Sinoatrial Node Structure, Mechanics, Electrophysiology and the Chronotropic Response to Stretch in Rabbit and Mouse.

The rhythmic electrical activity of the heart's natural pacemaker, the sinoatrial node (SAN), determines cardiac beating rate (BR). SAN electrical activity is tightly controlled by multiple factors, including tissue stretch, which may contribute to adaptation of BR to changes in venous return. In most animals, including human, there is a robust increase in BR when the SAN is stretched. However, the chronotropic response to sustained stretch differs in mouse SAN, where it causes variable responses, including decreased BR. The reasons for this species difference are unclear. They are thought to relate to dissimilarities in SAN electrophysiology (particularly action potential morphology) between mouse and other species and to how these interact with subcellular stretch-activated mechanisms. Furthermore, species-related differences in structural and mechanical properties of the SAN may influence the chronotropic response to SAN stretch. Here we assess (i) how the BR response to sustained stretch of rabbit and mouse isolated SAN relates to tissue stiffness, (ii) whether structural differences could account for observed differences in BR responsiveness to stretch, and (iii) whether pharmacological modification of mouse SAN electrophysiology alters stretch-induced chronotropy. We found disparities in the relationship between SAN stiffness and the magnitude of the chronotropic response to stretch between rabbit and mouse along with differences in SAN collagen structure, alignment, and changes with stretch. We further observed that pharmacological modification to prolong mouse SAN action potential plateau duration rectified the direction of BR changes during sustained stretch, resulting in a positive chronotropic response akin to that of other species. Overall, our results suggest that structural, mechanical, and background electrophysiological properties of the SAN influence the chronotropic response to stretch. Improved insight into the biophysical determinants of stretch effects on SAN pacemaking is essential for a comprehensive understanding of SAN regulation with important implications for studies of SAN physiology and its dysfunction, such as in the aging and fibrotic heart.

Mitral valve leaflet remodelling during pregnancy: insights into cell-mediated recovery of tissue homeostasis.

Little is known about how valvular tissues grow and remodel in response to altered loading. In this work, we used the pregnancy state to represent a non-pathological cardiac volume overload that distends the mitral valve (MV), using both extant and new experimental data and a modified form of our MV structural constitutive model. We determined that there was an initial period of permanent set-like deformation where no remodelling occurs, followed by a remodelling phase that resulted in near-complete restoration of homeostatic tissue-level behaviour. In addition, we observed that changes in the underlying MV interstitial cell (MVIC) geometry closely paralleled the tissue-level remodelling events, undergoing an initial passive perturbation followed by a gradual recovery to the pre-pregnant state. Collectively, these results suggest that valvular remodelling is actively mediated by average MVIC deformations (i.e. not cycle to cycle, but over a period of weeks). Moreover, tissue-level remodelling is likely to be accomplished by serial and parallel additions of fibrillar material to restore the mean homeostatic fibre stress and MVIC geometries. This finding has significant implications in efforts to understand and predict MV growth and remodelling following such events as myocardial infarction and surgical repair, which also place the valve under altered loading conditions.

Alkyne Ligation Handles: Propargylation of Hydroxyl, Sulfhydryl, Amino, and Carboxyl Groups via the Nicholas Reaction.

The Nicholas reaction has been applied to the installation of alkyne ligation handles. Acid-promoted propargylation of hydroxyl, sulfhydryl, amino, and carboxyl groups using dicobalt hexacarbonyl-stabilized propargylium ions is reported. This method is useful for introduction of propargyl groups into base-sensitive molecules, thereby expanding the toolbox of methods for the incorporation of alkynes for bio-orthogonal reactions. High-value molecules are used as the limiting reagent, and various propargylium ion precursors are compared.

Pregnancy-induced remodeling of heart valves.

Recent studies have demonstrated remodeling of aortic and mitral valves leaflets under the volume loading and cardiac expansion of pregnancy. Those valves' leaflets enlarge with altered collagen fiber architecture, content, and cross-linking and biphasic changes (decreases, then increases) in extensibility during gestation. This study extends our analyses to right-sided valves, with additional compositional measurements for all valves. Valve leaflets were harvested from nonpregnant heifers and pregnant cows. Leaflet structure was characterized by leaflet dimensions, and ECM composition was determined using standard biochemical assays. Histological studies assessed changes in cellular and ECM components. Leaflet mechanical properties were assessed using equibiaxial mechanical testing. Collagen thermal stability and cross-linking were assessed using denaturation and hydrothermal isometric tension tests. Pulmonary and tricuspid leaflet areas increased during pregnancy by 35 and 55%, respectively. Leaflet thickness increased by 20% only in the pulmonary valve and largely in the fibrosa (30% thickening). Collagen crimp length was reduced in both the tricuspid (61%) and pulmonary (42%) valves, with loss of crimped area in the pulmonary valve. Thermomechanics showed decreased collagen thermal stability with surprisingly maintained cross-link maturity. The pulmonary leaflet exhibited the biphasic change in extensibility seen in left side valves, whereas the tricuspid leaflet mechanics remained largely unchanged throughout pregnancy. The tricuspid valve exhibits a remodeling response during pregnancy that is significantly diminished from the other three valves. All valves of the heart remodel in pregnancy in a manner distinct from cardiac pathology, with much similarity valve to valve, but with interesting valve-specific responses in the aortic and tricuspid valves.

Conditions for a Rh(I)-catalyzed [2 + 2 + 1] cycloaddition reaction with methyl substituted allenes and alkynes.

The direct installation of the C4 and C10 methyl groups present in the 6,12-guaianolide framework using a Rh(I)-catalyzed cyclocarbonylation reaction of methyl subsituted allenes and alkynes is described. High yields of bicyclo[5.3.0]decanes are afforded when low reaction concentrations involving syringe pump addition of the allene-yne to the catalyst are used.

Biaxial Creep Resistance and Structural Remodeling of the Aortic and Mitral Valves in Pregnancy.

Pregnancy produces rapid, dramatic volume-overload changes to the maternal circulation. This paper examines pregnancy-induced structural-mechanical changes in bovine aortic and mitral heart valve leaflets. Valve leaflets were harvested from non-pregnant heifers and pregnant cows. Dimensions, biaxial extensibility and creep resistance were assessed and related to changes in the collagen network: histological leaflet and anatomic layer thicknesses plus collagen crimp, and biochemical collagen content. Collagen stability and crosslinking were assessed thermomechanically. Pregnancy altered both aortic and mitral valve leaflets. Both valves demonstrated biphasic changes in leaflet stretch, decreasing in early pregnancy and recovering by late pregnancy. Creep in leaflets from both valves was minimal and decreased even further with pregnancy in the mitral valve. There were valve-specific changes in preconditioning areal extension with pregnancy: increasing in the aortic valve and decreasing in the mitral valve. Leaflet area increased dramatically (84% aortic, 56% mitral), with thickening mainly in the fibrosa, accompanied by increases in collagen content (8% aortic, 16% mitral): together suggesting synthesis of new collagen. Collagen crimp was almost completely lost in pregnancy, with the denaturation temperature decreased by approximately 2 °C. Mature and total crosslinking increased, curiously without a significant increase in immature crosslinking. Mature aortic and mitral heart valve leaflets in the maternal cardiovascular system remodel substantially and similarly-despite their different embryological origins.

Pregnancy-induced remodeling of collagen architecture and content in the mitral valve.

Pregnancy produces rapid, non-pathological volume-overload in the maternal circulation due to the demands of the growing fetus. Using a bovine model for human pregnancy, previous work in our laboratory has shown remarkable pregnancy-induced changes in leaflet size and mechanics of the mitral valve. The present study sought to relate these changes to structural alterations in the collagenous leaflet matrix. Anterior mitral valve leaflets were harvested from non-pregnant heifers and pregnant cows (pregnancy stage estimated by fetal length). We measured changes in the thickness of the leaflet and its anatomic layers via Verhoeff-Van Gieson staining, and in collagen crimp (wavelength and percent collagen crimped) via picrosirius red staining and polarized microscopy. Collagen concentration was determined biochemically: hydroxyproline assay for total collagen and pepsin-acid extraction for uncrosslinked collagen. Small-angle light scattering (SALS) assessed changes in internal fiber architecture (characterized by degree of fiber alignment and preferred fiber direction). Pregnancy produced significant changes to collagen structure in the mitral valve. Fiber alignment decreased 17% with an 11.5° rotation of fiber orientation toward the radial axis. Collagen fiber crimp was dramatically lost, accompanied by a 53% thickening of the fibrosa, and a 16% increase in total collagen concentration, both suggesting that new collagen is being synthesized. Extractable collagen concentration was low, both in the non-pregnant and pregnant state, suggesting early crosslinking of newly-synthesized collagen. This study has shown that the mitral valve is strongly adaptive during pregnancy, with significant changes in size, collagen content and architecture in response to rapidly changing demands.

Physiological remodeling of the mitral valve during pregnancy.

There is growing evidence that heart valves are not passive structures but can remodel with left ventricular dysfunction. To determine if these tissues remodel under nonpathological conditions, we examined the mirtal valve anterior leaflet during the volume loading and cardiac expansion of pregnancy using a bovine model. We measured leaflet dimensions, chordal attachments, and biaxial mechanical properties of leaflets collected from never-pregnant heifers and pregnant cows (pregnancy duration estimated from fetal length). Hydrothermal isometric tension (HIT) tests were performed to assess the denaturation temperature (T(d)) associated with collagen molecular stability and the load decay half-time (t(1//2)) associated with intermolecular cross-linking. Histological changes were examined using Verhoeff-van Gieson and picrosirius red staining with polarized light. We observed striking changes to the structure and material properties of the mitral anterior leaflet during pregnancy. Leaflet area was increased 33%, with a surprising increase (nearly 25%) in chordae tendinae attachments. There was a biphasic change in leaflet extensibility: it rapidly decreased by 30% and then reversed to prepregnant values by late pregnancy. The 2°C decrease in T(d) in pregnancy was indicative of collagen remodeling, whereas the 70% increase in HIT t(1/2) indicated an increase in collagen cross-linking. Finally, histological results suggested transient increases in leaflet thickness and transient decreases in collagen crimp. This remodeling may compensate for the increased loading conditions associated with pregnancy by normalizing leaflet stress and maintaining coaptation. Understanding the mechanisms of mitral valve physiological remodeling in pregnancy could contribute to alternative treatments of pathological remodeling associated with left ventricular dysfunction.

Differential changes in the molecular stability of collagen from the pulmonary and aortic valves during the fetal-to-neonatal transition.

During the fetal-to-neonatal transition, transvalvular pressures (TVPs) on the aortic and pulmonary valves change dramatically-but differently for each valve. We have examined changes in the molecular stability and crosslinking of collagen during this transition. Aortic and pulmonary valves were harvested from fetal and neonatal cattle. Using differential scanning calorimetry (DSC), denaturation of valvular collagen was examined and, using HPLC, the types and quantities of enzymatic crosslinks were examined. No difference in hydrothermal stability was found between the collagens in the fetal aortic and pulmonary valves; this was expected since the TVP is approximately the same across both valves before birth. Only in the neonatal samples was the collagen from aortic valves (higher TVP) less stable than that from pulmonary valves (lower TVP). Surprisingly, the enthalpy of denaturation did not differ either between valve type or with age, suggesting an entropic mechanism of altered molecular stability. A significant difference in immature-to-mature crosslink ratio was found between neonatal aortic and pulmonary valves: a difference absent in fetal valves. This ratio-indicative of remodeling rate-parallels (and may be a function of) the changing in vivo load. This study highlights the relationship between in vivo load and both (i) molecular stability and (ii) collagen remodeling in heart valves.

Differential biomechanical development of elastic tissues in the bovine fetus.

Mechanical loading conditions are important factors in the gestational development of fetal tissues. However, little is known about how mechanical loading during development modulates the structure and function of elastic tissues. We hypothesized that developing elastic tissues functionally adapt to their loading conditions. To test this hypothesis, we assessed the changes in the composition, viscoelasticity, and thermoelastic properties of elastic tissue from bovine aortas (functional during gestation) and nuchal ligaments (nonfunctional during gestation). Clear differences in the developmental timeline of elastic tissue structure and function were observed between aortic and ligament elastic tissue. Elastic tissue in the aorta developed earlier than that of the nuchal ligament, indicating a role for loading conditions in the timeline of development. Ligament elastic tissue, however, underwent rapid remodeling in late gestation-likely as a preadaptation to the sudden-onset of tensile load it experiences at birth. Finally, while the same fundamental structure-mechanical relationships were seen in both tissues, there was a clear difference in mechanical properties between the elastic tissues from the adult nuchal ligament and the adult aorta, indicating that postnatal loading conditions continue to influence tissue structure and mechanical properties, tailoring them to their functional roles in adult life.

Changes in the mechanical properties and residual strain of elastic tissue in the developing fetal aorta.

Formed almost exclusively during development, arterial elastic fibers must function for the lifetime of the animal. We have observed dramatic structural and mechanical changes in aortic elastic tissue during gestational and postnatal development. Elastic tissue was isolated from bovine aortas: (i) during late pregnancy and (ii) in adults. Changes in the relative content of aortic elastic tissue were assessed, as were the viscoelastic properties and residual strains of purified aortic elastic tissue rings. As aortic elastic tissue content increased during development, its circumference and thickness increased-but with circumference rising faster than wall thickness, causing a relative thinning of the elastic tissue. At the same time, elastic tissue stiffness increased while viscoelastic behavior decreased. Much of these changes were concentrated during late gestational development, such that the changes observed during the short span of late gestation examined (~60 days) were similar in magnitude to those occurring over the much longer postnatal period (approximately 1-2 years). Finally, we observed an approximately threefold increase in residual strain in aortic elastic tissue from fetal to adult life, with most of this increase again occurring in late gestation. These results suggest that rapid remodeling, as well as accumulation, of aortic elastic tissue occurs during late gestation. These changes significantly alter both fetal aortic mechanical properties and residual stresses.

Ramped versus stepwise thermoelastic testing of latex and elastic tissues.

Thermoelastic testing assesses the elastic mechanisms of polymers through measurement of the retractive force (f) of constrained samples with increasing temperature (T). f contains an entropic (fs) and an internal energy component (fe), where f= fs +fe. The elastic mechanism is normally described by the energetic contribution (fe/f). We have produced a novel thermoelastic testing device capable of performing "stepwise" or "ramped" temperature profiles and have shown excellent agreement between these two techniques for both latex and bovine elastin. Experiments on latex produced an fe/f= 0.18 +/- 0.05 (mean +/-SD, n=15, ramped protocol) that was independent of extension ratio and temperature. These results demonstrate the highly entropic elastic mechanism in this well-defined material. In agreement with previous studies, thef-T curves for elastin were non-linear, leveling off above approximately 60 degrees C. Previous studies quote fe/f for elastin within the 50-70 degrees C range where volume changes (via loss of water) of elastin are thought to be negligible. While we observed a mean fe/f for elastin of 0.18 +/- 0.04 at 70 degrees C (not significantly different from that of latex), the fe/f values for elastin were highly temperature-dependent over the entire experimental temperature range (20-90 degrees C). These observations may reflect a continuous water loss with increasing temperature in our samples. However, since thermoelastic analysis assumes that force depends only on temperature, other complicating factors must also be considered: e.g. thermal transitions such as microfibril denaturation. These complications call into question the physical meaning of fe/f reported for elastin at any temperature.

Cyclic loading response of bioprosthetic heart valves: effects of fixation stress state on the collagen fiber architecture.

Biologically derived, chemically modified collagenous tissues are being increasingly used to fabricate cardiac valve prostheses and as biomaterials in cardiovascular repair. A stress-free state during chemical modification has been shown to preserve the collagen fiber architecture of the native tissue, potentially preserving native mechanical properties and improving prostheses durability. However, it is not known if the native collagen fiber architecture is stable during long-term in vivo operation. To address this question, we obtained porcine aortic valves chemically treated at (i) 0 mmHg transvalvular pressure (with 40 mmHg aortic pressure) and (ii) 4 mmHg transvalvular pressure, then subjected the valves to 0, 1 x 10(6), 50 x 10(6), and 200 x 10(6) in vitro accelerated wear testing (AWT) cycles. The resulting changes in collagen fiber architecture were quantified using small angle light scattering analysis (SALS). SALS measurements indicated that collagen fibers in the 0 mmHg pressure-fixed leaflets became more aligned between 1 x 10(6) and 50 x 10(6) AWT cycles. In contrast, only minor changes (not statistically significant) in collagen fiber orientation occurred in the 4 mmHg pressure-fixed valvular tissue with cycling. It was also noted that although the 0 mmHg group was fixed without transvalvular pressure, distention of the root induced significant changes in collagen structure of the leaflets. Overall, our observations suggest that the native collagen fiber crimp of the 0 mmHg pressure-fixed leaflets were rapidly lost after only 50 x 10(6) AWT cycles (equivalent to approximately 1.6 patient years) and thus may not be maintained over a sufficient period of time to be clinically beneficial. Further, the collagen structure of the native aortic valve is exquisitely sensitive to dimensional change in the aortic root-independent of the presence of transvalvular pressure. Our findings also suggest that without in vivo remodeling, any collagenous tissue used to fabricate BHV may undergo similar degenerative, irreversible changes in vivo.

Effects of fixation pressure on the biaxial mechanical behavior of porcine bioprosthetic heart valves with long-term cyclic loading.

Zero transvalvular pressure fixation is thought to improve porcine bioprosthetic heart valve (BHV) durability by preserving the collagen fiber architecture of the native tissue, and thereby native mechanical properties. However, it is not known if the native mechanical properties are stable during long-term valve operation and thus provide additional durability. To address this question, we examined the biaxial mechanical properties of porcine BHV fixed at 0 and 4mmHg transvalvular pressure following 0, 1 x 10(6), 50 x 10(6), and 200 x 10(6) in vitro accelerated test cycles. At 0 cycles, the extensibility and degree of axial cross-coupling of the zero-pressure-fixed cusps were higher than those of the low-pressure-fixed cusps. Furthermore, extensibility of the zero-pressure-fixed tissue decreased between 1 x 10(6) and 50 x 10(6) cycles, approaching that of the low-pressure-fixed tissue, whose extensibility was unchanged over 0-200 x 10(6) cycles. The decrease in extensibility of the zero-pressure-fixed tissue between 1 x 10(6) and 50 x 10(6) cycles may be attributable to the ability of its collagen fibers to undergo larger changes in orientation and crimp with cyclic loading. These observations suggest that the collagen fiber architecture of the 0-mmHg-fixed porcine BHV, although locked in place by chemical fixation, may not be maintained over a sufficient number of cycles to be clinically beneficial. This study further underscores that chemically treated collagen fibers can undergo conformational changes under long-term cyclic loading not associated with damage.