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Background/Introduction Alterations in cellular bioenergetic events owing to metabolic dysfunctions are well demonstrated in heart failure (HF). Cardiomyocytes derived from human-induced pluripotent stem cells (hiPSC-CMs) have the potential to model metabolic alterations as observed in the human failing hearts, which however requires precise arrangements of the respiratory chain complexes. Purpose Develop a method to improve the hiPSC-CMs architecture by using a micropatterned substrate (repeated lines of 30 μm-width separated with a cell repellant) that promotes elongation and anisotropy. Methods Cardiomyocyte differentiation was performed using a chemically defined method and 35 days later the generated hiPSC-CMs were cultured on unpatterned vs. micropatterned substrates for 7 additional days. Energy metabolic profiles were then measured using the Seahorse analyzer and mitochondrial components were analyzed by fluorescence microscopy and western blot. Results Cultures on micropatterned anisotropic substrates resulted in the generation of elongated cell morphology, rod-shaped hiPSC-CMs with improved sarcomere organization scores, and more aligned Z-lines. We first found a specific increase in oxidation phosphorylation (OXPHOS) activity in micropatterned CMs with a significant increase in the oxygen consumption rate (OCR) linked to basal respiration (58.4±24.0 vs 40.6±21.0 pmol/min, p Conclusion(s) We developed a novel method for modeling metabolic activity in hiPSC-CMs based on micropatterned cultures. Our data show that the linear architectural pattern drives the formation and efficiency of OXPHOS function in cardiomyocytes with a mitochondrial structural reorganization. Funding Acknowledgement Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Agence Nationale de la Recherche

BackgroundMyocardial infarction (MI) induces a repair response that ultimately generates a stable fibrotic scar. Although the scar prevents cardiac rupture, an excessive profibrotic response impairs optimal recovery.ObjectiveTo explore the regulation of fibroblasts proliferation through a paracrine action of cardiac stromal cells post-MIMethodsWe carried out a bioinformatic secretome analysis of cardiac stromal PW1+ cells isolated from normal and post-MI mouse hearts to identify novel secreted proteins. Functional assays were used to screen secreted proteins that promote fibroblast proliferation. The expressions of secreted proteins candidates were subsequently analyzed in mouse and human hearts and plasmas. The relation between levels of circulating protein candidates and adverse post-MI cardiac remodeling was examined in a cohort of 80 patients with a first ST-elevation MI and serial cardiac magnetic resonance imaging (MRI) evaluations.ResultsCardiac stromal PW1+ cells undergo a change in paracrine behavior post-MI and secrete factors that promote fibroblast proliferation. Among these factors, growth differentiation factor 3 (GDF3), a member of the transforming growth factor-β family, was markedly upregulated in the ischemic hearts and induced fibroblast proliferation at high level. In humans, GDF3 was detected in the plasma at day 4 post-MI and GDF3 circulating levels were significantly associated with an increased risk of adverse remodeling 6-month post-MI (adjusted Odds Ratio (OR) = 1.76 [1.03 - 3.00], p = 0.037).ConclusionsOur findings define a mechanism for the pro-fibrotic action of cardiac stromal cells through secreted cardiokines, such as GDF3, a candidate marker of adverse fibrotic remodeling following MI.

Introduction Hypertrophic cardiomyopathy (HCM) is a relatively frequent (1/500) heart disease that could result in arrhythmia and sudden death. It is mainly caused by sarcomere gene mutations. Disease mechanisms are still poorly known due to limited access to humanized experimental models. Cardiomyocytes derived from iPSc of mutated patients could serve as a platform for modeling the disease and help identifying novel therapeutic strategies. Objective To derive and differentiate into cardiomyocytes an iPSc line from a patient with an HCM hot-spot mutation and its isogenic control. Method From patient's fibroblasts we derived an iPSC line harboring the heterozygous MYH7 R403L (c.1208G > T) mutation (Okita et al). The mutation was corrected using CRISPR/Cas9 editing to produce an isogenic pair. Genomic editing, pluripotency and cardiogenicity of the clones were evaluated. Results The 2 derived iPSc clones proved to be pluripotent after phosphatase alkaline labelling, RT-qPCR on specific mRNA and proteins immunolabelling (Oct4, Nanog, Sox2, SSEA4). High TaqMan hPSC Scorecard confirmed tri-lineage differentiation potential. Normal karyotype indicated genomic integrity and STR analysis of donor fibroblasts and iPSc lines proved filiation of the clones. The mutated clone was electroporated with spCas9 and a gRNA targeting a PAM 3-bp away from the mutated allele and a correcting ssODN DNA donor. Homologous recombination events were selected by allele-specific ddPCR and reversion of the mutation was confirmed by Sanger sequencing. The 2 lines were able to differentiate into beating cellular sheets and cells stained positive for aACTN2 indicating cardiomycocyte differentiation. FACS on cTNT positive cells counts 75% of cardiomyocytes in independent differentiation experiment after 3 weeks. Conclusion We established an isogenic pair of iPSc lines from a HCM patient with MYH7 mutation defining a model for cellular and molecular study of HCM.

Background: Lower risk of COVID-19 was reported in men with prostate cancer receiving androgen deprivation therapy while low levels of testosterone (T) were associated with a more severe disease and poor clinical outcomes in COVID-19 male patients (pts). In the latter case, it is unclear whether low levels of T and dihydrotestoserone (DHT) are risk factors or consequences of COVID-19. Here, we investigated T and DHT levels impact on COVID-19 severity in ambispective cohorts of symptomatic SARS-CoV-2 infected males. Methods: Both prospective (European Hospital Georges Pompidou patients, P-cohort) and retrospective (French COVID-19 cohort, REacting project, R-cohort) cohorts included male pts admitted for severe COVID-19. The P-cohort included pts admitted in a medical unit (non-ICU) or in ICU immediately (ICU-I). The R-cohort included pts admitted to a medical unit, ICU-I or to ICU secondarily (ICU-S). The size of ICU-S pts group in P-cohort was insufficient to include their data in the analysis. We collected information on pts demographics and COVID-19-related outcomes. T, DHT levels and inflammation markers were measured. Wilcoxon-Mann-Whitney test and chi2-test (or Fisher’s exact test, if appropriate) were performed. All tests were two-sided at 0.05 significance level. Results: The P-cohort included 71 pts (median age: 64 years) and the R-cohort 89 pts (median age: 62 years). The median duration between admission and measurement of hormone levels was 2 days (range: 0-16) and 0.5 days (range: 0-11) respectively. T and DHT levels were low in all pts as compared to standards and even lower in ICU pts (Table). In the R-cohort, T and DHT lowest values were observed for ICU-I pts and median values for ICU-S pts. [Formula presented] Conclusions: Low T and DHT levels were associated with the severity of the disease and the poorest clinical outcomes in males with severe COVID-19. This suggests that COVID-19 may cause a rapid and profound decrease in androgens levels and that T and DHT serum levels may be used as prognostic markers. Legal entity responsible for the study: Pr. Stephane Oudard. Funding: Has not received any funding. Disclosure: All authors have declared no conflicts of interest.

Background Activated cardiac fibroblasts produce extracellular matrix proteins that accumulate during cardiac fibrosis. We have recently shown that PW1 is expressed in a subset of cardiac stromal cells and that cardiac PW1+ cells represent a cellular source of fibroblasts in the ischemic hearts. Purpose We aimed to further identify new cell surface markers expressed by cardiac PW1+ cells and to investigate their role in the fibrogenic behavior of these cells. Methods and results We first performed transcriptomic and proteomic profiling of FACS-isolated cardiac PW1+ from normal and ischemic hearts. RNA-sequencing output files were processed with bioinformatics algorithms to identify 378 specific cell-surface markers for cardiac PW1+ cells. By comparing these candidates with the proteomic profile, we then cross-identified 9 cell surface proteins primarily involved in cell motility, adhesion to the matrix, inflammatory response and response to wounding. One of these candidates (i.e., aV-integrin or CD51) was expressed in almost all cardiac PW1+ cells (93±1%), and was predominantly found in cells expressing PW1 in the myocardium. Cardiac PW1+ cells showed a predominant expression of aVβ1 complex which is known to mediate fibrosis through TGF-beta activation in a number of tissues. The transfer of isolated cardiac PW1+CD51+ cells into ischemic hearts was associated with fibrosis development. We further demonstrated that inhibition of aV-integrin in cardiac PW1+ cells reduces their profibrotic gene expression profile and their ability to differentiate into fibroblasts. Lastly, a pharmacological blockade of aV-integrin improved cardiac function and animal survival following myocardial infarction coupled with a reduced infarct size and fibrotic lesion. Conclusions These data identify a targetable pathway that regulates cardiac fibrosis in response to an ischemic injury and demonstrate that pharmacological inhibition of aV-integrin leads to reduced pathological outcomes following cardiac ischemia. Acknowledgement/Funding Fondation Leducq (grant 13CVD01, CardioStemNet project), Fédération Française de Cardiologie and Era-CVD (ANR-16-ECVD-0011-03, Clarify project)

Introduction Hypertension is one of the major contributors to cardiac diastolic dysfunction as observed in patients with heart failure and preserved ejection fraction. Different therapeutics strategies have been proposed to control blood pressure (BP) but their independent impact on cardiac function remain undetermined. In patients referred to our center for uncontrolled hypertension, we compared changes in cardiac parameters between two strategies consisting of sequential nephron blockade with intensive diuretics (NBD) or sequential renin angiotensin blockade (RAB). Purpose To determine evolution of BNP levels and echocardiographic (TTE) parameters of diastolic dysfunction according to strategies to control BP. Methods After 4-weeks treatment with 300mg irbesartan + 12.5mg hydrochlorothiazide + 5mg amlodipine, 140 patients with uncontrolled hypertension were randomized to either complete nephron blockade with sequential introduction of 25mg spironolactone, 20 to 40mg furosemide and 5mg amiloride (NBD group, n=74) or complete RAAS blockade with 5 to 10mg ramipril and 5 to 10mg bisoprolol (RAB group, n=67). BNP levels and TTE parameters according to ESC guidelines were assessed at baseline and week 12. Results BNP levels were not significantly different in the two groups at baseline (mean±SD 30.1±37.0pg/mL in NBD versus 22.6±22.3pg/mL in RAB, p=0.147) but significantly decreased in NBD at week 12 (20.1±22.7pg/mL in NBD versus 55.2±60.4pg/mL in RAB, p35pg/mL versus 37 (55%) in RAB (p Concordantly, TTE parameters such as left ventricular end-diastolic diameter (mean±SD, 49±4mm in NBD and 52±5mm in RAB, p=0,004) and left atrial area (16.9±3.6cm2 in NBD and 19.1±3.7cm2 in RAB, p=0,003) improved significantly in NBD group. The proportions of patients presenting ≥2 criteria of diastolic dysfunction according to ESC guidelines in NBD and RAB group respectively were 31.2% and 19.3% at baseline vs. 3.1% and 32.2% after 12 weeks (p=0.001). In a multivariate analysis, NBD treatment was significantly associated with a decline in BNP levels (p Conclusion In patients with uncontrolled hypertension, intensive nephron blockade with sequential addition of diuretics improves markers of diastolic dysfunction independently of BP lowering.

Introduction Myocardial injury is frequently observed in patients hospitalized with Coronavirus disease 2019 (COVID-19) pneumonia. Different cardiac abnormalities have been reported during the acute COVID-19 phase but there is limited information on late cardiac sequelae in patients who have recovered from acute COVID-19 illness. Objective To document the presence and quantify the extent of myocardial functional alterations in patients hospitalized 6 months earlier for COVID-19 infection. Methods and results We conducted a prospective echocardiographic evaluation of 48 patients (58 ± 13 years, 69% male) hospitalized 6 ± 1 month earlier for a laboratory-confirmed and symptomatic COVID-19. Thirty-two (66.6%) had pre-existing cardiovascular risks factors (hypertension, T2DM or dyslipidemia) and three patients (6.2%) had a known prior myocardial infarction. Sixteen patients (33.3%) experienced myocardial injury during the index COVID-19 hospitalization as identified by a rise in cardiac troponin levels. Six months later, 60.4% of patients still reported clinical symptoms including exercise dyspnea for 56%. Echocardiographic measurements under resting conditions were not different between patients with vs. without myocardial injury during the acute COVID-19 phase. In contrast, low-level exercise (25 W for 3 minutes) induced a significant increase in the average E/e’ ratio (10.1 ± 4.3 vs. 7.3 ± 11.5, P = 0.01) and the systolic pulmonary artery pressure (33.4 ± 7.8 vs. 25.6 ± 5.3 mmHg, P = 0.02) in patients with myocardial injury during the acute COVID-19 phase. Sensitivity analyses showed that these alterations of left ventricular diastolic markers were observed regardless of cardiovascular disorders indicating SARS-CoV-2 infection as a primary cause. Conclusions Six months after the acute COVID-19 phase, significant cardiac diastolic abnormalities are observed in patients who experienced myocardial injury but not in patients without cardiac involvement.

Introduction Pw1/Peg3 is a parentally imprinted gene unique to placental mammals, suggesting a specific role in the placenta. Although Pw1/Peg3 is highly expressed in the placenta, the effects of a loss of function during placental development have not been well characterized. Objective To determine Pw1/Peg3 function during placental vascular formation as well as placental remodeling and how this impacts fetal intra-uterine growth in response to hypoxia. Method A comprehensive study was performed in order to analyze the placental phenotype: at the macroscopic (weight), histological (layers size and composition) and cellular (isolation of specific cell populations) levels. The functional impact of the vascular alteration of the Pw1KO placenta was accessed by the study of placental ability to remodel under hypoxia conditions. Results We observe that the Pw1/Peg3 is highly expressed in the placental labyrinth zone, i.e. fetal capillaries and syncytiotrophoblast cells, which is the site for fetal-maternal exchange. The mutant placenta displayed an overall lower weight and size coupled with an increase in endothelial and stromal cells and a decrease in pericytes, leading to an altered vascular structure. In order to determine if this altered vascular in the mutant placenta also had a functional impact, we placed pregnant dames in 10% O2 (hypoxic) environment during late fetal development. We observed that Pw1/Peg3 mutants exhibit significantly smaller placentas and embryos with asymmetric intrauterine growth restrictions as compared to wildtypes. These results demonstrate that Pw1/Peg3 is required to regulate placental plasticity in response to environmental challenges, which are critical for fetal and neonatal health, through the establishment of a functional labyrinth vascular network. Conclusion The loss of Pw1/Peg3 function impairs the development of the vascular network in the labyrinth layer and governs placental remodeling in response to hypoxia.

Introduction Certain individuals have increased propensity to develop prolonged QT interval and consequent life-threatening ventricular arrhythmias in response to drugs that alter cardiac ion currents, notably IKr. We recently performed a transcriptomic profiling of induced pluripotent stem cells-derived cardiomyocytes (iPSC-CM) in patients with high risk to develop diLQTS and identified 5 molecules mechanistically connected to QT prolongation including 4 up-regulated (DLG2, KCNE4, PTRF, and HTR2 C) and 1 down-regulated (CAMKV) genes. Objective To confirm the role the 5 identified candidates in modulating cardiac repolarization in response to drugs. Methods We used iPSC-CM from 10 subjects with the highest sensitivity (S + ) to sotalol (ie, those with the highest increase in QT interval) and from 8 individuals with the lowest sensitivity to sotalol (S-) to perform molecular and electrophysiological investigations. Results In line with the recorded QT prolongation after Sotalol administration in patients, S+ iPSC-CM displayed 2 and 3 Fold increase of field potential duration after adding 30 and 100 μM of Sotalol respectively. Among the 5 candidate genes, we found that PTRF presented the highest transcriptomic level in human ventricular tissue in GTEx publicly available bank. PTRF mRNA and protein expression levels were increased by 40% in the S+ iPSC-CM as compared to the S- iPSC-CM. PTRF has been reported as a key regulator of calveolae. To assess the implication of PTRF in the exaggerated reaction to Sotalol, we aimed to induce PTRF overexpression in the S- iPSC-CM and to repress its translation in the S+ iPSC-CMs using shRNA. Electrophysiological tests will be then used to assess the changes in response to Sotalol after manipulation of PTRF expression. Conclusion Altogether, this work identified PTRF as a candidate for diLQTS. Further understanding of its contribution may pave the way for personalized drug prescription to avoid undesirable cardiotoxicity.