Ann Thorac Surg 2003;75:1937-1941
© 2003 The Society of Thoracic Surgeons
Original article: cardiovascular
Fetal myocardial protection is markedly improved by reduced cardioplegic calcium content
Sunil P. Malhotra, MDa,
Stephan Thelitz, MDb,
R. Kirk Riemer, PhDc,
V. Mohan Reddy, MDc,
Sam Suleman, BSc,
Frank L. Hanley, MD*a,b,c
a Department of Surgery, New York University School of Medicine, New York, New York, USA
b Department of Cardiothoracic Surgery, University of Cologne Medical Center, Cologne, Germany
c Department of Cardiothoracic Surgery, Stanford University School of Medicine, Stanford, California, USA
Accepted for publication December 31, 2002.
* Address reprint requests to Dr Malhotra, New York University School of Medicine, Department of Surgery, 530 First Avenue, NB-15N1, New York, NY 10016, USA
e-mail: spmalhotra{at}yahoo.com
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Abstract
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BACKGROUND: Fetal cardiac surgery holds a clear therapeutic benefit in the treatment of lesions that increase in complexity due to pathologic blood flow patterns during development. Fetal and neonatal myocardial physiology differ substantially, particularly in the regulation of myocardial calcium concentration. To examine issues of calcium homeostasis and fetal myocardial protection, a novel isolated biventricular working fetal heart preparation was developed.
METHODS: Hearts from 20 fetal lambs, 115 to 125 days gestation, were harvested and perfused with standard Krebs-Henseleit (K-H) solution. The descending aorta was ligated distal to the ductal insertion and the branch pulmonary arteries were ligated to mimic fetal cardiovascular physiology. Hearts were arrested for 30 minutes with normocalcemic (n = 8), hypocalcemic (n = 6), or hypercalcemic (n = 6) cold crystalloid cardioplegia before reperfusion with K-H solution.
RESULTS: Compared with normocalcemic cardioplegia, hypocalcemic cardioplegia improved preservation of left ventricular (LV) systolic function (88% ± 2.2% vs 64% ± 15% recovery of end-systolic elastance, p = 0.02), diastolic function (12% ± 21% vs 38% ± 11% increase in end-diastolic stiffness, p = 0.04), and myocardial contractility (97% ± 9.6% vs 75.2% ± 13% recovery of preload recruitable stroke work [PRSW], p = 0.04). In contrast, the fetal myocardium was sensitive to hypercalcemic arrest with poor preservation of LV systolic function (37.5% ± 8.4% recovery of elastance), diastolic function (86% ± 21% increased stiffness), and overall contractility (32% ± 13% recovery of PRSW). Myocardial water content was reduced in hearts arrested with hypocalcemic cardioplegia (79% ± 1.8% vs 83.7% ± 0.9%, p = 0.0006).
CONCLUSIONS: This study demonstrates the sensitivity of the fetal myocardium to cardioplegic calcium concentration. Hypocalcemic cardioplegia provides superior preservation of systolic, diastolic, and contractile function of the fetal myocardium.
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Introduction
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Fetal surgical intervention has been successful in the treatment of several life-threatening defects including congenital diaphragmatic hernia and sacrococcygeal teratoma [1, 2]. In utero repair of selected congenital cardiac lesions may also be beneficial for defects that increase in complexity during the prenatal period. Currently, fetal echocardiography can accurately diagnose certain congenital heart defects as early as 10 to 12 weeks of gestation [3]. Development of techniques to perform fetal cardiac surgery will enable prenatal repair of appropriate lesions.
Many complex structural defects evolve during development due to alterations in intracardiac blood flow. A primary lesion, such as valvular stenosis, can progress to a more severe lesion, such as hypoplasia of a heart chamber or great vessel, due to the pathologic derangements in blood flow patterns [4]. Addressing the primary defect during the fetal period is advantageous because normal intracardiac flow patterns can be restored. The fetal heart can recover during this period and prepare for the transition to postnatal circulation. It is well documented that complex congenital heart defects such as hypoplastic left heart syndrome, hypoplastic right heart syndrome, and the univentricular heart are well tolerated during the fetal period, primarily due to the parallel arrangement of the fetal ventricles [5, 6].
However, before fetal cardiac surgery becomes a clinical reality, the unique characteristics of the developing myocardium need to be recognized to develop appropriate strategies for fetal myocardial protection. Although the heart is functional early in gestation, many of its cellular components remain immature. The sarcoplasmic reticulum (SR) in fetal myocytes, essential for myocardial force generation, differs significantly in morphology and function from adult myocytes. Myocardial SR content is markedly reduced in the fetal myocardium [7]. Furthermore, fetal SR structure is immature due to low concentrations of calcium ATPase and calsequestran, a calcium storage protein [8]. In addition, functional differences exists between fetal and adult forms of phospholamban, a protein involved in SR membrane calcium transport [8]. As a result, myocardial calcium uptake and release is impaired in fetal myocytes [9]. Immature isoforms of contractile proteins, such as heavy chain myosin, are predominant in the fetal ventricle [10]. The adult form of heavy chain myosin is only found in atrial tissue during fetal life and only begins to be expressed in the ventricle postnatally [10]. These findings may explain the observed impairment of the Frank-Starling mechanism and diastolic compliance of the fetal ventricle [11].
Calcium concentration during cardioplegia is a significant issue in myocardial protection. Due to the immaturity of myocardial calcium regulation, the fetal heart may be particularly sensitive to cardioplegic calcium concentration. In this study we examined the affect of calcium concentration on preservation of fetal myocardial function using an isolated, biventricular fetal working heart preparation that closely simulates fetal cardiovascular physiology.
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Material and methods
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Experimental preparation
Hearts from 20 fetal lambs (119 ± 3.1 days gestation, 80% gestational age, 2.5 ± 0.4 kg) were harvested and mounted on a controlled perfusion apparatus (Fig 1).
Pregnant ewes were sedated with intramuscular ketamine (10 mg/kg). Ewes were then anesthetized with inhaled isoflurane (2%), intubated, and mechanically ventilated. Following maternal laparotomy and uterotomy, the fetal chest was exposed. A midline sternotomy was performed and the great vessels were identified; 1500 U of heparin was administered directly into the superior vena cava (SVC) to account for both the fetus and placenta. The descending aorta was transected distal to the ductal insertion. The heart was then removed following transection of the inferior vena cava (IVC), SVC, and innominate artery. The heart was excised along with a cuff of lung tissue to facilitate identification of the pulmonary arteries and veins during mounting. The heart was placed in Krebs-Henseleit (K-H) bicarbonate buffer equilibrated with 95% O2 and 5% CO2 and transported to the water-jacketed perfusion apparatus (Radnotti Glass Technology, Inc., Monrovia, CA). To simulate the hypoxic conditions of the fetal heart, the perfusate was maintained at a pO2 of 30 using blood gas measurements.
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Fig 1. Diagram of the isolated biventricular fetal heart preparation. Orientation for Langendorff (solid arrows) and working heart (dashed arrows) perfusion modes illustrated. (IVC = inferior vena cava; LA = left atrium; LV = left ventricle; PA = pulmonary artery; RA = right atrium; RV = right ventricle; SVC = superior vena cava.)
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During continuous aortic perfusion with K-H at 37°C, the innominate artery was cannulated with a flexible plastic cannula. A similar perfusion cannula was placed through the SVC into the right atrium (RA). A pressure catheter was placed into the transected descending aorta into the aortic root. The aorta was then ligated distal to the ductus arteriosus. To approximate the high fetal pulmonary vascular resistance, the branch pulmonary arteries were ligated. Clips were applied to close the transected pulmonary veins. An additional pressure catheter was secured in the RA through the IVC. To measure changes in ventricular dimension, sonomicrometry crystals (Sonometrics Corp., London, Ontario, Canada) were fixed to the epicardium with cyanoacrylic glue. Two crystals were placed at opposite ends of the short axis of the left ventricle (LV) and an additional two crystals were similarly placed on the short axis of the RV. High-fidelity transducer-tip catheters (Millar Instruments, Houston, TX) were inserted into both the LV and RV.
All animals received humane care in compliance with the "Principles of Laboratory Animal Care" formulated by the National Society of Medical Research and the "Guide for the Care and Use of Laboratory Animals" prepared by the Institute of Laboratory Animal Resources and published by the National Institutes of Health. The protocol was approved by the Committee on Animal Research at the University of California, San Francisco.
Data acquisition
Pressure-dimension (PD) loops were generated using the Sonoview software package (Sonometrics Corp.) from data acquired from the crystals and the transducer-tip catheters. Inflow occlusion was used to generate descending PD loops. Heart rate, aortic root pressure and RA pressure (Table 1)
were measured using Transpal pressure transducers (Abbot Labs, North Chicago, IL). Myocardial temperature was measured with an electronic temperature probe (YSI).
Experimental protocol
Following 10 minutes of direct aortic perfusion (Langendorff mode), perfusion through the SVC into the right atrium was initiated (working heart mode). Baseline hemodynamic measurements were taken after 15 minutes in the working heart mode. The hearts were arrested for 30 minutes by a single infusion of normocalcemic (n = 8), hypocalcemic (n = 6), or hypercalcemic (n = 6) crystalloid cardioplegia (20 mL/kg) at 6°C into the aortic root at a pressure of 20 mm Hg. Plegisol (Abbot Labs) was used for the normocalcemic cardioplegia (Table 2).
Aortic perfusion was then restarted through the aortic root and continued for 10 minutes. The working mode was resumed and postarrest measurements were taken after 15 minutes.
Calculations of myocardial function
Systolic, diastolic, and overall contractile function was calculated from PD loops using the Cardiosoft software package (Sonometrics Corp.). End-systolic elastance (ESE) is an index of systolic contractility calculated from the slope of the regression line that fits the end-systolic points on the PD loops. Diastolic stiffness is the end-diastolic pressure-dimension relationship, obtained from the exponential fit of the end-diastolic pressure-dimension points on the PD loop. Preload recruitable stroke work (PRSW) is a preload-independent index of contractility. PRSW represents the linear relationship between stroke work and end-diastolic volume.
Myocardial water determinations
Upon completion of the study, the LV free wall was excised, placed in a preweighed dish, and the wet weight was obtained. To obtain the dry weight, the sample was heated at 80°C until the weight remained constant.
Statistical analysis
All data are reported as mean ± SD. Analysis of statistical significance was performed using Student’s t-test. All tests were paired and two-tailed. Significance was accepted at p less than 0.05. Significant differences between the experimental and control groups were confirmed by ANOVA. Analyses were performed with the Statview statistical package (SAS, Inc, Cary, NC).
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Results
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Figures 2 through 4
depict the degree of postarrest recovery of fetal
myocardial function. The prearrest and postarrest x intercepts
(V0) were unchanged for elastance, stiffness, and PRSW. Accordingly, recovery of end-systolic elastance, diastolic compliance, and preload-recruitable stroke work are expressed as changes in slope compared with prearrest measurements.
Recovery of systolic function
Use of hypocalcemic cardioplegia resulted in superior preservation of biventricular systolic function (Fig 2). Postarrest recovery of left ventricular ESE was 88% ± 2.2%, compared with 64% ± 15% in the normocalcemic group (p = 0.02). Similar protection of RV systolic function was observed with hypocalcemic cardioplegia with 88% ± 4.8% recovery of ESE with only 60% ± 8.5% recovery with normocalcemic cardioplegia (p = 0.002). Increased calcium concentration resulted in a marked impairment in the systolic function of both ventricles (% recovery: LV -27.5 ± 8, RV -39 ± 12).
Protection from diastolic dysfunction
Optimal preservation of LV diastolic compliance was achieved with hypocalcemic cardioplegic arrest (Fig 3). LV diastolic stiffness increased 38% ± 11% following normocalcemic arrest but only 12% ± 7% when hypocalcemic cardioplegia was used (p = 0.04). Interestingly, no difference in protection of RV diastolic function between the normocalcemic and hypocalcemic groups was observed (22% ± 2.1% vs 20% ± 4.1%, respectively. p = 0.5). Diastolic dysfunction was most pronounced in the hypercalcemic arrest group with an increase of 86% ± 21% in LV stiffness (p = 0.006) and 85% ± 13% in RV stiffness (p = 0.001).
Preservation of overall myocardial function
Hypocalcemic cardioplegia provided nearly complete protection of global biventricular myocardial function with 97% ± 9.6% (p = 0.04) and 91% ± 1.1% (p < 0.001) recovery of LV and RV PRSW, respectively (Fig 4). Recovery following normocalcemic arrest was 75% ± 13%. Again, hypercalcemic cardioplegia provided poor protection of contractility, with only 32% ± 13% recovery of LV PRSW (p = 0.006) and 47% ± 6% recovery of RV contractile function (p = 0.001).
Myocardial edema
Myocardial water content was lowest in the hypocalcemic cardioplegia group (Fig 5).
Myocardial edema was most pronounced following hypercalcemic arrest.
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Fig 5. Myocardial water content determinations. Hypercalcemic arrest caused the greatest degree of myocardial edema with water content of 88% ± 1.1% (p = 0.004). Low calcium cardioplegia resulted in the lowest myocardial water content of the three groups (79.1% ± 1.8%, p = 0.0006).
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Comment
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These results demonstrate the exquisite sensitivity of the fetal myocardium to cardioplegic calcium concentration. Hypocalcemic cardioplegia provided optimal preservation of overall fetal myocardial function. Several studies examining the ideal cardioplegic calcium level for neonatal myocardial protection have demonstrated improved postischemic recovery with normocalcemic cardioplegia [12, 13]. Bolling and coworkers [14] reported equivalent protection of the neonatal myocardium with different calcium cardioplegic concentrations under normal conditions, but indicated improved protection of the hypoxically stressed myocardium with hypocalcemic cardioplegia. As the fetal heart has a lower threshold for ischemic stress [15], those findings may be consistent with the superiority of hypocalcemic cardioplegia in fetal cardioprotection.
Calcium influx has been implicated in cellular damage during reperfusion injury. The immaturity of the fetal myocardial calcium regulatory apparatus undoubtedly magnifies the deleterious effects of increased intracellular calcium during periods of ischemia. The sensitivity of the fetal myocardium to cardioplegic calcium concentration was demonstrated by the profound postischemic myocardial dysfunction observed with hypercalcemic cardioplegia.
Hypocalcemic cardioplegia improved postischemic systolic and contractile function of both fetal ventricles. Interestingly, right ventricular diastolic function was not improved with hypocalcemic cardioplegia. The right ventricle may be more compliant than the left, and as a result, may be less prone to ischemic diastolic dysfunction. As the fetal right ventricle contributes up to two-thirds of the combined ventricular output, this finding holds potentially significant implications for myocardial protection in the fetus.
Due to the diminished reserve of the fetal myocardium, optimal myocardial protection is essential for successful fetal cardiac intervention. This model is designed to mimic in vivo fetal cardiac dynamics by including the ductus in the circuit and ligating the branch pulmonary arteries so both ventricles are performing in parallel as in fetal life. Accordingly, this preparation is well suited to study other important issues of fetal myocardial protection, including the efficacy of fibrillation, blood cardioplegia, and multidose cardioplegia.
It is important to note that using an isolated preparation allows fetal cardiovascular physiology to be studied without the influence of extrinsic factors, such as the placenta. Numerous studies have demonstrated the adverse effects of fetal intervention and fetal cardiac bypass on the placenta, resulting in cardiovascular decompensation [16, 17]. By studying the fetal heart independently, a more accurate assessment of fetal myocardial function under various conditions can be made.
Furthermore, this preparation provides a valid model to study the chronically hypoxic myocardium. Attempts to create models mimicking clinical situations such as tetralogy of Fallot have been criticized because these models have created hypoxic conditions in an otherwise healthy organism existing previously under normoxic conditions. In the clinical setting, patients with cyanotic lesions rarely experience normoxia, bringing into question the applicability of existing models to these clinical scenarios. Under normal conditions, the fetal heart is subject to chronic hypoxia, as normal fetal arterial pO2 levels range from 18 to 24 mm Hg. Although isolated models cannot account for the effects of noncoronary collaterals or bronchial blood flow, this fetal heart preparation can provide valuable insights into the responses of chronically cyanotic myocardium to ischemia and cardioplegia.
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Acknowledgments
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Dr Malhotra is a recipient of the National Research Service Award (F32 HL 10339-01) from the National Heart, Lung, and Blood Institute of the National Institutes of Health.
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References
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Abstract
Introduction
= normal [Ca]; 
= low [Ca]; 
= high [Ca].)
