© 2000 American Heart Association, Inc.
Integrative Physiology |
Synthetic Strands of Neonatal Mouse Cardiac Myocytes
Structural and Electrophysiological Properties
From the Department of Physiology (S.P.T., L.B.-L., J.Z., A.G.K.), University of Bern, Switzerland, and the Center for Cardiovascular Research and the Departments of Pathology and Medicine (S.A.T., J.E.S.), Washington University, St. Louis, Mo.
Correspondence to André G. Kléber, MD, Department of Physiology, University of Bern, Bühlplatz 5, CH-3012 Bern, Switzerland. E-mail kleber{at}pyl.unibe.ch
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Abstract |
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Abstract—The aim of the present study was to morphologically and electrically characterize synthetic strands of mouse ventricular myocytes. Linear strands of mouse ventricular myocytes with widths of 34.7±4.4 µm (W1), 57.9±2.5 µm (W2), and 86.4±3.6 µm (W3) and a length of 10 mm were produced on glass coverslips with a photolithographic technique. Action potentials (APs) were measured from individual cells within the strands with cell-attached microelectrodes. Impulse propagation and AP upstrokes were measured with multisite optical mapping (RH237). Immunostaining was performed to assess cell-cell connections and myofibril arrangement with polyclonal antisera against connexin43 and N-cadherins and monoclonal antibodies against cardiac myosin. Light microscopy and myosin staining showed dense growth of well-developed elongated myocytes with lengths of 34.2±4.2 µm (W1), 36.9±5.8 µm (W2), and 43.7±6.9 µm (W3), and length/width ratios of 3.9±0.2. Gap junctions were distributed around the cell borders (3 to 4 junctions/µm2 cell area). Each cell was connected by gap junctions to 6.5±1.1 neighboring cells. AP duration shortened with time in culture (action potential duration at 50% repolarization: day 4, 103±34 ms; day 8, 16±3 ms; P<0.01). Minimum diastolic potential and AP amplitude were 71±5 and 97.2±7.6 mV, respectively. Conduction velocity and the maximum dV/dt of the AP upstroke were 43.9±13.6 cm/s and 196±67 V/s, respectively. Thus, neonatal ventricular mouse myocytes can be grown in continuous synthetic strands. Gap junction distribution is similar to the neonatal pattern observed in the hearts of larger mammals. Conduction velocity is in the range observed in adult mice and in the higher range for mammalian species probably due to the higher dV/dtmax. This technique will permit the study of propagation, AP, and structure-function relations at cellular resolution in genetically modified mice.
Key Words: strands • mice • cardiomyocytes • connexin43 • conduction
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Introduction |
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Propagation of the cardiac impulse is a complex process that depends on the active electrical properties of the cardiac cells and the passive properties of the cellular network. Thus far, analysis of impulse propagation at a cellular level has been possible only through computer simulations1 2 or in experiments in which high-resolution optical mapping of transmembrane potential has been performed under high magnification in cultured myocytes grown in patterned arrays.3 Results of these experiments have provided insight into the role of discontinuous tissue structure, membrane ion channels, and cell-to-cell connections in propagation.4 5 6
Experimental elucidation of the functional role in impulse propagation of specific proteins that form ion channels, ion exchangers, ion pumps, and gap junction channels requires interventions that selectively and specifically inhibit or activate cellular functions. Steady-state conditions are often difficult to achieve with drugs. An alternative approach is to study genetically engineered animals to define the functional consequences of deletion or overexpression of specific genes. Until further advances occur in the development of methods and reagents for large animal transgenic technology, genetic engineering of mammals will remain practical only in mice. A number of transgenic mice with defined alterations in the expression of genes critical in depolarization, repolarization, and cell-to-cell communication have been reported.7
In the present report, we describe the production of synthetic strands of neonatal mouse ventricular myocytes and the characterization of structural and electrophysiological features pertinent to impulse propagation in this synthetic preparation. Mouse myocyte strands were produced with a technique similar to that previously described for neonatal rat myocytes.8 To relate structural features of these artificial strands to electrical function and to calibrate measurements made with voltage-sensitive dyes, optical measurements were combined with direct microelectrode recordings, and the preparations were subsequently analyzed morphometrically and immunohistochemically to assess the 2-dimensional structure of the multicellular strand and the expression of intercellular junction proteins. This experimental system has far-reaching potential for elucidation of the determinants of impulse propagation and arrhythmogenesis with cells from mice with defined molecular pathology.
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Materials and Methods |
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Cell Cultures
Hearts were rapidly excised from neonatal (1 to 2 days old) ddY mice (Japan SLC, Shizuoka Laboratory Animal Center). The ventricles were minced and placed in a dissociating solution (Ca2+- and Mg2+-free Hanks’ balanced salt solution [GIBCO] containing 0.5 mg/L trypsin [Boehringer-Mannheim], 10 mg/L pancreatin [Sigma Chemical Co], and 10 mmol/L HEPES). Harvested cells were centrifuged, and the sediment was resuspended in 6 mL M199 (GIBCO) containing 20 U/mL penicillin, 20 mg/L streptomycin, 0.1 mmol/L bromodeoxyuridine, and 10% newborn calf serum (Bio Concept). The fibroblast content of the cell suspension was reduced with a differential attachment technique.9 Cell-rich medium (28±7x104 cells/mL) was placed in a multiwell plate (2 mL/well) containing glass coverslips that had been prepared for patterned cell growth with a photolithographic technique described in detail previously.8 In the present study, cells were cultured in parallel strands 1 cm in length. Culture medium was changed after 24 hours and every 2 days thereafter with M199 containing 5% neonatal calf serum, bromodeoxyuridine (0.1 mmol/L), and epinephrine (10 µmol/L for the first 3 days). A total of 114 mice were used for 14 culture preparations. The study complies with the ethical principles described by the Swiss Academy of Medical Science and institutional guidelines were observed.
Optical Mapping and Analysis of Propagation
The technique of multiple-site optical recording of
transmembrane potential and the staining of cell cultures with the
voltage-sensitive dye RH237 have been described in detail
elsewhere.10 11 The cultures were stimulated >1 mm
from the recording site (cycle length 500 ms), and
isochronal maps were calculated as previously
described.12
Microelectrode measurements were performed on days 4 (n=5), 6 (n=7), 7 (n=4), and 8 (n=4) in culture. Glass microelectrodes with a tip diameter of 1 µm were filled with a solution containing (in mmol/L) K gluconate 120, KCl 20, HEPES 20, and Mg · ATP 5, with pH adjusted to 7.2. Signals were recorded with an Axoprobe 1A amplifier (Axon Instruments) in the current clamp mode and MacLab AD conversion and analysis (sampling rate 20 kHz). Junction potentials were measured and computed (correction factor -11.8 mV) with the method described by Neher.13 Recordings stable over >60 seconds were used for analysis.
Immunohistochemistry
Myocytes on coverslips were fixed in 4%
paraformaldehyde in PBS for 15 minutes and rinsed 3
times in PBS. Immunostaining was performed with an
affinity-purified polyclonal rabbit anti–connexin43 (Cx43) antiserum
(Zymed) diluted 1:200 in blocking buffer (PBS containing 0.1% Triton
X-100, 3% normal goat serum, and 1% BSA) and/or a polyclonal rabbit
antiserum against a conserved sequence in the N-cadherins (Sigma
Chemical Co) diluted 1:400 in blocking buffer. In selected experiments,
cells were incubated with a monoclonal antibody specific for mouse
cardiac myosin (kindly provided by Dr Stacy Smith) to determine whether
significant contamination by nonmyocyte cells was present.
All immunostaining procedures, including the use of
controls for nonspecific binding, have been described in detail in a
previous report.14 Immunostained cells were
mounted onto glass slides and examined with a Sarastro model 2000 laser
scanning confocal microscope (Molecular Dynamics).
Confocal Microscopy
Three to five high-power fields of strands of different widths
in 6 separate cultures were examined at a magnification of x400 as
previously described.14 The proportion of total cell area
occupied by Cx43 immunoreactive signal was defined as the number of
high-signal-intensity pixels divided by the total number of pixels
occupied by cells. The total number and mean size of individual spots
of high intensity signal, operationally defined as individual gap
junctions, were measured according to methods previously
validated.14 Confocal microscopy was also used to
visualize the distribution of N-cadherin immunoreactive signal to
delineate the locations of fascia adherens junctions. Cultures stained
with antibodies against cardiac-specific myosin were examined to define
the relative proportions of myocytes (identified by intense staining in
a sarcomeric pattern) and nonmyocytic cells (fibroblasts and
endothelial cells) that do not express cardiac myosin.
Confocal microscopy was also used to determine cell thickness in
cultures stained with Fluo-3.
Measurement of Cell Size, Cell Shape, and Connectivity
The outlines of individual cells were readily delineated in
cultures stained with antibodies against Cx43 or N-cadherins.
Accordingly, these preparations were used for morphometric studies to
measure the length and width of cells in strands, the number and
relative end-to-end or side-to-side orientation of cells interconnected
by gap junctions to an individual cell within strands, the width of the
strands, and the number of cells across strands of different widths.
The extent of intercellular connectivity was determined by selecting
index cells within the center of strands and counting the total number
of neighboring cells connected to each index cell by
immunohistochemically identified gap junctions. The spatial orientation
of the interconnected cells was defined according to system used in
previous studies.15
Statistical Analysis
Trends in morphology in relation to strand width were calculated
with ANOVA. Multiple regression was used to determine the effect of
strand width and age in culture on conduction velocity (
) and
maximum dV/dt of the AP upstroke. Continuous variables are
expressed as mean±SD. The cell orientation analysis was
performed using the Mann-Whitney U test.
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Results |
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Structural Features of Synthetic Mouse Cardiac Myocyte Strands
The arrangement of cells in strands was clearly demonstrated in cultures stained with an antibody against cardiac myosin (Figure 1
). These preparations showed closely
packed, elongated cardiac myocytes aligned with their long axes
parallel to the long axis of the strand. Alignment of cells was almost
complete in the narrow strands. Thus, the absolute value of the angle
between the long axis of the cells and the strand axis was 3±1° in
the small strands and 15±4° in the large strands
(P=0.001). The cells exhibited robust staining in a
sarcomeric pattern, indicating well developed contractile organelles.
An examination of both strands and peripheral areas
composed of confluent monolayers of cells revealed no cells that failed
to demonstrate myosin staining (Figure 1). Thus, contamination
with nonmyocytic cells was not detectable.
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Table 1 summarizes the results of
morphometric analyses. Cells were grown in strands of 3 widths:
34.7±4.4 (W1), 57.9±2.5
(W2), and 86.4±3.6
(W3) µm. The average dimensions of
individual cells within strands varied as a function of strand width
(Table 1). Both cell length and width increased with increasing
strand width. In contrast, there was a trend toward an inverse
relationship between cell thickness and stand width that did not
achieve statistical significance. The number of cells across each
strand fell within a narrow range of 3 or 4 cells across small strands
to 8 or 9 cells across the largest strands (Table 1). Cell
dimensions were independent of duration in culture between days 4 and
8.
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The number of cells interconnected by gap junctions to an individual cell located within the center of a strand was 6.5±1.1. Of the neighbors interconnected to an average index cell, 2.5 or 38% were connected in a purely or predominantly side-to-side orientation, whereas 4.0 or 62% were connected purely or mainly in an end-to-end juxtaposition. No differences were observed in the number or spatial orientation of interconnected neighbors in strands of different widths or with duration of cells in culture between 4 and 8 days.
Immunohistochemistry and Confocal Microscopy of Intercellular
Junction Proteins
The Cx43 immunoreactive signal in mouse myocytes in strands was
analyzed with quantitative confocal microscopy. Figure 2 shows representative
confocal images of immunostained strands of different
widths. In all cases, the Cx43 signal appeared as discrete spots of
high-intensity signal against a dark background. Table 2 shows the results of quantitative
analysis of the amount of Cx43 immunoreactive signal as well as
the number of individual spots of high-intensity signal (operationally
defined as individual gap junctions) and the mean size of individual
gap junctions. These parameters were independent of strand
width.
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Figure 3 shows cultured neonatal mouse
ventricular myocytes incubated simultaneously
with anti-Cx43 antibodies and with an antibody against a conserved
sequence in the N-cadherins that stains fascia adherens junctions in
the intercalated disk. Patches or linear arrays of discrete spots of
high-intensity signal were seen at the edges of neighboring cells where
junctional membranes had formed. Simultaneous visualization
of fascia adherens junctions and gap junctions in double-label
preparations revealed an intimate spatial relationship between these 2
types of junctions. There was minimal overlap in the distribution of
the 2 signals, however, indicating that each of these organelles
occupied separate discrete regions of the junctional membrane (Figure 3).
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Electrical Properties of Synthetic Cardiac Myocyte Strands
A major goal of the present study was to characterize APs and
propagation with multisite optical recordings. Because the
fluorescence change of the voltage-sensitive dye does not
indicate the absolute value of the voltage change, it was necessary to
calibrate the optical signals with microelectrode measurements. As
illustrated in Figure 4, the AP duration
and shape of the plateau phase were dependent on the age of cells in
culture. At day 4, the plateau phase was present and repolarization
was relatively delayed. By day 8, the AP had a short spiked appearance,
similar to that seen in adult mice.16 17 The amplitude of
the AP and the minimum diastolic potential, averaged over
all recording days, were 97.2±7.6 and 71±5 mV, respectively.
The AP upstroke velocity was 196±67 V/s; higher than that previously
observed in synthetic strands of rat ventricular
myocytes.8 The propagation velocity recorded with
optical mapping was 43.9±13.6 cm/s and was independent of strand
width. Figure 5 illustrates propagation,
measured optically, in a medium-sized (W2)
street.
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There was an increase in maximum dV/dt of the AP upstroke with
increasing age of the culture. This trend was clearly apparent in the
microelectrode recordings but did not achieve statistical
significance. However, the optical measurements of upstroke velocity
confirmed this trend (P=0.01). The lower recorded
amplitude of dV/dtmax in the optical signals is
due to low-pass filtering at 1.5 kHz. The change in maximum velocity of
the AP upstroke with increasing age of the culture was not associated
with a significant increase in despite an apparent slight trend in
the mean values (Table 3).
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Discussion |
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Gap Junction Distribution and Connectivity
Cell shape, orientation, and connections between adjacent myocytes are major determinants of the passive properties of conduction within multicellular networks. Therefore, the description of cell morphology, connectivity, and Cx43 distribution are key components of this work. In normal adult humans, ventricular myocyte gap junctions are distributed anisotropically. In these structures, there is colocalization of gap junctions and fascia adherens junctions predominantly at the cell poles.18 19 This is not the case in human neonates, in whom Cx43 in myocytes has a uniform punctate distribution throughout the appositional cell membrane. Initially, gap junctions and adhering junctions are not usually closely associated. The adult pattern develops progressively during the first 6 years of life.20 Previous studies have shown that Cx43 in neonatal rat ventricular myocyte cultures exhibit a uniform distribution pattern within the cell membrane similar to that of the human neonate.21 In the present study, we showed that neonatal mouse ventricular myocytes have a similar distribution of Cx43. Changes in connexin distribution are also part of the remodeling process observed in several disease states. In the border zone between necrotic and healthy tissue after myocardial infarction, marked alterations of Cx43 distribution have been observed.22 23 A similar pattern of gap junction distribution was recently observed in the hypertrophied right ventricles of rats with pulmonary hypertension24 and in hypertrophic cardiomyopathy.25
The length/width ratio and connectivity of cardiac myocytes are important determinants of anisotopic conduction. The number of cells connected to an individual cell was 6.5±1.1. This value is comparable to the connectivity in adult canine ventricular tissue when the 2-dimensional structure is taken into consideration.26 As in the intact human ventricle, end-to-end connections dominate. In the adult canine ventricle, purely or predominantly end-to-end connections form 53% of connections compared with 62% in our preparations.15 By contrast, the length/width ratio was lower than that seen in adult dogs and neonatal rat cultures.11 These factors need to be taken into account if these cell cultures are to be used to investigate aspects of anisotropic conduction (see later).
Transmembrane APs
In the present study, APs from dense cultured monolayer
networks of mouse ventricular myocytes were recorded
for the first time. Minimum diastolic membrane potentials
and AP amplitudes were comparable to previous recordings of
resting and diastolic ventricular APs from
isolated cells27 28 29 30 31 32 or intact tissue
fragments.17 33 34 35 In intact ventricular
tissue fragments from neonatal and adult mice, the resting membrane
potentials were -76 to -81 mV.17 35 The AP amplitude was
similar to that observed in the present study and slightly lower
than that of isolated cells.27 30 31 The maximum upstroke
velocity of the AP in tissue fragments was similar to that observed in
the present study.17 35 Wang et al35
found no difference in the resting membrane potential or the maximum
upstroke velocity of phase 0 of the AP between neonatal and adult
mice.
In contrast to the absence of depolarization changes during postnatal development, Wang and Duff34 and Wang et al35 demonstrated a progressive reduction in ventricular AP duration in the mouse right ventricle with normal postnatal development. The action potential duration at 50% repolarization (APD50) was 43±14 ms in hearts from 1-day-old neonates, 18±6 ms after 3 days, and 10±3 ms in adult hearts. Studies in isolated cells indicated that changes in the density and inactivation kinetics of Ito contribute to this development.34 Babij et al33 showed that Ikr did not increase during postnatal development. Another study that involved the targeted disruption of the gene minK, which encodes a subunit of IKs, showed no QT prolongation in neonatal or adult mice.36 In the present study, a similar dramatic reduction in AP duration was observed for the first time in cultured networks. This finding suggests that developmental changes in the cultured network mimic those seen in vivo. We demonstrated an 85% reduction in APD50 and a 52% reduction in action potential duration at 90% repolarization (APD90) between days 4 and 8. If AP durations of myocytes freshly prepared from neonatal mice of varying postnatal ages35 are compared with myocytes after a similar number of days in culture, we observe that the APs are longer in our preparations and that the reduction in duration develops more slowly.
Propagation Velocity
Several differences between the cultures described here and adult
myocytes in vivo would be expected to affect . Conduction velocity
is determined not only by depolarizing ion channels4 5 and
cell-to-cell connections5 but, as demonstrated in a recent
study by Spach et al,37 also by cell size. The small cell
size and distribution of gap junctions may suggest that in these
preparations would be low. Instead, we found that was in the upper
range for ventricular conduction in larger
mammals.38 39 Clearly, other factors may have affected
in this model. The rapid AP upstroke velocity that suggests a high
density of Na+ channels may at least in part
compensate for the relatively small cell size.
Cell morphology was not uniform across the different strand widths
assessed in this study. We observed that the cell length and width were
greater in the large strands. By contrast, there was a trend toward an
increase in cell thickness in small strands. This suggests that the
difference in thickness may have compensated for the tendency of the
larger cell length to increase . Moreover, the greater alignment of
cells in the smaller strands with the direction of propagation also may
have attenuated the effect of a decrease in cell length on . Our
failure to detect any significant difference in between the strand
widths suggests that within the range of strand widths studied, these
opposing effects are small or that they cancel each other out.
Implications
This model allows the combination of several powerful tools for
the investigation of impulse propagation in cardiac tissues: genetic
engineering in mice, patterned growth of synthetic strands of neonatal
ventricular myocytes, and high-resolution optical mapping
of transmembrane potentials. These methods will contribute to the
investigation of the basic mechanisms of propagation and repolarization
at a cellular level.
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Acknowledgments |
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This work was supported by grants from the Swiss National Science Foundation, the Swiss Heart Foundation, a Cardiovascular Lipid Research Grant from Parke Davis (Division of Warner Lambert Ltd) and Pfizer/Australia Ltd, and NIH Grant HL-58507. The authors would like to thank Franco del Principe for assisting with the cell thickness measurements and Karen Green for technical assistance with morphometry and immunohistochemistry.
Received June 19, 2000; revision received July 20, 2000; accepted July 21, 2000.
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B. London Staying Connected Without Connexin43: Can You Hear Me Now? Circ. Res., July 23, 2004; 95(2): 120 - 121. [Full Text] [PDF]
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P. Beauchamp, C. Choby, T. Desplantez, K. de Peyer, K. Green, K. A. Yamada, R. Weingart, J. E. Saffitz, and A. G. Kleber Electrical Propagation in Synthetic Ventricular Myocyte Strands From Germline Connexin43 Knockout Mice Circ. Res., July 23, 2004; 95(2): 170 - 178. [Abstract] [Full Text] [PDF]
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 A. G. KLEBER and Y. RUDY Basic Mechanisms of Cardiac Impulse Propagation and Associated Arrhythmias Physiol Rev, April 1, 2004; 84(2): 431 - 488. [Abstract] [Full Text] [PDF]
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S. P. Thomas, J. P. Kucera, L. Bircher-Lehmann, Y. Rudy, J. E. Saffitz, and A. G. Kleber Impulse Propagation in Synthetic Strands of Neonatal Cardiac Myocytes With Genetically Reduced Levels of Connexin43 Circ. Res., June 13, 2003; 92(11): 1209 - 1216. [Abstract] [Full Text] [PDF]
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I. Kehat, A. Gepstein, A. Spira, J. Itskovitz-Eldor, and L. Gepstein High-Resolution Electrophysiological Assessment of Human Embryonic Stem Cell-Derived Cardiomyocytes: A Novel In Vitro Model for the Study of Conduction Circ. Res., October 18, 2002; 91(8): 659 - 661. [Abstract] [Full Text] [PDF]
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R. C. Pimentel, K. A. Yamada, A. G. Kleber, and J. E. Saffitz Autocrine Regulation of Myocyte Cx43 Expression by VEGF Circ. Res., April 5, 2002; 90(6): 671 - 677. [Abstract] [Full Text] [PDF]
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D. L Lerner, M. A Beardslee, and J. E Saffitz The role of altered intercellular coupling in arrhythmias induced by acute myocardial ischemia Cardiovasc Res, May 1, 2001; 50(2): 263 - 269. [Full Text] [PDF]
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R. C. Pimentel, K. A. Yamada, A. G. Kleber, and J. E. Saffitz Autocrine Regulation of Myocyte Cx43 Expression by VEGF Circ. Res., April 5, 2002; 90(6): 671 - 677. [Abstract] [Full Text] [PDF]
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