Jeffrey W. Holmes

Chronic Unloading by Left Ventricular Assist Device Reverses Contractile Dysfunction and Alters Gene Expression in End-Stage Heart Failure

BackgroundLeft ventricular (LV) assist devices (LVADs) can improve contractile strength and normalize characteristics of the Ca2+ transient in myocytes isolated from failing human hearts. The purpose of the present study was to determine whether LVAD support also improves contractile strength at different frequencies of contraction (the force-frequency relationship [FFR]) of intact myocardium and alters the expression of genes encoding for proteins involved in Ca2+ handling. Methods and ResultsThe isometric FFRs of LV trabeculae isolated from 15 patients with end-stage heart failure were compared with those of 7 LVAD-supported patients and demonstrated improved contractile force at 1-Hz stimulation, with reversal of a negative FFR after LVAD implantation. In 20 failing hearts, Northern blot analysis for sarcoplasmic endoreticular Ca2+-ATPase subtype 2a (SERCA2a), the ryanodine receptor, and the sarcolemmal Na+-Ca2+ exchanger was performed on LV tissue obtained before and after LVAD implantation. These paired data demonstrated an upregulation of all 3 genes after LVAD support. In tissue obtained from subsets of these patients, Western blot analysis was performed, and oxalate-supported Ca2+ uptake by isolated sarcoplasmic reticular membranes was determined. Despite higher mRNA for all genes after LVAD support, only SERCA2a protein was increased. Functional significance of increased SERCA2a was confirmed by augmented Ca2+ uptake by sarcoplasmic reticular membranes isolated from LVAD-supported hearts. ConclusionsLVAD support can improve contractile strength of intact myocardium and reverse the negative FFR associated with end-stage heart failure. The expression of genes encoding for proteins involved in Ca2+ cycling is upregulated (reverse molecular remodeling), but only the protein content of SERCA2a is increased.

Mechanical load initiates hypertrophic scar formation through decreased cellular apoptosis

Hypertrophic scars occur following cutaneous wounding and result in severe functional and esthetic defects. The pathophysiology of this process remains unknown. Here, we demonstrate for the first time that mechanical stress applied to a healing wound is sufficient to produce hypertrophic scars in mice. The resulting scars are histopathologically identical to human hypertrophic scars and persist for more than six months following a brief (one‐week) period of augmented mechanical stress during the proliferative phase of wound healing. Resulting scars are structurally identical to human hypertrophic scars and showed dramatic increases in volume (20‐fold) and cellular density (20‐fold). The increased cellularity is accompanied by a four‐fold decrease in cellular apoptosis and increased activation of the prosurvival marker Akt. To clarify the importance of apoptosis in hypertrophic scar formation, we examine the effects of mechanical loading on cutaneous wounds of animals with altered pathways of cellular apoptosis. In p53‐null mice, with down‐regulated cellular apoptosis, we observe significantly greater scar hypertrophy and cellular density. Conversely, scar hypertrophy and cellular density are significantly reduced in proapoptotic BclII‐null mice. We conclude that mechanical loading early in the prolifer‐ative phase of wound healing produces hypertrophic scars by inhibiting cellular apoptosis through an Akt‐dependent mechanism.—Aarabi S., Bhatt, K. A., Shi, Y., Paterno, J., Chang, E. I., Loh, S. A., Holmes, J. W., Longaker, M. T., Yee, H., Gurtner G. C. Mechanical load initiates hypertrophic scar formation through decreased cellular apoptosis. FASEB J. 21, 3250–3261 (2007)

Contribution of extracellular matrix to the mechanical properties of the heart

Extracellular matrix (ECM) components play essential roles in development, remodeling, and signaling in the cardiovascular system. They are also important in determining the mechanics of blood vessels, valves, pericardium, and myocardium. The goal of this brief review is to summarize available information regarding the mechanical contributions of ECM in the myocardium. Fibrillar collagen, elastin, and proteoglycans all play crucial mechanical roles in many tissues in the body generally and in the cardiovascular system specifically. The myocardium contains all three components, but their mechanical contributions are relatively poorly understood. Most studies of ECM contributions to myocardial mechanics have focused on collagen, but quantitative prediction of mechanical properties of the myocardium, or changes in those properties with disease, from measured tissue structure is not yet possible. Circumstantial evidence suggests that the mechanics of cardiac elastin and proteoglycans merit further study. Work in other tissues used a combination of correlation, modification or digestion, and mathematical modeling to establish mechanical roles for specific ECM components; this work can provide guidance for new experiments and modeling studies in myocardium.

Creating alignment and anisotropy in engineered heart tissue: role of boundary conditions in a model three-dimensional culture system.

Electrical and mechanical anisotropy arise from matrix and cellular alignment in native myocardium. Generation of anisotropy in engineered heart tissue will be required to match native properties and will provide immediate opportunities to investigate the genesis and structural determinants of functional anisotropy. We investigated the influence of geometry and boundary conditions on fibroblast alignment in thin collagen gels. Consistent with previous reports, we found that human dermal fibroblasts align parallel to free edges in partially constrained gels; in contrast to at least one report, fibroblasts in fully constrained gels remained randomly aligned independent of geometry. These experiments allowed us to distinguish between two possible mechanisms for such alignment. Mean orientations that followed the shape of the free edges and stronger alignment nearest the free edges in gels with a variety of geometries suggested that cells align parallel to a local free boundary rather than to local lines of tension. These findings focus attention on the presence of voids and free surfaces such as the endocardium and epicardium, cleavage planes, and blood vessels in governing cell and fiber alignment in developing and remodeling myocardium, myocardial scar tissue, and engineered heart constructs.

LV volume quantification via spatiotemporal analysis of real-time 3-D echocardiography

This paper presents a method of four-dimensional (4-D) (3-D+Time) space-frequency analysis for directional denoising and enhancement of real-time three-dimensional (RT3D) ultrasound and quantitative measures in diagnostic cardiac ultrasound. Expansion of echocardiographic volumes is performed with complex exponential wavelet-like basis functions called brushlets. These functions offer good localization in time and frequency and decompose a signal into distinct patterns of oriented harmonics, which are invariant to intensity and contrast range. Deformable-model segmentation is carried out on denoised data after thresholding of transform coefficients. This process attenuates speckle noise while preserving cardiac structure location. The superiority of 4-D over 3-D analysis for decorrelating additive white noise and multiplicative speckle noise on a 4-D phantom volume expanding in time is demonstrated. Quantitative validation, computed for contours and volumes, is performed on in vitro balloon phantoms. Clinical applications of this spatiotemporal analysis tool are reported for six patient cases providing measures of left ventricular volumes and ejection fraction.

The Development of Structural and Mechanical Anisotropy in Fibroblast Populated Collagen Gels

An in vitro model system was developed to study structure-function relationships and the development of structural and mechanical anisotropy in collagenous tissues. Fibroblast-populated collagen gels were constrained either biaxially or uniaxially. Gel remodeling, biaxial mechanical properties, and collagen orientation were determined after 72 h of culture. Collagen gels contracted spontaneously in the unconstrained direction, uniaxial mechanical constraints produced structural anisotropy, and this structural anisotropy was associated with mechanical anisotropy. Cardiac and tendon fibroblasts were compared to test the hypothesis that tendon fibroblasts should generate greater anisotropy in vitro. However, no differences were seen in either structure or mechanics of collagen gels populated with these two cell types, or between fibroblast populated gels and acellular gels. This study demonstrates our ability to control and measure the development of structural and mechanical anisotropy due to imposed mechanical constraints in a fibroblast-populated collagen gel model system. While imposed constraints were required for the development of anisotropy in this system, active remodeling of the gel by fibroblasts was not. This model system will provide a basis for investigating structure-function relationships in engineered constructs and for studying mechanisms underlying the development of anisotropy in collagenous tissues.

Theoretical Quality Assessment of Myocardial Elastography with In Vivo Validation

Myocardial elastography (ME), a radio frequency (RF)-based speckle tracking technique with one-dimensional (1-D) cross correlation and novel recorrelation methods in a 2-D search was proposed to estimate and fully image 2-1) transmural deformation field and to detect abnormal cardiac function. A theoretical framework was first developed in order to evaluate the performance of 2-D myocardial elastography based on a previously developed 3-D finite-element model of the canine left ventricle. A normal (control) and an ischemic (left-circumflex, LCx) model, which more completely represented myocardial deformation than a kinematic model, were considered. A 2-D convolu-tional image formation model was first used to generate RF signals for quality assessment of ME in the normal and ischemic cases. A 3-D image formation model was further developed to investigate the effect of the out-of-plane motion on the 2-D, in-plane motion estimation. Both orthogonal, in-plane displacement components (i.e., lateral and axial) between consecutive RF frames were iteratively estimated. All the estimated incremental 2-D displacements from end-diastole (ED) to end-systole (ES) were then accumulated to acquire the cumulative 2-D displacements, which were further used to calculate the cumulative 2-D systolic finite strains. Furthermore, the cumulative systolic radial and circumferential strains, which were angle-and frame-rate independent, were obtained from the 2-D finite-strain components and imaged in full view to detect the ischemic region. We also explored the theoretical understanding of the limitations of our technique for the accurate depiction of disease and validated it in vivo against tagged magnetic resonance imaging (tMRI) in the case of a normal human myocardium in a 2-D short-axis (SA) echocardiographic view. The theoretical framework succeeded in demonstrating that the 2-D myocardial elastography technique was a reliable tool for the complete estimation and depiction of the in-plane myocardial deformation field as well as for accurate identification of pathological mechanical function using established finite-element, left-ventricular canine models. In a preliminary study, the 2-D myocardial elastography was shown capable of imaging myocardial deformation comparable to equivalent tMRI estimates in a clinical setting.

Evolution of scar structure, mechanics, and ventricular function after myocardial infarction in the rat

The mechanical properties of the healing scar are an important determinant of heart function following myocardial infarction. Yet the relationship between scar structure, scar mechanics, and ventricular function remains poorly understood, in part because no published study has tracked all of these factors simultaneously in any animal model. We therefore studied the temporal evolution of scar structure, scar mechanics, and left ventricular (LV) function in large anterior myocardial infarcts in rats. At 1, 2, 3, and 6 wk after left anterior descending coronary ligation, we examined LV function using sonomicrometry, infarct mechanical properties using biaxial mechanical testing, infarct structure using polarized light microscopy, and scar collagen content and cross-linking using biochemical assays. Healing infarcts in the rat were structurally and mechanically isotropic at all time points. Collagen content increased with time and was the primary determinant of scar mechanical properties. The presence of healing infarcts influenced systolic LV function through a rightward shift of the end-systolic pressure-volume relationship (ESPVR) that depended on infarct size, infarct collagen content, and LV dilation. We conclude that in sharp contrast to previous reports in large animal models, healing infarcts are structurally and mechanically isotropic in the standard rat model of myocardial infarction. On the basis of the regional strain patterns we observed in healing rat infarcts in this study and in healing pig infarcts in previous studies, we hypothesize that the local pattern of stretching determines collagen alignment in healing myocardial infarct scars.

Impact of Mechanical Activation, Scar, and Electrical Timing on Cardiac Resynchronization Therapy Response and Clinical Outcomes

OBJECTIVES Using cardiac magnetic resonance (CMR), we sought to evaluate the relative influences of mechanical, electrical, and scar properties at the left ventricular lead position (LVLP) on cardiac resynchronization therapy (CRT) response and clinical events. BACKGROUND CMR cine displacement encoding with stimulated echoes (DENSE) provides high-quality strain for overall dyssynchrony (circumferential uniformity ratio estimate [CURE] 0 to 1) and timing of onset of circumferential contraction at the LVLP. CMR DENSE, late gadolinium enhancement, and electrical timing together could improve upon other imaging modalities for evaluating the optimal LVLP. METHODS Patients had complete CMR studies and echocardiography before CRT. CRT response was defined as a 15% reduction in left ventricular end-systolic volume. Electrical activation was assessed as the time from QRS onset to LVLP electrogram (QLV). Patients were then followed for clinical events. RESULTS In 75 patients, multivariable logistic modeling accurately identified the 40 patients (53%) with CRT response (area under the curve: 0.95 [p < 0.0001]) based on CURE (odds ratio [OR]: 2.59/0.1 decrease), delayed circumferential contraction onset at LVLP (OR: 6.55), absent LVLP scar (OR: 14.9), and QLV (OR: 1.31/10 ms increase). The 33% of patients with CURE <0.70, absence of LVLP scar, and delayed LVLP contraction onset had a 100% response rate, whereas those with CURE ≥0.70 had a 0% CRT response rate and a 12-fold increased risk of death; the remaining patients had a mixed response profile. CONCLUSIONS Mechanical, electrical, and scar properties at the LVLP together with CMR mechanical dyssynchrony are strongly associated with echocardiographic CRT response and clinical events after CRT. Modeling these findings holds promise for improving CRT outcomes.

Scar remodeling and transmural deformation after infarction in the pig.

Changes in stress and tissue material properties have been proposed as important mechanical factors that may influence infarct expansion and subsequent healing. Because such changes will be reflected by alterations in the finite deformation of the tissue, we examined the direction and magnitude of myocardial deformation after coronary ligation in the pig. Methods and ResultsGold beads were implanted in the left ventricular free wall of five pigs. After ligation of the coronary supply to the region containing the markers, we used biplane cineradiography to reconstruct the three-dimensional deformations of the myocardium during single cardiac cycles as well as the remodeling deformations that occurred over time. Deformations were studied at 1 and 3 weeks after infarction. The analysis of single cardiac cycles revealed permanent loss of systolic shortening immediately after ligation. However, significant passive systolic wall thickening (P < .001) and large shears were observed at 3 weeks in regions composed almost entirely of collagen. The analysis of remodeling deformations at 1 week revealed infarct expansion with a predominant axis that varied widely. At 3 weeks, a 30% to 60% reduction in local tissue volume was measured in the infarct region, with the principal direction of scar shrinkage nearly circumferential in all animals (range, −2° to 35°). ConclusionsWe conclude that infarct expansion and scar shrinkage may be controlled by different factors. In addition, we conclude that measurement of systolic wall thickening alone is not always adequate to assess postinfarction regional contractile function.