Page A.W. Anderson

The prostaglandin receptor EP4 triggers remodelling of the cardiovascular system at birth

Survival of newborn placental mammals depends on closure of the ductus arteriosus (DA), an arterial connection in the fetus which directs blood away from the pulmonary circulation and towards the placenta where oxygenation occurs. Here we show that morphological changes resulting in closure of the DA in mice are virtually identical to those observed in larger mammals, including humans, and that maintenance of the DA in the open, or patent, state in fetal mice is dependent on prostaglandin synthesis. This requirement is absent in mice lacking the prostaglandin E2 EP4 receptor (EP4(−/−) mice). In EP 4(−/−) mice of the 129 strain, remodelling of the DA fails to occur after birth, resulting in a left-to-right shunt of blood and subsequently in death. This suggests that the neonatal drop in prostaglandin E2 (refs 3,4,5,6,7) that triggers ductal closure is sensed through the EP4 receptor. In contrast, 5% of EP4(−/−) mice of mixed genetic background survive, and selective breeding of these mice leads to a 21% survival rate, suggesting that alleles at other loci can provide an alternative mechanism for ductal closure.

Troponin T isoform expression in humans. A comparison among normal and failing adult heart, fetal heart, and adult and fetal skeletal muscle.

The expression of troponin (Tn) T, a thin-filament regulatory protein, was examined in left ventricular myocardium from normal and from failing adult human hearts. The differences in isoform expression between normal and failing myocardium led us to examine the ontogenic expression of TnT in human striated muscle. Left ventricular samples were obtained from patients with severe heart failure undergoing cardiac transplantation and normal adult organ donors. Fetal muscle was obtained from aborted fetuses after 14-15 weeks of gestation, and adult skeletal muscle was obtained from surgical biopsies. Western blots of normal and failing adult heart proteins demonstrated that two isoforms, TnT1 and TnT2, are expressed in different amounts, with TnT2 being significantly greater in failing hearts (p less than 0.004). Western blots of two-dimensional gels of these proteins resolved two predominant spots of both TnT1 and TnT2 and several minor TnT species. Alkaline phosphatase treatment converted the two major spots of each isoform into the single more basic spots. A comparison of the ATPase activities and the TnT2 percentage of total TnT in individual failing and normal adult hearts demonstrated an inverse and negative relation (r = 0.7, p less than 0.02). In the fetal heart, four TnT isoforms were found, two of which had the same electrophoretic mobilities as the adult cardiac isoforms TnT1 and TnT2. Fetal skeletal muscle expressed two of the four fetal cardiac TnT isoforms, one of which comigrated with adult cardiac TnT1. These cardiac isoforms were expressed in low abundance in fetal skeletal muscle relative to seven fast skeletal muscle TnT isoforms. No cardiac isoforms were present in adult skeletal muscle. Because many etiologies caused heart failure in the transplant patients, we propose that the disease-associated increased expression of the TnT isoform TnT2 is an adaptation to the heart failure state and a partial recapitulation of the fetal expression of cardiac TnT isoforms.

Contemporary Outcomes After the Fontan Procedure: A Pediatric Heart Network Multicenter Study

OBJECTIVES We characterized a large cohort of children who had a Fontan procedure, with measures of functional health status, ventricular size and function, exercise capacity, heart rhythm, and brain natriuretic peptide (BNP). BACKGROUND The characteristics of contemporary Fontan survivors are not well described. METHODS We enrolled 546 children (age 6 to 18 years, mean 11.9 years) and compared them within pre-specified anatomic and procedure subgroups. History and outcome measures were obtained within a 3-month period. RESULTS Predominant ventricular morphology was 49% left ventricular (LV), 34% right ventricular (RV), and 19% mixed. Ejection fraction (EF) was normal for 73% of subjects; diastolic function grade was normal for 28%. Child Health Questionnaire mean summary scores were lower than for control subjects; however, over 80% of subjects were in the normal range. Brain natriuretic peptide concentration ranged from <4 to 652 pg/ml (median 13 pg/ml). Mean percent predicted peak O2 consumption was 65% and decreased with age. Ejection fraction and EF Z score were lowest, and semilunar and atrioventricular (AV) valve regurgitation were more prevalent in the RV subgroup. Older age at Fontan was associated with more severe AV valve regurgitation. Most outcomes were not associated with a superior cavopulmonary connection before Fontan. CONCLUSIONS Measures of ventricular systolic function and functional health status, although lower on average in the cohort compared with control subjects, were in the majority of subjects within 2 standard deviations of the mean for control subjects. Right ventricular morphology was associated with poorer ventricular and valvular function. Effective strategies to preserve ventricular and valvular function, particularly for patients with RV morphology, are needed.

Troponin I Phosphorylation in the Normal and Failing Adult Human Heart

BACKGROUND In the failing human heart myofibrillar calcium sensitivity of tension development is greater and maximal myofibrillar ATPase activity is less than in the normal heart. Phosphorylation of the cardiac troponin I (cTnI)-specific NH2-terminus decreases myofilament sensitivity to calcium, while phosphorylation of other cTnI sites decreases maximal myofibrillar ATPase activity. METHODS AND RESULTS We examined cTnI phosphorylation in left ventricular myocardium collected from failing hearts at the time of transplant (n=20) and normal hearts from trauma victims (n=24). The relative amounts of actin, tropomyosin, and TnI did not differ between failing and normal myocardium. Using Western blot analysis with a monoclonal antibody (MAb) that recognizes the striated muscle TnI isoforms, we confirmed that the adult human heart expresses only cTnI. A cTnI-specific MAb recognized two bands of cTnI, designated cTnI1 and cTnI2, while a MAb whose epitope is located in the cTnI-specific NH2-terminus recognized only cTnI1. Alkaline phosphatase decreased the relative amount of cTnl1, while protein kinase A and protein kinase C increased cTnI1. The percentage of cTnI made up of cTnI1, the phosphorylated form of TnI, is greater in the normal than the failing human heart (P<.00). CONCLUSIONS This phosphorylation difference could underlie the reported greater myofibrillar calcium sensitivity of failing myocardium. The functional consequence of this difference may be an adaptive or maladaptive response to the lower and longer calcium concentration transient of the failing heart, eg, enhancing force development or producing ventricular diastolic dysfunction.

Adult-Derived Stem Cells from the Liver Become Myocytes in the Heart in Vivo

Recent evidence suggests that adult-derived stem cells, like their embryonic counterparts, are pluripotent. These simple, undifferentiated and uncommitted cells are able to respond to signals from their host tissue microenvironment and differentiate, producing progeny that display a phenotype characteristic of the mature cells of that tissue. We used a clonal stem cell line (termed WB-F344) that was derived from an adult male rat liver to investigate the possibility that uncommitted stem cells from a nonmyogenic tissue source would respond to the tissue microenvironment of the heart in vivo and differentiate into cardiac myocytes. Male WB-F344 cells that carry the Escherichia coli beta-galactosidase gene were identified in the left ventricular myocardium of adult female nude mice 6 weeks after transplantation. We confirmed the presence of a rat Y-chromosome-specific repetitive DNA sequence exclusively in the beta-galactosidase-positive myocytes by polymerase chain reaction and fluorescence in situ hybridization. Immunohistochemistry, using a cardiac troponin T-specific monoclonal antibody, and ultrastructural analysis confirmed a cardiac myocyte phenotype of the stem cell-derived myocytes. The beta-galactosidase-positive myocytes ranged from < 20 microm to 110 microm in length. The longer of these cells contained well-organized sarcomeres and myofibrils, and formed intercalated disks and gap junctions with endogenous (host-derived) myocytes, suggesting that WB-F344-derived myocytes participate in the function of the cardiac syncytium. These results demonstrate that adult liver-derived stem cells respond to the tissue microenvironment of the adult heart in vivo and differentiate into mature cardiac myocytes.

Force-pCa relation and troponin T isoforms of rabbit myocardium.

We have previously reported the existence of at least four troponin T isoforms in rabbit ventricular muscle and described the changes in their distribution with development. In this report we test whether the proportions of the troponin T isoforms are related to the sensitivity of the myofilaments to calcium. We measured the force-pCa relations in 12 detergent-skinned ventricular strands of cardiac muscle from newborn (2-5-day-old) rabbits. We determined from each strand the amount of each troponin T isoform relative to the total amount of troponin T by using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and densitometric scans of Western blots probed with a cardiac-specific troponin T monoclonal antibody, MAb 13-11. To assess the presence of different relative amounts of cardiac and slow skeletal troponin I among the strands, we determined the amount of cardiac troponin I relative to tropomyosin. We determined the Hill coefficient and the pCa for half-maximal force, pCa50, for each strand. pCa50 was related directly to the relative amount of troponin T2 (pslope = 0.037). Our results do not indicate a relation between the Hill coefficient and troponin T2. We also did not find a relation between pCa50 and the cardiac troponin I/tropomyosin ratio, which suggests that the correlation between pCa50 and troponin T2 was not a result of changes in the relative amounts of cardiac and slow skeletal muscle troponin I. Our findings indicate that a relation exists between the force-pCa characteristics of rabbit myocardium and the troponin T isoforms that it expresses, suggesting a role for troponin T in modulating the sensitivity of cardiac myofilaments to calcium.

Developmental changes in the ultrastructure and sarcomere shortening of the isolated rabbit ventricular myocyte.

Sarcomere shortening and ultrastructure of intact isolated myocytes from ventricles of three-week-old and adult rabbits were examined. Cells were fixed and embedded, and after measuring their sarcomere shortening in response to electrical stimulation, they were examined in serial thin sections by electron microscopy. This structure-function analysis showed that adult cells were significantly larger, had longer rest sarcomere lengths, greater amount and velocity of sarcomere shortening, greater velocity of reextension, and shorter contraction duration than immature cells. In immature myocytes, a thin outer shell of myofibrils enveloped a central mass of mitochondria and nuclei, but in adult cells, the cytoskeleton divided the cell into compartments with the mitochondria arranged around and interspersed among the myofibrils. The different arrangement of the organdies and the cytoskeleton at the two ages may account for the shorter rest sarcomere length in the young myocytes and may confer differing internal loads that contribute to their smaller amount and velocity of sarcomere shortening. The corbular and longitudinal sarcoplasmic reticulum were less demarcated in immature than in adult cells. Myocytes from both ages showed postextrasystolic potentiation, suggesting that the sarcoplasmic reticulum modulates calcium at both ages. Restitution of contractility between contractions, obtained by measuring sarcomere shortening of interpolated extrasystoles, was faster in immature than in adult cells and may reflect the structural differences in the sarcoplasmic reticulum. The developmental differentiation in the sarcoplasmic reticulum suggests that changes in compartmentalization of calcium and in the distribution of putative calcium-release sites contribute to the increased contractility of adult myocytes.

Multiple regional differences in cellular properties that regulate repolarization and contraction in the right atrium of adult and newborn dogs.

Recent studies of isolated cardiac myocytes have generated the need for detailed information about regional electrophysiological differences in the atrium. We measured the spatial distribution of action potentials in adult and newborn canine right atria. Multiple regional differences in action potential shape and duration were found. The multiple regional differences produced an overall simple pattern: the longest action potentials occurred in the area of the sinus node, and the action potential duration decreased with increasing distance from the sinus node area. To account for the overall pattern, we tested factors considered important in causing atrial action potential shape differences (e.g., electronic interactions). None of the factors tested accounted for the regional differences. We then found regional differences in the responses to pauses, which suggested that differences in the properties of individual cells accounted for the regional repolarization differences. If so, genetic regulation of the regional differences may produce the overall pattern on a developmental basis. Experiments in newborn atria showed that only in the upper crista was the spatial pattern similar to that of the adult; there was little variability in action potential shape and duration in the other areas. As a further test for associated regional differences in cell properties, we examined for differences in the isoform expression of troponin T (TnT1, TnT2, TnT3, and TnT4), a protein important in excitation-contraction coupling. In adults, the greatest proportion of TnT1 occurred in the area of the sinus node, and its proportion decreased with increasing distance from the sinus node area in association with a relative increase in the proportion of TnT2. In newborn atria the relative amount of TnT1 was greatest in the upper crista (similar to adult), but little difference was found in the distribution of the isoforms in the other regions. The correspondence between the regional differences in repolarization and in the expression of the troponin T isoforms in adult and newborn atria suggests that 1) cellular ionic mechanisms vary regionally to coordinate differences in action potential configuration with differences in cell properties that regulate contractility and 2) genetic expression of the systems that regulate repolarization and mechanical cellular properties are under similar developmental and regional control.

The Pediatric Heart Network: A Primer for the Conduct of Multicenter Studies in Children with Congenital and Acquired Heart Disease

Most contemporary diagnostic and treatment strategies for pediatric patients with cardiovascular disease are not supported by evidence from clinical trials but instead are based on expert opinion, single-institution observational studies, or extrapolated from adult cardiovascular medicine. In response to this concern, the National Heart, Lung, and Blood Institute established the Pediatric Heart Disease Clinical Research Network (PHN) in 2001. The purposes of this article are to describe the initiation, structure, and function of the PHN; to review the ongoing studies; and to address current and future challenges. To date, four randomized clinical trials and two observational studies have been launched. Design and conduct of complex, multicenter studies in children with congenital and acquired heart disease must address numerous challenges, including identification of an appropriate clinically relevant primary endpoint, lack of preliminary data on which to base sample size calculations, and recruitment of an adequate number of subjects. The infrastructure is now well developed and capable of implementing complex, multicenter protocols efficiently and recruiting subjects effectively. The PHN is uniquely positioned to contribute to providing evidence-based medicine for and improving the outcomes of pediatric patients with cardiovascular disease.

Noninvasive detection of retrograde descending aortic flow in infants using continuous wave Doppler ultrasonography Implications for diagnosis of aortic run-off lesions

Continuous wave Doppler ultrasonography was utilized to detect and characterize descending aortic blood flow velocity patterns in 27 preterm and term infants with or without an aortic run-off lesion proximal to the descending aorta. The effects of coexistent intracardiac defects on the velocity time profiles were evaluated by comparing records from patients with no intracardiac defects or systemic run-off lesions to those with a variety of intracardiac defects but no systemic run-off lesion. No significant alterations were noted. In all patients, the velocity tracing in systole was triangular in shape. During diastole, however, there were qualitative and quantitative differences between patients with and without a proximal run-off lesion. In those without a run-off lesion, retrograde descending aortic flow was present only in early diastole, and aortic flow velocity oscillated around the zero baseline during mid and late diastole. In those with a run-off lesion, retrograde flow present in early diastole continued throughout diastole. Quantitatively, the ratio of the area under the retrograde flow portion of the tracing to the forward flow portion was significantly greater in those with a run-off lesion. The velocity time profiles obtained with CW Doppler were similar to those previously obtained invasively with electromagnetic flow probes of catheter-mounted velocitometers. CW Doppler ultrasonograpy provides a reliable, noninvasive method for describing descending aortic blood flow velocity in infants.