SRSF3 is a key regulator of epicardial formation

The epicardium is a fundamental regulator of cardiac development, functioning to secrete essential growth factors and to produce epicardium-derived cells (EPDCs) that contribute most coronary vascular smooth muscle cells and cardiac fibroblasts. The molecular mechanisms that control epicardial formation and proliferation have not been fully elucidated. In this study, we found that the RNA-binding protein SRSF3 is highly expressed in the proepicardium and later in the epicardial layer during heart development. Deletion of Srsf3 from the murine proepicardium using the Tg(Gata5-Cre) or embryonic day (E) 8.5 induction of Wt1CreERT2 led to proliferative arrest and impaired epithelial-to-mesenchymal transition (EMT), which prevented proper formation and function of the epicardial layer. Induction of Srsf3 deletion with the Wt1CreERT2 after the proepicardial stage resulted in impaired EPDC formation and epicardial proliferation at E13.5. Single-cell RNA-sequencing showed SRSF3-depleted epicardial cells were removed by E15.5 and the remaining non-recombined cells became hyperproliferative and compensated for the loss via up-regulation of Srsf3. This research identifies SRSF3 as a master regulator of cellular proliferation in epicardial cells.

Global, segmental and layer specific analysis of myocardial involvement in Duchenne muscular dystrophy by cardiovascular magnetic resonance native T1 mapping

Background Progressive cardiomyopathy accounts for almost all mortality among Duchenne muscular dystrophy (DMD) patients.‍ Thus, our aim was to comprehensively characterize myocardial involvement by investigating the heterogeneity of native T1 mapping in DMD patients using global and regional (including segmental and layer-specific) analysis across a large cohort. Methods We prospectively enrolled 99 DMD patients (8.8 ± 2.5 years) and 25 matched male healthy controls (9.5 ± 2.5 years). All subjects underwent cardiovascular magnetic resonance (CMR) with cine, T1 mapping and late gadolinium enhancement (LGE) sequences. Native T1 values based on the global and regional myocardium were measured, and LGE was defined. Results LGE was present in 49 (49%) DMD patients. Global native T1 values were significantly longer in LGE-positive (LGE +) patients than in healthy controls, both in basal slices (1304 ± 55 vs. 1246 ± 27 ms, p < 0.001) and in mid-level slices (1305 ± 57 vs. 1245 ± 37 ms, p < 0.001). No significant difference in global native T1 was found between healthy controls and LGE-negative (LGE−) patients. In segmental analysis, LGE + patients had significantly increased native T1 in all analyzed segments compared to the healthy control group. Meanwhile, the comparison between LGE− patients and healthy controls showed significantly elevated values only in the basal anterolateral segment (1273 ± 62 vs. 1234 ± 40 ms, p = 0.034). Interestingly, the epicardial layer had a significantly higher native T1 in LGE− patients than in healthy controls (p < 0.05), whereas no such pattern was noticed in the global myocardium. Epicardial layer native T1 resulted in the highest diagnostic performance for distinguishing between healthy controls and DMD patients in receiver operating curve analyses (area under the curve [AUC] 0.84 for basal level and 0.85 for middle level) when compared to global native T1 and endocardial layer native T1. Conclusions Myocardial regional native T1, particularly epicardial native T1, seems to have potential as a novel robust marker of very early cardiac involvement in DMD patients. Trial registration: Chinese Clinical Trial Registry (http://www.chictr.org.cn/index.aspx) ChiCTR1800018340, 09/12/2018, Retrospectively registered.

The usefulness of echocardiography in the diagnosis of myocarditis in a young male after sudden cardiac arrest

The diagnosis of acute myocarditis (AM) remains challenging because of its diverse clinical manifestations. Thus, a wide range of diagnostic tests may be warranted. Although cardiac magnetic resonance (CMR) is the preferred imaging technique, it may not be applicable in the acute AM phase. Our case report highlights the usefulness and diagnostic accuracy of echocardiographic examination. In the first 2-dimensional echocardiography, the focal echobright was presented. A reduced value of global longitudinal strain, and regional disturbances of segmental myocardial strain, both longitudinal and circumferential, in the epicardial layer, were detected with a good correlation with CMR results.

Thermal modulation of epicardial Ca2+ dynamics uncovers molecular mechanisms of Ca2+ alternans

Ca2+ alternans (Ca-Alts) are alternating beat-to-beat changes in the amplitude of Ca2+ transients that frequently occur during tachycardia, ischemia, or hypothermia that can lead to sudden cardiac death. Ca-Alts appear to result from a variation in the amount of Ca2+ released from the sarcoplasmic reticulum (SR) between two consecutive heartbeats. This variable Ca2+ release has been attributed to the alternation of the action potential duration, delay in the recovery from inactivation of RYR Ca2+ release channel (RYR2), or an incomplete Ca2+ refilling of the SR. In all three cases, the RYR2 mobilizes less Ca2+ from the SR in an alternating manner, thereby generating an alternating profile of the Ca2+ transients. We used a new experimental approach, fluorescence local field optical mapping (FLOM), to record at the epicardial layer of an intact heart with subcellular resolution. In conjunction with a local cold finger, a series of images were recorded within an area where the local cooling induced a temperature gradient. Ca-Alts were larger in colder regions and occurred without changes in action potential duration. Analysis of the change in the enthalpy and Q10 of several kinetic processes defining intracellular Ca2+ dynamics indicated that the effects of temperature change on the relaxation of intracellular Ca2+ transients involved both passive and active mechanisms. The steep temperature dependency of Ca-Alts during tachycardia suggests Ca-Alts are generated by insufficient SERCA-mediated Ca2+ uptake into the SR. We found that Ca-Alts are heavily dependent on intra-SR Ca2+ and can be promoted through partial pharmacologic inhibition of SERCA2a. Finally, the FLOM experimental approach has the potential to help us understand how arrhythmogenesis correlates with the spatial distribution of metabolically impaired myocytes along the myocardium.

Understanding inter-tissue crosstalk during zebrafish cardiovascular development

My PhD work employed genetic and pharmacological manipulations, coupled with highresolution live imaging, to understand intercellular communications during zebrafish cardiovascular development. The heart is the first organ to form, and it is composed of several tissues, among which interactions are crucial. I identified two important interactions between muscular and non-muscular tissues in poorly characterized contexts, and the molecules required for the signalling. First, I discovered an important cellular and molecular crosstalk orchestrating the development of the cardiac outflow tract (i.e., the aortic root in mammals). Endothelial-derived TGF-beta signalling controls the generation of the local extracellular matrix (ECM). The ECM in turn affects endothelial proliferation as well as smooth muscle cell organization (Boezio et al, 2020; Bensimon-Brito*, Boezio* et al, 2020). In my second project, I investigated the crosstalk between the epicardial layer and the myocardial wall. By generating epicardial-impairment models, I identified a novel role for the epicardium in regulating cardiomyocyte volume during heart development (Boezio et al, 2021). Ultimately, this research contributed to our understanding of how paracrine signalling controls the multicellular interactions integral to organogenesis.

Electrophysiological remodeling of the right ventricle in experimental heart failure of different etiologies

The aim of the study was to evaluate electrophysiological remodeling of the right ventricle in rats in experimental heart failure of different etiologies. Materials and methods. Isadrin-, doxorubicin- and monocrotaline-induced heart failure models were developed. Unipolar epicardial electrograms of the ventricles (256 recording sites) were recorded using a 144-channel system. The cardiac output and pressure in both ventricles of the heart were measured. Activation-recovery intervals were used as an index of duration of local repolarization, and the general and local dispersions of activation-recovery intervals were used as an index of heterogeneity of ventricular repolarization. Results. In all models of heart failure, the following were identified: 1) non-uniform prolongation of repolarization with the greatest elongation at the apex of the right ventricle; 2) an increase in apicobasal differences of repolarization with the greatest change in the right ventricle; 3) an increase in the heterogeneity of the repolarization of the epicardial layer of the ventricles with heterogeneous changes in the local heterogeneity of repolarization and a decrease in the interregional differences in the heterogeneity of the electrophysiological properties of the myocardium; 4) more pronounced changes in the repolarization of the right ventricle than in the repolarization of the left ventricle. Conclusion. Thus, irrespective of the cause of the heart failure, the following changes occur: 1) prolongation of the right ventricular repolarization occurs non-uniformly (mostly due to the apical area), which results in an increase in the right ventricular repolarization heterogeneity; 2) an increase in the heterogeneity of right ventricular repolarization is observed, which causes an increase in the overall heterogeneity of the ventricular epicardial surface.

Spatiotemporal analysis reveals significant overlap of key proepicardial markers in the developing murine heart

During embryonic development, the epicardium provides a source of multipotent progenitors for cardiac lineages, including pericytes, fibroblasts and coronary smooth muscle cells. The epicardium originates from a region of splanchnopleural mesoderm known as the proepicardial organ (PEO). The potential of the epicardium to contribute to coronary endothelium has been disputed, due to conflicting lineage tracing results with different PEO Cre lines. Controversy also surrounds when epicardial cell fate becomes restricted. Using single-cell RNA-sequencing, microscopy and flow cytometry-based single molecule RNA in situ hybridisation techniques, we systematically investigated the expression of five widely used epicardial markers, Wt1, Tcf21, Tbx18, Sema3d and Scx, over the course of development. We show co-expression of all markers in the PEO and epicardial layer until E13.5, then sequential downregulation as it undergoes quiescence. Markers also decrease in invading epicardium-derived progenitors, with the exception of Tcf21, lost only in epicardium-derived mural cells. Moreover, we demonstrate that the epicardium does not significantly contribute coronary endothelium. Our findings clarify a number of prevailing discrepancies in the field and support the notion that epicardial fate is not pre-determined within the PEO.Summary statementAssessing expression of five principal (pro)epicardial markers reveals their complete overlap during early embryonic development, challenging previous dogma regarding the existence of sub-compartments and the pre-committed fate model.

Epicardial prestrained confinement and residual stresses: a newly observed heart ventricle confinement interface

The heart epicardial layer, with elastin as the dominant component, has not been well investigated, specifically on how it contributes to ventricular biomechanics. In this study, we revealed and quantitatively assessed the overall status of prestraining and residual stresses exerted by the epicardial layer on the heart left ventricle (LV). During porcine heart wall dissection, we discovered that bi-layered LV surface strips, consisting of an epicardial layer and cardiac muscle, always curled towards the epicardial side due to epicardial residual stresses. We hence developed a curling angle characterization technique to intuitively and qualitatively reveal the location-dependency and direction-dependency of epicardial residual stresses. Moreover, by combining prestrain measurement and biaxial mechanical testing, we were able to quantify the epicardial prestrains and residual stresses on the unpressurized intact LV. To investigate the potential mechanical effect of epicardial prestraining, a finite-element (FE) model has been constructed, and we demonstrate that it is the prestraining of the epicardial layer, not the epicardial layer alone, providing an additional resistance mechanism during LV diastolic expansion and ventricular wall protection by reducing myocardial stress. In short, our study on healthy, native porcine hearts has revealed an important phenomenon—the epicardial layer, rich in elastin, acts like a prestrained ‘balloon’ that wraps around the heart and functions as an extra confinement and protection interface. The obtained knowledge fills a gap in ventricular biomechanics and will help design novel biomimicking materials or prosthetic devices to target the maintenance/recreation of this ventricle confinement interface.

Abstract 15570: Direct Evidence of Endo-Epicardial Dissociation of the Atrial Wall in Patients With Longstanding Persistent Atrial Fibrillation

Introduction: Transition of paroxysmal to longstanding persistent atrial fibrillation (LsPAF) is associated with progressive longitudinal dissociation in conduction and a higher incidence of focal fibrillation waves. The aim of this study was to provide direct evidence that the substrate of LsPAF consists of an electrical double-layer of dissociated waves, and that focal fibrillation waves are caused by endo-epicardial breakthrough. Hypothesis: LsPAF in humans is caused by electrical dissociation of the endo- and epicardial layer. Methods: Intra-operative mapping of the endo- and epicardial right atrial wall was performed in 9 patients with induced (N=4), paroxysmal (N=1), persistent (N=2) or longstanding-persistent AF (N=2). A clamp of two rectangular electrode-arrays (128 electrodes; inter-electrode distance 2mm) was introduced through an incision in the right atrial appendage. Series of 10 seconds of AF were analyzed and the incidence of endo-epicardial dissociation (≥15ms) was determined for all 128 endo-epicardial recording sites. Results: In patients with LsPAF the averaged degree of endo-epicardial dissociation was highest (24.9% vs. 5.9%). Using strict criteria for breakthrough (presence of an opposite wave within 4mm and <15ms before the origin of the focal wave), the far majority (77%) of all focal fibrillation waves could be attributed to endo-epicardial excitation. Conclusions: During LsPAF considerable differences in activation of the right endo- and epicardial wall exist. Endo-epicardial fibrillation waves that are out of phase, may conduct transmurally and create breakthrough waves in the opposite layer. This may explain the high persistence of AF and the low succes rate of ablative therapies in patients with LsPAF.

Abstract 12797: A Global Longitudinal Strain in Endocardial Layer Selectively Correlated With Left Ventricular Ejection in Systemic Autoimmune Disorder Patients: A Multi-layer Transthoracic Echocardiography Study

Introduction: In patients with systemic autoimmune disorders (SAD), hemodynamic abnormalities such as pulmonary hypertension occur. Hypothesis: Some specific myocardial characteristics in SAD patients may precipitate hemodynamic abnormalities. Using myocardial multi-layer transthoracic echocardiography (TTE) analysis, we determine left ventricular (LV) myocardial characteristics in SAD patients, and compared LV ejection fraction (LVEF), estimated pulmonary arterial systolic pressure (ePASP) on TTE and serum brain natriuretic protein (BNP). Methods: Twenty SAD patients (18 female; mean age 49±18 years; systemic lupus erythematosus 35%; vasculitis 20%; scleroderma 5%, rheumatoid arthritis 5%, mixed connective tissue disease 5%) underwent TTE (Vivid E9, GE Healthcare). Apical 2-, 3-, and 4-chamber GLS views and parasternal short axis GCS view at the level of the papillary muscle was acquired. GLS was defined by averaging all 17 LV segments. GCS was defined as averaged LV segments at the level of papillary muscle. Furthermore strain measurements (absolute values) of whole, endocardial, and epicardial layers were performed using Echo PAC version 113 (GE Healthcare). Results: There was no significant correlation between GLS (whole, endocardial, and epicardial layers) and LVEF, ePASP, and BNP, respectively. GCS was significantly negatively correlated with serum BNP (R=-0.606 (whole), -0.452 (endocardial) and -0.447 (epicardial layer)). GCS in whole and epicardial layer was significantly negative correlated with ePASP (-0.528 (whole) and -0.457 (epicardial layer)). Only GCS in endocardial layer was significantly positive correlated with LVEF (R=0.466). Conclusions: In SAD patients, GCS significantly correlated with cardiac function and, especially in the endocardial layer selectively and positively correlated with LVEF. These specific myocardial characteristics in SAD patients may precipitate hemodynamic abnormalities.