Effects of a contoured articular prosthetic device on tibiofemoral peak contact pressure: a biomechanical study

Many middle-aged patients are affected by localized cartilage defects that are neither appropriate for primary, nor repeat biological repair methods, nor for conventional arthroplasty. This in vitro study aims to determine the peak contact pressure in the tibiofemoral joint with a partial femoral resurfacing device (HemiCAP®, Arthrosurface Inc., Franklin, MA, USA). Peak contact pressure was determined in eight fresh-frozen cadaveric specimens using a Tekscan sensor placed in the medial compartment above the menisci. A closed loop robotic knee simulator was used to test each knee in static stance positions (5°/15°/30°/45°) with body weight ground reaction force (GRF), 30° flexion with twice the body weight (2tBW) GRF and dynamic knee-bending cycles with body weight GRF. The ground reaction force was adjusted to the living body weight of the cadaver donor and maintained throughout all cycles. Each specimen was tested under four different conditions: Untreated, flush HemiCAP® implantation, 1-mm proud implantation and 20-mm defect. A paired sampled t test to compare means (significance, P ≤ 0.05) was used for statistical analysis. On average, no statistically significant differences were found in any testing condition comparing the normal knee with flush device implantation. With the 1-mm proud implant, statistically significant increase of peak contact pressures of 217% (5° stance), 99% (dynamic knee bending) and 90% (30° stance with 2tBW) compared to the untreated condition was seen. No significant increase of peak contact pressure was evaluated with the 20-mm defect. The data suggests that resurfacing with the HemiCAP® does not lead to increased peak contact pressure with flush implantation. However, elevated implantation results in increased peak contact pressure and might be biomechanically disadvantageous in an in vivo application.
Introduction
In 50% of the patients undergoing routine arthroscopy, articular cartilage defects are detected [6]. The majority of cartilage lesions are observed in patients over the age of 40 years [6, 11]. Approximately 20% of these lesions were categorized as full thickness cartilage defects (Grade IV) [6]. The average defect size in 1,000 knee arthroscopies was 2.1 cm2 with 42% of these defects being between 2 to 4 cm2 in area [11]. Location of the lesions are predominantly on the medial femoral condyle [6, 11]. Clinical and experimental studies have shown that a relationship exists between larger defect size and degeneration of the articular surface. Linden et al. [19] demonstrated that the presence of a significant chondral defect is associated with a much higher incidence of osteoarthritis than occurs in the general population. Hughston et al. [13] showed that larger osteochondral defects are associated with a poorer clinical outcome than smaller defects.
The treatment for articular cartilage defects offers a great variety of options depending on the severity of the damage. The main factors to be considered are the patient age, the size of the lesion, the location of the defect, and further damage to the joint. Biological repair techniques, such as autologeous chondrocyte transplantation, osteochondral transplantation (OATS, Mosaicplasty) and marrow stimulation techniques are proposed for the treatment of localized cartilage defects in younger patients with a normal weight bearing axis and no further damage to the joint [3, 8]. However, many middle aged patients are affected by localized cartilage damage and are neither fit for biological repair methods, nor for traditional resurfacing techniques such as unicompartimental or total joint replacement. A contoured articular prosthetic unicondylar femoral resurfacing prosthesis (HemiCAP®, Arthrosurface Inc., Franklin, MA, USA) was developed to offer a treatment option among the currently used modalities. It is intended for use as a partial resurfacing device of the femoral condylar surface of the knee when only one compartment is affected by posttraumatic, degenerative disease or necrosis associated with large unstable articular defects with significant subchondral bone exposure.
However, it is unknown if the femoral resurfacing device leads to increased peak contact pressure to the opposing biological structures such as meniscus and articular cartilage of the tibial plateau.
The purpose of our study was to determine peak contact pressures in the medial compartment of human cadaveric knees under different loading conditions comparing the normal articular surface to flush and proud HemiCAP® implantation and defect. Based on the experimental study, biomechanical device safety on opposing structures can be assessed and a guideline might be given to the clinician for prosthetic implantation while avoiding potentially damaging effects.
Materials and methods
Knee joints
A total of eight fresh-frozen knee cadaver specimens (3 pairs, 2 single) were used for data collection in this study. The specimens were obtained from donors, who consented in writing during their lifetime to the use of their body for research and education. The average age of the seven male and one female specimen was 71 years (range 61–81) with an average weight of 71 kg (range 62–85). Specimens were selected after inspection of the medial compartment according to the following criteria: Intact femorotibial cartilage, intact meniscus, and intact collateral and cruciate ligaments. Thirteen specimens were excluded following these criteria. Another six specimens were excluded due to specimen failure during the testing procedure: The reason for exclusion was fracture of the femur or tibial plateau (2 specimens), rupture of the anterior cruciate ligament (2 specimens) or rupture of the patella tendon (2 specimens). All specimen failures were female human cadaver knees.
Technology
A specially designed knee simulator was used for this study (Fig. 1). Similar to in vivo conditions, the main system composed of artificial muscle, force transducer sensor, the joint angle detection and the ground reacting force form a closed loop. The ground reaction force is adjustable according to the donor’s weight. The knee simulator consists of a loading frame (MTS 858 Bionix, MTS Systems, Eden Prairie, MN, USA) with a long stroke main actuator driven by a hydraulic pump unit (MTS 505.11 silent flow) to simulate body weight and the vertical hip displacement in the mechanical axis of the lower limb. Ankle joint simulation is performed with a hinge joint with one free motion axis. The possible rotation during the movement occurs in the artificial hip joint as if standing with fixed shoe contact. A load transducer is fixed between the mounting plate and ankle joint to detect the vertical ground reacting force (U3 load cell, Hottinger-Baldwin, Darmstadt, Germany). Two smaller actuators apply loads, which simulate the quadriceps force. The tendons of the quadriceps muscle are attached to customized curved cryo-clamps, which avoid patella tilting. These cryo-clamps are connected to a waterproof force transducer (SSM-AJ 500, Interface, Scottsdale, AZ, USA) and connected to an artificial muscle (Fluidic muscle MAS, Festo, Esslingen, Germany). The mathematical model shows that the properties of this fluidic muscle is comparable with a skeletal muscle [28].
Fig. 1Schematic drawing of the knee simulator that was used for this study
A 0.1 mm thin electronic pressure measuring sensor (K-scan 4000, Tekscan, Boston, MA, USA) was placed in the medial compartment above the menisci and fixed with sutures in a manner that no displacement was possible. The sensor consists of load-sensing regions oriented in a grid with 1.27 mm spacing between rows and columns. Each region is referred to a sensel with piezo-resistive pigments to determine the total compressive load within that region. The size of the sensor is 28 × 33 mm with 62 sensels/cm2. The K-scan sensor was successfully used in comparable applications by several authors [18, 21, 25, 31].
The HemiCAP® implant (Arthrosurface Inc., Franklin, MA, USA) is a contoured articular prosthetic (CAP) unicondylar femoral resurfacing prosthesis consisting of two components, a fixation component and an articular component, that mate together via a taper interlock to provide stable and immobile fixation of the implant and stress-bearing contact at the bone/prosthetic interface. The fixation component is a modified titanium cancellous screw with a tapering distal tip, a full-length cannulation, and a proximal female taper bore. The articular component is a dome-shaped component manufactured of a Cobalt–Chromium–Molybdenum alloy with titanium plasma spray coverage on the underside for bony in-growth. Each diameter comes in a variety of incremental offset sizes which correspond to the superior/inferior and medial/lateral radius of curvatures at the implant site. The size of the implant used in this study was 20 mm in diameter with different offset sizes, matching the individual specimen joint curvature.
Testing protocol
The specimens were aligned using a fixed laser beam to achieve correct alignment in the mechanical axis of the lower limb. The mechanical axis was defined by a line through the center of the head of the artificial hip joint, the center of the knee joint and the center of the hinge joint representing the artificial ankle. For calibration of the sensor the ankle hinge joint was secured with two aluminum plates perpendicular to the ground. Thereby the knee was fixed in full extension. Each sensor was individually preconditioned and calibrated intra-articulary with a two-point calibration method at 700 and 1,500 N according to manufacturer’s guidelines. Definitions of the correct angles of the actual knee position were adjusted with a custom-made goniometer and by the displacement-controlled main rod. During the test cycles the cadaver were sprayed with saline solution to prevent dehydration.
The specimens were tested in four different conditions: (1) Untreated knee, (2) Flush HemiCAP® (20 mm) implanted in the central weight bearing area of the medial femoral condyle, (3) 1 mm proud implantation to adjacent cartilage, (4) 20 mm defect (Fig. 2a–d). Each knee was tested in static knee stance positions (5°, 15°, 30°, 45°) with body weight ground reaction force, 30° flexion with two times body weight ground reaction force and during a knee-bending dynamic cycle (10 cycles) with body weight ground reaction force. The setting of ground reaction force to be achieved during the trial was adjusted to the living body weight of the donator of the cadaver (e.g. 70 kg∼700 N). The loading rate for exploring the dynamic contact pressures was 0.1 Hz/s.
Fig. 2Different testing conditions (a Untreated; b Flush; c 1 mm; d Defect)
Operative technique
All specimens were released at their femoral fixation during preparation of a new testing condition. The exact position was marked and recorded to maintain specimen position in the knee simulator across all testing conditions. The position of the sensor did not change during the preparation. All procedures were performed by the same investigator (CB). The medial femoral condyle was exposed by a medial parapatellar incision. The knee was flexed to 90° to expose the central weight-bearing portion. A drill guide was used to place a pin perpendicular to the joint surface representing the center of the defect. The center of the defect was determined by measuring the condylar width and bisecting the distance. The cannulated instrumentation set ensured that the vertical axis was maintained throughout the procedure. After drilling a pilot hole, the fixation component was inserted. A contact probe determined the radius of curvature in two planes. With a matching reamer, the site for the implantation was prepared and a sizing trial with corresponding offsets inserted. The selected device was oriented in the correct planes and connected to the anchoring screw with a tapered lock. In order to allow for careful removal of the articular component, the guide wire was initially drilled through the condyle and remained inside the cannulated screw during the trial. After testing with flush implantation (Fig. 2b), the device was removed with the guide wire. The anchoring screw was elevated with a counter-clockwise quarter turn representing 1 mm and the device re-inserted for positioning 1 mm above the adjacent cartilage (Fig. 2c). Defect condition was tested after removing the guide wire and device leaving a 20-mm osteochondral defect of 3–4 mm depth (Fig. 2d).
Data analysis
Data were obtained using I-Scan software 4.23 (Tekscan, Boston, MA, USA). Peak contact pressure was assessed and recorded as the highest value at each stance position and during the dynamic knee-bending cycle. Mean, median and standard deviation values were evaluated using MS-Excel 2003 (Microsoft Inc., Redmond, WA, USA) and SPSS 11.0 (SPSS Inc., Chicago, IL, USA). For statistical analysis a paired sampled t test to compare means (significance, P ≤ 0.05) was used.
Results
Continuous data were obtained at every trial. No difference in the quality of data collection was seen comparing the stance positions or dynamic testing.
The highest mean peak contact pressures were noted with the implant being 1 mm proud (Fig. 3; Table 1). Statistically significant increase of peak contact pressures of 217% (5° stance; P ≤ 0.004), 99% (dynamic knee bending; P ≤ 0.02) and 90% (30° stance with 2tBW; P ≤ 0.03) compared to the untreated condition was seen. Average results of 45° static testing demonstrated only slightly higher values compared to the other testing positions, indicating that the implant was non-weight bearing and lost contact to the sensor at this position (Fig. 3; Table 1).
Fig. 3Mean peak contact pressures with one standard deviationTable 1Peak contact pressures at different testing conditionsTesting positionTesting conditionPeak contact pressure (Mpa)Mean difference to untreated (%)P value5°Untreated3.09 ± 0.86 (3.05, 1.92–4.86)N/AN/AFlush3.98 ± 2.19 (3.59, 2.15–9.02)29NS1 mm proud9.80 ± 4.37 (9.36, 4.70–18.37)217≤0.004Defect3.35 ± 1.39 (3.39, 1.90–5.83)8NS15°Untreated3.01 ± 0.81 (2.95, 2.01–4.11)N/AN/AFlush3.69 ± 1.79 (3.89, 1.38–7.06)23NS1 mm proud 9.19 ± 9.27 (5.87, 2.10–30.49)205NSDefect2.94 ± 1.07 (2.84, 1.69–5.09)−2NS30°Untreated3.18 ± 0.94 (3.52, 1.46–4.26)N/AN/AFlush2.89 ± 0.85 (3.02, 1.03–3.83)−9NS1 mm proud 7.07 ± 5.97 (5.24, 1.03–19.81)122NSDefect3.31 ± 1.06 (3.11, 2.05–5.34)4NS45°Untreated4.77 ± 1.85 (4.23, 2.66–7.90)N/AN/AFlush4.96 ± 2.02 (4.18, 2.79–7.92)4NS1 mm proud 5.79 ± 2.76 (5.54, 2.01–11.82)21NSDefect4.94 ± 1.81 (4.65, 2.05–7.54)4NSDynamicUntreated5.84 ± 2.12 (5.14, 3.85–9.82)N/AN/AFlush6.02 ± 2.05 (5.21, 3.46–8.97)3NS1 mm proud 11.61 ± 6.39 (10.50, 6.22–25.46)99≤0.02Defect5.68 ± 1.76 (5.11, 3.76–8.24)−3NS30° 2tBWUntreated6.57 ± 2.31 (6.01, 4.09–11.15)N/AN/AFlush6.05 ± 1.40 (5.82, 4.68–8.97)−8NS1 mm proud 12.49 ± 8.02 (9.67, 5.83–30.49)90≤0.03Defect7.38 ± 4.68 (5.31, 4.75–17.70)12NSValues given as mean ± SD (median, range)NS not significant
On average, comparison of the untreated normal knee with flush device implantation demonstrated neither statistically significant differences in peak contact pressure during the dynamic knee-bending cycle nor static testing (5°, 15°, 30°, 45°) or two times body weight GRF at 30° static testing (Table 1). Six out of eight specimens demonstrated similar peak contact pressures in both testing conditions, two specimens showed a noticeable increase in peak contact pressures (flush device implantation) averaging 29% during 5° static testing and 23% in 15° static testing. Data evaluation of the two respective trials with outlying measurements demonstrated edge loading at the border of the implant to the adjacent cartilage (Figs. 4b, 5b).
Fig. 4Peak contact pressure at 15° static knee stance position with single body weight of specimen No. 2. The color represents the spectrum of pressures (high pressure-red; low pressure-blue). From left to right the different testing conditions are displayed (a Untreated, b Flush, c 1 mm proud, d Defect). Marked increase of peak contact pressure at the edge of the implant to the adjacent cartilage is demonstrated (b)Fig. 5Picture of specimen No. 2 before testing with flush implantation of the device in front view (a) and top view (b). The arrow indicates the area of peak contact pressure displayed in Fig. 4b
No significant increase of peak contact pressures was evaluated with the 20-mm osteochondral defect. However, average increase by 8% (5° stance) and 12% (30° stance with 2tBW) were found compared to untreated-condition (Table 1). Furthermore, maximum values were higher compared to untreated condition (except 45° stance position and dynamic testing cycle).
Discussion
The patient aged over 40 years with a full thickness chondral or osteochondral defect reflects a serious problem for the orthopedic surgeon. Considered as being too old for biological repair of the defect, primarily the patients are mostly managed with conservative, non-surgical treatment including weight reduction, physical therapy to increase and support musculature, unloading braces and medications such as NSAID’s, intraarticular injections (Corticosteroids, Hyaluronic acid, etc.) and dietary supplements.
However, conservative treatment at best ameliorates the symptoms. Biomechanical studies have shown that untreated osteochondral defects may result in increased contact pressures [4, 7, 17]. Animal models proved that untreated osteochondral defects undergo progressive degenerative changes [5, 14]. Whereas smaller defects might have the capacity for healing [5], it was shown that larger defects resulted in resorption of the osseous walls of the defect, the formation of a large cavitary lesion, and the collapse of the surrounding articular cartilage and subchondral bone as well as to degeneration of the opposing tibial articular surface [5, 14]. A“threshold” effect for localized full thickness defects was described with rim concentration around the lesion becoming a factor for defects greater than 10 mm in diameter (0.79 cm2) [7].
Defect repair is therefore regarded crucial to prevent or delay progressive degenerative joint destruction. However, unicompartimental or total knee arthroplasty represent procedures of final resort for some of the affected patients. The HemiCAP® resurfacing prosthesis (Arthrosurface Inc., Franklin, MA, USA) offers an interim or alternative treatment strategy for the middle-aged patient with a full thickness cartilage defect. However, effects of a metallic implant articulating with intact opposing tibial articular cartilage and meniscus remain largely unanswered to date. An experimental study assessing the functional and biological response to its use in a goat model resulted in good clinical outcomes. One year after implantation, gross necropsy and histologic data implied the biocompatibility and functionality of the implant. No ongoing degenerative joint disease was apparent [15]. Macroscopic and histological analysis showed that the cartilage around the implant was largely intact although some focal fraying and erosion and limited meniscal damage was observed. The opposing tibial plateau cartilage surface was generally intact but exhibited some focal erosions of variable depth [15]. The outcome was substantially better by comparison to other reported experimental animal studies in goats with untreated full-thickness defects [14, 29]. A patellar resurfacing prosthesis resulted at an average of 8.1 years in 71% good or excellent cases with no progressive degenerative changes in the non-resurfaced apposing femoral articular cartilage as long as 16 years [9]. A successful clinical outcome with a metallic distal femoral prosthesis articulating directly against the menisci and proximal tibial plateau over 30 years was documented in a case report [12].
Our results revealed significant increase of peak contact pressures with the implant being proud to the surrounding cartilage compared to the untreated condition with an average maximum increase of 217% and 205% at 5° and 15° stance compared to untreated knees. Results in a biomechanical model using osteochondral plugs for the treatment of osteochondral defects demonstrated increased contact pressures up to 57% compared to intact condition in elevated or angled plugs with an edge placed higher than the adjacent cartilage [16, 17]. Thus, increased peak contact pressures may suggest biomechanical disadvantages and may cause damage to opposing structures. Several in vitro studies have shown increased chondrocyte apoptosis and matrix deformation after peak stress loading of bovine and human articular cartilage [2, 20, 22, 23]. However, quantitative thresholds above which elevated pressure is detrimental are not defined yet.
Flush implantation showed no statistical increase of peak contact pressures compared to untreated. However, in two specimens we found marked increase of peak contact pressures at the edge of the implant. Evaluation of the pictures of the specimen after the trial showed that the implant did not appear to be proud to the adjacent cartilage in the front view (Fig. 5a). However, in the top view it seemed that the implant was in level with the adjacent cartilage or even slightly elevated at the point of increased peak contact pressure (Fig. 5b). Reduced quality of the adjacent cartilage directly next to the measured peak contact pressure might have lead to less resistive cartilage capacity allowing for edge loading. Thus, it appears to be crucial to have healthy surrounding cartilage around the implant and to spend special care during the implantation process so that the implant is not elevated above the adjacent cartilage at any point. Koh et al. [16] showed in their biomechanical study, that slightly recessed osteochondral grafts with the highest edge placed flush to neighboring cartilage demonstrated nearly normal contact pressures, whereas elevated angled grafts produced increased contact pressures. Our results confirm the observation concluding that slightly recessed implantation should be considered if flush implantation cannot be accomplished.
Defect situation in our study did not result in significant increase of peak contact pressures. Rim stress concentration has been described by several authors in their biomechanical models evaluating the contact pressures in full thickness cartilage defects [4, 5, 7, 16, 17, 26]. However, results were uncertain whether the peak contact pressure increases at the rim of the defect. Whereas Koh et al. [17] reported average increase of peak contact pressure by 23% compared to the untreated condition, Raimondi et al. [26] and Nelson et al. [24] found no elevated contact stresses at the rim of the defect. These different observations might be caused by the different deformation of defect rims under a given load, especially in larger defects than in smaller defects. The relationship of defect size to the condylar surface may be also a factor [7]. The methodology concerning the model (animal or human being cadaver), the experimental setup (loading mechanics, dynamic or static, etc.) and the pressure-sensitive sensor and performance of the trial have also to be considered.
Limitations of this study have to be considered and include the following. (1) This is a human cadaver study and biomechanical model. Only approximation of the living system can be achieved. However, the pre-selection of the applied load by the ground reaction force gave the opportunity to load the specimens with the known body weight of the donor, and thus better approximate the forces that occur in the living system [30]. Furthermore, the knee bending dynamic measurement may have provided a more accurate reproduction of physiologic weight-bearing activity. However, the complexity of weight-bearing motions including all the muscle groups for knee motion could not be reproduced. Applied forces were too high for female human cadaver knees resulting in a high specimen’s failure rate. (2) Peak contact pressures were determined by an electronic pressure sensitive sensor. The reliability of the K-scan sensor was verified in several studies [10, 18, 21, 31]. In comparison to other measuring devices (Fuji Photo Film), the Tekscan K-sensor proved to be superior [10, 27]. Limitations of the sensor include the thickness (0.1 mm), its sensitivity to temperature changes, its disposition for crinkling and the establishment of the position [1]. Although the position of the sensor was accurately secured, a small amount of displacement and crinkling could not be excluded. A certain amount of loss of sensitivity during the different testing conditions and imprecise calibration due to the different surface materials (metal on cartilage, cartilage on cartilage) have to be considered. Some drop of data point was observed at some specimen. However, peak contact values seemed not to be affected. (3) Stable, reproducible ground reaction force in full extension could not be established by the simulator. The quadriceps tendon could not be adequately tensioned in this position. (4) Implantation of the device was performed in the central weight-bearing area. Results might be different for the implantation in different areas of the medial femoral condyle or lateral condyle.
In conclusion, the data suggest that resurfacing with the HemiCAP® with flush implantation does not lead to significantly increased peak contact pressure. However, elevated implantation results in significantly increased peak contact pressure and might be biomechanically disadvantageous in an in vivo application. Further research is necessary to evaluate the effects of the prosthetic device on contact pressures after loss of meniscus function and in longer continuous dynamic testing.

articular prosthetic device
peak contact pressure
tibiofemoral joint
osteochondral defect
knee biomechanics


Preconception Care for Improving Perinatal Outcomes: The Time to Act

Introduction
A healthy baby and a healthy mother are valued hopes and dreams of families of all cultural heritages. National health goals in many countries around the world prioritize infant and maternal mortality and morbidity. In the United States, as in many other industrialized countries, pregnancy outcomes and maternal and infant health indicators have improved dramatically. At the turn of the 20th century, 7.28 women and 96 infants died for every 1,000 babies born alive [1]. By the end of the year 2000, the maternal mortality rate had decreased to less than 0.08 and infant mortality had decreased to less than 7 deaths per 1,000 live births [2]. These changes were characterized as one of the ten “great public health achievements” of the 20th century [3]. Much of this progress can be attributed to changes in social and living conditions as well as the development and implementation of effective medical interventions. Despite this progress, during the last two decades of the 20th century, even in the presence of significant breakthroughs in medical technology and its application, improvements in maternal and infant pregnancy ouctomes slowed down significantly, and in some cases, outcomes deteriorated. For example, from 1960 to 1980, the maternal mortality rate decreased from 32.1 to 9.4 deaths per 100,000 live births, a decrease of 70.7%. From 1980 to 2000, the maternal mortality rate decreased only 12.8%, from 9.4 to 8.2 (Figure 1) [2]. At the same time, from 1980 to 2000, the proportion of babies born preterm, very preterm, low birth weight, and very low birth weight increased by 26%, 8.2%, 14.7% and 25.9%, respectively (Figure 2) [4]. However, unlike other maternal and infant health indicators, from 1980 to 2000 the infant mortality rate continued to decrease almost at a similar pace as that from 1960 to 1980, dropping by 45.2% from 12.6 to 6.9 infant deaths per 1,000 live births, compared with a drop of 51.5% from 26.0 to 12.6 per 1,000 live births for 1960 to 1980 (Figure 1) [2]. Finally, it is estimated that the proportion of babies born in the United States who have a serious or major structural defect that can have adverse effects on their health or development continues to be about 3% [5].
Associated with this slowing rate of improvement (and, in some cases, deterioration) in pregnancy outcomes and maternal and infant health indicators is a shift in the leading causes of infant mortality. In 1960, maternal complications of pregnancy (including complications of placenta, cord, and the membranes) did not appear on the list of the 10 leading causes of infant mortality [1]. By 1980, maternal complications of pregnancy became the fifth leading cause of infant mortality, and the third leading cause of infant death in 2002, after congenital anomalies and low birth weight/preterm delivery [6, 7]. In 2002, congenital anomalies, low birth weight, preterm delivery, and maternal complications of pregnancy accounted for 46.4% of all infant deaths in the United States (12,996 infant deaths) (Figure 3) [7]. Although some of these infant deaths might have been prevented through interventions targeted at improving the health of mothers and modifying behaviors contributing to adverse pregnancy outcomes, poor maternal health, behaviors contributing to adverse pregnancy outcomes, and maternal complications of pregnancy continue to be prevalent. For example, 28.6% of women who gave birth between 1993 and 1997 were reported to have an obstetric complication, 4.1% had a preexisting medical condition, and 43.0% experienced some form of maternal morbidity (i.e., an obstetric complication, a preexisting medical condition, a cesarean section, or any combination of the three types of morbidity) [8]. In 2002, 26.1% of all deliveries were performed using cesarean section, presumably because of maternal or infant complications [8].
Fig. 1Maternal and infant mortality rates, United States, 1960–2002Fig. 2Percent of all live births that are preterm delivery, very preterm delivery, low birth weight, and very low birth weight, United States, 1980–2002
Early prenatal care is too late
One of the reasons that progress in improving pregnancy outcomes has slowed down, and in some cases reversed direction, is that we have failed to intervene before pregnancy to detect, manage, modify, and control maternal behaviors, health conditions, and risk factors that contribute to adverse maternal and infant outcomes. Although we know many interventions that, if delivered before pregnancy, could improve pregnancy outcomes, we have failed to make those services and interventions available to couples and women in need. Women of childbearing age suffer from a variety of chronic conditions that could potentially contribute to adverse pregnancy outcomes. For example, in 2002, the U.S. Department of Health and Human Services reported that 6.1% of women of reproductive age have asthma, 5% are obese, 3.4% have cardiac disease, 3.0% are hypertensive, 9.3% are diabetic, and 1.4% have thyroid disorder [9]. Moreover, a substantial proportion of women continue to enter pregnancy with risks proven to contribute to adverse pregnancy outcomes: in 2002, 11.4% of pregnant women smoked during pregnancy, a risk factor for low birth weight [4]; at the same time, 10.1% of pregnant women and 54.9% of women at risk of getting pregnant consumed alcohol, a risk factor for fetal alcohol syndrome [10]. Finally, using the Perinatal Periods of Risk approach, researchers in three cities (New York City; Tulsa, Oklahoma; and Kansas City, Missouri) concluded that racial and ethnic disparities in feto-infant mortality were largely related to maternal health, and, interventions to reduce feto-infant mortality should include preconception care and improvements in women's health [11–13].Fig. 3Leading causes of infant mortality, United States, 1960, 1980, and 2002. *IMR =  Infant Mortality Rate
The prenatal care “revolution” of the 1980s resulted in an increase in the proportion of women receiving early prenatal care (defined as prenatal care begun in the first trimester) [2]. After decreasing from 76.3% in 1980 to 75.8% in 1990, the proportion of women receiving early prenatal care increased to 83.2% in 2000 [2]. However, for many women, “early prenatal care is too late” [14]. By the time a pregnant woman makes it to her first early prenatal visit, most fetal organs are already been formed, and many interventions to prevent birth defects or adverse maternal and infant outcomes come too late to have any effect. As a result, many national organizations now recommend routine preconception care. For example, the March of Dimes recommends that “as the key physician/primary care provider, the obstetrician/gynecologists must take advantage of every health encounter to provide preconception care and risk reduction before and between conceptions—the time when it really can make a difference” [14]. The American Academy of Pediatrics (AAP) and the American College of Obstetricians and Gynecologists (ACOG) recommend that “all health encounters during a woman's reproductive years, particularly those that are a part of preconception care, should include counseling on appropriate medical care and behavior to optimize pregnancy outcomes” [15].
Preconception interventions work
Interventions designed to lower preconception risks are known collectively as preconception care. These interventions are characterized by the need to start—and sometimes complete—a designated intervention before conception occurs. To simplify matters, ACOG and AAP have grouped the main components of preconception care under four categories of interventions: maternal assessment (e.g., family history, behaviors, obstetric history, general physical exam); vaccinations (e.g., rubella, varicella and hepatitis B); screening (e.g., HIV, STD, genetic disorders); and counseling (e.g., folic acid consumption, smoking and alcohol cessation, weight management) [15]. Many of these interventions are currently available to women and, when indicated, to couples; however, they are not systematically delivered. A comprehensive review of the literature identified a long list of risk factors suggested to be included in comprehensive preconception care (Table 1). The strength of evidence supporting the efficacy of the various components of preconception care varies greatly [16]. Clinical practice guidelines (CPGs) have been developed for reducing the risk related to some of these factors. We identified 14 conditions for which CPGs exist and for which there is scientific evidence demonstrating effectiveness in improving pregnancy outcomes (Table 2) [17--74].
Table 1Risk factors (by Category) identified in the literature as needing attention during the preconception period1Chronic diseases: Diabetes; heart disease; high blood pressure; thyroid disease; asthma; anemia; kidney disease; metabolic and hematological disorders; depression and other mental disorders; autoimmune disease; and physical disability (access)2Infectious diseases: Vaccine-preventable diseases (rubella, hepatitis B, varicella, influenza, and tetanus); HIV/AIDS; syphilis, chlamydia, and other sexually transmitted diseases; periodontal disease; toxoplasmosis, and cytomegalic inclusion virus3Reproductive concerns: Unplanned pregnancies; contraception; infertility; adverse past pregnancy outcomes (preterm delivery, birth defects, fetal/infant death, maternal complications)4Genetic/inherited conditions: Sickle cell anemia; thalassemia; Tay-Sachs disease; fragile X syndrome; Down syndrome; cystic fibrosis; muscular dystrophy; hearing and vision loss associated with genetic predisposition5Medications and medical treatment: Prescription medications contraindicated in pregnant women (FDA's Category X Drugs, 117 products in 2001 PDR, antiepileptic drugs, oral anticoagulants for maternal clotting disorders, and Accutane); diagnostic radiation exposures6Personal behaviors and exposures: Smoking; alcohol consumption; illicit drug use; overweight/underweight; folic acid supplement use; domestic violence; eating disorders; exposure to infections; exposures to chemicals and other environmental toxins; consumption of over-the-counter medications; hyperthermia (e.g., from sauna use)Note. This list is not exhaustiveTable 2Selected preconception risk factors for adverse pregnancy outcomes for which clinical practice guidelines have been developed•Folic acid. Daily use of vitamin supplements containing folic acid has been demonstrated to reduce the occurrence of neural tube defects by two thirds [17–24]•Rubella seronegativity. Rubella vaccination provides protective seropositivity and prevents the occurrence of congenital rubella syndrome [25–27]•Diabetes (preconception). The three-fold increase in the prevalence of birth defects among infants of women with type 1 and type 2 diabetes is substantially reduced through proper management of diabetes [28–31]•Hypothyroidism. The dosages of Levothyroxine™ required for treatment of hypothyroidism increases in early pregnancy. Levothyroxine™ dosage needs to be adjusted for proper neurologic development [32–34]•HIV/AIDS. If HIV infection is identified before conception, timely treatment can be administered and women (or couples) can be given additional information that can influence the timing of the onset of pregnancy [35–41]•Maternal phenylketonurea (PKU). Women diagnosed with PKU as infants have infants with mental retardation. However, this adverse outcome can be prevented when mothers adhere to a low phenylalanine diet before conception and continue it throughout their pregnancy [42, 43]•Oral anticoagulant. Warfarin, which is used for the control of blood clotting, has been demonstrated to be a teratogen. To avoid exposure to warfarin during early pregnancy, medications can be changed to a nonteratogenic anticoagulant before the onset of pregnancy [15, 44–46]•Anti-epileptic drugs. Certain anti-epileptic drugs are known as teratogens. Before conception, women who are on a regimen of these drugs and who are contemplating pregnancy should be prescribed a lower dosage of these drugs [15, 47–51]•Isotretinoins (Accutane®): Use of isotretinoins in pregnancy to treat acne results in miscarriage and birth defects. Effective pregnancy prevention should be implemented to avoid unintended pregnancies among women with childbearing potential who use this medication [15, 52–54]•Smoking. Preterm birth, low birthweight, and other adverse perinatal outcomes associated with maternal smoking in pregnancy can be prevented if women stop smoking during early pregnancy. Because only 20% of women successfully control tobacco dependence during pregnancy, cessation of smoking is recommended before pregnancy [55–58]•Alcohol misuse. No time during pregnancy is safe to drink alcohol, and harm can occur early, before a woman has realized that she is or might be pregnant. Fetal alcohol syndrome and other alcohol-related birth defects can be prevented if women cease intake of alcohol before conception [59–64]•Obesity. Adverse perinatal outcomes associated with maternal obesity include neural tube defects, preterm delivery, diabetes, cesarean section, and hypertensive and thromboembolic disease. Weight loss before pregnancy reduces these risks [15, 65–69]•STD. Chlamydia trachomatis and Neisseria gonorrhea have been strongly associated with ectopic pregnancy, infertility, and chronic pelvic pain. STDs during pregnancy might result in fetal death or substantial physical and developmental disabilities, including mental retardation and blindness [70, 71]•Hepatitis B. Vaccination is recommended for men and women who are at risk for acquiring hepatitis B virus (HBV) infection. Preventing HBV infection in women of childbearing age prevents vertical transmission of infection to infants and eliminates risk for infection and sequelae, including hepatic failure, liver carcinoma, cirrhosis, and death [72–74]
However, the best evidence for the effectiveness of specific components of preconception care is seen when the focus was on a single intervention and often not in the context of improving pregnancy outcomes. For example, effectiveness documented by the U.S. Preventive Services Task Force for interventions related to smoking, alcohol misuse, and obesity are based on studies of interventions delivered in primary care settings that was not complicated by the additional delivery of other components of preconception care [56, 61, 66]. One study reported the effectiveness of comprehensive preconception care but inferences for the United States are limited because the setting of intervention delivery was in Hungary [75].
Priorities for preconception care and preconception health
The immediate priority is to ensure that evidence-based preconception interventions are implemented to further improve infant and maternal pregnancy outcomes. Many national professional organizations have developed and published CPGs for specific components of preconception care. For example, the American Diabetes Association developed CPGs to be started before pregnancy for women with preconception diabetes [28]. The American Association of Clinical Endocrinologists has developed preconception CPGs for women with hypothyroidism [34]. CPGs have also been developed for women being treated with teratogenic medications to guide the transition to safer medications. CPGs for women using antiepileptic drugs or oral anticoagulants have been developed by the American Academy of Neurology [50], the American Heart Association, and the American College of Cardiologists, respectively [44, 46]. Guidelines, recommendations, and strategies for reducing alcohol misuse and prevention of fetal alcohol syndrome have been developed and published by the U.S. Public Health Service, U.S. Preventive Service Taskforce, ACOG, and the National Taskforce on Fetal Alcohol Syndrome and Fetal Alcohol Effects [55–59].
However, moving forward towards more universal preconception care is not without its challenges. There is no national policy, nor do standard tools exist, for the delivery of these services. Some have suggested that challenges could be overcome by promoting preconception health to all women of reproductive age at each and every encounter with the health care system. The generally recommended mechanisms would include risk assessment (screening), health promotion (education and counseling), and intervention or referral. However, the existing recommendations to provide these three essential components have not been translated into practical tools for action and decision-making in the clinical setting. Stated another way, primary care providers do not have the tools they need to implement what is known to work for improving preconception health.
Challenges and opportunities
Whereas clinical practice and the promotion of preconception care services could potentially be shaped by guidelines, widespread adoption of the guidelines requires a more active approach [76, 77]. The literature on diffusion of innovation and translation of research into practice clearly indicates that changing primary care providers’ knowledge, attitudes, and practices requires multiple steps including consolidation and distillation of guidelines, active organizational support, clinically relevant decision tools, use of quality improvement techniques, and performance monitoring [78, 79]. A growing body of research indicates that providers are more likely to engage in evidence-based practices following participation in quality improvement projects (e.g., rapid improvement cycles or collaborative groups). The federally funded Health Disparities Collaborative, the Perinatal Care and Patient Safety Collaboratives, and similar quality improvement efforts for community health centers demonstrate how such approaches are being used to better serve low-income and uninsured patients.
The need to develop innovative approaches to deliver and finance bundles of preconception interventions must be viewed as a high priority. However, although strong evidence exists for many preconception care interventions and shows they are effective, discussion continues regarding the benefits of delivering comprehensive preconception care [16, 80, 81], and the effectiveness or added value of “packaging” or “bundling” these interventions remains to be determined. For example, preconception care interventions could potentially be bundled into behavioral modification services (e.g., smoking and alcohol cessation, weight control, exercise), screening and assessment (e.g., vaccines, general physical examination), and specialized care (e.g., management of diabetes and hypertension). Alternatively, interventions could be bundled into visits (e.g., do x at each well-woman visit, do y every 2 years, do z only for women at risk). Some evidence already suggests that bundling could be of added value. For example, an evaluation of the National Centers of Excellence in Women's Health found that, compared with other settings, women received better-quality primary health care at those centers having better integrated and coordinated services [82]. However, more such research, focused specifically on integration of preconception care components into primary care, is clearly warranted.
Efforts to promote healthy behavior and improve service delivery should be accompanied by improvements in health care coverage and financing for women of childbearing age. Affordability of care is a major concern for many women [83], and more can be done to improve access to preconception care. Many women under age 65 are uninsured, including 40% of poor women, one third of near-poor women (with income between 100% and 200% of the federal poverty level), 50% of women with disabilities, and 29% of young women ages 19–24 years [84]. Expanding health care coverage for low-income women, through public programs such as Medicaid, Medicaid waivers, the State Children's Health Insurance Program (SCHIP), is particularly important. Medicaid offers particularly important opportunities. In 2003, 12% of all women of childbearing age (15–44 years) and 37% of poor women in that age group relied on Medicaid for health care coverage [83]. Many low-income women do not, however, qualify for Medicaid because they do not have children under age 18, are not over age 65, or are undocumented. As states seek to expand Medicaid coverage to low-income, uninsured adults, women of childbearing age should receive high priority.
Although two thirds of women in Medicaid are of childbearing age [19–44, 85], many additional low-income women do not qualify for Medicaid benefits under current state eligibility rules unless they are pregnant. Without coverage before and between pregnancies, low-income women typically miss preventive visits [83]. Recognizing these gaps and missed opportunities for prevention, 21 states have developed special programs (under federal Medicaid waivers) that cover some women who do not otherwise qualify for Medicaid, particularly to offer family planning and interconception care. Specifically, these states extend eligibility to women who lose coverage after the birth of a baby or starting a job, while other states offer family planning coverage based on income status to men and/or women [86]. An evaluation of “family planning waiver” projects prepared for the federal Center for Medicare and Medicaid Services (CMS) found that they resulted in significant savings to both the federal and state governments [87]. Greater potential savings and prevention, however, could undoubtedly result if states offered more preconception risk screening, health promotion, and interventions; to do so, however, states need permission from the federal government to include such services in their waivers or need for Congress to approve interconception care as an optional benefit.
Finally, special attention should be given to the continuing, and sometimes increasing, racial gap in poor outcomes, especially in terms of access to services, and quality of care. These priorities can potentially complicate decision-making for public health policy. The best evidence of intervention effectiveness is often based on studies of health care systems. However, in developing programs and policies for the delivery of preconception services, one must consider the fact that poor access to health care can be a major determinant of health disparities.
Preconception care: the next maternal and child health frontier
The significant improvements in infant and maternal pregnancy outcomes during the past 40 years have resulted largely from the intensified focus on family planning in the 1960s and 1970s, on child health in the 1970s and 1980s, and on prenatal care in the 1980s and 1990s. Over the past 40 years, many public health and clinical care providers and their organizations have also recognized that, although it is important to offer family planning services to prevent unplanned pregnancies, it is not enough to worry about a pregnant woman's health only during her pregnancy or a child's health only after its birth, because many of the factors that contribute to the mother's and child's health can be identified and often successfully managed before pregnancy.
Today, the greatest opportunities for further improvement in pregnancy outcomes—in improving the health of women and their children—lie in prevention strategies that must be implemented prior to conception to be effective. In 1989, the U.S. Public Health Service Expert Panel identified preconception care as an essential part of prenatal care [88]. National organizations of health professionals, such as the AAP, ACOG, and the American Academy of Family Physicians (AAFP), all recommend specific components of preconception care that should be delivered within the context of health care systems [15, 89]. The time has come to move forward to the next maternal and child health frontier of “prevention” by acting on the recommendations of professional organizations and implementing scientifically proven interventions to further improve pregnancy outcomes.
Action is urgently required in an array of clinical settings, in health promotion campaigns, and in academic research settings. Because of the wide range of interventions included under the umbrella of preconception care, such interventions may be delivered in both primary care and specialty care practices. However, opportunities also exist to improve preconception health through wellness care, through maintenance care for women with chronic health conditions associated with increased preconception risk (e.g., maternal diabetes), and in settings where women seek medical support for one specific risk such as smoking or obesity. In addition, more research is needed to understand how a women's motivation for healthy behavior can be used to initiate the reduction of a number of preconception risks. For example, comparison of concurrent versus sequential interventions for smoking and weight control requires additional clarification [90].
Above all, health care providers and health agencies must understand that early prenatal care is too late for many women and babies. In particular, some evidence-based interventions recommended for implementation during pregnancy could be more effective and beneficial if implemented before conception. For example, although smoking cessation is recommended for all pregnant women, more than one attempt to stop may be necessary to achieve success. Only about 20% of pregnant women who smoke are able to stop smoking during pregnancy [90]. Starting smoking cessation treatment — and successfully completing it—should be recommended before pregnancy is begun. Similarly, the only definitive way of preventing fetal alcohol syndrome is to stop drinking even before pregnancy. In the same manner, HIV testing and genetic counseling are recommended during pregnancy, but additional options are available to women who use these interventions prior to conception, including the decision not to become pregnant.
Conclusion
There is ample evidence that individual elements of preconception care work. Equally important, however, is the evidence that some interventions work best and others only work if provided before pregnancy. Given these realities, to further improve perinatal outcomes, the United States must commit to improving preconception health and to providing preconception care to all women of reproductive age. Even though sufficient knowledge and evidence is present to take action now, further efforts are needed to identify best practices and the most effective means of delivering integrated preconception services.

preconception care
perinatal outcomes
women's health


Criteria for arrhythmogenicity in genetically-modified Langendorff-perfused murine hearts modelling the congenital long QT syndrome type 3 and the Brugada syndrome

The experiments investigated the applicability of two established criteria for arrhythmogenicity in Scn5a+/Δ and Scn5a+/− murine hearts modelling the congenital long QT syndrome type 3 (LQT3) and the Brugada syndrome (BrS). Monophasic action potentials (APs) recorded during extrasystolic stimulation procedures from Langendorff-perfused control hearts and hearts treated with flecainide (1 μM) or quinidine (1 or 10 μM) demonstrated that both agents were pro-arrhythmic in wild-type (WT) hearts, quinidine was pro-arrhythmic in Scn5a+/Δ hearts, and that flecainide was pro-arrhythmic whereas quinidine was anti-arrhythmic in Scn5a+/− hearts, confirming clinical findings. Statistical analysis confirmed a quadratic relationship between epicardial and endocardial AP durations (APDs) in WT control hearts. However, comparisons between plots of epicardial against endocardial APDs and this reference curve failed to correlate with arrhythmogenicity. Restitution curves, relating APD to diastolic interval (DI), were then constructed for the first time in a murine system and mono-exponential growth functions fitted to these curves. Significant (P < 0.05) alterations in the DI at which slopes equalled unity, an established indicator of arrhythmogenicity, now successfully predicted the presence or absence of arrhythmogenicity in all cases. We thus associate changes in the slopes of restitution curves with arrhythmogenicity in models of LQT3 and BrS.
Introduction
Mutations in the SCN5a gene encoding the α-subunit of the cardiac voltage-gated Na+ channel are well established to underlie hereditary arrhythmic syndromes which may result in sudden cardiac death. On the one hand, gain-of-function mutations which increase inward depolarising currents and consequently prolong the ventricular action potential are associated with arrhythmogenicity in the congenital long QT syndrome type 3 (LQT3) [23]. On the other hand, loss-of-function mutations which decrease such currents are associated with arrhythmogenicity in the Brugada syndrome (BrS) [1]. In this case, spatial heterogeneities in the degree of shortening of the ventricular action potential result in characteristic electrocardiographic changes, namely a partial right bundle branch block pattern with ST segment elevation in the right precordial leads [2].
Despite the similar clinical outcomes of LQT3 and BrS, class 1A and 1C, anti-arrhythmic agents, exemplified by quinidine and flecainide respectively, exert contrasting effects on arrhythmogenicity in these two syndromes [31]. Thus in LQT3, quinidine is pro-arrhythmic [33] and flecainide is anti-arrhythmic [22]. In contrast, in BrS quinidine is anti-arrhythmic [24], while flecainide is pro-arrhythmic [7] and used to unmask the arrhythmic phenotype in asymptomatic patients. These contrasts are precisely reflected in results from our murine models of LQT3 [39] and BrS [38], emphasizing the translatability of results obtained from such systems. Furthermore, both quinidine and flecainide are known to have the potential for pro-arrhythmic effects [12, 30].
These similarities and differences have parallels in the physiological changes thought to underlie arrhythmogenicity in LQT3 and BrS. Thus circus-type re-entry [2], requiring both an initiating trigger and a re-entrant substrate, has been implicated in arrhythmogenicity in both these conditions. Of these requirements, both conditions are associated with triggering extrasystoles [3]. In LQT3, these are initiated by early after-depolarizations attributable to action potential prolongation [42], while in BrS they are initiated by phase 2 re-entry [20, 46]. Furthermore, in both LQT3 and BrS, arrhythmia occurs against a re-entrant substrate [3]. In LQT3 this is provided by increased transmural dispersion of repolarization [13, 27, 42]. While increased transmural dispersion of repolarization has also been demonstrated in some studies on BrS [3], increased epicardial dispersion of repolarization [2] and slowed conduction [29] are also implicated. Thus studies in murine hearts have associated alterations in transmural dispersion of repolarization, reflected in relationships between epicardial and endocardial action potential durations, with arrhythmogenicity in models of LQT3 but failed to do so in models of BrS [21, 33, 38, 39, 42].
Arrhythmogenicity has often been associated with increases in the slopes of restitution curves, plotting action potential duration against the preceding diastolic interval, to greater than unity [14, 17–19, 28, 40]. Possible mechanisms that might underlie this association have been discussed in detail on a number of occasions [11, 25, 43, 44]. Furthermore, decreases in the slopes of such curves are associated with the anti-arrhythmic effects of several clinically important agents including amiodarone, bretylium and procainamide [11, 26, 32]. Such associations have previously been demonstrated in a pharmacological model of LQT2 [45] but has not been assessed in LQT3 or in BrS.
The present study compares an analysis examining transmural dispersion of repolarization, assessed by comparing epicardial and endocardial action potential durations obtained over a range of heart rates and previously established to predict arrhythmogenicity in hypokalaemic Langendorff-perfused murine hearts [34], with another examining effects on restitution curve parameters. This represents the first occasion on which restitution curves have been constructed in a murine system, in relation to BrS or LQT3, or indeed in any genetic model of an hereditary arrhythmic syndrome. To this end, the monophasic action potential (MAP) technique and an established Langendorff-perfused system [15, 34–36] are used to study genetically modified mice modelling LQT3 (Scn5a+/Δ) [13] and BrS (Scn5a+/−) [29] before and after exposure to quinidine and flecainide. We proceed to demonstrate that while the former criterion fails, the latter produces predictions in full agreement with the arrhythmogenic findings in every case and thus suggest that alterations in the slopes of restitution curves are associated with arrhythmogenicity in both LQT3 and BrS.
Materials and methods
Experimental animals
Wild-type (WT), Scn5a+/Δ and Scn5a+/− 129 Sv mice aged 3–6 months were housed at 21 ± 1°C with 12 h light/dark cycles. They were fed sterile chow (RM3 Maintenance Diet, SDS, Witham, Essex, UK) and had free access to water. All procedures complied with the UK Animals (Scientific Procedures) Act 1986.
Solutions
Solutions were based on bicarbonate-buffered Krebs-Henseleit solution (119 mM NaCl, 25 mM NaHCO3, 4 mM KCl, 1.2 mM KH2PO4, 1 mM MgCl2, 1.8 mM CaCl2, 10 mM glucose and 2 mM Na-pyruvate; pH adjusted to 7.4) and were bubbled with 95% O2/5% CO2 (British Oxygen Company, Manchester, U.K). Drug-containing solutions were prepared by adding flecainide (Sigma-Aldrich, Poole, UK) to final concentration of 1 μM and quinidine (Sigma-Aldrich, Poole, UK) to final concentrations of 1 and 10 μM: these concentrations were chosen to reflect clinically therapeutic values (effective free and total plasma concentrations of 0.79 and 1.48 μM, respectively, for flecainide and 0.74 and 7.9 μM, respectively, for quinidine) [9].
Preparation
A Langendorff-perfusion protocol previously adapted for murine hearts [4] was used. Briefly, mice were killed by cervical dislocation [Schedule 1: UK Animals (Scientific Procedures) Act 1986] before hearts were quickly excised and placed in ice-cold bicarbonate-buffered Krebs-Henseleit solution. Short sections of aorta were cannulated under the surface of the solution and attached to a custom-made 21-gauge cannula filled using an aneurysm clip (Harvard Apparatus, Edenbridge, Kent, UK), within 90 s of excision. Fresh Krebs-Henseleit solution was passed through 200- and 5-μm filters (Millipore, Watford, UK) and warmed to 37°C using a water jacket and circulator (Techne model C-85A, Cambridge, UK). This solution was then used for constant-flow retrograde perfusion, thereby circumventing any changes in coronary vascular resistance and therefore flow that might otherwise result from the effects of the drugs studied [10], at 2–2.5 ml/min, driven by a peristaltic pump (Watson-Marlow Bredel model 505S, Falmouth, Cornwall, UK). Hearts were regarded as suitable for experimentation if they regained a healthy pink colour and began to contract spontaneously on re-warming.
Electrophysiological measurements
An epicardial MAP electrode (Hugo Sachs, Harvard Apparatus, UK) was placed against the basal left ventricular epicardium. A custom-made endocardial MAP electrode consisting of two twisted strands of high-purity Teflon-coated silver wire of 0.25 mm diameter (Advent Research Materials, UK) was manufactured. A small access window was created in the interventricular septum to allow access to the left ventricular endocardium [8]. The Teflon coating was removed from the distal 1 mm of the electrode; this was then galvanically chlorided to eliminate DC offset, inserted and placed against the septal endocardial surface. MAPs were amplified, band-pass filtered (0.5 Hz to 1 kHz: Gould 2400S, Gould-Nicolet Technologies, Ilford, Essex, UK) and digitised at a sampling frequency of 5 kHz (micro1401, Cambridge Electronic Design, Cambridge, UK). Analysis of MAPs was performed using Spike II (Cambridge Electronic Design, Cambridge, UK).
Experimental protocol
A bipolar platinum stimulating electrode (1 mm inter-pole spacing) was placed on the basal surface of the right ventricular epicardium. Square-wave stimuli (Grass S48 stimulator, Grass-Telefactor, Slough, UK) of 2 ms duration and amplitudes of twice the excitation threshold were initially applied to hearts at a constant baseline cycle length (BCL) of 125 ms until MAPs showed stable baselines, rapid upstroke phases that reached consistent amplitudes and smooth repolarization phases [16] and for at least 10 min. Before subsequent recordings were made, hearts were then exposed to test solutions for 20 min while stimulation was continued.
In initial experiments, MAPs were recorded during regular stimulation at a BCL of 125 ms. Arrhythmogenicity was then assessed both during regular stimulation and using an extrasystolic stimulation protocol [37] previously adapted for the study of arrhythmogenesis in murine hearts incorporating the Scn5a+/Δ and Scn5a+/− mutations [13, 38] and described below. Hearts were then subjected to an adapted dynamic pacing protocol [17] explained in detail below, and the data obtained used to construct restitution curves.
Data analysis
All data are presented as means ± standard errors of the means and include the number of hearts studied. Comparisons between data sets used analysis of variance (significance threshold set at P ≤ 0.05). Curve fitting of particular functions to data sets used a Levenberg-Marquardt algorithm (OriginPro 7.5, OriginLab, MA, USA).
Results
The experiments made direct measurements of MAPs from intact Langendorff-perfused murine hearts. They compared the extents to which relationships between epicardial and endocardial action potential durations and the slopes of restitution curves are associated with arrhythmogenicity in genetic models of the congenital LQT3 (Scn5a+/Δ) and of the BrS (Scn5a+/−), and in wild-type (WT) controls. They began by successfully demonstrating that these experimental models recapitulated arrhythmic phenotypes observed clinically as well as confirming the anticipated effects of treatment with clinically-relevant [6] concentrations of flecainide (1 μM) and quinidine (1 or 10 μM). Hearts were exposed to solutions for 20 min while stimulated at a constant BCL of 125 ms before recording was commenced.
Arrhythmogenicity in WT, Scn5a+/Δ and Scn5a+/− hearts subject to both regular and extrasystolic stimulation recapitulates clinical observations
Figures 1 and 2 show results obtained from WT (Figs. 1a, 2a), Scn5a+/Δ (Figs. 1b, 2b) and Scn5a+/− (Figs. 1c, 2c) hearts treated with control solution (A) and with solutions containing 1 μM flecainide (B), 1 μM quinidine (C) and 10 μM quinidine (D). Figure 1 illustrates MAP waveforms recorded from the epicardia of hearts during the extrasystolic stimulation procedure showing arrhythmic waveforms in situations where arrhythmic activity was observed in >50% of cases; Fig. 2 quantifies the incidence of such arrhythmic activity both during regular stimulation (i) and during the extrasystolic stimulation procedure (ii).
Fig. 1Arrhythmic activity resulting from extrasystolic stimulation. Epicardial monophasic action potential (MAP) recordings illustrating the result of the application of extrasystolic stimuli to WT (a), Scn5a+/Δ (b) and Scn5a+/− (c) hearts treated with control solution (A) and with solutions containing 1 μM flecainide (B), 1 μM quinidine (C) and 10 μM quinidine (D). Traces show arrhythmic activity where this was observed in ≥50% of casesFig. 2Incidences of arrhythmic activity in WT, Scn5a+/Δ and Scn5a+/− hearts subjected to the extrasystolic stimulation procedure. Percentage of WT (a), Scn5a+/Δ (b) and Scn5a+/− (c) hearts demonstrating arrhythmic activity during regular stimulation (i) and during the extrasystolic stimulation procedure (ii). In both these cases, MAPs were obtained during treatment with control solution (A) and with solutions containing 1 μM flecainide (B), 1 μM quinidine (C) and 10 μM quinidine (D). Hashing indicates hearts demonstrating arrhythmic activity, and dots indicate hearts not demonstrating such activity
The frequencies of arrhythmic phenomena in hearts subjected to regular stimulation at a BCL of 125 ms were relatively low irrespective of the genetic or pharmacological conditions but were nevertheless consistent with clinical expectations. First, zero out of seven WT hearts (~0%, Fig. 2a, i) exposed to control solution (A) demonstrated arrhythmic activity, while treatment with flecainide (B) led to arrhythmic activity in only one out of six (~17%) cases. Furthermore, such hearts never showed arrhythmic activity during treatment with 1 μM quinidine (C, zero out of six hearts, ~0%) and again did so in only one out of six cases (~17%) with 10 μM quinidine (D). Secondly, arrhythmic activity did occur in one out of six (~17%) Scn5a+/Δ hearts (Fig. 2b) treated with control solution (A) and was absent in all cases (zero out of six hearts, ~0%) during treatment with flecainide (B). Arrhythmic activity was not observed (zero out of four hearts, ~0%) during treatment with 1 μM quinidine (C) but did occur in one out of five hearts (~20%) exposed to 10 μM quinidine (D). Thirdly, two out of nine Scn5a+/− hearts (~22%, Fig. 2c) exhibited arrhythmic activity during exposure to control solution (A). Such arrhythmic activity was also observed in one out of five hearts (~20%) treated with flecainide (B). Arrhythmic activity was then absent after treatment with either 1 μM (C) or 10 μM (D) quinidine (zero out of five, 0%, and zero out of six, 0%, hearts, respectively).
In contrast, extrasystolic stimulation (Figs. 1 and 2, ii) revealed clearer trends in arrhythmogenicity in full agreement with clinical expectations. First, while zero out of seven WT hearts (0%, Fig. 2a, ii) exposed to control solution (A) exhibited arrhythmic activity, treatment with flecainide (B) increased the incidence of such events to two out of six hearts (~33%). Furthermore, exposure to 1 μM quinidine (C) resulted in arrhythmic activity in four out of eight hearts (~50%), while exposure to 10 μM quinidine (D) resulted in arrhythmic activity in three out of six hearts (~50%). Secondly, one out of five Scn5a+/Δ hearts (~20%, Fig. 2b, ii) demonstrated arrhythmic activity during exposure to either control solution (A) or flecainide (B). However, exposure to quinidine resulted in arrhythmic activity in three out of five hearts (~60%) at 1 μM (C) and four out of four hearts (~100%) at 10 μM (D). Thirdly, Scn5a+/− hearts (Fig. 2c, ii) demonstrated such arrhythmic activity in three out of nine cases (~33%) during treatment with control solution (A) but in five out of six cases (~83%) during treatment with flecainide (B). Furthermore, arrhythmic activity was observed in only one out of six such hearts (~17%) during treatment with 1 μM quinidine (C) and in zero out of six hearts during exposure to 10 μM quinidine (D).
Graphical analyses of arrhythmogenic tendency
In an effort to explore the possible basis for these arrhythmic phenomena, subsequent experiments made systematic measurements of epicardial and endocardial action potential durations (at 90% repolarization, APD90s) during a dynamic pacing protocol [17]. This consisted of cycles each comprising 100 stimuli, initially delivered at a BCL of 175 ms. BCL was then decremented in 50-ms steps with each subsequent cycle until a reproducible sequence of consistently shaped MAP waveforms was no longer obtained. Steady states were consistently reached within the first 50 responses, and therefore mean values of APD90 were obtained from the final 50 action potentials in each cycle. This approach provided data that could be used to evaluate over the full range of BCLs the relationship between arrhythmogenicity and two possible electrophysiological properties. Thus it was possible first to evaluate transmural repolarization gradients and secondly to evaluate slopes of restitution curves relating APD90 to the preceding DI, given by BCL minus APD90 [17, 25, 28, 43].
Relationships between epicardial and endocardial action potential durations are not consistently associated with either the presence or absence of arrhythmogenicity
Alterations in relationship between epicardial and endocardial APD90 determined at a single BCL has previously been associated with arrhythmogenicity in hearts obtained from our Scn5a+/Δ mice [42]. The present study now extends this analysis from a single BCL to a range of BCLs and applies it to Scn5a+/− hearts for the first time, plotting endocardial APD90 on the abscissa and epicardial APD90 on the ordinate over the full range of BCLs investigated (triangles, Fig. 3). Such plots have previously been used to assess arrhythmogenicity in hypokalaemic WT murine hearts [34] and provided possible criteria for arrhythmogenicity. The control plot obtained from WT hearts perfused with control solution (six hearts, Fig. 3a, A) gave a quadratic relationship between endocardial and epicardial APD90 (solid line), in agreement with previous work [34]. Thus the curve y = ax2 + bx + c, where a = 0.0097 ± 0.0028, b = 1.46 ± 0.27 and c = −5.06 ± 6.34, gave a reduced χ2 of 0.49. Repeating the regression analysis this time fitting the line y = mx + c gave a reduced χ2 of 0.86, and permitted the derivation of an F statistic for the appropriate, N + 1 = 17 data points, given by  in which the subscripts on the right-hand side of the equation denote the number of parameters involved [5]. This yielded a probability of exceeding F of <0.05, suggesting that inclusion of the quadratic term was justified, again in full agreement with the previous study [34].
Fig. 3Relationships between epicardial and endocardial action potential durations (at 90% repolarization, APD90s) obtained over a range of baseline cycle lengths. WT (a), Scn5a+/Δ (b) and Scn5a+/− (c) hearts were exposed to control solution (A) and to solutions containing 1 μM flecainide (B), 1 μM quinidine (C) and 10 μM quinidine (D) and stimulated at baseline cycle lengths of between 80 ms and 175 ms. Broken lines indicate reference curves constructed by least-squares fitting of the equation y = ax2 + bx + c to data points obtained from wild-type hearts treated with control solution
Data points obtained under the range of genetic and pharmacological conditions studied could then be compared with this reference curve (broken lines, Fig. 3) with the expectation that the departure of points to the left of this curve might be associated with arrhythmogenicity [34]. However, data sets obtained before and after the pharmacological manoeuvres satisfied this expectation in WT but clearly failed to do so in the remaining genetically modified hearts.
First, treatment of WT hearts (Fig. 3a) with flecainide (five hearts, B), 1 μM quinidine (six hearts, C) and 10 μM quinidine (six hearts, D) all resulted in points falling to the left of the reference curve, in parallel with the occurrence of arrhythmic activity under these conditions (Fig. 2a, B, C and D). Secondly, data points obtained from Scn5a+/Δ hearts (Fig. 3b) treated with control solution (five hearts, A) again fell to the left of the reference curve in contrast with the low incidence of arrhythmic activity in this setting (Fig. 2b, A). This pattern was maintained when such hearts were exposed to flecainide (five hearts, Fig. 3b, B), once again in contrast with the low frequency which arrhythmic activity was observed under these conditions (Fig. 2b, B). Exposure to 1 μM quinidine (five hearts, Fig. 3b, C) and 10 μM quinidine (six hearts, D) again both resulted in points falling to the left of the control curve, this time in parallel with the high incidence of arrhythmic activity in these settings (Figs. 2b, C and 3b, D). Thirdly, data points obtained from Scn5a+/− hearts (Fig. 3c) treated with control solution (six hearts, A) consistently fell on the reference curve, in contrast with the occurrence of arrhythmic activity in this setting (Fig. 2c, A). Furthermore, treatment with flecainide (six hearts, Fig. 3c, B) failed to result in a left-shift of data points, in contrast with the large increase in the incidence of arrhythmic activity (Fig. 2c, B). In addition, points obtained during treatment with either 1 μM quinidine (five hearts, Fig. 3c, C) or 10 μM quinidine (seven hearts, D) again fell to the left of the reference curve, in contrast with the anti-arrhythmic effect of this agent demonstrated in Fig. 2c, C and D.
Alterations in the slopes of restitution curves correlate with the presence and absence of arrhythmic activity
Restitution curves (data points, left ordinate) were then plotted using the APD90 (left ordinate) and DI data obtained during the dynamic pacing protocol from the epicardia (circles, Fig. 4) and endocardia (squares, Fig. 5) of WT (Figs. 4a, 5a), Scn5a+/Δ (Figs. 4b, 5b) and Scn5a+/− (Figs. 4c, 5c) hearts for the first time. Hearts were studied during treatment with control solution (A) and with solutions containing flecainide (B), 1 μM quinidine (C) or 10 μM quinidine (D). Under all conditions, epicardial and endocardial APD90 both decreased as DI decreased. Increases or decreases in the slopes of such relationships were in full agreement with the presence or absence of arrhythmic activity under all conditions studied.
Fig. 4Epicardial restitution curves plotting action potential duration (at 90% repolarization, APD90) against preceding diastolic interval (DI). Data were obtained from the epicardia (circles) of WT (a), Scn5a+/Δ (b) and Scn5a+/− (c) hearts exposed to control solution (A) and to solutions containing 1 μM flecainide (B), 1 μM quinidine (C) and 10 μM quinidine (D). Curves are fitted with mono-exponential growth functions obtained by least squares fitting to the experimental values of APD90 and DI (solid lines, left ordinates). Gradients were obtained by differentiation of the fitted functions (broken lines, right axes). Shaded boxes indicate ranges of DI values at which such gradients exceed unityFig. 5Endocardial restitution curves plotting action potential duration (at 90% repolarization, APD90) against preceding diastolic interval (DI). Data were obtained from the endocardia (squares) of WT (a), Scn5a+/Δ (b) and Scn5a+/− (c) hearts exposed to control solution (A) and to solutions containing 1 μM flecainide (B), 1 μM quinidine (C) and 10 μM quinidine (D). Curves are fitted with mono-exponential growth functions obtained by least squares fitting to the experimental values of APD90 and DI (solid lines, left ordinates). Gradients were obtained by differentiation of the fitted functions (broken lines, right axes). Shaded boxes indicate ranges of DI values at which such gradients exceed unity
Restitution curves yield parameters predictive of arrhythmogenicity
To permit quantitative analysis, simple mono-exponential growth functions, as used previously [28], were fitted to the experimental data. These took the form:
where y represents APD90, x represents DI, and y0, A and τ are constants obtained by least squares fitting to the experimental values of APD90 and DI in each case. These fitted parameters, along with corresponding reduced χ2 values, are shown in Tables 1 (epicardium) and 2 (endocardium). Curve fits performed on data sets obtained by separating successive odd- and even-numbered action potentials yielded statistically indistinguishable (P > 0.05) parameters in all cases (Tables 3 and 4) thus eliminating the possibility of error arising as a result of averaging. The corresponding gradients were then given by:
and take their largest values at the shortest BCLs studied.
Table 1Fit parameters for epicardial restitution curvesConditiony0 (ms)A (ms)τ (ms)χ2WT (6 hearts)1.6 ± 4.456.2 ± 2.458.4 ± 9.81.4WT + flecainide (5 hearts)−16.3 ± 9.4104.7 ± 7.182.8 ± 28.05.9WT + 1 μM quinidine (6 hearts)−38.3 ± 17.492.4 ± 14.745.1 ± 9.42.5WT + 10 μM quinidine (6 hearts)−863.0 ± 1,387.1981.6 ± 1,369.127.1 ± 15.9113.3Scn5a+/Δ (5 hearts)−19.6 ± 7.290.0 ± 4.453.1 ± 10.714.0Scn5a+/Δ + flecainide (5 hearts)−16.1 ± 6.582.2 ± 12.395.3 ± 42.13.5Scn5a+/Δ + 1 μM quinidine (5 hearts)−677.1 ± 306.1748.0 ± 304.715.2 ± 2.210.0Scn5a+/− (6 hearts)−674.8 ± 329.9729.4 ± 329.114.2 ± 2.18.4Scn5a+/− + flecainide (6 hearts)−56,359.8 ± 2,559.752,677.3 ± 2.27.8 ± 2.514.8Scn5a+/− + 1 μM quinidine (5 hearts)−118.3 ± 57.4202.5 ± 49.436.4 ± 10.516.7Scn5a+/− + 10 μM quinidine (7 hearts)−1.7 ± 44.7131.9 ± 62.3158.4 ± 262.210.8Results of least-squares fits to the function  under the conditions indicatedTable 2Fit parameters for endocardial restitution curvesConditiony0 (ms)A (ms)τ (ms)χ2WT (6 hearts)−11.8 ± 3.773.8 ± 1.765.2 ± 8.41.2WT + flecainide (5 hearts)−769.0 ± 244.1827.3 ± 243.714.5 ± 1.31.4WT + 1 μM quinidine (6 hearts)−65.8 ± 100.4113.5 ± 98.324.9 ± 12.011.7WT + 10 μM quinidine (6 hearts)−41.9 ± 71.578.2 ± 68.732.8 ± 19.313.6Scn5a+/Δ (5 hearts)−31.9 ± 9.0109.9 ± 5.149.6 ± 9.010.3Scn5a+/Δ + flecainide (5 hearts)−561.1 ± 1.2631.6 ± 15.118.7 ± 0.36.7Scn5a+/Δ + 1 μM quinidine (5 hearts)−499.7 ± 143.8563.0 ± 142.916.3 ± 1.656.0Scn5a+/− (6 hearts)−281.7 ± 9.843.5 ± 6.448.5 ± 19.04.3Scn5a+/− + flecainide (6 hearts)−1,050.6 ± 1,558.51,100.1 ± 1,557.811.7 ± 4.010.0Scn5a+/− + 1 μM quinidine (5 hearts)−261.2 ± 117.9350.5 ± 107.834.2 ± 9.321.7Scn5a+/− + 10 μM quinidine (7 hearts)−220.1 ± 322.0319.1 ± 316.826.2 ± 12.745.5Results of least-squares fits to the function  under the conditions indicatedTable 3Fit parameters for endocardial restitution curves separating successive odd- and even-numbered action potentialsConditionOddEveny0 (ms)A (ms)τ (ms)χ2y0 (ms)A (ms)τ (ms)χ2WT (6 hearts)13.4 ± 7.640.5 ± 12.950.3 ± 21.46.38.1 ± 11.258.3 ± 25.448.2 ± 32.34.6WT + flecainide (5 hearts)−14.7 ± 8.9105.2 ± 9.088.0 ± 31.35.8−44.6 ± 18.5117.4 ± 14.249.0 ± 9.91.5WT + 1 μM quinidine (6 hearts)−20.6 ± 9.975.8 ± 6.256.4 ± 13.23.8−34.2 ± 11.789.2 ± 9.345.9 ± 7.83.3WT + 10 μM quinidine (6 hearts)−1,391.7 ± 3,195.91,496.0 ± 3,182.021.1 ± 14.3117.9−393.1 ± 5.3.0532.8 ± 481.741.1 ± 27.6115.7Scn5a+/Δ (5 hearts)−23.9 ± 8.392.0 ± 5.347.8 ± 9.012.4−24.4 ± 8.692.6 ± 5.647.6 ± 8.911.8Scn5a+/Δ + flecainide (5 hearts)14.6 ± 6.377.1 ± 8.484.1 ± 32.23.4−33.2 ± 46.4110.1 ± 41.936.4 ± 13.12.0Scn5a+/Δ + 1 μM quinidine (5 hearts)−250.2 ± 12.8483.0 ± 24.518.8 ± 7.812.4−254.7 ± 321.2328.1 ± 318.320.0 ± 7.75.3Scn5a+/− (6 hearts)−850.4 ± 282.7904.2 ± 282.113.5 ± 1.34.3−913.3 ± 283.3968.6 ± 282.713.7 ± 1.23.7Scn5a+/− + flecainide (6 hearts)−1,473.8 ± 3,144.51,534.3 ± 2,141.214.7 ± 7.634.3−2,108.8 ± 4,489.92,171.3 ± 4,486.814.0 ± 6.735.0Scn5a+/− + 1 μM quinidine (5 hearts)−46.8 ± 51.2146.8 ± 21.359.5 ± 43.59.4−46.7 ± 50.6149.2 ± 20.261.0 ± 44.29.3Scn5a+/− + 10 μM quinidine (7 hearts)7.3 ± 34.2167.7 ± 264.8272.8 ± 78.310.82.8 ± 44.6143.1 ± 121.3192.2 ± 405.812.0Results of least-squares fits to the function  under the conditions indicatedTable 4Fit parameters for endocardial restitution curves separating successive odd- and even-numbered action potentialsConditionOddEveny0 (ms)A (ms)τ (ms)χ2y0 (ms)A (ms)τ (ms)χ2WT (6 hearts)−17.4 ± 5.373.8 ± 2.867.3 ± 13.54.7−38.9 ± 15.491.6 ± 13.046.5 ± 8.51.8WT + flecainide (5 hearts)−49.2 ± 26.2113.8 ± 23.035.7 ± 8.27.6−367.3 ± 171.3426.9 ± 170.617.7 ± 2.41.3WT + 1 μM quinidine (6 hearts)−393.2 ± 368.6437.7 ± 368.614.9 ± 4.318.1−1,337.3 ± 1,206.41,384.5 ± 1,205.711.5 ± 2.514.7WT + 10 μM quinidine (6 hearts)−17.7 + −39.053.2 ± 35.042.6 ± 28.911.2−28.0 ± 62.766.6 ± 58.239.6 ± 29.717.3Scn5a+/Δ (5 hearts)−149.6 ± 131.4221.0 ± 128.626.4 ± 7.60.9−42.3 ± 10.5115.3 ± 8.240.8 ± 5.02.6Scn5a+/Δ + flecainide (5 hearts)−1,133.8 ± 784.21,201.4 ± 783.115.2 ± 2.62.0−2,164.0 ± 1,195.72,233.3 ± 1,195.013.6 ± 1.61.4Scn5a+/Δ + 1 μM quinidine (5 hearts)−2,720.4 ± 3,917.82,779.7 ± 3,917.210.4 ± 2.83.4−3,527.2 ± 4,674.783,603.0 ± 4,674.111.1 ± 2.73.9Scn5a+/− (6 hearts)−134.6 ± 28.9195.3 ± 28.022.8 ± 2.13.3−149.1 ± 33.5212.0 ± 32.622.9 ± 2.23.6Scn5a+/− + flecainide (6 hearts)−13.8 ± 29.266.2 ± 27.132.5 ± 11.11.8−55.4 ± 55.5108.6 ± 54.623.6 ± 5.91.2Scn5a+/− + 1 μM quinidine (5 hearts)−1,979.9 ± 1,709.02,051.5 ± 1,704.615.9 ± 2.813.4−108.4 ± 15,101.810,911.4 ± 15,098.311.8 ± 3.014.8Scn5a+/− + 10 μM quinidine (7 hearts)−332.1 ± 447.2431.4 ± 439.825.2 ± 12.7116.0−160.0 ± 289.5262.2 ± 281.920.1 ± 18.148.1Results of least-squares fits to the function  under the conditions indicated
Figures 4 and 5 thus superimpose optimisations of Eq. 1 to the data shown in Tables 1 and 2 (solid lines, left-hand ordinates). Furthermore, they show the corresponding gradients calculated using Eq. 2 (broken lines, right-hand axes). Shaded regions where present indicate DIs less than the critical DI at which these gradients exceed unity: the existence of such a region is an established criterion for arrhythmogenesis [25]. Critical DI was calculated as:
Figure 6 proceeds to show these values of critical DI derived from the fitted values of A and τ for the epicardia (filled bars) and endocardia (open bars) under each of the above conditions. In contrast to the findings from the analysis of the relationships between epicardial and endocardial APD90s described above, the present analysis of epicardial and endocardial restitution properties together provided datasets in complete agreement with the observed incidences of arrhythmic activity.
Fig. 6Critical diastolic intervals obtained from restitution curves. Critical diastolic intervals at which gradients equalled unity obtained from epicardial (filled bars) and endocardial (open bars) restitution curves shown in Figs. 4 and 5 in WT (a), Scn5a+/Δ (b) and Scn5a+/− (c) hearts exposed to control solution (A) and to solutions containing 1 μM flecainide (B), 1 μM quinidine (C) and 10 μM quinidine (D). Asterisks indicate values that are significantly (P < 0.05) larger than those recorded in WT hearts exposed to control solution
First, in WT hearts (Fig. 6a) exposed to control solution (A) the critical DI took a value of −63.63 ± 14.41 ms in the epicardium and −15.43 ± 4.97 ms in the endocardium. Treatment with flecainide (B) significantly (P < 0.05) increased these values to 19.45 ± 9.39 ms in the epicardium and 58.75 ± 18.71 ms in the endocardium. This critical DI was attained in the endocardium (shaded region in Fig. 5a, B), in parallel with the increased incidence of arrhythmogenesis observed during the extrasystolic stimulation procedure under these conditions (Fig. 2, ii). Treatment with 1 μM quinidine (Fig. 6a, C) also significantly (P < 0.05) increased the critical DI in both the epicardium and endocardium (32.30 ± 7.80 ms and 27.80 ± 14.53 ms respectively) in parallel with the observed arrhythmogenicity on extrasystolic stimulation under these conditions (Fig. 2, ii), although such values were not attained during the dynamic pacing protocol. Furthermore, treatment with 10 μM quinidine (Fig. 6a, D) again significantly (P < 0.05) increased these values to 97.20 ± 57.90 ms in the epicardium and to 28.50 ± 34.52 ms in the endocardium. This DI was attained in the epicardium in parallel with the observed arrhythmogenicity during the extrasystolic stimulation procedure (Fig. 2, ii). Thus, increases in the critical DI are precisely paralleled with arrhythmogenicity in WT hearts treated with flecainide and quinidine.
Secondly, critical DIs obtained from Scn5a+/Δ hearts (Fig. 6b) were always significantly greater than those in WT controls (P < 0.05 in all cases) in parallel with the increased incidence of arrhythmic activity observed during the extrasystolic stimulation procedure (Fig. 2, ii). Thus, in hearts treated with control solution (Fig. 6b, A) the critical DI increased to 28.04 ± 8.08 ms in the epicardium and 39.38 ± 10.28 ms in the endocardium: These values were attained in both the epicardium and endocardium in association with the increased incidence of arrhythmic activity on extrasystolic stimulation observed (Fig. 2, ii) and in full agreement with clinical expectations [23]. Treatment with flecainide (Fig. 6b, B) decreased the epicardial value (−14.09 ± 9.05 ms, P < 0.05 as compared to hearts treated with control solution) but did not significantly affect the endocardial value (21.41 ± 8.58 ms, P > 0.05 as compared to hearts treated with control solution), having no effect on the observed incidence of arrhythmic activity on extrasystolic stimulation (Fig. 2, ii). However, treatment with 1 μM quinidine (Fig. 6b, C) significantly (P < 0.05) increased both epicardial and endocardial values (59.09 ± 26.91 ms and 57.64 ± 16.68 ms, respectively) as compared to those obtained from such hearts treated with control solution, in association with the observed increases in the incidence of arrhythmic activity resulting from extrasystolic stimulation (Fig. 2, ii) and again in agreement with expectations from clinical work [33]. It was not possible to obtain MAPs over a sufficiently wide range of BCLs to permit the construction of such curves in Scn5a+/Δ hearts treated with 10 μM quinidine.
Thirdly, critical DI values were also increased in Scn5a+/− hearts (Fig. 6c) in association with the increased incidence of arrhythmic activity observed. Thus, in hearts treated with control solution (A), the critical DIs were significantly (P < 0.05) increased as compared to WT controls in both the epicardium and endocardium to 56.00 ± 27.79 ms and 52.59 ± 9.50 ms, respectively. These values were attained in both the epicardium and endocardium, fulfilling clinical expectations [1]. Treatment with flecainide (B) did not significantly alter these values (68.14 ± 24.09 ms in the epicardium and 58.56 ± 14.75 ms in the endocardium). These were again attained in both epicardium and endocardium in association with the arrhythmic activity observed on extrasystolic stimulation (Fig. 2, ii). Furthermore, treatment with quinidine at a concentration of 1 μM (Fig. 6c, C) had no significant effect on critical DIs in either the epicardium or endocardium (62.45 ± 29.74 ms and 79.63 ± 39.31 ms, respectively). These values were again attained in both epicardium and endocardium. However, treatment with quinidine at 10 μM significantly decreased both epicardial and endocardial critical DIs, such that they became statistically indistinguishable from WT controls (29.01 ± 9.27 ms and −80 ± 3.58 ms, respectively). This was associated with the complete absence of arrhythmogenesis during the extrasystolic stimulation procedure (Fig. 2, ii).
Thus, while relationships between epicardial and endocardial action potential durations obtained over a range of cycle lengths are poorly correlated with arrhythmogenicity, restitution curves are, in general, predictive of arrhythmogenicity under the genetic and pharmacological conditions studied.
Discussion
The congenital LQT3 and the BrS constitute two important examples of genetic causes for ventricular arrhythmia and sudden cardiac death [3]. These conditions result from similarly targeted mutations in the SCN5a gene coding for the cardiac Na+ channel: gain-of-function mutations are characteristic of LQT3, while loss-of-function mutations are characteristic of BrS [1, 23].
The present study began by using the MAP technique to verify that Langendorff-perfused murine hearts modelling LQT3 (Scn5a+/Δ) [13] and BrS (Scn5a+/−) [29] recapitulated features of the incidence of arrhythmic activity reported clinically. These results were in full agreement with previous physiological and pharmacological findings from our LQT3 [39] and BrS [38] models. Epicardial and endocardial MAP recordings were then used to evaluate the applicability of two established criteria in predicting arrhythmogenicity in these models over the full physiological range of heart rates (quantified as baseline cycle lengths, BCLs).
Transmural dispersion of repolarization was studied over a range of BCLs by comparing epicardial and endocardial action potential durations (at 90% repolarization, APD90s) in the two genetic models. Such an analysis had previously been demonstrated to reveal arrhythmogenicity in hypokalaemic Langendorff-perfused murine hearts [34]. Furthermore, changes in this relationship investigated at a single BCL had previously been associated with arrhythmogenicity in our Scn5a+/Δ hearts [42]. Plots of epicardial against endocardial APD90 in WT hearts treated with control solution gave reference relationships best fitted by a quadratic function, in full agreement with previous work [34]. Treatment of such WT hearts with either quinidine or flecainide resulted in the data points falling to the left of the reference curves, reflecting increased transmural repolarization gradients, in association with increased incidences of arrhythmic activity and again in full agreement with a previous study [34]. However, such plots failed to consistently predict the presence or absence of arrhythmogenicity in the Scn5a+/Δ and Scn5a+/− hearts. Such left-shifting of points was observed in Scn5a+/Δ hearts under all pharmacological conditions studied, despite the established anti-arrhythmic effect of flecainide in this setting. Furthermore, data points obtained from Scn5a+/− hearts treated with control solution fell on the reference curve despite the increased incidence of arrhythmic activity observed. Treatment with flecainide failed to result in a left-shift of points, despite its established pro-arrhythmic effect; treatment with quinidine did result in such a shift, despite its anti-arrhythmic effect.
These discrepancies prompted an investigation for alternative criteria for arrhythmogenicity. We therefore explored the applicability of a second approach involving restitution curves plotting APD90 against the preceding DI. Arrhythmic activity occurring in a range of settings has previously been associated with increases in the slopes of such curves to greater than unity [14, 17–19, 28, 40]. Such an association has been demonstrated in a canine pharmacological model of the congenital long QT syndrome type 2 [45] but has not previously been studied in any models of LQT3 or BrS in any species. Furthermore, decreases in the slopes of such curves have been associated with the effects of clinically important anti-arrhythmic drugs, including the class 1A agent procainamide in a canine model [32]. However, such slopes have not been examined in relation to either quinidine or flecainide, nor have any such studies previously been attempted in murine models. This study accordingly proceeded to construct epicardial and endocardial restitution curves for the first time in a murine system and in relation to LQT3 or BrS in any species.
In contrast to the previous criterion, epicardial and endocardial restitution curves taken together yielded parameters in precise agreement with the presence or absence of arrhythmic phenomena demonstrated. In WT hearts, treatment with either quinidine or flecainide significantly increased the critical DI at which the slopes of the restitution curves exceeded unity, in full agreement with the increased incidences of arrhythmic activity observed. Not only did the Scn5a+/Δ and Scn5a+/− mutations result in significant increases in critical DI in association with increased rates of arrhythmic activity, but quinidine and flecainide exerted contrasting effects on this critical DI in agreement with clinical expectations in both cases. Thus, in Scn5a+/Δ hearts, treatment with quinidine increased critical DIs in association with its pro-arrhythmic effect. Furthermore, in Scn5a+/− hearts, treatment with quinidine decreased critical DIs in association with the decreased incidence of arrhythmic activity observed, while treatment with flecainide had the opposite effect. Thus class 1A agents may result in either increases (quinidine in WT and Scn5a+/Δ murine hearts) or decreases (quinidine in Scn5a+/− murine hearts and procainamide in canine hearts) [32] in the critical DI: Such changes are in full agreement with effects on arrhythmogenicity in all cases.
Taken together, these results demonstrate that criteria considering effects on the slopes of restitution curves successfully account for arrhythmogenicity in murine models of arrhythmic syndromes resulting from mutations in the Scn5a Na+ channel, as well as for the effects of quinidine and flecainide on such arrhythmogenicity. These observations might form the basis for future clinical explorations of the utility of restitution curves obtained using the MAP technique [28, 40] in the assessment of arrhythmic propensity in hereditary arrhythmic syndromes. Such a robust means of assessing arrhythmogenicity might be particularly useful given the known variable expressivity of the arrhythmic phenotype in patients harbouring such mutations [41].

heart
action potential
arrhythmia
heart rate
heart excitation
venticular muscle
ventricle