The hypothesis that the VP contributes significantly to LV passive mechanical properties was primarily derived from our gross dissection experience and is supported by our observations of its microscopic three-dimensional composition and configuration as well as its location at the geometrically advantageous outer edge of the heart. Experimental results supported this hypothesis. First, the opening angle of the LV slices, which reflects the level of myocardial residual stress, was highly dependent on the presence of the VP. Second, mechanical uncoupling of the VP in the intact heart led to a modest rightward shift of the pressure-volume relationship and a decrease in LV passive stiffness at lower volumes. Based on these results, we conclude that the VP plays a significant role in the passive mechanical properties of the LV in early diastolic filling.
Several previous lines of evidence support the role of the VP in the passive mechanical properties of the heart. The VP tensile strength is more than an order of magnitude greater than that of any other portion of the myocardium, which agrees with the greater hydroxyproline content found in this layer (26
). In addition, the epicardium with the VP has greater stiffness than the intact endocardium (16
The VP may also have a role in storing elastic energy during systole and releasing it in early diastole to aid the relaxation and untwisting of the LV. In human hearts, the systolic deformation of the epicardial surface is characterized by a longitudinal shortening of 10% (37
), shear due to ventricular twisting of 20° from the base to apex (19
), and a small amount circumferential shortening. Although direct measurements of circumferential shortening of the epicardial surface are not available, geometric considerations and direct myocardial measurements have shown a decrease of circumferential strain from endocardial to epicardial layers and the strain on the epicardial surface is likely low by the trend of these measurements (2
). Using these typical values and assuming that they are uniform over most of the epicardial surface, we calculated the composite deformation of the epicardial surface and the associated shortening or stretching of a segment of elastin fiber at a range of fiber angles from −90 to 90°. We modeled the two cases of no appreciable circumferential shortening and 2% shortening. The result is shown in . Indeed, given the measured fiber angles in this study (), the elastin fibers should be stretched during systole. The resulting elastic return force should actively contribute to the relaxation and untwisting of the LV. The epicardial location of the VP is again advantageous in two aspects: the circumferential shortening is minimum and, correspondingly, the elastin fiber lengthening is maximum; the torque from the elastic recoil is also maximized. The epicardial location of the VP also permits this function with minimal impact on the contractile apparatus within the wall by not being interleaved with the contractile elements. The VP can also serve as a final safety factor limiting the expansion of the heart beyond critical limits due to its tensile strength. Clinical cases of near-myocardial rupture resulting from acute myocardial infarction have been documented (12
), where the myocardium had separated and only the VP remained resisting the ventricular pressure, also attesting to the VP tensile strength and its ability to provide a final barrier to myocardial rupture.
Fig. 8 Elastin fiber length change during systole versus fiber angle. Two estimates are shown for no appreciable circumferential shortening (Ecc)on the epicardial surface or 2% Ecc. The range of measured elastin fiber angles fell in the region where stretching (more ...)
It is of note that that the VP has a very low hydraulic water permeability (Lp
) as well as low diffusional water permeability (1
). In addition, the pressure gradient at the surface of the heart has been estimated to be very steep (32
), suggesting that the very low filtration coefficient of the VP may also prevent plasma filtration across the heart wall into the pericardial sack. No “solid” macromolecular structure, such as the elastic lamina in arterial walls, was observed in the VP. Thus, the low Lp
is likely due to the extensive tight junctions in the endothelial layer (13
) and not a function of the macromolecular structure.
Although the opening angle may not be a complete description of the residual stress, it does reflect the overall level of residual stress in the myocardium (24
). The opening angle is a measure of two-dimensional residual strain, which quantifies the circumferential deformation of the LV slice when the residual stress is relieved. A zero opening angle indicates no residual stress, and the angle positively correlates with the residual stress (23
). The primary source of residual stress has been considered to arise from deformation of cellular (e.g., titin) (4
) and extracellular elements (e.g., perimysial collagen fibers along myofibers) (5
) within the myocardium from the stress-free state to the unloaded state (28
). However, our results clearly demonstrate that the VP has a significant impact on the opening angle and, therefore, must make a major contribution to the residual stress of the LV. The increased opening angles found in epicardial portions of myocardium slices are consistent with the VP being an important factor in the residual stress found in the resting heart (24
). This concept is also supported by the report by Omens et al. (25
), which showed that the opening angle does not change during pressure overload despite significant cardiomyocyte hypertrophy. We speculate that earlier studies of the opening angle in physiological and pathological states may have been confounded by modifications of the VP that need to be accounted for beyond the changes within the myocardium itself. For example, the stiffness of the VP increases within the infarcted region of the myocardium during an acute period after myocardial infarction associated with small changes in total hydroxyproline content (26
). Alterations in the VP composition and structure could play a role in numerous pathological states associated with passive mechanical properties that warrant further investigation.
In the intact heart, we found that mechanically uncoupling the VP only modestly decreased the passive stiffness of the LV at lower volumes, with no effect at higher volumes (). In comparison, the PP has been shown to impact many mechanical aspects of the myocardium, including the diastolic pressure-volume curve as well as RV-LV contraction interactions (33
). Since we were unable to work with a contracting myocardium, we could only compare the diastolic pressure-volume effect of the PP with our data on the VP disruption. In addition, no study on the role of the PP in the porcine heart is available, which is of some concern since the structure and mechanics of the PP are species dependent (22
). Data on PP removal are primarily from canines and somewhat quantitatively variable depending on the laboratory and experimental methods. In an early study by Spotnitz and Kaiser (34
) on in vitro canine hearts, the ventricle stiffness [dP/dV (in mmHg/ml)] was 0.98 in control and 0.67 without the pericardium over very modest increases in ventricular volumes (from 0 to ~18 ml) and pressures (from ~0 to 15 mmHg), representing a 46% decrease in stiffness with the removal of the PP. This effect in the in vitro canine heart was comparable with what was observed at low volumes with the removal of the VP, which also decreased the stiffness by ~50% (see ). However, the removal of the PP has been shown to dramatically reduce the stiffness of the myocardium at very high volumes in numerous studies (9
), which was not observed in this study (see and ). This comparison implies that the VP is important in the elastic component of the myocardium, as indicated by the effects at low ventricular volumes, its dramatic compression on dissection, and the impact on opening angle of the myocardial slices, but has little impact on the limitation of ventricular volume compared with the myocardium itself or the PP in vivo. Currently, no information is available on the role of the encasing VP on the LV and RV pressure interactions that have been well characterized as a consequence of an intact PP (9
The endocardium was compared with the structure of the VP using two-photon excitation and classical histological approaches. Due to the highly irregular surface of the endocardium and its location, we did not attempt to strip this layer in our opening angle experiments or mechanically uncouple it in the intact heart experiments. However, the structures of the two layers were similar with more elastin and less collagen found in the endocardial layers (see –), consistent with the more extensive opening angles found with endocardial sections than with epicardial sections by Omens et al. (24
), which we confirmed in this study (data not shown). Thus, the endocardial layer of elastin may also contribute to the net residual strain of the myocardium; however, we were not able to confirm this directly due to the inability to dissect this layer free from the wall.
The optical properties of the VP present some interesting challenges for using a variety of optical techniques to examine the intact heart. The strong fluorescence of VP elastin significantly interferes with the detection of NAD(P)H or any blue fluorescence from the surface of the heart using conventional one-photon excitation schemes. The spectral emission of the elastin is also essentially identical to tissue NAD(P)H fluorescence (), providing no spectral analysis solution. The strong UV absorbance of the VP acts as a very significant primary filter that severely limits the penetration of UV light to myocytes in confocal experiments on extracted tissues blocks (). With regard to two-photon excitation, we suggest that the scattering properties of collagen severely disrupted the point-spread function of the infrared light as a function of depth through the VP. This notion was supported by measurements of transmission characteristics of the dissected VP that revealed a strong light-scattering spectrum (). In, addition we found that we could consistently optically penetrate to the myocytes from the endocardial surface but less frequently from the epicardial surface, which was consistent with the relative collagen content of these two structures. Thus, the scattering and emission properties of the VP will generate significant problems with regard to obtaining optical signals from cardiac myocytes, in vivo.
Fig. 9 A: elastin and NAD(P)H fluorescence collected using the META detector of the Zeiss LSM 510 microscope. The similar absorbance spectra caused difficulty in optically separating the emissions. In B, the scattering spectrum of the VP layer, once it had been (more ...)
In conclusion, the microstructures of macromolecules of the endocardium and epicardium have been determined. The VP was found to significantly contribute to the residual strain of the myocardium but not impact overall passive properties of the intact myocardium to the same extent as the PP. The VP apparently has its largest effect on elastic components of the heart at low ventricular volumes, or early diastole. We suggest that the composition and mechanical properties of the VP should be considered in mechanical models of myocardium function and, more importantly, in a variety of clinical conditions with demonstrated alterations in diastolic function that might be reflecting changes in this diminutive structure.