Regional variations in ex-vivo diffusion tensor anisotropy are associated with cardiomyocyte remodeling in rats after left ventricular pressure overload

Systemic hypertension is a major health challenge, affecting approximately 45% of adults in the United States based on current guidelines [1]. The well-known response of the left ventricle (LV) to such pressure overload is concentric hypertrophy, which is characterized by the addition of sarcomeres in parallel within cardiomyocytes, resulting in LV wall thickening without chamber dilation. Such remodeling in pressure overload hypertrophy is considered a compensatory mechanism to maintain cardiac output despite increased downstream resistance to blood flow. This compensatory state, however, often leads to impaired diastolic filling, accompanied by wall thinning, chamber dilation, impaired systolic function, and eventual heart failure [2, 3].

Many studies have described effects of pressure overload on structural features, e.g. cardiomyocyte hypertrophy, laminar cardiomyocyte sheet architecture, proteins (both myocyte and extracellular matrix), molecular signaling, and gene expression networks [4‐9]. However, it is unclear whether remodeling of structural features in the heart during pressure overload hypertrophy, such as fibrosis and myocyte hypertrophy, occurs uniformly in all LV regions. Spatial gradients of structure and mechanical function are found in the normal and diseased LV, especially transmurally [10]. These transmural gradients, together with other factors, are thought to contribute to normalizing the otherwise large gradients in stress and strain along the long-axis of the myocytes (sometimes referred to as “fiber” stress or strain) during both diastolic filling and systolic contraction [10‐15].

For example, a paper on myocyte dimensions reported a transmural gradient in myocyte geometry in isolated cells from normal hearts, with larger myocyte volume and cross-sectional area (CSA) in the sub-endocardium (ENDO) compared to the midwall (MID) or sub-epicardium (EPI) [16]. In pressure overload hypertrophy, this transmural gradient was “abolished” [17]. However, others have shown different transmural patterns of myocyte geometry in both normal and hypertrophied rat LV [18, 19]. These conflicting results could be due to differences in loading conditions, different strains of rat, experimental processes such as fixation, or other factors. The primary goal of this study was to improve characterization of the transmural remodeling response to pressure overload by combined assessment of myocyte cell hypertrophy and sheet structure.

Measuring variations in these structural features is not trivial and usually involves histological methods, including tissue sectioning, staining, imaging, and careful measurement. Not only is this time-consuming and tedious, but such experiments cannot be performed in vivo, are destructive, and are usually only performed in small sub-regions of an organ or tissue. Non-destructive imaging is a much more appealing approach, especially methods which can relatively quickly and accurately capture the information in the entire organ or tissue of interest, while preserving 3D geometry. Cardiovascular magnetic resonance (CMR) diffusion tensor imaging (DTI) has emerged as one such technique to measure local structural orientations from eigenvectors, such as the local myocyte orientation described by the “helix” or “fiber” angle, and local laminar sheet orientations [20‐23]. The diffusion tensor not only provides orientation information in its eigenvectors, but also the apparent diffusivities along those eigenvectors in the eigenvalues. Some recent studies have described transmural variations in fractional anisotropy (FA), which is derived from the eigenvalues and indicates the degree of self-diffusion anisotropy [24‐26]. Others have shown an increase in secondary and tertiary diffusivities and sheet reorientation in heart failure patients [27, 28]. These results are also somewhat conflicting and unclear, and even less well understood are the contributions of different structural features to variations in 3D diffusivity. Thus, a secondary objective of this study was to determine whether regional and pathophysiological variations in these structural features could be detected consistently using DTI.

In this work, we have shown that several microstructural features in ex-vivo rat myocardium, including myocyte aspect ratios, correlated significantly with parameters derived from DTI in the same regions of the same hearts. Most of these correlations held true regardless of surgical group (TAC vs SHAM), whereas the association of others were modulated by pressure overload (Table 3). Our results also may support the conclusion that transmural gradients in myocyte geometry, including CSA and aspect ratios, are present in the normal rat LV but are reduced in pressure overload hypertrophy. This normal transmural gradient has been characterized by larger myocyte CSA in ENDO than EPI [17]. A similar trend appears to be present in our data, however, further study with larger sample sizes is required to confirm this pattern and its association with DTI.

We also observed a difference in the transmural patterns of several other structural features, as measured from DTI. One notable example is the orientation and dispersion of sheet angles. In agreement with our hypothesis, we observed that the transmural gradients in both the mean sheet angle and the dispersion of those angles about the mean, were more transmurally uniform in the LV of rats with pressure overload hypertrophy. This does not indicate that sheet angles are globally reoriented to become, for example, more radially aligned as others have observed [28]. Rather, transmural variation is reduced so that across the wall, the orientation and dispersion of sheet structures were more similar. Also of note, CP varied in the equatorial LV free wall, with a different pattern in SHAM than TAC hearts. In this region, CP decreased from EPI to ENDO in SHAM hearts, with a low point in MID, whereas in TAC hearts, CP was lower in EPI and ENDO regions, with a high point in MID (Fig. 2). Given that CP correlated negatively with MFA and L:W ratio, it may be that CP is altered (increased) in hypertrophied myocytes, due to anisotropic fibrosis or cleavage plane remodeling.

Others have reported transmural gradients and responses to pressure overload hypertrophyin the rat LV with different patterns than the present study [18, 19]. There are many factors that could contribute to these differences, including rat strain, the method of inducing pressure overload, and differences in measurement methods. However, our results appear to agree with those of Campbell et al., who showed a transmural gradient in myocyte CSA which is normalized in pressure overload hypertrophyin the rat [16, 17]. Others have reported that transmural gradients in mitochondrial respiratory chain activity and oxidative stress are normalized in pressure overload hypertrophy [40]. As sarcomeres are added and myocyte volume increases, the metabolic capacity must also increase to facilitate the energetic demands of those additional sarcomeres. It is clear that a remodeling balance must be achieved between the need to respond to the increase in downstream resistance to blood flow and the mechanical optimization of maintaining uniform myocyte long-axis stress and strain under such conditions. Several structural features have been shown to exhibit a transmural gradient in normal tissue, but remodel during pressure overload hypertrophy, which may be associated with the observed changes presented here [10].

Although in its infancy, the in vivo application of DTI in human hearts has already begun to show promise for detecting structural features in healthy tissue and in remodeled tissue during disease [28, 41‐43]. If our approach were to be applied in vivo and/or to other species, several points would be important to consider. For example, our results, especially regarding helix and sheet angle dispersions, may depend strongly on image resolution, which required long scan times (> 11 h) at high field strengths to achieve the high-fidelity DTI data presented here. Additionally, differences between species, e.g. perfusion, may impact the structural remodeling observed in pressure overload hypertrophyor other diseases [44, 45].

Previous studies have shown transmural gradients in FA in normal hearts of sheep, human (in-vivo), and rat [24‐26]. In sheep hearts, FA was lower in ENDO compared to MID or EPI regions. In the human study, FA was higher in MID than ENDO or EPI. Finally, in normal rat LV, a gradient of increasing FA from EPI to ENDO was observed [24]. We observed an opposite trend to that study, namely, with generally increasing values of FA from ENDO to EPI. Although Giannakidis et al. state that their result is consistent with measurements of myocyte CSA by McCrossan et al. [18], it is unclear how a decrease in L:W ratio would result in an increase in FA. Conversely, our data suggest a more logical correspondence between a decrease in FA when the L:W ratio decreases, as it does from EPI to ENDO (data not shown), however this correspondence did not reach statistical significance. In other words, diffusion would be more isotropic in voxels containing myocytes with a lower aspect ratio compared to more elongated myocytes. The same argument holds when comparing our results to those described by Campbell et al., who showed that in normal Sprague-Dawley rats, myocytes in the ENDO had larger CSA than EPI [16]. However, we did not observe a significant reduction of the gradient of FA in TAC, as one might expect from the results of a follow-up study [17]. Due to the similar transmural gradients in FA from DTI and MFA from confocal measurements, as well as similar definitions albeit from different sources, we expected there might be a correlation, however, this surprisingly did not reach statistical significance. This may be due to the somewhat limited study size, or perhaps the dip in FA near the epicardial surface, which might be a result of partial volume effects in that region.

To our knowledge, this is the first study to show correlations between DTI-derived parameters and myocyte geometry measurements in the same hearts. Others have used a similar approach to validate that the eigenvectors correspond with local helix and sheet orientations [21, 22], and more recently, that the eigenvalues correlate with collagen content [27, 46]. The correlations we observed between variables from DTI and measurements from histology were statistically significant, which may indicate that the DTI variables are influenced by myocyte geometry. Additionally, however, several of those same variables derived from DTI correlated significantly with the measured helix angle, which features a roughly linear transmural gradient. This may indicate that both myocyte geometry and some other structural features, which contribute to variations in DTI-derived parameters, also vary transmurally, giving rise to the moderate correlations we observed. In reality, the restriction to self-diffusion of water at the 100-μm scale is highly complex [47] so it is likely that structural features other than myocyte geometry (such as collagen content and composition) influence the diffusion weighted signal. However, we postulate that myocyte geometry plays an important role at the 100-μm resolution of our DTI scans. Potential methods to understand the contributions of different structural features to variation in DTI include simulation [48‐50], and dPFG or mPFG pulse sequences or qMAS diffusion encoding [51].

Several limitations to this study are worth consideration. First, eight rats in each group is a relatively small number, and a better understanding of the differences between TAC and SHAM animals would require more. However, even with a relatively small N we were able to observe statistically significant, regionally varying differences in many DTI-derived parameters, as well as significant correlations with measurements from histological sections. Second, the region for correlating between DTI and histological measurement was limited to the LV free wall. This region was chosen because it is the most commonly studied. The nature of sectioning, staining, and imaging limits the expanse of regions that can be analyzed using this method. An interesting follow-up study would be to predict with a statistical degree of certainty myocyte geometry based on DTI in a different region, such as the septum. Third, due to the direction of sectioning for confocal imaging, we could not validate sheet angle measurements. However, this has previously been done and consistent agreement was found between DTI-derived sheet angles and those from histological measurement [20, 22] or synchrotron radiation imaging [52]. Lastly, all image data acquisition was performed on hearts fixed in the diastolic, unloaded state. The process of fixation alters the microstructural environment by cross-linking proteins, and tissue shrinkage is a known side-effect. Thus, 3D diffusivity may not be completely representative of that in vivo. Additionally, further shrinkage is known to occur during processing of samples for histology. Despite these structural changes in tissue structure, we observed significant correlations in multiple variables, indicating that the essence of tissue anisotropy and microstructure was preserved during fixation and histological processing.

Regional variations in tissue microstructural features are evident in normal hearts, and remodeling during pressure overload hypertrophy is spatially non-uniform, with apparently more myocyte remodeling in MID and ENDO regions than EPI. Regional remodeling can be readily detected using cardiac DTI, with indications that parameters such as sheet angle dispersion and measures of anisotropy correlate significantly with variations in myocyte geometry, such as L:W ratio and MFA. As it is refined and developed further for in-vivo use in humans, cardiac DTI appears to be a promising method to non-invasively and comprehensively detect regional variations in cardiac tissue microstructure and its remodeling during disease.