Elsevier

Journal of Biomechanics

Volume 50, 4 January 2017, Pages 11-19
Journal of Biomechanics

Spatial phenotyping of the endocardial endothelium as a function of intracardiac hemodynamic shear stress

https://doi.org/10.1016/j.jbiomech.2016.11.018Get rights and content

Abstract

Despite substantial evidence for the central role of hemodynamic shear stress in the functional integrity of vascular endothelial cells, hemodynamic and molecular regulation of the endocardial endothelium lining the heart chambers remains understudied. We propose that regional differences in intracardiac hemodynamics influence differential endocardial gene expression leading to phenotypic heterogeneity of this cell layer. Measurement of intracardiac hemodynamics was performed using 4-dimensional flow MRI in healthy humans (n=8) and pigs (n=5). Local wall shear stress (WSS) and oscillatory shear indices (OSI) were calculated in three distinct regions of the LV – base, mid-ventricle (midV), and apex. In both the humans and pigs, WSS values were significantly lower in the apex and midV relative to the base. Additionally, both the apex and midV had greater oscillatory shear indices (OSI) than the base. To investigate regional phenotype, endocardial endothelial cells (EEC) were isolated from an additional 8 pigs and RNA sequencing was performed. A false discovery rate of 0.10 identified 1051 differentially expressed genes between the base and apex, and 321 between base and midV. Pathway analyses revealed apical upregulation of genes associated with translation initiation. Furthermore, tissue factor pathway inhibitor (TFPI; mean 50-fold) and prostacyclin synthase (PTGIS; 5-fold), genes prominently associated with antithrombotic protection, were consistently upregulated in LV apex. These spatio-temporal WSS values in defined regions of the left ventricle link local hemodynamics to regional heterogeneity in endocardial gene expression.

Introduction

Endothelial cell (EC) structure and function is strongly influenced by the frictional force of wall shear stress (WSS) at the blood-EC interface. The effects of WSS on EC phenotype are well described to play an important role in blood vessel physiology and pathology. In regions where undisturbed WSS dominates, ECs are healthy. Conversely, ECs in regions of disturbed WSS, characterized by flow separation and transient flow reversals, have a pro-inflammatory, pro-oxidative stress phenotype and represent sites where atherosclerosis preferentially develops (Civelek et al., 2009, Davies et al., 2013).

Endocardial endothelial cells (EECs) line the heart chambers and represent an important barrier between the circulation and underlying myocardium. Studies in rat and pig demonstrate that EECs, which may originate from precursor vascular ECs, differ from mature vascular ECs with regard to morphology, gene and protein expression and labile molecule production (Hendrickx et al., 2004, Mebazaa et al., 1995). EECs are important in regulating cardiac contraction; endocardial denudation results in loss of contractile strength (Brutsaert et al., 1988, Mebazaa et al., 1993, Shen et al., 2013, Smith et al., 1991). This is attributed to the production and release of paracrine signaling agents such as nitric oxide (NO) (Smith et al., 1991). Unlike vascular ECs, the role of hemodynamics in the regulation of EEC biology has not been carefully studied.

Preservation of flow patterns throughout the LV is important to maintain its efficient function (Gharib et al., 2006). For example, diastolic vortex formation prevents the dissipation of blood kinetic energy and therefore reduces the myocardial force required to eject blood during systole (Kilner et al., 2000). Recent advances in cardiac imaging have enabled more detailed descriptions of intracardiac hemodynamics (Rodriguez Muñoz et al., 2012). In particular, the development of four-dimensional (4D) flow MRI enables 3D velocity encoding with ECG-gating, providing accurate spatial and temporal information. This technology has demonstrated that flow patterns within the LV change with age and gender and that blood residence times vary within the LV (Föll et al., 2013, Hendabadi et al., 2013). The influence of spatio-temporal differences of hemodynamics on EEC biology in the LV is poorly understood.

Here we calculated regional LV WSS using 4D flow MRI in humans and pigs to show that WSS varies regionally throughout the left ventricle of both species. We then isolated EECs from sites matched to the regional hemodynamic characteristics in pig LV and profiled gene expression by RNA sequencing to demonstrate significant regional differences of EEC phenotypes.

Section snippets

4D Flow MRI acquisition

Four-dimensional (4D) flow MRI (4D flow MRI) was performed to assess intracardiac hemodynamics in humans and pigs. For human MRI, IRB approval and informed consent were obtained from all 4D flow MRI study participants. The specifics of image acquisition and analysis were as previously described (Markl et al., 2012, Markl et al., 2016 McCormick et al., 2016). Studies were performed on 8 healthy volunteers (mean age: 24±1.8 years; four females and four males, heart rate 63.5±beats per minute, LV

Regional differences in LV wall shear stress and oscillatory shear index

To investigate regional WSS in healthy humans and pigs, we selected three heart regions: base, inferior to the aortic valve on the posterior heart wall, mid-ventricle (midV), inferior to the mitral valve on the free wall, and the apex (Fig. 1A). Following selection of two-dimensional planes, the images were segmented to define the LV lumen and 12 reference points were positioned. Hemodynamic characteristics were calculated as previously described (Stalder et al., 2008, Markl et al., 2012,

Discussion

The endocardial lining of the heart represents a greatly understudied endothelial compartment, particularly with regard to its phenotypic relationship with WSS. Here we report regional WSS and oscillatory (OSI) flow dynamics throughout the human and pig cardiac cycles, and establish corresponding spatial relationships with endocardial endothelial cell gene expression profiles.

Preservation of blood flow patterns within the LV is important in maintaining its function (Gharib et al., 2006). The

Conflict of interest

The authors declare no conflicts of interest.

Acknowledgments

Supported by NIH NRSA Training Grant T32 HL07954 (MEM), AHA Postdoctoral Fellowship16POST29110001 (MEM), AHA Postdoctoral Fellowship13POST14070010 (YJ), NIH, United States R00 HL108157 (WRTW), NIH, United States R01 HL115828 (MM), NIH, United States K25 HL119608 (AJB) and NIH, United States P01 HL06220 (PFD).

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