Hydrothermal isometric tension (HIT) testing and high-performance liquid chromatography were used to assess the molecular stability and cross-link population of collagen in the four valves of the adult bovine heart. Untreated and NaBH(4)-treated tissues under isometric tension were heated in a water bath to a 90 degrees C isotherm that was sustained for 5 h. The denaturation temperature (T(d)), associated with hydrogen bond rupture and molecular stability, and the half-time of load decay (t(1/2)), associated with peptide bond hydrolysis and intermolecular cross-linking, were calculated from acquired load/temperature/time data. An unpaired group of samples of the same population was biochemically assayed for the types and quantities of enzymatic cross-links present. Tissues known to endure higher in vivo transvalvular pressures had lower T(d) values, suggesting that molecular stability is inversely related to in vivo loading. The treated inflow valves (mitral and tricuspid) had significantly lower t(1/2) values than did treated outflow valves (aortic and pulmonary), suggesting lower overall cross-linking in the inflow valves. Inflow valves were also found to fail during HIT testing significantly more often than outflow valves, also suggestive of a decreased cross-link population. Inflow valves may be remodeling at a faster rate and may be at an earlier state of molecular "maturity" than outflow valves. At the molecular level, the thermal stability of collagen is associated with in vivo loading and may be influenced by the mature, aldimine-derived cross-link, histidinohydroxylysinonorleucine. We conclude that the valves of the heart utilize differing, location-specific strategies to resist biomechanical fatigue loading.

Soft tissue injuries are poorly understood at the molecular level. Previous work using differential scanning calorimetry (DSC) has shown that tendon collagen becomes less thermally stable with rupture. However, most soft tissue injuries do not result in complete tissue rupture but in damaging fiber overextension. Covalent crosslinking, which increases with animal maturity and age, plays an important role in collagenous fiber mechanics. It is also a determinant of tissue strength and is hypothesized to inhibit the loss of thermal stability of collagen due to mechanical damage. Controlled overextension without rupture was investigated to determine if overextension was sufficient to reduce the thermal stability of collagen in the bovine tail tendon (BTT) model and to examine the effects of aging on the phenomenon. Baseline data from DSC and hydrothermal isometric tension (HIT) techniques were compared between two groups: steers aged 24-30 months (young group), and skeletally mature bulls and oxen aged greater than five years (old group). Covalent crosslinks were quantified by ion exchange chromatography. Overextension resulted in reduced collagen thermal stability in the BTT model. The Young specimens, showing detectably lower tissue thermomechanical competence, lost more thermal stability with overextension than did the old specimens. The effect on old specimens, while smaller, was detectable. Multiple overextension cycles increased the loss of stability in the young group. Compositional differences in covalent crosslinking corresponded with tissue thermomechanical competence and therefore inversely with the loss of thermal stability. HIT testing gave thermal denaturation temperatures similar to those measured with DSC. The thermal stability of collagen was reduced by overextension of the tendon--without tissue rupture--and this effect was amplified by increased cycles of overextension. Increased tissue thermomechanical competence with aging seemed to mitigate the loss of collagen stability due to mechanical overextension. Surprisingly, the higher tissue thermomechanical competence did not directly correlate with the concentration of endogenous enzymatically derived covalent crosslinking on a mole per mole of collagen basis. It did, however, correlate with the percentage of mature and thermally stable crosslinks. Compositional changes in fibrous collagens that occur with aging affect fibrous collagen mechanics and partially determine the nature of mechanical damage at the intermolecular level. As techniques develop and improve, this new information may lead to important future studies concerning improved detection, prediction, and modeling of mechanical damage at much finer levels of tissue hierarchy than currently possible.

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.