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American Journal of Physiology - Regulatory, Integrative and Comparative Physiology logoLink to American Journal of Physiology - Regulatory, Integrative and Comparative Physiology
. 2015 Sep 9;309(12):R1490–R1498. doi: 10.1152/ajpregu.00040.2015

Increased systolic load causes adverse remodeling of fetal aortic and mitral valves

Frederick A Tibayan 1,2,, Samantha Louey 1, Sonnet Jonker 1, Herbert Espinoza 1, Natasha Chattergoon 1, Fanglei You 1, Kent L Thornburg 1, George Giraud 1,3
PMCID: PMC4698413  PMID: 26354842

Abstract

While abnormal hemodynamic forces alter fetal myocardial growth, little is known about whether such insults affect fetal cardiac valve development. We hypothesized that chronically elevated systolic load would detrimentally alter fetal valve growth. Chronically instrumented fetal sheep received either a continuous infusion of adult sheep plasma to increase fetal blood pressure, or a lactated Ringer's infusion as a volume control beginning on day 126 ± 4 of gestation. After 8 days, mean arterial pressure was higher in the plasma infusion group (63.0 mmHg vs. 41.8 mmHg, P < 0.05). Mitral annular septal-lateral diameter (11.9 mm vs. 9.1 mm, P < 0.05), anterior leaflet length (7.7 mm vs. 6.4 mm, P < 0.05), and posterior leaflet length (P2; 4.0 mm vs. 3.0 mm, P < 0.05) were greater in the elevated load group. mRNA levels of Notch-1, TGF-β2, Wnt-2b, BMP-1, and versican were suppressed in aortic and mitral valve leaflets; elastin and α1 type I collagen mRNA levels were suppressed in the aortic valves only. We conclude that sustained elevated arterial pressure load on the fetal heart valve leads to anatomic remodeling and, surprisingly, suppression of signaling and extracellular matrix genes that are important to valve development. These novel findings have important implications on the developmental origins of valve disease and may have long-term consequences on valve function and durability.

Keywords: animal model, heart valve, mechanical load, heart development, fetus


acquired diseases of the heart valves lead to over 90,000 heart surgeries and 22,000 deaths in the United States each year (14, 33). In addition, abnormalities of the heart valves make up 25–30% of cardiovascular malformations, the most common type of congenital defect (25). While overt structural defects of valves that present themselves at birth arise from faulty developmental processes, the underlying mechanisms that lead to valve pathology in later life are less obvious. Prenatal nutrition, growth, and oxygen supply are well known to be powerful risk factors for subsequent cardiovascular disease (47, 11, 17, 28, 3032). The connection between the prenatal environment and valvular heart disease, however, remains unexplored. Given the high morbidity, mortality, and costs associated with valvular heart disease, we must better understand the processes underlying valve disease at every stage of life. The human heart valves withstand roughly 3 billion cycles of tension, flexion, and shear stress over a lifetime. With each cardiac cycle, greater left ventricular and aortic pressures lead to greater stresses on the mitral and aortic valve leaflets. However, the degree to which increased systolic load alters valve growth and the expression of genes responsible for the composition of the extracellular matrix from which the valve is formed has not been elucidated.

Perturbed valve development during the critical perinatal window may impart vulnerability to valvular disease in adult life. Fetal growth restriction, which impacts up to 10% of births each year in the United States, is associated with increased fetal afterload and cardiovascular mortality in adulthood (10, 27). Increased cardiac load places greater stress on the heart muscle and heart valves (10, 27). Mechanical stress is a powerful modulator of cardiovascular growth across the life span, starting in the embryonic period (23, 38). In addition, abnormal cardiac load during the embryonic period leads to abnormal valve formation (1820, 44). It has been postulated that fetal and neonatal valves remodel in response to the mechanical environment, but the impact of increased mechanical load during late gestation, when the immature leaflets remodel to prepare for extrauterine life, is unknown (3).

We reasoned that increased load on the growing fetal heart valves would stimulate adaptive remodeling, making the valves more adept at bearing the increased load. We hypothesized that increased systolic load modifies the normal processes of valve development and maturation by stimulating expression of genes that regulate the composition of the extracellular matrix and the signaling pathways through which those genes are regulated. To test this hypothesis, we used a fetal ovine model of increased systolic load on the heart (13). We measured changes in aortic and mitral valve morphology and gene expression in fetuses with elevated aortic pressures, and thus, we increased load on the aortic and mitral valves, compared with controls. Herein, we present the first report of the impact of altered hemodynamic load on valve development in the late fetal window of maturation.

MATERIALS AND METHODS

Animals.

All surgical and experimental methods were approved by the Institutional Animal Care and Use Committee. Time-bred ewes of mixed Western breeds were obtained from a commercial supplier and acclimatized to the laboratory. Ewes carrying twins were used for mRNA expression studies after 8 days of infusion (n = 8 control, n = 8 plasma infusion) or 2 days of infusion (n = 5 control, n = 5 plasma infusion). In addition, ewes carrying singletons were used for valve morphology studies (n = 5 control, n = 5 plasma infusion). All fetuses underwent the same surgical and in vivo experimental procedures.

Animal preparation.

Details of animal surgery have been published previously (12, 22). In brief, anesthesia was induced by administering an intravenous mixture of diazepam (0.13 mg/kg) and ketamine (5 mg/kg) solution. The ewe was intubated and mechanically ventilated; anesthesia was maintained using 1–2% isoflurane in a carrier gas mixture of 70/30 oxygen and nitrous oxide. The isoflurane concentration was adjusted as necessary, and additional doses of diazepam or ketamine were administered to ensure a surgical level of anesthesia in both the ewe and the fetus(es). A catheter was placed in the carotid artery of the fetus and the tip advanced to the level of the aorta. A catheter was placed in the jugular vein and the tip advanced to a level just cranial to the right atrium. A catheter was placed in the amniotic fluid space. All catheters were secured to the fetal skin. The catheters exited the uterus, and the uterus was closed forming a tight seal of the amniotic cavity. The catheters were tunneled through the ewe's abdominal wall, and the abdomen was closed. The catheters were then tunneled under the ewe's skin emerging at the ewe's flank and were stored in a pouch sewn to the skin. One million units of penicillin G (Bristol-Meyers Squibb, Princeton, NJ) were instilled into the amniotic space at the conclusion of the procedure, anesthesia was terminated, and the ewe was allowed to recover. The ewes received routine postoperative pain medication (0.6 mg sc bupremorphine, twice a day) for 2 days. Animals were placed in metabolic crates after 4 days of recovery from surgery.

Experimental protocol.

Baseline measurements were made at mean gestational age 126 ± 4 days. Daily arterial blood gas partial pressures, pH, hemoglobin concentrations, and hemoglobin oxygen content were measured in a Radiometer ABL 720 blood analyzer at 39°C. A refractometer was used to obtain plasma protein concentrations.

Fetal aortic and right atrial pressures were continuously measured with Abbott Transpac pressure transducers (Abbott Park, IL) and a computerized recording system (AD Instruments, Colorado Springs, CO; Apple, Cupertino, CA). The system was calibrated against a mercury manometer and rezeroed daily for drift before each measurement, and all fetal intravascular pressures were referred to amniotic fluid pressure as zero. Heart rate was derived from arterial pressure recordings. Daily measurements taken over a 60-min period were averaged.

On day 0 after baseline measurements were made, an intravenous infusion of adult sheep plasma (to increase fetal pressures and the mechanical load on the heart valves) or lactated Ringer solution (as an equal volume control) was started using a Minipuls 3 roller pump (Gilson, Middleton, WI). Initial plasma infusion rate was calculated to deliver 18.75 g protein/day (324 ± 23 ml/day) initially, and increased by 3.5% per day to correspond to expected fetal growth, as previously described (22). After 8 days, the plasma infusion group had received a total 2,509 ± 147 ml fluid. In the two-day infusion group, the plasma infusion group had received a total of 800 ± 25 ml fluid. Each control received an identical volume of lactated Ringer solution. Fetuses were randomly assigned to the experimental or control group without regard to sex.

Plasma source.

Sheep plasma was obtained from control ewes from other studies that were due to be euthanized. The ewes were anesthetized with ketamine (400 mg iv) and supplemented as necessary to ensure deep anesthesia. Ten thousand units of heparin were administered intravenously. Each animal was exsanguinated via large-bore catheter in the carotid artery into 1-liter sterile bottles containing 10,000 units of heparin or 130 ml of sodium citrate. After blood collection was completed, the ewes were euthanized by intravenous injection of pentobarbital sodium (Somnasol, ∼80 mg/kg; Butler Schein Animal Health, Dublin, OH). The blood was centrifuged to separate the plasma, which was then serially ultrafiltered through sterile cellulose acetate filters (0.22 μm).

Tissue preparation.

At the end of each experiment, the ewe was euthanized by intravenous injection of pentobarbital sodium (Somnasol, ∼80 mg/kg; Butler Schein Animal Health, Dublin, OH). The ewe's abdomen and uterus were opened, exposing the fetus. The fetuses received 10,000 U heparin and then 10 ml of saturated potassium chloride via umbilical vein to arrest the heart in diastole. The fetuses were weighed and sexed. The fetal hearts were then harvested in a standardized fashion and weighed. The hearts of the singletons were perfused with PBS and pressure fixed with 4% paraformaldehyde for 1 h at the mean arterial pressure recorded for that day. For the twin fetuses (2 day and 8 day), aortic and mitral valve tissues (all leaflets of the entire valve, from the annulus to the free edge) were dissected; half of each valve was stored in RNAlater (Life Technologies, Grand Island, NY) at 4°C for 24 h before storage at −20°C, and the remainder was flash frozen in liquid nitrogen and stored at −80°C.

Valve morphology.

Valves in perfusion-fixed hearts were measured in situ with calipers (Mitutoyo, Aurora, IL). Aortic valve diameter was measured at the level of the valve annulus. Sinus of Valsalva dimension was also measured between the valve and the sinotubular junction. Aortic valve leaflets are too thin and friable for reliable and accurate measurement at this stage of development. Mitral annular diameters were measured in the septal-lateral and commissure-commissure dimension. Anterior mitral leaflet length and the lengths of the P1, P2, and P3 scallops of the posterior mitral leaflet were also measured (Fig. 1).

Fig. 1.

Fig. 1.

Schematic of the mitral valve as seen from the left atrium (surgeon's view). Sustained increased systolic load leads to an enlarged mitral valve. P1–P3, posterior leaflet scallops P1–P3. Data show mean valve measurements; see text for more details and SEs. *P < 0.05 control vs. increased load group.

Anterior mitral leaflets of the perfusion fixed valves were then cut radially in the central region from the aorto-mitral continuity to the free edge of the anterior mitral leaflet and embedded in paraffin. Sections were cut at 6-µm thickness and stained with Masson's trichrome to stain for collagen and Verhoeff-van Gieson for elastin. Images were captured with a microscope (Zeiss Axiophot), and the area fraction stained for collagen and elastin was quantified using ImageJ (National Institutes of Health, Bethesda, MD).

Tissue processing, RNA isolation, and first-strand cDNA synthesis.

Fetal valve tissue preserved in RNAlater (Ambion, Grand Island, NY) was homogenized using the TissueLyser LT (Qiagen, Valencia, CA), and RNA was isolated via the TRIzol method (Ambion). RNA samples were cleaned using the RNeasy mini kit (Qiagen) prior to analysis on a Synergy H1 hybrid multi-mode microplate reader (Gen5 Data Analysis Software, BioTek Winooski, VT) to ensure integrity of RNA prior to first-strand cDNA synthesis. Total first-strand cDNA was synthesized (high-capacity cDNA reverse transcription kit; Applied Biosystems, Grand Island, NY) per the manufacturer's protocol (10 min at 25°C, 120 min at 37°C, 5 min at 85°C, hold at 4°C), with the addition of 2.5 mM oligo dT.

Quantitative polymerase chain reaction assays and data analysis.

Candidate genes from genomic databases were identified by analysis of sequence alignments of sheep and cow (Ovis aries and Bos taurus) target sequences. Quantitative PCR (qPCR) primers were designed using the Primer 3 software program (San Diego Biology Workbench 3.2, http://workbench.sdsc.edu/). All primers (Eurofins MWG Operon, Huntsville, AL) were tested to determine appropriate annealing temperatures and ensure single-product formation. Each primer pair produced a single PCR product, as evidenced by melt curve analysis and gel electrophoresis. Amplification products were sequenced at Eurofins MWG Operon, and the sequences were compared with other DNA sequences using BLAST in The Gene Index database (TGI: http://compbio.dfci.harvard.edu/tgi/ncbi/blast/blast.html) to confirm amplification of the target gene. The BLAST queries resulted in correct gene hits with BLAST e-values <10−15. We also compared similar sequences with lesser e-values before assigning identities to the target genes.

After PCR product validation and assay optimization, qPCR assays were conducted in a 96-well format under the optimal PCR conditions with Applied Biosystems Power SYBR Green master mix (Applied Biosystems) and were quantified using the relative standard curve method on a Stratagene Mx3005P QPCR System and MxPro software (Agilent Technologies, Santa Clara, CA). Reactions for each cDNA sample were performed in triplicate, including the standard curve, which was generated from pooled samples and quantified before serial dilution. PCR amplifications were performed for 1 cycle at 95°C for 10 min, 42 cycles with denaturation at 94°C for 20 s, annealing at optimum temperature for primers (55–58°C) for 30 s, and extension at 72°C for 30 s followed by melt curve analysis. Expression for each gene was normalized to a housekeeping gene [β-2 microglobulin (B2M)] and expressed as a ratio of the gene of interest:B2M for each sample. B2M was chosen for normalization after determining that it was not affected by changing experimental conditions.

The primer sequences used for PCR are found in Table 1.

Table 1.

Primers for quantitative PCR studies

Gene Primer Sequence Accession Number (species)
Bone morphogenic protein-1 S: ATGAGGCACTGGGAGAAGC KC007442.1 (Bubalus bubalis);
AS: GTGGACCACGATGCCAAAC XM_004005260.1 (Ovis aries)
Collagen alpha-1(I) chain S: GAAGGCCAGGAATCAACCAC AF129287.1 (Ovis aries);
AS: TTGTCCAGGGATGCCATCTC NM_001034039.2 (Bos taurus)
Elastin S: CTTTCCTGGCTTCGGAGAC BC149367.1 (Bos taurus); XM_004021248.1 (Ovis aries);
AS: GGGCCTAATCCAAACTGGG
Notch-1 S: CGGCCAGCAGATGATCTTCC EF999923.1 (Ovis aires); XM_004023059.1 (Ovis aries)
AS: CACTGCCGGTTGTCGATCTC
Transforming growth factor-β2 S: AGGAATACTACGCCAAGGAGG AY656797.1 (Ovis aries); NM_001113252.1 (Bos taurus)
AS: GGCTTTCGGGTTCTGTAAACG
Versican S: AGTGTGGAGGTGGTTTAC NM_181035.2 (Bos taurus);
AS: AGTGGGTGAGACAGTTTC XM_004009070.1 (Ovis aries)
β2 microglobulin S: ATCCAGCGTATTCCAGAG EF489536.1 (Ovis aries)
AS: GGGACAGAAGGTAGAAAG

AS, antisense; S, sense.

Statistics.

Comparisons between groups for the twins were made using paired t-tests. Comparisons between groups for the singletons were made using unpaired t-tests. Significance was defined as P < 0.05. Data are expressed as means ± SE. All statistical analyses were performed with GraphPad Prism 6 (San Diego, CA).

RESULTS

Effects of increased load on fetal vascular pressures, body weight, and heart weight.

Hemodynamics, body weight, and heart weights for the 8-day infusion groups (twins) are summarized in Table 2 and Fig. 2. Fetal arterial pressure, atrial pressure, and heart rate were not different between the control and increased load groups on day 0 before infusions. After 8 days, mean arterial pressure was 54% higher in the increased load group compared with the control group. After 8 days, right atrial pressure was 44% greater in the increased load group compared with the control group. After 8 days, heart rate was 13% higher in the increased load group compared with the control group. Increased cardiac load caused an increase in cardiac mass; hearts were 49% heavier in the increased load group compared with the control group. Fetuses were 21% heavier in the increased load group compared with the control group. Heart weight-to-body weight ratios were 24% greater in the increased load group compared with the control group.

Table 2.

Fetal hemodynamics, body, and heart weights

Control Group Increased Load Group
Day 0
    Mean arterial pressure, mmHg 40.5 ± 0.7 41.3 ± 0.5
    Mean right atrial pressure, mmHg 2.1 ± 0.5 1.6 ± 0.2
    Heart rate, beats/min 168 ± 3.6 167 ± 3.4
Day 8
    Mean arterial pressure, mmHg 41.8 ± 0.6 63.0 ± 2.0*
    Mean right atrial pressure, mmHg 2.5 ± 0.3 3.6 ± 0.3*
    Heart rate, beats/min 142 ± 4.3 160 ± 2.7*
    Body weight, kg 3.8 ± 0.2 4.6 ± 0.2*
    Heart weight, g 24.3 ± 2.2 36.1 ± 3.2*
    Heart weight/body weight ratio, g/kg 6.3 ± 0.2 7.8 ± 0.3*

Values are expressed as means ± SE. For both groups, n = 8.

*

P < 0.05.

Fig. 2.

Fig. 2.

Mean arterial pressure measurements in the control and increased load group (twins) over the 8-day study period.

In the 8-day infusion groups (singletons), aortic pressure was similar at baseline between the increased load and control groups (39.2 ± 1.2 vs. 39.6 ± 0.9 mmHg) and increased after 8 days of infusion (61.4 ± 2.5 vs. 42.9 ± 0.7 mmHg). Right atrial pressure was similar at baseline (1.8 ± 0.4 vs. 1.3 ± 0.3 mmHg) and after 8 days (3.5 ± 0.2 vs. 3.3 ± 0.6 mmHg) in the increased load and control groups. Heart rate was also not different at baseline (167 ± 5 vs. 174 ± 8 beats per minute) and after 8 days of infusion (162 ± 5 vs. 153 ± 6 beats per minute). Heart weight (38.9 ± 1.7 vs. 30.4 ± 0.8 g), body weight (4.4 ± 0.2 vs. 4.1 ± 0.1 kg), and heart weight-to-body weight ratio (8.8 ± 0.7 vs. 7.4 ± 0.2 g/kg, P < 0.05 for all) were all increased in the increased load group.

For the twins infused for 2 days, aortic pressure was similar at baseline between the increased load and control groups (42.5 ± 1.1 vs. 40.7 ± 1.5 mmHg) and increased after 2 days of infusion (51.9 ± 2.1 vs. 41.2 ± 1.5 mmHg). Heart rate was similar at baseline [174 ± 6 vs. 175 ± 7 beats per minute (bpm)], and after two days of infusion (174 ± 6 vs. 167 ± 3 bpm). Right atrial pressure was also similar at baseline (3.7 ± 0.2 vs. 3.6 ± 0.1 mmHg) and after two days of infusion (2.7 ± 0.6 vs. 3.0 ± 0.5 mmHg). Heart weight (24.4 ± 1.6 vs. 23.1 ± 1.7 g), body weight (3.7 ± 0.2 vs. 3.6 ± 0.1 kg), and heart weight-to-body weight ratio (6.5 ± 0.3 vs. 6.4 ± 0.4 g/kg) were not different between the two groups.

Effects of increased load on mitral valve annular and leaflet size.

Table 3 and Fig. 1 summarize mitral and aortic valve morphology in this study. After 8 days of increased cardiac load, the septal to lateral diameter of the mitral annulus was 31% larger in the increased load group compared with the control (Fig. 1, Table 3). There was no difference in mitral commissure-commissure diameter in the load vs. the control group. Sustained increased cardiac load was associated with a 20% lengthening of the anterior mitral leaflet compared with controls. On average, the P1, P2, and P3 scallops of the posterior leaflet were lengthened by 45% in the increased load group compared with the control group. There were no differences in mean aortic valve annular diameter or diameter at the sinuses of Valsalva in the increased load vs. the control group.

Table 3.

Mitral and aortic valve geometry

Control Group Increased Load Group
Mitral valves
    Septal-lateral diameter, mm 9.1 ± 1.7 11.9 ± 1.5*
    Commissure-commissure diameter, mm 11.6 ± 1.3 13.2 ± 2.1
    Anterior mitral leaflet, mm 6.41 ± 0.68 7.71 ± 0.71*
    P1, mm 1.76 ± 0.24 2.82 ± 0.50*
    P2, mm 2.98 ± 0.51 3.96 ± 0.55*
    P3, mm 2.26 ± 0.64 3.18 ± 0.55*
Aortic valves
    Aortic annulus, mm 6.44 ± 1.41 6.19 ± 0.35
    Sinotubular junction, mm 9.12 ± 1.13 9.24 ± 1.66

Values are expressed as means ± SE. P1–P3, posterior leaflet scallops 1–3. For both groups, n = 5.

*

P < 0.05 by unpaired t-test.

Effects of increased load on anterior mitral leaflet collagen and elastin deposition.

After 8 days of increased cardiac load, the area fraction of the anterior mitral leaflet stained for collagen was not statistically different between the increased load and control groups (0.34 ± 0.15 vs. 0.42 ± 0.02; P = 0.3). Area fraction of the anterior mitral leaflet stained for elastin was also not statistically different between the increased load and control groups (0.04 ± 0.03 vs. 0.09 ± 0.10, P = 0.3).

Suppression of extracellular matrix and cell signaling gene expression.

After 8 days of increased cardiac load, mRNA levels of Notch-1, TGF-β2, Wnt-2b, and BMP-1 were lower in the increased load group compared with the controls for both the aortic and mitral valve leaflets (Fig. 3). mRNA expression levels of elastin, α1 type 1 collagen, and versican were all lower in the leaflets of the aortic valve in the increased load group compared with the control group (Fig. 4). For the mitral valve leaflets, levels of versican were lower in the increased load group compared with the control group.

Fig. 3.

Fig. 3.

mRNA expression of key signaling molecules involved in valve remodeling, 8 days of increased load vs. control. Data are expressed relative to a housekeeping gene (β2 microglobulin), and are shown as means ± SE; *P < 0.05.

Fig. 4.

Fig. 4.

mRNA expression of key extracellular matrix molecules involved in valve remodeling, 8 days of increased load vs. control. Data are expressed relative to a housekeeping gene (β2 microglobulin), and are shown as means ± SE; *P < 0.05.

Temporal pattern of gene expression.

To better understand how fetal pressure loading affects cellular signaling genes over time, we measured gene expression in aortic valve and mitral valve leaflet tissue after only 2 days of increased pressure load. Aortic valve versican expression was almost three-fold greater in the increased load group (1.9 ± 0.4 vs. 0.7 ± 0.2, P < 0.05), while the 66% greater expression of α1 type I collagen (4.8 ± 1.4 vs 2.9 ± 1.1) and more than twofold greater expression of BMP (2.5 ± 0.5 vs 1.1 ± 0.4) were not significant. In the mitral valve, expression of versican (0.9 ± 0.1 vs 0.7 ± 0.2, increased load vs. control), α1 type I collagen (5.3 ± 3.1 vs. 6.3 ± 3.5), and BMP (1.5 ± 0.3 vs. 1.5 ± 0.5) were not significantly different.

DISCUSSION

This study for the first time links altered late-gestation fetal hemodynamic load to geometric valve remodeling. Valve enlargement was accompanied by unexpected suppression of key genes involved with valve remodeling, indicating that prenatal hemodynamic stress dysregulates valve development at this critical window.

Mitral valve annular dilation and leaflet lengthening.

Sustained (8 days) of increased systolic load increased mitral septal-lateral diameter to more than 30% larger than controls. Both anterior leaflet and every scallop of the posterior leaflet were significantly lengthened. These findings support the speculation of other researchers that fetal valvular tissue adapts to environmental stimuli, particularly mechanical load (3, 34).

In previous studies, we found annular dilation and leaflet lengthening of the mitral valve in response to elevated mechanical load in the adult heart (35, 36, 40). These morphological changes in valve structure have important consequences. In a sheep model of chronic ischemic mitral regurgitation, annular dilation and leaflet lengthening increased anterior and posterior leaflet radii of curvature (41). Why might this be important? The mitral annulus, leaflets, and chordal apparatus bear the load generated by the pressure in the left ventricle. Leaflet stress is known to be a stimulus that is likely to drive valve remodeling, due to the LaPlace relationship (Fig. 5). The LaPlace relationship estimates the mechanical effect of such changes on a curved surface: Sw = (Pt/2) × (r/h), where Sw is leaflet wall stress, Pt is transmural pressure, r is radius of curvature, and h is wall thickness.

Fig. 5.

Fig. 5.

Schematic of contributors to wall stress on control and pressure-loaded mitral valves. Cross-sectional view of the mitral valve in control (left) and increased load (right) conditions. Left: curved lines depict the anterior (AML) and posterior (PML) mitral leaflets. Solid arrows represent pressure, and dashed lines signify the radius of curvature of the leaflet. Right: with increased load, the mitral leaflets become longer. Thicker solid arrows denote higher pressure, and the dashed lines are longer, representing longer radii of curvature for the leaflets. These changes both tend to increase wall stress according to the Law of LaPlace, wherein wall stress is directly proportional to the pressure and the radius.

In this study, transmural pressure (Pt) increased 54%, and the observed annular dilation, and leaflet lengthening increased leaflet radii of curvature, which would tend to increase leaflet stress (41). Leaflet thickness was not measured in the present study. However, even if thickness increased to maintain leaflet stress, this too could be maladaptive, as studies of mitral valve function in adult sheep hearts have shown that leaflet thickening decreases leaflet extensibility and exacerbates mitral regurgitation (15). Aikawa and Grande-Allen (2) proposed a vicious cycle of disadvantageous remodeling, wherein leaflet lengthening leads to increased leaflet tensile stress, resulting in further remodeling. Annular dilation in adult mitral valves has important functional and clinical consequences, such as mitral regurgitation and heart failure (42, 43, 47). Indeed, geometric changes in the annulus and leaflets similar in magnitude to those in the present study have been associated with functional mitral regurgitation in larger, adult sheep hearts (41). Thus, the valvular remodeling demonstrated in this study is physiologically significant and may represent an anatomic basis for vulnerability to valvular disease in adult life.

Interestingly, the aortic valve annular and sinus of Valsalva geometry were not different compared with controls after 8 days of increased pressure load. We speculate that the aortic valve annulus is more resistant to dilation because it is supported by the fibrous skeleton of the heart and because it is surrounded by the mitral, tricuspid, and pulmonary valves. This is consistent with the clinical observations that, in the absence of connective tissue disorders, dilation of the aortic valve is uncommon, and aortic stenosis is much more prevalent in the adult population than aortic regurgitation (14, 29). In the setting of heart failure, mitral annular dilation and resultant functional mitral regurgitation are much more common than aortic valve dilation and aortic insufficiency. Our findings show that the aortic valve is resistant to load-induced dilation even during fetal life.

Fetal valve gene expression.

Very little is known about the signaling cascades coordinating valve maturation in late gestation, when the valve must prepare for the stresses of extra-uterine life (24). Abnormal hemodynamic forces perturb early heart and valve development in chick and zebrafish models, but the impact of increased load on gene expression is largely unknown (20, 37). The genes investigated in this study are all involved in early valve development and are also implicated in adult valvular disease (1, 9, 16, 21, 26, 48). While the effects of increased pressure load on in vivo adult valve gene expression are poorly understood, BMP, TGF-β, Notch, and collagen have been shown to be regulated by mechanical strain in cultured valvular cells (8). In an effort to bridge this crucial knowledge gap, we measured expression of genes for signaling molecules known to be important in early valvulogenesis, or in regulating growth and remodeling of extracellular matrix, to see whether they are altered by increased load. Early, after two days of increased pressure load, there was a trend toward increased expression of BMP in the aortic valve. However, this tendency toward upregulation was not sustained with continued pressure overload. After 8 days of increased pressure load, mRNA levels of Notch-1, Wnt-2b, BMP-1, and TGF-β2 were lower in both aortic and mitral valves. Because each of these pathways impacts extracellular matrix deposition and is implicated in adult valve disease, we speculate that their suppressed expression may disrupt proper prenatal valve formation and maturation.

Expression of extracellular matrix genes in the setting of increased pressure load also varied with time. For instance, there was a many-fold increase in versican expression in the aortic valve after two days of increased load, while after 8 days of increased pressure load, versican expression was reduced in both the aortic and mitral valves. Similarly, collagen gene expression was not different after two days of increased cardiac load in the aortic valve but was lower in the aortic valve after 8 days of increased load. Fetal valve gene regulation in the face of altered mechanical load is complex, and the response at different time points is likely different for different genes.

We hypothesized that increased load would stimulate these regulatory pathways and the expression of extracellular matrix genes, enabling the valve to withstand greater mechanical stress; the observed downregulation of signaling and extracellular matrix genes at 8 days of sustained arterial load surprised us. This downregulation could be a response to altered expression of regulatory genes at an earlier time point, increased extracellular matrix deposition, or changed valve morphology. The first possibility seems unlikely, however, as after 2 days of increased load, the only change in gene expression that we observed was in aortic valve versican. The second possibility also seems unlikely because we did not find increased elastin or collagen staining in our histological studies. Finally, mitral valve morphology was significantly altered, but we did not observe similar changes in aortic valve size to drive the changes in gene expression observed there. The question remains open as to what is driving gene expression in the chronically pressure-loaded fetal valve. We speculate that the downregulation is a protection mechanism against excessive stiffening of the developing valve. In the developing aorta, maintaining the elastic modulus, a measure of stiffness, within a narrow range may be the primary variable driving tissue remodeling, and the same may be true in the leaflet (45). As too much collagen may result in a very stiff valve, the proposed fetal adaptation may suppress these pathways driving matrix deposition. While valve tissue may adaptively remodel in response to physiological increases in load by stimulating gene expression and deposition of extracellular matrix, we propose that more severe, sustained increases in load exceed the capability of the fetal valve to favorably remodel.

Suppressed fetal expression of extracellular matrix genes may have long-term functional consequences in valves.

The durability and function of heart valves depend on the complex, precise, trilaminar architecture of the extracellular matrix. An elastin-rich layer allows the valve tissue to extend during opening and recoil during closing. In our study, gene expression of elastin was decreased by 45% in aortic valve leaflets of the increased load group. On the opposite side of the valve, a collagen-rich layer confers stiffness and structural strength. We expected increased pressure to stimulate collagen expression, but, surprisingly, in the aortic valve, it resulted in significantly decreased expression of type I collagen, the most abundant fibrillar collagen. Between the elastin and collagen layers, a proteoglycan layer absorbs compressive forces and lubricates the sliding of the outer layers over each other. Versican is an important proteoglycan in this layer that is crucial for heart valve formation (46). We found that versican expression was also more than 30% lower in the aortic valves of the increased fetal load group. In the mitral leaflets, versican expression also was decreased, while collagen and elastin only trended toward lower expression. Similarly, in a perinatal study of porcine valves, Stephens et al. (39) observed extracellular matrix composition changes in aortic valves, but not in mitral valves. They hypothesized that aortic leaflets, which bear greater diastolic pressures (on the aortic side) after the transition from fetal to neonatal circulation compared with the mitral leaflets, are more responsive to hemodynamic loading conditions. Suppression of these extracellular matrix genes may disrupt the precise organization, composition, and architecture of the extracellular matrix with important implications for valve development, function, and durability. These perturbations in extracellular matrix genes during such a critical window of development may give insights into the fetal and neonatal roots of valve disease in later life.

Limitations.

While intrauterine growth restriction due to placental vascular insufficiency and coarctation of the aorta are thought to significantly increase systolic load in the fetus, because of the lack of invasive fetal hemodynamic monitoring, we do not know whether the degree to which the changes in aortic pressure observed in this study qualitatively match these pathological states in the human. We speculate that smaller increases in cardiac load will have qualitatively similar effects in valve remodeling.

Perspectives and Significance

Sustained increased systolic load on the fetal valve leads to anatomic remodeling and, surprisingly, suppression of signaling and extracellular matrix genes that are important to valve development. These novel findings have important implications on the developmental origins of valve disease and open new avenues to explore the pathogenesis and treatment of adult valvular dysfunction. The mitral annular dilation and leaflet lengthening may set up a cycle of altered leaflet stress and progressive remodeling. We speculate that the observed gene suppression may be part of a protective mechanism against excessive valve stiffening. Future work will seek to identify the epigenetic mechanisms coordinating this response. Such epigenetic features may exert a programming effect by limiting the ability of the adult valve to respond to hemodynamic alterations later in life. These findings are clinically significant, as fetal growth restriction, which affects up to 10% of births every year, is linked to increased fetal pressure loading (10, 27).

GRANTS

This research was supported by The American Association for Thoracic Surgery Michael DeBakey Research Scholarship, the Medical Research Foundation New Investigator Grant, and the Eunice Kennedy Shriver National Institute of Child Health and Human Development under award numbers P01-HD-34430 and R01-HD-071068.

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the authors.

AUTHOR CONTRIBUTIONS

Author contributions: F.A.T., S.L., S.S.J., N.N.C., K.L.T., and G.D.G. conception and design of research; F.A.T., S.L., S.S.J., H.M.E., N.N.C., F.Y., and G.D.G. performed experiments; F.A.T., S.L., S.S.J., H.M.E., N.N.C., F.Y., K.L.T., and G.D.G. analyzed data; F.A.T., S.L., S.S.J., N.N.C., K.L.T., and G.D.G. interpreted results of experiments; F.A.T., S.L., and S.S.J. prepared figures; F.A.T. drafted manuscript; F.A.T., S.L., S.S.J., H.M.E., N.N.C., F.Y., K.L.T., and G.D.G. edited and revised manuscript; F.A.T., K.L.T., and G.D.G. approved final version of manuscript.

ACKNOWLEDGMENTS

The authors would like to acknowledge the technical assistance of Isa Lindgren, Loni Socha, Robert Webber, Elizabeth Clarke, Sean Lamb, and Marjorie Grafe in the Knight Cardiovascular Institute Histopathology Core Facility. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

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