consistent with BAV aortopathy (cystic medial necrosis, ECM fragmentation and mucopolyssacharide deposition) and molecular mediators of ECM regulation (TGFb-1, MMP, TIMP). All BAV patients studied had adjacent aortic regions with elevated and normal areas of WSS suggesting hemodynamic heterogeneity. Within the same patients’ aorta, aortic media subjected to elevated WSS demonstrated increased mucopolyssacharide composition (Figure 1C, white arrows) and elastin fragmentation (Figure 1D, black arrows) as compared to adjacent areas with normal WSS. Multiplex protein analyses revealed a 1.49-fold ( 0.71SD) increase in the profibrotic transforming growth factor b-1 (TGFb-1) in elevated WSS regions as compared to normal (P1⁄40.045). Mean fold increases in ECM-proteases MMP-1 (1.62 0.84; P1⁄40.057), MMP-2 (1.49 1.00; P1⁄40.18), MMP-3 (1.23 0.36; P1⁄40.18), MMP-7 (1.57 0.75; P1⁄40.067), and TIMP-2 (1.26 0.33; P1⁄40.038) were observed in aortic wall subjected to elevated WSS regions as compared to normal WSS regions within the same patients’ ascending aorta. CONCLUSION: In the BAV aorta, regional WSS corresponds with local histological abnormalities and ECM dysregulation. These novel data strongly implicate local hemodynamics as a mediator of BAV aortopathy. With further validation, 4DMRI could be used to guide personalized resection strategies for patients with BAV aortopathy.

s S155 BACKGROUND: Allo-reactive memory T cells are a recognized threat to solid-organ transplants, and yet there is limited information regarding their development and activation following solid-organ transplantation. Using an animal model of cardiac transplantation we previously demonstrated that CD8+ T cells were critical for the development of chronic rejection in the presence of calcineurin inhibitor (CNI) immunosuppression. Clinically, CNI initiation is often delayed post-transplant in an attempt to prevent the development of renal insufficiency. Based on recent reports concerning the development of memory CD8+ T cells we hypothesized that the degree of CNI delay could strongly influence the development of allo-reactive memory CD8+ T cells post-transplant. This was investigated in the current study using a murine model of allogeneic cell priming. METHODS: Allo-reactive memory T cells are a recognized threat to solid-organ transplants, and yet there is limited information regarding their development and activation following solid-organ transplantation. Using an animal model of cardiac transplantation we previously demonstrated that CD8+ T cells were critical for the development of chronic rejection in the presence of calcineurin inhibitor (CNI) immunosuppression. Clinically, CNI initiation is often delayed post-transplant in an attempt to prevent the development of renal insufficiency. Based on recent reports concerning the development of memory CD8+ T cells we hypothesized that the degree of CNI delay could strongly influence the development of allo-reactive memory CD8+ T cells post-transplant. This was investigated in the current study using a murine model of allogeneic cell priming. RESULTS: Effector and memory CD8+ T cell development was prevented when CyA was initiated at 0 or 2d post-prime (p

s S153 CONCLUSION: In this pilot study there were no observable histological differences in human right atrial tissue from individuals at highand low-risk for OSA. Further investigation would be required for more definitive results. Canadian Cardiovascular Society (CCS) Oral