Adolescent Feline Heart Contains a Population of Small, Proliferative Ventricular Myocytes With Immature Physiological Properties
Recent studies suggest that rather than being terminally differentiated, the adult heart is a self-renewing organ with the capacity to generate new myocytes from cardiac stem/progenitor cells (CS/PCs). This study examined the hypotheses that new myocytes are generated during adolescent growth, to increase myocyte number, and these newly formed myocytes are initially small, mononucleated, proliferation competent, and have immature properties. Ventricular myocytes (VMs) and cKit+ (stem cell receptor) CS/PCs were isolated from 11- and 22-week feline hearts. Bromodeoxyuridine incorporation (in vivo) and p16INK4a immunostaining were measured to assess myocyte cell cycle activity and senescence, respectively. Telomerase activity, contractions, Ca2+ transients, and electrophysiology were compared in small mononucleated (SMMs) and large binucleated (LBMs) myocytes. Heart mass increased by 101% during adolescent growth, but left ventricular myocyte volume only increased by 77%. Most VMs were binucleated (87% versus 12% mononucleated) and larger than mononucleated myocytes. A greater percentage of SMMs was bromodeoxyuridine positive (SMMs versus LBMs: 3.1% versus 0.8%; P<0.05), and p16INK4a negative and small myocytes had greater telomerase activity than large myocytes. Contractions and Ca2+ transients were prolonged in SMMs versus LBMs and Ca2+ release was disorganized in SMMs with reduced transient outward current and T-tubule density. The T-type Ca2+ current, usually seen in fetal/neonatal VMs, was found exclusively in SMMs and in myocytes derived from CS/PC. Myocyte number increases during adolescent cardiac growth. These new myocytes are initially small and functionally immature, with patterns of ion channel expression normally found in the fetal/neonatal period
The possibility that resident progenitor cells are present in the developing and adult heart1 suggests that new myocytes are continuously formed to replace dying cells. Based on this paradigm, a positive balance with a prevailing generation of myocytes would be expected to occur during the transition from the adolescent to the adult heart phenotype to accommodate the increase in body mass, blood flow, and cardiac workload. To support this dynamic view of the heart, a significant degree of structural and functional myocyte heterogeneity should be present during periods of cardiac growth.
Myocyte maturation involves a progressive increase in cell size and structural complexity2 that is coupled with the loss of replicative potential.3 Conversely, recently formed myocytes may have the properties of small amplifying, functionally immature cells that retain the ability to divide and concurrently differentiate.1 At the completion of this process, myocytes become terminally differentiated and can only undergo cellular hypertrophy.4 As myocytes become hypertrophic and senescent, they may die by apoptosis.5
To test whether this emerging model of cardiac biology holds merit and might be applicable to large mammals including humans, we determined the cellular mechanisms implicated in the increase in mass of the adolescent feline heart. Myocyte formation, myocyte hypertrophy, and myocyte apoptosis were concurrently examined because cell death, cell regeneration, and cell differentiation modulate tissue homeostasis and growth in self-renewing organs regulated by a pool of resident progenitor cells.5 The possibility that these fundamental cellular processes are operative in the maturing and adult myocardium raises some critical questions concerning the presence and functional integration of newly formed immature myocytes together with adult terminally differentiated and senescent cells.1 By inference, the electrophysiological, contractile, and Ca2+ handling properties of these classes of parenchymal cells may be profoundly distinct.1 This variability in myocyte function predicts an unprecedented dynamism of the heart and poses complex questions about the cellular physiological basis of ventricular performance.
The hypotheses tested in the present research are that myocyte number increases during normal adolescent cardiac growth and these newly formed myocytes are functionally immature. The respective contributions of myocyte growth (hypertrophy) and increased myocyte number (hyperplasia) to the increase in cardiac mass during normal adolescent growth was determined. Newly formed myocytes were identified by bromodeoxyuridine (BrdUrd) incorporation and markers of cell cycle activity, and then the electrophysiological, contractile, and Ca2+ handling properties of new versus mature myocytes were compared. As an initial step toward determining the source of these new myocytes, we also studied whether the young adult feline heart contains a resident cKit+ (stem cell factor) cardiac stem/progenitor cell (CS/PC) that can differentiate into new cardiac myocytes (in vitro).
Our results show that the increase in the mass of the adolescent feline heart involves an increase in both myocyte size and number. BrdUrd was incorporated into a small percentage of cardiac myocytes, with preferential incorporation into small mononucleated myocytes (SMMs) that also expressed markers of cell cycle activity (Ki67) and had greater telomerase activity than large binucleated myocytes (LBMs). SMMs had a less organized T-tubular system and more slowly rising, prolonged Ca2+ transients than LBMs. In addition, SMMs uniquely expressed T-type Ca2+ channels (TTCCs), which are involved in the differentiation and proliferation6,7 of other cell types. We also found that the normal feline heart contains a population of resident c-Kit+ CS/PCs that, when cocultured with rat neonatal myocytes, differentiate into cardiac myocytes that also express T-type Ca2+ currents. These data strongly support the idea that cardiac myocyte number increases during adolescent growth, with newly formed myocytes initially having immature physiological properties.
Materials and Methods
Ventricular myocytes (VMs)8 and CS/PCs9 were isolated from adolescent feline hearts. BrdUrd incorporation,10 nucleation, and cell volume11,12 were determined in isolated myocytes. Myocyte surface area, Ki67 expression,13 p16ink4a expression,5 telomerase activity,13 and structural14 and functional properties15,16 were compared in small mononucleated myocytes versus in binucleated ventricular myocytes.
An expanded Materials and Methods section can be found in the online data supplement at http://circres.ahajournals.org.
Heart Size Increases More Than Myocyte Size During Adolescent Growth
Heart mass of adolescent felines increased by 101% between 11 and 22 weeks of age (7.46±0.28 g [N=6] versus 14.98±0.18 g [N=16]; P<0.001). Myocyte volume fraction did not change during this time (81.8±0.66% at 11 weeks and 80.9±0.9% at 22 weeks, n=3 at both ages). If no new myocytes were generated during adolescent growth, then myocyte volume should increase by 101%. Myocyte volume, determined by Coulter Z2 particle size analysis,11 using populations of isolated cells, increased from 11 782±438 to 20 871±909 μm3 in these hearts, a 77% increase (P<0.005). Identical increases in myocyte volume during adolescent growth were found by measuring myocyte 2D surface area and cell depth (with confocal microscopy) and then computing myocyte volume12 (see below). Collectively, these data show that the increase in myocyte size during adolescent cardiac growth is not sufficient to account for the associated increase in cardiac mass, consistent with the hypothesis that new myocytes are generated during adolescent growth and result in an increase in myocyte number.
Number and Size of Mononucleated and Binucleated Ventricular Myocytes
The adolescent (11-week) and young adult (22-week) feline hearts had a significantly greater percentage of binucleated (87.3±1.2%) than mononucleated (11.5±1.3%) left ventricular myocytes (23 280 myocytes from 12 hearts; Figure 1A). No difference in nucleation was detected at 11 versus 22 weeks. The size of these 2 myocyte populations was significantly different in both adolescent and young hearts. Two dimensional surface area (SA) (Figure 1) and cell depth of mononucleated myocytes were significantly smaller than that of binucleated myocytes from the adolescent (11 weeks) hearts (SA=1262.19±65.55 μm2 [n=157] in mononucleated versus SA=2007.14±111.58 μm2 [n=333] in binucleated; N=3; P<0.0001; cell depth=4.99 μm in mononucleated and cell depth=5.83 μm in binucleated myocytes; P<0.01) and the young adult (22 weeks) hearts (SA=1534.41±25.44 μm2 [n=573] in mononucleated versus SA=2566.26±43.20 μm2 [n=1250] in binucleated; N=11; P<0.0001; Figure 1A; cell depth=6.65±0.14 μm [n=31] in mononucleated myocytes and cell depth=7.76±0.09 μm [n=34] in binucleated myocytes; P<0.01). The myocyte volume calculated from these morphological measurements was 10 921.8 μm3 in the 11-week hearts and increased by 70% in the 22-week feline hearts (18 558.5 μm3). These average myocyte volumes are almost identical to those determined by Coulter analysis. Because mononucleated myocytes were significantly smaller than binucleated myocytes, we refer to mononucleated myocytes as small mononucleated myocytes (SMMs) and binucleated myocytes as large binucleated myocytes (LBMs).
Bromodeoxyuridine Incorporation in Adolescent Ventricular Myocytes
The percentage of BrdUrd+ mono- and binucleated myocytes was measured (Figure 1A) on VMs isolated from adolescent (22-week-old) animals in which BrdUrd minipumps had been inserted (7 days of subcutaneous infusion; 10 mg/kg per day). BrdUrd incorporation was observed in myocyte nuclei in tissue sections (Figure 1B), but the percentage of BrdUrd+ nuclei was quantified in isolated myocytes, so that we could unambiguously determine whether these cells were mono- or binucleated. The percentage of BrdUrd+ isolated myocytes was 1.04% (242/23 280 myocytes; N=12 hearts), but a greater percentage of mononucleated (3.1±0.8%) than binucleated (0.8±0.4%) myocytes were found (P=0.01; N=12 hearts) (Figure 1 A and 1C through 1H). Ki67 staining was observed in a small percentage of myocytes, and the majority (>95%) of these were small mononucleated myocytes (Figure 1J and 1K). These studies strongly support the idea that new myocytes are generated in the normal young adult heart and that these myocytes are small and mononucleated.
The number of myocytes within the 11- and 22-week hearts computed11,12 using measured myocyte volumes (Coulter or morphological measurements), heart weights, and myocyte volume fraction increased from 5.48×108 to 6.16×108, consistent with an average increase in new myocytes of 8.8×105 per day. Incrementing myocyte number in the 11-week heart by 1.04% per week (the percentage of BrdUrd+ myocytes per week) for 11 weeks predicts that the myocyte number will increase to 6.11×108, almost identical to the number determined independently by morphological analyses.
Senescence Markers and Telomerase Activity in Small Versus Large Ventricular Myocytes
Newly formed small mononucleated myocytes would be expected to be “younger” than their binucleated counterparts. This ideas was explored by staining ventricular myocytes for the cell senescence marker p16INK4a (Figure 2A and 2B).5 A small fraction of these myocytes were p16INK4a+ in 11- and 22-week hearts (8.1% and 7.1%, respectively), and most (>95%) were LBMs.
Newly formed myocytes might also retain the ability to proliferate. To test this idea, isolated myocytes were separated by size and the telomerase activity of small and large myocytes was measured. This assay was performed because telomerase activity is commonly found in activated stem cells and early committed cells undergoing rapid growth and differentiation.5 In fact, when cells are mature, they became telomerase incompetent and the downregulation of the catalytic function of the ribonucleoprotein is a necessary step for the acquisition of the terminally differentiated phenotype.5 Telomerase activity was observed in all myocyte populations but was significantly higher in the pool of smallest myocytes obtained from 5 cat hearts at 22 weeks of age (Figure 3A and 3B). The enhanced enzyme activity in small myocytes is consistent with the higher level of cell replication documented in this cell subset by BrdUrd incorporation and Ki-67 labeling. Telomere length (QFISH) was not significantly different in 11-week (20.2±2.4kb) and 22-week (20.9±3.1kb) myocytes, consistent with telomerase-dependent preservation of telomere length in these young animals.
Apoptosis in the Adolescent Heart
An increase in myocyte number during adolescent growth could take place in the absence of myocyte death or could occur in addition to ongoing myocyte turnover. A significant number of TUNEL+ myocyte nuclei were observed in both 11- and 22-week animals, with no significant differences found at these ages (11 weeks versus 22 weeks: 0.083±0.015% versus 0.077±0.017% TUNEL+ myocyte nuclei). These finding suggest that to increase myocyte number during adolescent growth, new myocyte formation must be in excess of the rate of myocyte apoptosis.
SMMs Contract Slower and Relax Slower Than LBM
Contractions (Figure 4A) in SMMs (7.6±0.6%) and LBMs (8.1±0.9%) were not significantly different (Figure 4B). However, the maximum shortening and relaxation rates were significantly slower in SMMs (Figure 4C). The times to 50% shortening, to peak shortening, and to 50% relaxation were all significantly longer in SMMs (Figure 4D).
Ca2+ Transients in SMMs Have Two Phases and Are Slowly Rising
The peak amplitude of the Ca2+ transient in SMMs was significantly lower than in LBMs (1.83±0.11, n=26, N=6 versus 2.29±0.16, n=25, N=6, respectively, P<0.05; Figure 5A and 5B). The time to peak Ca2+ in SMMs was significantly longer than in LBMs (165.9±14.2/ms, n=26, N=6 versus 101.1±14.2/ms, n=25, N=5, P<0.01) and the maximal rate of rise of the Ca2+ transient was significantly slower in SMMs versus in LBMs ([F/F0]/ms: 0.122±0.019/ms versus 0.057±0.011/ms) (Figure 5D and 5E). The rising phase of the Ca2+ transient in SMMs had an initial rapidly rising phase and a secondary, slower, rising phase. There was primarily a rapidly rising portion in LBMs (Figure 5A and 5C). The configuration of the Ca2+ transient in SMMs is similar to the type reported in neonatal myocytes.2 The duration of the fast rising phase (SMMs versus LBMs: 29.8±2.0 ms, n=26, N=6 versus 44.8±6.9ms, n=25, N=5, P<0.05) was significantly shorter and the contribution of the rapidly rising phase to the total amplitude (SMMs versus LBMs: 71.9±4.7%, n=26, N=6 versus 92.2±2.5%, n=25, N=5, P<0.05) was significantly smaller in SMMs (Figure 5E). The time between the peak of the rapidly rising phase and the peak of the slowly rising phase was significantly longer (P<0.01) in SMMs (136.1±1.7ms, n=26, N=6) than in LBMs (56.1±0.8 ms, n=25, N=5).
The decay of the Ca2+ transient also has 2 portions that could be fit with a monoexponential function.17 The time constant of the initial decay period was significantly slower in SMMs than in LBMs (248.2±25.0 ms, n=26, N=6 versus 180.1±17.9 ms, n=25, N=6, P<0.001; Figure 5F). These results indicate that the excitation/contraction (EC) coupling machinery (the T-tubular system and the SR) is not fully developed in SMMs.
Ca2+ Release During the Early Phase of the Ca2+ Transient Is Less Synchronized in SMMs
Ca2+ release is less well spatially synchronized within fetal and neonatal than in adult ventricular myocytes.18 This is primarily attributable to a more rudimentary T-tubule/sarcoplasmic reticulum (SR) coupling, which is essential for normal EC coupling in the adult heart.18 We explored the idea that a less well organized T-tubular system is responsible for a less well organized SR Ca2+ release in SMMs by measuring Ca2+ transients with confocal microscopy (line-scan imaging) in myocytes paced at 0.5 Hz. SR Ca2+ release was spatially less well organized in SMMs, revealed by the uneven wave front of their Ca2+ transients (Figure 5G and 5H). The synchrony of Ca2+ release, assessed by the percentage of pixels with intensity over half maximum intensity (%F>F50),16 was significantly smaller in SMMs than LBMs (Figure 5I). These results suggest that components necessary for the synchronized SR Ca2+ release of normal adult cardiac myocytes are not fully developed in SMMs.
Two processes that could produce this less synchronized SR Ca2+ release are a less well organized T-tubular system (the absence of some release sites) and/or electrophysiological differences that would cause a fraction of release sites to fail to release their stored Ca2+.16 A decrease in the density of the transient outward potassium current (Ito) elevates the early portion of the cardiac action potential and reduces Ca2+ entry through the L-type calcium channel.16 We have previously shown that in hypertrophied ventricular myocytes, this can cause dyssynchronous SR Ca2+ release.16 There were no significant differences in resting membrane potentials, phase 0 maximum rising rates, peak or plateau voltages, or the duration of the action potentials (APs) between SMMs and LBMs (Figure 6A). However, there were significant differences in the early and late AP repolarization phases, which are largely dictated by K+ currents.19 Phase 1 repolarization was significantly less prominent in SMMs (15.3±2.1 mV, n=18, N=5) than in LBMs (22.2±2.5 mV, n=18, N=5, P<0.05) and the maximum repolarization rate of phase 3 was significantly slower in SMMs (−1.29±0.18 mV/ms, n=18 versus −1.87±0.22 mV/ms in n=18 LBMs, P<0.05). The 4-aminopyridine–sensitive, transient outward current (Ito) that controls phase 1 repolarization16 was smaller in SMMs than in LBMs (Figure 6D through 6F). The T-tubule density (T-tubular index, as described by He et al14) in 1-(3-sulfonatopropyl)-4-[β[2-(di-n-octylamino)-6-naphthyl]vinyl]pyridinium betaine (Di-8-ANEPPS)–labeled myocytes (Figure 6G through 6I) was significantly smaller in SMMs (15.1±1.4% versus 24.3±1.5% in LBMs, P<0.01). These differences in T-tubule and Ito density should contribute to the spatial disorganization of Ca2+ release2,16 observed in SMMs.
SMMs Have T-type Calcium Current
The functional roles of TTCC currents (ICa-T) in adult VMs are still not fully understood. ICa-T is present in embryonic and neonatal VMs but not in most adult VMs, implying a developmental role for Ca2+ flux through this channel.6 We showed many years ago that pressure overload induces a “reexpression” of T-type Ca2+ channels in adult feline VMs.20 A number of related studies in noncardiac cells have shown that the T-type calcium channel plays an important role in differentiation, growth, and proliferation.6 We suggest that the TTCC may have a role in the generation of new cardiac myocytes and/or in the maturation of newly formed into fully functional VMs. As a first test of these ideas, we measured ICa-T and ICa-L (L-type calcium current, the major Ca2+ channel current in cardiac myocytes) in SMMs and LBMs (Figure 7A and 7B) using established techniques based on differences in their voltage dependence of activation and inactivation6 (Figure 7D, 7E, and 7G). ICa-T (−3.0±0.6 pA/pF) was found in 11 of 19 SMMs (57.6±4.0 pF). No detectable ICa-T was found in any of the 18 LBMs (139.9±9.6 pF) examined, similar to what we have reported previously.20 ICa-L was present in all myocytes and its density was not different in SMMs and LBMs (Figure 7D and 7E). ICa-T (and ICa-L) was also observed in 3 myocytes derived from cardiac c-Kit+ stem cells maintained in coculture with neonatal rat cardiomyocytes (Figure 7C and 7F). The T-type Ca2+ current in the SMMs and cKit+ CS/PC-derived myocytes likely reflects Ca2+ influx through the α1H TTCC gene, because these currents were blocked by low concentrations of Ni2+ and were insensitive to Cd2+ (Figure 7H and 7I).21 These studies support an association between Ca2+ influx through the TTCC and new myocyte generation.
The present study was performed in adolescent animals undergoing a rapid growth phase, near the time of sexual maturity, because it was our view that if new myocyte formation were part of the normal physiology of the postnatal heart, then the capacity to generate these new myocytes should be evident during this period of robust physiological cardiac growth. Our results show that (1) the increase in the size of the adolescent heart is greater than the enlargement of its individual myocytes, supporting myocyte hyperplasia above and beyond the rate of myocyte apoptosis; (2) the young adult heart has a population of small mononucleated ventricular myocytes that are BrdUrd+, Ki67+, P16INK4a− and have telomerase activity; and (3) SMMs have physiological properties reminiscent of fetal/neonatal myocyte.
What Is the Source of Newly Formed Ventricular Myocytes?
Although we clearly show that new myocytes are small and mononucleated with immature functional properties that are reminiscent of fetal/neonatal myocytes,2 we cannot discriminate between CS/PCs and small proliferative myocytes as the source of the new myocytes in the adolescent heart. The recognition that telomerase activity is present in small cycling myocytes is consistent with the notion that these dividing cells represent a population of amplifying myocytes derived from a primitive pool of c-kit+ CS/PCs. Our in vitro studies have shown that this class of undifferentiated progenitor cells can acquire the cardiomyogenic fate leading to the generation of functionally competent myocytes.1 Together, these results indicate that the adolescent feline heart retains a remarkable degree of developmental plasticity with the inherent ability to create a significant number of new myocytes to accommodate its increasing hemodynamic demands.
A number of laboratories have identified populations of resident CS/PCs that have the capacity to differentiate into new cardiac myocytes both in vitro and in vivo.1,22–24 These include cells expressing the receptor tyrosine kinase c-Kit,1 stem cell antigen-1 (Sca-1),23 side population (SP) cells that can exclude the DNA-binding dye Hoechst 33258, and even cells expressing the transcription factor isl-1.22 The role of each of these cell types in the generation of new myocytes in the adolescent heart is yet to be determined.
SMMs Have Immature Functional Properties
In a previous study, we tested the idea that cardiac contractility varied with cell size in both the normal and hypertrophied (pressure overload) heart.25 This study clearly showed that myocytes from hypertrophied hearts were functionally distinct from those in the normal heart, but, in neither the normal or hypertrophied hearts, was myocyte function related to myocyte size. Although a broad range of myocyte sizes were examined, the smallest myocytes from both the normal and hypertrophied hearts were not studied. We characterized the physiological properties of SMMs for the first time and determined that they are functionally immature and distinct from the LBMs, which make up the majority of the young adult heart.
The functional properties of cardiac myocytes derived from embryonic stem cells (ESCs),26 hematopoietic stem cells,27 and cardiac stem cells (CSCs) in culture are also functionally immature. In cardiac myocytes derived from ESCs,28 the transient outward K+ and L-type Ca2+ currents are expressed first, followed by cardiac-specific Na+ current, the delayed rectifier K+ current, and If currents, and, in the latest stages, inwardly rectifying K+ and ATP-sensitive K+ currents are observed. These changes in ion channel expression are similar to those seen during the development of cardiac myocytes in vivo and resemble those of cardiac myocytes in embryonic heart tubes.29 Matsuura et al30 reported that oxytocin induces Sca-1+ CSCs from the adult murine heart to differentiate into functional, spontaneously beating, immature cardiomyocytes with Ca2+ transients. These cells had positive inotropic responses to isoproterenol via β1-adrenergic receptor signaling. ESC-derived cardiac myocytes also responded to isoproterenol and carbachol.31 Collectively, these studies show that ESCs and CS/PC-derived cardiac myocytes express the ion channels seen in the developing, but immature heart.
SMMs Express TTCCs
The present experiments show that SMMs with immature functional properties express TTCCs. Because the TTCC is not an effective inducer of SR Ca2+ release,32 the functional significance of Ca2+ influx through the TTCC in VMs is, in our view, still not known. The TTCC is present in fetal/neonatal ventricular myocytes,33 in cardiac myocytes derived from mouse embryonic stem cells,34 but not in most adult ventricular myocytes,33 suggestive of some developmental function.35 The disappearance of T-type calcium current (ICa-T) after birth parallels the withdrawal of VMs from the cell cycle,6 suggesting it may have some role in cell cycle regulation. We previously showed that ICa-T reemerges in adult feline VMs under pathological cardiovascular stress,20 which we proposed was related to induction of a fetal gene program. We now hypothesize that that the presence of TTCCs in SMMs is associated with the fact that these VMs were recently formed. These ideas are supported by studies in other cell types (see below) but need to be examined in detail in cardiac myocytes.
Ca2+ influx through the TTCC plays a critical role in the proliferation of blastocyte-derived embryonic stem cells, fibroblasts, vascular smooth muscle,6 and even neonatal ventricular myocytes.33 Blockade of ICa-T inhibits proliferation of smooth muscle as well as differentiation of neuronal stem cells.6 We speculate that TTCCs play an important signaling function during the generation of newly formed ventricular myocytes in the adolescent heart. Whether TTCCs are expressed on CSCs during their conversion to the myocyte lineage and their subsequent maturation are important unanswered questions. Because TTCCs appear to modulate the growth and differentiation of smooth muscle and endothelial cells,6 they may be a key regulator of the commitment of multipotent CS/PCs to the various cardiac cell classes.
Many of the critical experiments in this study used isolated myocytes, with the assumption that we isolated myocytes that were representative of the entire heart. Although we think the possibility is unlikely, there would be bias in the cell volume calculations if selective subpopulations of myocytes were not routinely isolated.
The generation of new myocytes is potentially a groundbreaking therapy for the treatment of cardiac diseases, especially those that involve a critical reduction in the number of cardiac myocytes, such as myocardial infarction and congestive heart failure.1 A number of recent studies in human patients and in animal models1 suggest that delivery of exogenous stem cells to the damaged heart leads to improved cardiac function. Although the mechanism by which nonresident progenitor cells results in improved function remains unclear,1 our findings point to the role that resident progenitor cells and their early committed progeny may have in cardiac growth and repair.1 The generation of new myocytes in the young heart is a normal physiological process and if confirmed later in life would strongly suggest that the understanding of the regulatory mechanisms of myocyte formation in the adult organ may be of fundamental biological importance and could have significant translational impact on patients with debilitating cardiac disorders.
Sources of Funding
Supported by NIH RO1 grants HL61495 and HL33921.
↵*Both authors contributed equally to this study.
Original received November 10, 2006; revision received January 8, 2007; accepted January 22, 2007.
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