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Ann Thorac Surg 2005;80:189-197
© 2005 The Society of Thoracic Surgeons

Original article: Cardiovascular

Structural Characterization of the Chordae Tendineae in Native Porcine Mitral Valves

Jennifer Ritchie, MSa, James N. Warnock, PhDb, Ajit P. Yoganathan, PhDa,*

a Wallace H. Coulter Department of Biomedical Engineering, Georgia Institute of Technology & Emory University, Atlanta, Georgia
b George W. Woodruff School of Mechanical Engineering, Georgia Institute of Technology, Atlanta, Georgia

Accepted for publication February 1, 2005.

* Address reprint requests to Dr Yoganathan, Wallace H. Coulter Department of Biomedical Engineering, Georgia Institute of Technology & Emory University, 313 Ferst Dr, Room 2119, Atlanta, GA 30332-0535 (Email: ajit.yoganathan{at}bme.gatech.edu).


   Abstract
Top
 Abstract
Introduction
 Material and Methods
 Results
 Comment
 Acknowledgments
 References
 
BACKGROUND: This study was aimed to characterize the different mitral valve chordae tendineae to provide additional understanding of their function.

METHODS: Mitral valve chordae tendineae from fresh porcine hearts were stained for collagen and elastin using either a Verhoeff and van Gieson stain or Verhoeff light green stain. Cellular distribution was determined using a hematoxylin and eosin stain. Immunohistochemistry was used to verify the findings of vasculature. Biochemical assays were performed to quantify DNA, collagen, and elastin content of each of the six different types of chordae tendineae.

RESULTS: Blood vessels were observed in the longitudinal and circumferential directions of the chordae. The strut chordae on the anterior leaflet of the mitral valve showed an increased degree of vascularization compared with the other chordae. All chordae had an inner layer characterized by a high concentration of collagen and an outer layer that was mostly elastin with interwoven collagen fibers. The collagen microstructure was characterized by directional crimping. Hematoxylin and eosin staining showed fibroblasts evenly distributed throughout the inner and outer layer of the chordae tendineae. Quantitative analysis showed significantly higher levels of DNA and collagen content in the anterior and posterior marginal chordae compared with the other chordae.

CONCLUSIONS: The chordae tendineae were seen to have different microstructures according to chordal type. The presence of vessels characterized the chordae tendineae as complex living components that work in coordination with the papillary muscles and mitral valve leaflets to prevent mitral valve prolapse and regurgitation. They may also function to supply nutrients to the valve leaflets.


   Introduction
Top
 Abstract
 Introduction
Material and Methods
 Results
 Comment
 Acknowledgments
 References
 
The mitral valve has a finely tuned closing mechanism composed of the anterior and posterior mitral leaflets, chordae tendineae, and papillary muscles that work together to ensure proper valve closure, hence, preventing mitral valve regurgitation. The chordae tendineae of the mitral valve function to transmit the contractions of the papillary muscles to the leaflets of the mitral valve complex. They also serve to secure the leaflets to maintain valve closure and prevent mitral valve prolapse. In order to perform these functions, the chords must contain a high degree of elasticity, as well as considerable strength and endurance. These tendinous chords are composed of collagen and elastin fibers arranged in parallel. A study conducted by Millington-Sanders and colleagues [1] showed that the chordae tendineae are composed of multiple layers of elastic fibers, an inner collagen core, and an outer layer of endothelial cells. The arrangement and morphology of the chordae tendineae can be classified according to their insertion sites (Fig 1)[2].



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  		Fig 1. Porcine mitral valve cut along the posterior leaflet showing the chordae insertion pattern. All six chordae shown here were used for the histologic and biochemical studies.

 
The chordae must withstand the large repetitive forces that are encountered in the left ventricle during the cardiac cycle. The arrangement of their elastic fibers and wavy collagen increases the mechanical properties of the chordae and allows them to withstand these forces. The wavy arrangement of collagen that is surrounded by elastic fibers is well adapted to the cyclic stresses to which the chordae are continuously subjected. This arrangement also provides a mechanism for smooth transfer of forces to the valve leaflets, thus protecting the structural components of the valve that have to withstand rapidly applied forces at systole. During stretching, when papillary muscle contraction straightens the chords, the collagenous wavy pattern disappears and transfers the peak stress during contraction to the leaflets. The sleeve-shaped elastic network that surrounds the internal collagen suggests that on relaxation of tension, the elastic fibers would tend to restore the collagen to their wavy configuration. This layer of elastic fibers also serves as a protective barrier between the collagen and the delicate layer of the endocardium.

Duran and Gunning [3] found that the chordae in fetuses and calves contained vessels running from the papillary muscles to the insertion site in the valve leaflet. Their study did not delineate which chordae contained vessels or the difference in the degree of vascularization. The study presented here focuses on the difference between the six types of chordae and the importance of their structure to their specific function during valve closure.

Mitral valve pathologies are serious conditions that cardiac surgeons are now trying to repair without replacing the mitral valve. Recent mitral repair procedures such as chordal cutting and chordal translocation have been used to correct mitral valve dysfunction and pathologies such as ischemic heart disease. The chordae were targeted in repair procedures because they had been characterized as simple collagenous structures that prevent mitral valve prolapse during systole and aid in ventricular function. Further understanding of the mechanical and histologic characteristics of the chordae tendineae is required to optimize these repair procedures. The objective of this study was to characterize the different chordae tendineae using histologic techniques and provide additional understanding of their function.


   Material and Methods
Top
 Abstract
 Introduction
 Material and Methods
Results
 Comment
 Acknowledgments
 References
 
Fresh porcine hearts were obtained from a local abattoir (Holifield’s Farm, Covington, GA) and transported to the laboratory on ice. Upon arrival the chordae tendineae were excised from the mitral valves according to insertion location. After extraction, the chordae tendineae were fixed in 10% neutral buffered formalin for 24 hours and then transferred to 70% ethanol until tissue processing. The tissue was processed in paraffin using the Shandon Pathcentre (Thermo Electron Corp, Waltham, MA), and then embedded in paraffin wax in both the longitudinal and radial directions. This allowed the tissue to be cut into 5 µm sections with a Microtome Microm HM355S and placed on specially treated slides (Superfrost, Plus, Shandon). The tissue sections where dried overnight in a 37°C incubator and then utilized for different staining protocols. Standard deparaffinization was performed on each slide before staining.

A standard hematoxylin and eosin stain was performed to determine cellular distribution. A Verhoeff stain followed by a van Gieson stain was performed to determine collagen, elastin, and cellular distribution. A Verhoeff stain followed by a light green stain allowed for observing elastin distribution. After de-paraffinization and rehydration to distilled water, the sections were placed in Verhoeff’s elastin stain [4] until the sections appeared jet black. Ferric chloride was used to microscopically differentiate the sections until the elastic fibers were distinct and the background was colorless. The sections were placed in sodium thiosulfate to remove the iodine. Depending on the stain, the sections were then counterstained either with van Gieson’s stain or light green. Due to the picric acid contained in van Gieson’s stain, the elastin fibers continued to differentiate; therefore, a light green was used as a counterstain to Verhoeff’s stain. Arterial cross sections were used as a control during the Verhoeff and van Gieson and Verhoeff light green staining processes.

After staining, the sections were dehydrated through ascending grades of alcohol, cleared in xylene, and cover slips where mounted with Permount (Biomeda Corp, Foster City, CA). All slides were observed with either conventional brightfield microscopy or a 100 watt mercury lamp fluorescence light source using the Nikon Eclipse E600 microscope imaging system (Nikon Corp, Japan) with magnifications of 4x, 10x, or 20x. All images had a final image resolution of 1,280 x 1,024 and a 48-bit intensity resolution.

Immunohistochemistry was performed on all six types of chordae tendineae in the radial direction to verify the finding of vasculature. After standard deparaffinization and rehydration to water, the sections were washed in phosphate buffered saline (PBS, Sigma, St. Louis, MO) for 5 minutes. This wash in the PBS stabilized the antigen-antibody interactions. The sections were pretreated with 100 µg/mL of protease for 10 minutes to better expose the antigen of interest. During pretreatment, the sections were incubated in a humid container. Sections were washed in PBS to remove the excess enzyme. Endogenous peroxidase (false positives) was blocked using 0.3% H2O2 in methanol for 15 minutes. After rehydration in PBS, the tissue was blocked with 1% gelatin and PBS mixture to neutralize the tissue to prohibit nonspecific binding. The first antibody, rabbit {alpha}-von Willebrand factor (Product F3520, Sigma, St. Louis, MO) was applied and the sections were placed in a humid chamber for 1 hour. After washing in the PBS, a biotinylated secondary antibody (goat anti-rabbit immunoglobulin G) was applied, and the sections were incubated for 30 minutes in a humid chamber. Once the slides were washed in the PBS, the ABC mixture (avidine-biotin-peroxidose) was applied to the slides and incubated in a humid chamber for 1 hour. The avidine binds with the biotin, which was contained in the second antibody. After washing in the PBS and deionized water, diaminobenzidine was applied to each section for 4 minutes. The sections were then counterstained with hematoxylin, dehydrated through graded alcohols, xylene, and coverslipped with Permount. Arterial cross sections were used as controls. The sections were observed using brightfield microscopy with a Nikon Eclipse E600 microscope.

Assays were performed on fresh tissue of all six types of chordae tendineae to quantify the amount of DNA, collagen, and elastin contained in each of the different chordae. After dehydration for 48 hours, the tissue was digested according to the specific assay. For the DNA and elastin assays, the tissue was digested for approximately 6 days with proteinase K and incubated in a water bath at 55°C. Two digestion processes were utilized for the collagen assay as the chordae contained soluble and insoluble collagen components. The tissue was digested for approximately 24 hours with pepsin to digest the soluble collagen, and then it was placed in a water bath at 80°C to dissolve the insoluble collagen. A Hoechst fluorescent assay was used to determine the DNA content of the chordae tendineae. Standards were calculated by serially diluting calf thymus DNA stocked at 10 µg/mL to a final concentration of 2.5 µg/mL. The Hoechst dye solution was added as 10,000 x into 0.1 µg/mL buffer (10x TNE). Then 10 µL of each sample and 200 µL of Hoechst dye were pipetted into each well. The fluorescence was measured at an excitation of 365 nm and emission of 458 nm. The amount of acid-pepsin soluble collagen was quantified using the Sircol Collagen assay kit (Biocolor, Newtownabbey, Ireland). The elastin content for each chord was measured using the Fastin Elastin assay kit (Biocolor).

All data are reported as mean ± standard error of the mean, unless otherwise stated. Means were compared using t tests for paired comparisons. A p value of less than 0.05 was considered significant. Statistical analysis was computed using Minitab software (version 14) (Minitab Inc, State College, PA).


   Results
Top
 Abstract
 Introduction
 Material and Methods
 Results
Comment
 Acknowledgments
 References
 
Hematoxylin and Eosin
Hematoxylin and eosin stains were performed on each type of chord to determine cellular distribution. Cell nuclei were stained dark blue, collagen fibers were stained dark pink, and elastin fibers were stained light pink (Fig 2).



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  		Fig 2. Hematoxylin and eosin staining did not reveal any differences in the gross morphology between the various chords. Cell nuclei were stained dark blue, collagen fibers were stained dark pink, and elastin fibers were stained light pink. Sections are 5 microns in the radial directions. (Left) Anterior marginal chordae, 10x. (Right) Basal chordae, 10x. The middle layer of loose collagen can be seen separating from the inner collagen core.

 
The hematoxylin and eosin stained images were also viewed using fluorescent microscopy to allow the directional loading crimping to be observed, as shown in Figure 3A. The collagen fibers aligned in the direction of loading, which is characteristic of those tissues whose function is mainly to transmit tension [5]. The outer layer of loose collagen fibers did not exhibit the same directional loading as the central core. The fluorescent microscopy also exhibited the natural fluorescence of the elastin fibers. Figure 3B shows the elastin fibers in the radial direction and circumferential direction. Hematoxylin and eosin staining did not reveal any differences in the gross morphology between the various chords. However, due to the intense staining of the collagen, it was difficult to delineate the elastin fibers from the collagen; therefore, a Verhoeff counterstained with a light green stain was used to distinguish the elastin fibers.



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  		Fig 3. (A) Hematoxylin and eosin staining observed under fluorescent microscopy to observe directional crimping and (B, C) elastin fibers. The crimping is observed in the direction of loading. The middle layer of loose collagen does not contain the tight crimping as seen in the inner collagen core. Elastin fibers can be seen in the (A) longitudinal and (B) radial directions. Sections are 5 microns in each direction.

 
Verhoeff & Light Green Stain
A Verhoeff stain, counterstained with a light green stain, stained the elastin fibers and cell nuclei black and the remaining tissue light green. Figure 4 shows a representative sample of elastin fibers interwoven with the collagen fibers in the central collagen core. Elastin fibers were observed in both the inner collagen core and the outer loose layer of collagen. The elastin was found to exist as single fibers and not arranged in fibrils as normally found in soft tissues [5]. This observation was consistent between all chords examined.



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  		Fig 4. Anterior strut chordae stained with Verhoeff counterstained with light green. The cell nuclei and elastin fibers were stained black and the surrounding tissue were stained green. Sections are 5 microns thick in both the radial and longitudinal directions. (Left) Strut, longitudinal, 40x. (Right) Strut, radial, 40x.

 
Verhoeff & van Gieson
A Verhoeff stain, counterstained with a van Gieson stain, was performed to distinguish between the collagen, elastin, cellular nuclei, and the surrounding tissue. The collagen was stained red, the elastin black, the cell nuclei black, and the surrounding tissue yellow. As shown in Figure 5, the outer layer of the chordae stained red for collagen. This is in contrast to previous findings stating that the outer layer is composed entirely of elastin [1]. There were a few single elastin fibers that were also seen in the Verhoeff and light green stain. Areas in the outer layer of the chordae stained yellow, indicating tissue that was not composed of collagen or elastin. These structures were identified as blood vessels; this was verified with immunohistochemistry. The vessels were found to run in the longitudinal direction. This indicates that the vessels did not run directly up the chordae, but circled the chordae as it proceeded from the papillary muscle to the leaflet. Staining was performed on six samples of each chordae. Figure 5 below shows a representative sample of each chord stained with the Verhoeff and van Gieson stain in the radial direction. Figure 6 shows the vascular distribution between the six chordae. Each chord contained vessels; however it was found that the anterior strut chordae contained significantly more vessels than the other chordae. The anterior strut chordae contained 4.73 ± 0.94 vessels, the anterior marginal chordae contained 1.50 ± 0.73 vessels, the commissural chordae contained 1.50 ± 0.43 vessels, the basal posterior chordae contained 1.27 ± 0.52 vessels, the posterior intermediate chordae contained 1.43 ± 0.43 vessels, and the posterior marginal chordae contained 1.00 ± 0.58 vessels.



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  		Fig 5. Verhoeff and van Gieson stain of the chordae shows vessels in the middle layer of loose collagen and (right) in two vessels that were located in close proximity. The vessel with the thicker endothelial wall is considered an artery, and the other vessel is considered a vein. The collagen was stained red, the elastin and cell bodies were stained black, and the remaining tissue was stained yellow. Sections are 5 microns thick in the radial direction. (Left) Anterior marginal, radial, 10x, and (right) anterior strut, radial, 20x.

 


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  		Fig 6. Evidence of vascularization of the chordae tendineae of the mitral valve. (A) Number of vessels located in each chordae. Data represent mean values ± standard error of the mean (n = 11). *Shows significant difference (p < 0.05). (B, C) Immunohistochemistry performed with von Willebrand factor on the chordae. Sections are 5 microns in the radial direction. The endothelial cells were stained brown; other cell nuclei were stained blue; and surrounding tissue was stained light gray. (B) Endothelial surrounding the chordae. Strut chordae at 40x. (B) Endothelium lining the vessel wall. Strut chordae at 20x.

 
Immunohistochemistry
Immunohistochemistry was performed with von Willebrand factor to stain for porcine endothelial cells. The endothelial cells were stained dark brown, the surrounding tissue was stained a light gray, and the non-endothelial cell nuclei was stained blue (Fig 6). This stain was performed to verify the finding of vasculature in the chordae. In addition to the vascular endothelium, a single layer of endothelial cells surrounding the chordae was detected.

DNA Content
A Hoechst fluorescent assay was used to determine the DNA content of the chordae tendineae. The amount of DNA per mg of tissue for each chord is presented in Figure 7. The anterior and posterior marginal chordae contained statistically significantly more DNA per mg of tissue than the other chordae (p < 0.01). The anterior strut chord was found to contain significantly less DNA per mg of tissue (0.63 ± 0.03 µg of DNA per mg of tissue dry weight) than all the other chordae (p < 0.01). There was no significant differences between the amount of DNA per mg of tissue for the commissural chordae (1.26 ± 0.12 µg of DNA per mg of tissue dry weight), posterior intermediate chordae (1.01 ± 0.07 µg of DNA per mg of tissue dry weight), and basal posterior chordae (1.09 ± 0.07 µg of DNA per mg of tissue dry weight). There was also no significant differences between the anterior marginal chordae (1.90 ± 0.21 µg of DNA per mg of tissue dry weight) and posterior marginal chordae (2.70 ± 0.27 µg of DNA per mg of tissue dry weight).



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  		Fig 7. Biochemical assay results. Data represent mean values ± standard error of the mean. *Shows significant difference (p < 0.05). (A) DNA content in the mitral valve chordae per mg of dry tissue. DNA was measured using a Hoechst fluorescent assay (n = 21). (B) Elastin content in the mitral valve chordae per mg of dry tissue. Elastin was measured using the Fastin Elastin assay kit (Biocolor, Ireland) (n = 7). (C) Amount of collagen per mg of tissue. Collagen content in the mitral valve chordae per mg of dry tissue. Collagen was measured using the Sircol collagen assay kit (Biocolor, Ireland) (n = 16).

 
Collagen Content
The amount of acid-pepsin soluble collagen was quantified using a sircol red colorimetric assay kit. The amount of newly synthesized collagen per mg of tissue for each chord is presented in Figure 7. The results showed that the posterior marginal chord contained significantly more collagen per mg of tissue than the other chordae (p < 0.01). The posterior marginal chordae contained 14.89 ± 1.53 µg of collagen per mg of tissue dry weight. There was no significant difference between the other chordae. The anterior strut chordae contained 5.49 ± 0.91 µg of collagen per mg of tissue dry weight, the anterior marginal chordae contained 8.94 ± 0.76 µg of collagen per mg of tissue dry weight, the commissural chordae contained 6.24 ± 0.75 µg of collagen per mg of tissue dry weight, the basal chordae contained 6.71 ± 0.99 µg of collagen per mg of tissue dry weight, and the posterior intermediate chordae contained 6.23 ± 0.98 µg of collagen per mg of tissue dry weight.

Elastin Content
The elastin content for each of the chordae was measured using the Fastin Elastin assay (Biocolor). The amount of elastin per mg of tissue for each chord is presented in Figure 7. The anterior strut chordae contained 16.44 ± 4.50 µg of elastin per mg of tissue dry weight, the anterior marginal chordae contained 34.58 ± 11.73 µg of elastin per mg of tissue dry weight, the commissural chordae contained 48.11 ± 21.87 µg of elastin per mg of tissue dry weight, the basal chordae contained 23.09 ± 7.30 µg of elastin per mg of tissue dry weight, the posterior intermediate chordae contained 19.03 ± 15.55 µg of elastin per mg of tissue dry weight, and the posterior marginal chordae contained 27.84 ± 23.60 µg of elastin per mg of tissue dry weight.


   Comment
Top
 Abstract
 Introduction
 Material and Methods
 Results
 Comment
Acknowledgments
 References
 
Quantitative biochemical analysis revealed that the chordae tendineae of the mitral valve have different microstructures according to chordal type. The presence of vessels characterize the chordae tendineae as complex living components that must work in coordination with the papillary muscles and mitral valve leaflets to prevent mitral valve prolapse and regurgitation. They may also function to supply nutrients to the valve leaflets. Therefore, chordal translocation and cutting procedures must consider the presence of these biological structures as they may be essential in the long-term outcome of these procedures [6].

Vessels were located in the chordae tendineae of the mitral valve and support the conclusion that the chordae are more than simple collagenous structures, but living tissues that act to support and feed the mitral apparatus. The observed vessels were found in the middle layer of the chordae running in a longitudinal manner from the papillary muscle to the mitral apparatus. The vessels tended to twist around the chordae while ascending. The vessels appear to feed the mitral apparatus and not the chordae. This is supported by the observation that there was no branching vasculature observed from the major vessels running along the length of the chordae. Like other tendons, the chordae are believed to be fed through diffusion. The substances first diffuse into the tissue fluid that surrounds the cells and then into the cells through four methods: (1) gases and lipid-soluble substances cross the cell membrane by lipid diffusion; (2) water crosses by osmosis; (3) ions cross by facilitated diffusion; and (4) glucose and amino acids cross by active transport. Further investigation of this diffusion process is necessary to have a definite answer as to how the chordae obtain nutrients from the blood.

Previous studies have shown that the mitral leaflets do contain vessels in which to supply the leaflet tissue with nutrients [7]. These vessels were believed to have originated in the annulus of the mitral valve; however, the study presented here shows that some vessels that do insert into the mitral leaflets extend from the papillary muscles through the chordae and into the leaflets. Further investigation is necessary to determine the origin of the vessels in the mitral leaflets, such as dye injection studies.

The strut chordae, which inserts into the rough zone of the anterior leaflet, was found to have significantly more vessels than the other chordae. The marginal chordae may be too small in diameter to support numerous vessels. The size of the posterior leaflet as compared with the anterior leaflet may provide the reasoning behind the lack of vessels in the basal posterior and posterior intermediate chordae as compared with the strut chordae, even though they are similar in diameter [8]. The vessels through the strut chordae lead to the thinner regions of the leaflet, which are farther from the annulus. This may imply that the leaflets that lay closer to the annulus (ie, the commissural and posterior) obtain more blood from the annulus itself; whereas, the anterior leaflet receives blood from both the annulus and the chordae.

Previous morphology of the chordae showed an inner collagen core surrounded by an outer layer of elastic fibers [1]. Histologic examination conducted during this study showed that there were three distinct layers as previously reported; however, the middle layer was not completely composed of elastic fibers. The inner layer was composed mainly of densely packed, highly cross-linked collagen fibrils with few elastic fibers. The collagen fibers provided the mechanical strength and integrity for the chordae [9]. The middle layer was found to be composed of a loosely connected layer of collagen with elastic fibers interspersed within the collagen fibrils. In a loose configuration, the collagen fibrils do not exhibit the high degree of strength as found in the densely packed collagen found in the inner layer. The elastin fibers found in this middle layer provide the elasticity seen in the chordae at lower stress levels when the collagen fibers are uncrimping. Histologic examination showed that during a relaxed state, the collagen in the chordae had a crimped configuration and the elastic fibers were straight hence giving the chordae mechanical properties that are exhibited by a composite material [5]. A single layer of endothelial cells surrounds the entire chordae. A three-dimensional model of the chordae showing the three distinct layers is shown in Figure 8.



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  		Fig 8. Three-dimensional model of the mitral valve chordae tendineae describing the histologic composition. The outer layer is composed of endothelial cells, the second layer is composed of loose collagen with elastin fibers interwoven, and the inner layer is composed of tightly bound collagen. Vessels were found in the second layer of loose collagen running from the papillary muscle to the mitral apparatus.

 
Using biochemical assays to study the compositional makeup of the chordae, it was found that the anterior marginal and posterior marginal chordae contained significantly more DNA per milligram of tissue than the other chordae. The anterior marginal and posterior marginal chordae function to secure the anterior and posterior leaflets, respectively, during valve closure ensuring a line of coaptation. The larger chordae, such as the strut and basal chordae, function to prevent the leaflets from prolapsing during valve closure and withstand the highest mechanical load during the cardiac cycle [10]. The thinner chordae (anterior and posterior marginal chordae) function to transmit the contractions of the papillary muscles to the mitral leaflets to ensure proper coaptation configuration. The collagen structure in the chordae provides the tensile strength needed to perform these functions. In order to ensure the collagen structure does not degrade, fibroblasts must be present to repair and remodel the collagen matrix by producing extracellular proteins under mechanical loading conditions [11]. A study conducted on rabbit patellar tendon demonstrated that stress enhancement can affect the extracellular matrix of the patellar tendon without living cells [12]. This infers that intrinsic fibroblasts inhibit the mechanical deterioration and repair micro-damage of the extracellular matrix induced by stress enhancement. Sutker and colleagues [13] and Yang and colleagues [11] report that cyclic uniaxial mechanical stretching stimulates the collagen synthesis and proliferation of fibroblasts in vitro that further supports the findings of Tohyama and Yasuda [12]. The increase in collagen synthesis and proliferation of fibroblasts is important to the chordae tendineae. The higher concentration of DNA and newly synthesized collagen in the anterior and posterior marginal chordae suggests that these chordae have higher rates of collagen synthesis than the other chordae in order to maintain the chordae and prevent mechanical deterioration. Because these chordae are essential in the correct coaptation of the leaflets, it is feasible that they would contain more fibroblasts to ensure mechanical function.

The posterior marginal chordae contain more newly synthesized collagen per milligram of tissue than the other chordae. A study conducted by Liao and Vesely [14] described the total collagen content; whereas this study describes the amount of newly synthesized collagen that is important for maintaining the integrity of the chordae. The amount of newly synthesized collagen per milligram of tissue contained in the posterior marginal chordae is in agreement with the DNA results that showed significantly more DNA per milligram of tissue. The more collagen the chordae contain, the stiffer the chordae would be under a high stress [9]. This finding is in agreement with Liao and Veseley [15] who found that the thicker strut chordae were more extensible and less stiff than the thinner marginal chordae. During valve closure the posterior leaflet remains relatively constant, and the anterior leaflet moves to the posterior leaflet until the line of coaptation is formed. The posterior marginal chord is responsible for maintaining the placement of the posterior leaflet during valve closure. This chord needs a high degree of strength to maintain the position of the posterior leaflet during valve closure. There was no statistical difference in the amount of elastin per milligram of tissue contained in the six types of chordae measured. The trend in the elastin content results was similar to those of Liao and Vesely [14].

The collagen contained within the chordae mediates the visco-elastic properties exhibited during mechanical testing. The highly cross-linked collagen within the chordae contains the mechanisms to prevent creep due to the low amount of proteoglycans. The tissue in the chordae must act more as an elastic tissue, which is known to have minimal creep characteristics, such that there is no regurgitation. If the chordae experienced creep seen by most visco-elastic tissues, the chordae would become elongated during valve closure and malcoaptation would occur, leading to regurgitation. It is known that collagen has a significant amount of stress relaxation; however, elastin fibers tend to have low-stress relaxation characteristics [5]. Although the chordae are composed almost entirely of tightly cross-linked collagen fibers, there are elastin fibers interwoven throughout the chordae arranged parallel to the collagen fibers. These elastin fibers may function to prevent stress relaxation of the chordae during valve function in the normal physiologic range. It has also been postulated that the number of proteoglycan linkages contained in the chordae prevent the stress relaxation of the chordae [14].

The histologic and biochemical examinations of the chordae describe the differences between the types of chordae based on function and insertion site in the mitral valve. The differences in the structure-function relationship among the chordae must be considered during chordal translocation procedures as they may be essential in the long-term outcome of the procedure. Mitral valve repair is becoming a more attractive option instead of replacement to correct regurgitation. Currently silk, nylon, and e-polytetrafluoroethylene (Gortex) sutures are used for chordae replacement [16–22]. If these synthetic materials are used to replace the native chordae, it is possible that the leaflet tissue where the native chordae inserts may become necrotic due to lack of a nutrient supply.

The primary limitation of this study was the use of porcine tissue instead of human tissue due to availability. With the collagen turnover we are assuming that cell specific collagen synthesis is consistent between each chord, which is based on DNA content and collagen content. This method is slightly crude, and more accurate techniques should be considered, such as gene expression studies, to more accurately determine what is occurring. Future directions of this study include dye injection studies to determine the exact location and function of the vessels located in the chordae.

In conclusion, histologic examination indicated that previous studies had not adequately described the histologic composition of the chordae tendineae. Contrary to earlier belief, vessels were found in the chordae running from the papillary muscle to the insertion sites of the chordae on the mitral leaflets. These vessels do not provide blood to the chordae, but rather act as a supply of nutrients to other parts of the mitral apparatus. The presence of vessels characterize the chordae tendineae as complex living components that must work in coordination with the papillary muscles and mitral valve leaflets to prevent mitral valve prolapse and regurgitation. Therefore, chordal translocation and cutting procedures must consider the presence of these biological structures as they may be essential in the long-term outcome of these procedures.

Biochemical examination showed that the chordae contain different amounts of collagen, elastin, and DNA depending on chordal type. It was concluded that the amounts of these different components is related to the function and location of the chordae. The chordae have similar elastin content to prevent stress relaxation during constant strain, and the chordae that functions to ensure correct coaptation configuration contains more DNA and newly synthesized collagen. During chordal translocation procedures, surgeons must consider the biochemical composition of each chord as it is specific to its location and function in the mitral apparatus.

The findings in this study confirm that the mitral apparatus is composed of many components that work together in a complex, dynamic environment to ensure proper function. Further understanding of the mitral apparatus, including the chordae tendineae, will help better define surgical techniques aimed at repairing the mitral valve to its normal functioning state.


   Acknowledgments
Top
 Abstract
 Introduction
 Material and Methods
 Results
 Comment
 Acknowledgments
References
 
This research was primarily supported by a grant from the National Heart, Lung, and Blood Institute (HL No. 52009). Dr Warnock was supported by the National Science Foundation through the ERC Program at Georgia Tech (Award No. EEC-9731643). The authors would like to thank Drs Magdi Yacoub and Adrian Chester for their knowledge of the mitral valve, and Tracey Couse for her technical assistance with the histologic preparation. They are also grateful to Holifield’s Farm (Covington, GA) for supplying porcine tissue.


   References
Top
 Abstract
 Introduction
 Material and Methods
 Results
 Comment
 Acknowledgments
 References
 

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