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Article
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Metal Particulate Matter Components Affect Gene Expression and Beat Frequency of Neonatal Rat Ventricular Myocytes Donald W. Graff,1 Wayne E. Cascio,2,3 Joseph A.
Brackhan,2 and Robert B. Devlin1 1National Health and Environmental Effects Research Laboratory,
U.S. Environmental Protection Agency, Research Triangle Park,
North Carolina, USA; 2Division of Cardiology, Department of Medicine,
and 3Asthma and Lung Biology, The Center for Environmental Medicine,
University of North Carolina, Chapel Hill, North Carolina, USA Abstract Soluble particulate matter (PM) components (e.g., metals) have the potential to be absorbed into the bloodstream and transported to the heart where they might induce the expression of inflammatory cytokines and remodel electrical properties. We exposed cultured rat ventricular myocytes to similar concentrations of two metals [zinc (Zn) and vanadium (V) ] found commonly in PM and measured changes in spontaneous beat rate. We found statistically significant reductions in spontaneous beat rate after both short-term (4-hr) and long-term (24-hr) exposures, with a more substantial effect seen with Zn. We also measured the expression of genes associated with inflammation and a number of sarcolemmal proteins associated with electrical impulse conduction. Exposure to Zn or V (6.25-50 µM) for 6 hr produced significant increases in IL-6, IL-1 , heat shock protein 70, and connexin 43 (Cx43) . After 24 hr exposure, Zn induced significant changes in the gene expression of Kv4.2 and KvLQt (potassium channel proteins) , the 1 subunit of the L-type calcium channel, and Cx43, as well as IL-6 and IL-1 . In contrast, V produced a greater effect on Cx43 and affected only one ion channel (KvLQT1) . These results show that exposure of rat cardiac myocytes to noncytotoxic concentrations of Zn and V alter spontaneous beat rate as well as the expression of ion channels and sarcolemmal proteins relevant to electrical remodeling and slowing of spontaneous beat rate, with Zn producing a more profound effect. As such, these data suggest that the cardiac effects of PM are largely determined by the relative metal composition of particles. Key words: beat frequency, cardiac myocytes, cytokines, gap junctions, ion channels, metals, particulate matter. Environ Health Perspect 112:792-798 (2004) . doi:10.1289/txg.6865 available via http://dx.doi.org/ [Online 12 April 2004] Address correspondence to D. Graff, National Health and Environmental Effects Research Laboratory, U.S. EPA, MD 58D, Research Triangle Park, NC 27711 USA. Telephone: (919) 843-5155. Fax: (919) 966-6271. E-mail: graff.don@epa.gov We acknowledge L. Dailey and R. Silbajoris for their work on the RT-PCR experiments, and K. Dreher and J. Samet for their careful review of this manuscript. The information described in this article has been reviewed by the National Health and Environmental Effects Research Laboratory, U.S. Environmental Protection Agency, and has been approved for publication. Approval does not signify that the contents necessarily reflect the views and policy of the agency, nor does mention of trade names or commercial products constitute endorsement or recommendation of use. The authors declare they have no competing financial interests. Received 17 November 2003 ; accepted 7 April 2004. |
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Epidemiologic evidence links exposure to ambient air pollution particulate
matter (PM) to morbidity and mortality associated with adverse cardiovascular
health outcomes, including discharges of internal cardiac defibrillators and
hospitalizations and death due to arrhythmia and myocardial infarction [Peters
et al. 2000, 2001; U.S. Environmental Protection Agency (U.S. EPA) 1996]. Exposure
to PM may account for as many as 60,000 cardiopulmonary deaths each year in
the United States [National Resource Defense Council (NRDC) 1996; U.S. EPA
1996]. Reports of changes in heart rate and heart rate variability suggest
that altered cardiac electrophysiological processes play a role in PM-induced
morbidity and mortality (Liao et al. 1999; NRDC 1996; Peters et al. 2000).
Additionally, several studies have reported correlations between PM exposure
and cardiovascular inflammatory responses, which could lead to ischemic events
(Pekkanen et al. 2002; Peters et al. 2001; Ross 1999), or alternatively, could
induce electrical remodeling of the myocardium. It has been proposed that PM
exerts its cardiac effects indirectly through modulation of autonomic control
of the heart (Pope et al. 1999). An alternative hypothesis is that the production
of inflammatory mediators produced at sites distant from the heart (e.g., the
lungs or blood vessels) are transported to the heart, where they produce cardiac
pathology (Frampton 2001). However, it has also been suggested that particle
components may reach the systemic circulation (Nemmar et al. 2002) and can
reach the heart (Calderon-Garciduenas et al. 2001), where they may produce
direct effects on cardiac myocytes, resulting in inflammation, arrhythmia,
or myocardial infarction. Nevertheless, a definitive mechanism, particle, or
particle component responsible for the increase in cardiovascular morbidity
and mortality has yet to be identified.
PM is a complex mixture containing many different components including metallic
compounds such as iron, zinc (Zn), copper, vanadium (V), and nickel. Such metals
are particularly potent inducers of physiological effects in both animals and
humans. Two of these metals, Zn and V, are found in PM samples from a number
of sources and their water solubility in various chemical forms suggests the
potential for systemic absorption after inhalation. Previous studies have reported
that Zn and V can produce physiological changes in cardiac myocytes (Campen
et al. 2002; Evangelou and Kalfakakou 1993; Kodavanti et al. 2003; Werdan et
al. 1980). However, to our knowledge, a direct comparison of these two metals
in the same in vitro experimental system has not been reported previously.
Zn and V may well produce changes in cardiac function by altering the normal
gene expression of membrane proteins that contribute to electrical signal propagation
or through the stimulation of inflammation mediators that exert direct effects
on cardiac myocytes. A complex system of ion channels and biochemical transport
mechanisms function in concert to initiate and regulate the heartbeat and subsequent
impulse propagation throughout the heart. It is well known that disease states
such as hypertension and ischemic heart disease promote systemic and local
inflammatory processes, resulting in interstitial inflammation and myocyte
hypertrophy. These processes alter the normal cardiac structure and function
and induce changes in the expression of voltage-gated channels controlling
inward and outward membrane currents and intercellular currents. Changes in
gene expression and subsequent effects on membrane proteins and currents and
their cumulative effects on tissue electrical properties are known as electrical
remodeling and contribute to arrhythmogenesis.
In this study we observed that Zn and V produce important contrasting effects
on myocyte function and gene expression of several cardiac proteins at equimolar
concentrations. We report that Zn exposure produces a greater decrease in spontaneous
beat rate, a sensitive biosensor capable of detecting physiologically relevant
changes in active and passive membrane properties and a surrogate for heart
rate. We also note that Zn produces a more profound effect on the accumulation
of mRNAs coding for several potassium channels, a cardiac calcium channel,
and a gap junction protein, indicating that Zn may precipitate an electrical
remodeling process in the heart. Our data suggest that Zn and V can affect
the function of cardiac myocytes and thus provide biological plausibility to
previous epidemiologic studies linking PM exposure to negative cardiovascular
health outcomes. Furthermore, our cell culture model demonstrates that PM rich
in Zn may produce a greater direct effect on the heart than PM equally rich
in V.
Materials and Methods
Cell Culture
Rat ventricular myocytes were isolated from 1-day-old Sprague-Dawley rats
(CRL:CD; Charles River, Wilmington, MA) in compliance with guidelines established
by the National Institutes of Health (NIH 1996) and with the approval of the
Institutional Animal Care and Use Committee at the University of North Carolina
at Chapel Hill. After trypsin and collagenase digestions, the cells were resuspended
in medium 199 supplemented with 10% fetal calf serum (FCS) and penicillin/streptomycin
(20 U/mL and 0.02 mg/mL, respectively) and fibroblasts were removed by preplating
in 750-mL cell culture flasks. The flasks were then rinsed, and the resulting
myocyte suspensions were diluted with medium 199 supplemented with 10% FCS,
penicillin/streptomycin (20 U/0.02 mg/mL) and bromodeoxyuridine (5 µg/mL)
and plated in laminin-coated plastic dishes at a density of approximately 250,000
cells/cm2. Basic media and serum conditions were constant during
all experiments to avoid a potential stress response and cell death due to
serum depletion (Leicht et al. 2001). All cultures were maintained at 37°C
under an atmosphere containing 5% CO2. Experiments were conducted
using 11- to 13-day-old confluent monolayers of spontaneously beating myocytes.
The sulfate salts of each metal were used in the exposure experiments.
Toxicity Experiments
Cytotoxicity produced by varying concentrations of Zn and V were assessed
using the CytoTox 96 nonradioactive cytotoxicity assay (Promega, Madison, WI).
Briefly, lactate dehydrogenase (LDH) enzyme in culture supernatants was measured
with a 30-min coupled enzymatic assay that results in the conversion of a tetrazolium
salt into a red formazan product, with the amount of color formed being proportional
to the number of lysed cells. Cardiac ventricular myocytes were grown in six-well
plates under the conditions described above for 11 days. Aliquots of the supernatants
were collected after 24 hr in control media and immediately assayed for baseline
LDH release under nonstimulated conditions. The media was then removed and
replaced with media containing 0, 6.25, 12.5, 25, or 50 µM Zn or V. After
24-hr exposure, aliquots of the supernatants were assayed for LDH. The media
was removed and replaced with media containing 1% Triton X-100 for 30 min to
lyse the cells, thereby providing a measure of maximal LDH release. Aliquots
were again obtained and assayed for LDH. To compute percent cytotoxicity, we
divided the experimental and control absorbance values by the corresponding
maximal absorbance values.
Beat Rate Experiments
Measurements of spontaneous beat rate were conducted on cells grown in plastic
35-mm cell culture dishes in the conditions described above. Baseline beat
rate measurements (beats per min) were obtained using a Nikon DIC inverted
microscope (Nikon Instruments Inc., Melville, NY) connected to a Hitachi CCD
camera (Hitachi Denshi, Ltd., Woodbury, NY). Images were displayed on a Sony
video monitor (Sony Corp., New York, NY) and simultaneously recorded with a
JVC VHS video recorder (JVC Professional Products, Wayne, NJ). Freshly prepared
Zn or V sulfate stock solution was then added to the medium to reach the desired
final concentration (0, 6.25, 12.5, 25, or 50 µM), and the effect on beat
rate was measured at 0.5, 1, 2, 4, and 24 hr. Three measurements were taken
from each culture dish, which was marked with a pen in three randomly chosen
spots to ensure that repeat measurements were taken from the same groups of
cells. Temperature was constantly maintained at 37°C using an infrared
heating lamp in an incubation chamber surrounding the microscope stage.
Reverse Transcription and Real-Time Polymerase Chain Reaction
Cells were grown in 24-well plates as described previously and exposed to
control media or media containing Zn or V (6.25, 12.5, 25, or 50 µM) for
6 or 24 hr. Extraction of RNA, first-strand cDNA synthesis, and DNA amplification
were performed by methods described previously with minor modifications (Carter
et al. 1997). Cells were lysed in buffered guanidine isothiocyanate (6 M) and
sheared through a 25-gauge needle. RNA was pelleted by ultracentrifugation
onto a 5.7 M cesium chloride cushion for 2 hr, resuspended in Tris-EDTA
buffer, and precipitated overnight at -80°C in 70% ethanol and 0.15
M NaCl. RNA concentrations were determined using the Ribogreen assay (Molecular
Probes, Inc., Eugene, OR). cDNA was synthesized from 100 to 200 ng RNA using
a reverse transcriptase (RT) reaction in a total volume of 100 µL with
10 polymerase chain reaction (PCR) buffer, a dNTP mix, random hexamers, Rnasin,
and marine leukemia virus RT.
Quantitative real-time PCR was performed using TaqMan polymerase with detection
of FAM (6-carboxy-fluorescein) fluorescence on a sequence detector (ABI PRISM
7700; PerkinElmer Applied Biosystems, Foster City, CA). Rat oligonucleotide
primer pair sequences (Integrated DNA Technologies, Inc., Coralville, IA) for
each gene of interest are listed in Table 1. Serial dilutions of cDNA isolated
from unexposed rat ventricular myocyte cultures were analyzed and used for
standard curves. Mouse glyceraldehyde-3-phosphate dehydrogenase sequence served
as an internal control. cDNA samples were subjected to 40 cycles on the sequence
detector, and the threshold was set at a point consistent among the samples
and on the linear upslope. Only the resulting curves with a correlation coefficient
above 0.98 were used to assure the accuracy of the data.
Statistics
Statistical analyses were performed using GraphPad Prism software, version
3.02 (GraphPad Software, Inc., San Diego, CA). The 0- to 4-hr beat rate experiments
used a repeated measures analysis of variance (ANOVA), whereas 0- to 24-hr
beat rate measurements (performed in separate experiments) were compared using
a paired t test. Standard ANOVA was used for LDH and mRNA data comparisons.
Dunnett's test was used after ANOVA for post hoc analysis to determine the
treatment groups that differed from baseline or control where appropriate.
Values are expressed as means ± SEM. Differences were considered statistically
significant at p < 0.05.
Results
Figure 1. LDH release after stimulation
with 0, 6.25, 12.5, 25, 50 µM Zn or V for 24 hr. Data from four experiments
are expressed as a mean (± SEM) percentage of the maximal LDH release
stimulated by 1% Triton X-100 treatment. Using ANOVA, no difference from
control was found at any concentration of either
metal.
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Table 1.
![Table 1](tab1sm.jpg)
|
Figure 2. Zn- and V-induced changes in the
spontaneous beat rate of myocytes in culture after exposure to 12.5,
25, or 50 µM Zn (A)
or V (B). Data are shown as mean percent of baseline rate ± SEM
of 9-17 measurements per dosing group. The 0.5- to 4-hr and 24-hr
experiments were carried out separately. For a frame of reference, mean
baseline beat rates were 211 (0.5-4 hr) and 242 (24 hr) bpm for
Zn experiments and 198 and 216 bpm, respectively, for V experiments.
There were no time-related changes related to control medium or the 6.25-µM
concentration of either metal.
*p < 0.05; **p < 0.0001. |
Figure 3. Changes in cardiac mRNA expression of (A)
inflammatory and stress markers and (B) ion channel proteins
after 6-hr exposure to 12.5, 25, or 50 µM Zn. Exposure to 6.25 µM
Zn had no effect on any transcript. Values are mean ± SEM of three
to four experiments, each comprising two to four independent measurements.
*p < 0.05; **p < 0.0001.
|
Figure 4. Changes in cardiac mRNA expression of (A) inflammatory
and stress markers and (B) ion channel proteins after 6-hr exposure
to 12.5, 25, or 50 µM V. Exposure to 6.25 µM V had no effect
on any transcript. Values are mean ± SEM of three to four experiments,
each comprising two to four independent measurements.
*p < 0.05; **p < 0.0001.
|
Figure 5. Changes in cardiac mRNA expression of (A) inflammatory
and stress markers and (B) ion channel proteins after 24-hr
exposure to 12.5, 25, or 50 µM Zn. Exposure to 6.25 µM Zn had
no effect on any transcript. Values are mean ± SEM of three to four
experiments, each comprising two to four independent measurements.
*p < 0.05; **p < 0.0001. |
Figure 6. Changes in cardiac mRNA expression of (A) inflammatory
and stress markers and (B) ion channel proteins after 24-hr exposure
to
12.5, 25, or 50 µM V. Exposure to 6.25 µM V had no effect on any transcript.
Values are means ± SEM of three to four experiments, each comprising two
to four independent measurements.
*p < 0.05; **p < 0.0001. |
To determine whether Zn or V affect the physiologic function of cardiac myocytes
in culture, we exposed cells to 6.25- to 50-µM concentrations of each
metal, then measured the spontaneous beat rate of the myocytes at various times
after exposure. These concentrations were chosen partly because they did not
induce significant cellular injury as determined by release of LDH (Figure
1). The effects of Zn exposure on the spontaneous beat rate of myocytes are
shown in Figure 2A. A 30-min exposure to 50 µM Zn sulfate produced a significant
decrease in spontaneous beat rate compared with baseline, an effect that persisted
for 24 hr. Lower concentrations of Zn also caused a decrease in spontaneous
beat rate, although longer exposure times were required. Figure 2B shows that
V exposure also resulted in a decrease in spontaneous myocyte beat rate, albeit
to a lesser extent than Zn.
The rate of spontaneous beating of cardiac cells in culture is determined
by a number of factors, including the rate of spontaneous depolarization of
latent pacemaker cells and the passive resistive properties of the cellular
syncitium. These factors are directly influenced by changes in voltage-gated
channels and structural proteins such as gap junctions. Therefore, in subsequent
experiments we exposed cardiac myocytes to concentrations of Zn and V similar
to those used in the beat rate experiments to determine whether these metals
also produce dissimilar effects on ion channel, gap junction protein, and inflammatory
mediator mRNA accumulation. Exposure of cells for 6 hr to Zn resulted in significant
changes in gene expression of two inflammatory cytokines (IL-6, IL-1 ) as well
as heat shock protein (HSP)70 (Figure 3A). Zn exposure also increased connexin
43 (Cx43) gene transcripts by approximately 50% but otherwise did not affect
the mRNA accumulation of genes encoding Cx40 or other ion channels studied
(Figure 3B). Exposure of cells to V for 6 hr also resulted in increased levels
of mRNAs coding for IL-6, IL-1 , and HSP70 (Figure 4A) but not Cx43 (Figure
4B).
Exposure of cells to Zn and V for 24 hr yielded results quite different from
those seen at 6 hr. As shown in Figure 5A, cells exposed to Zn had an even
greater increase in accumulation of mRNAs coding for IL-6 and HSP70 than seen
after 6-hr exposure, although IL-1 gene expression was no longer significantly
altered (Figure 5A). However, marked changes in gene expression of several
ion channel and gap junction proteins were also observed (Figure 5B). Zn exposure
resulted in statistically significant dose-dependent increases in three of
the four potassium channels studied (Kv1, Kv4.2, KvLQT1), the 1 subunit
of the L-type calcium channel, and Cx43. Exposure of cells to V for 24 hr resulted
in a significant dose-dependent increase in IL-6 and IL-1 gene expression
but not HSP70 expression (Figure 6A). Cx43 gene expression was also increased
at 24 hr, but in marked contrast to Zn, V exposure did not cause an increase
in ion channel gene expression (Figure 6B). One ion channel (KvLQT1) actually
had significantly decreased mRNA accumulation.
Discussion
The results of this study indicate that the soluble metal composition of
PM may be particularly important when one is assessing cardiac toxicity associated
with PM exposure. Both Zn and V modulate the function of cardiac myocytes by
slowing their spontaneous beat rate, analogous to slowing of heart rate previously
demonstrated in rats instilled with residual oil fly ash containing high amounts
these metals (Campen et al. 2002; Wichers et al. 2004). However, compared with
V, the decrease induced by Zn was significantly more pronounced and occurred
at lower concentrations with shorter exposure times. The distinct responses
to these two metals is not surprising, as earlier work has shown that Zn and
V produce dissimilar effects in guinea pig heart contractile rate (Evangelou
and Kalfakakou 1993; Kalfakakou et al. 1993) and rat myocyte beat rate (Werdan
et al. 1980).
Numerous transcriptional and posttranslational modifications may account
for the changes in spontaneous beat rate that we observed. Rapid changes (those
occurring within 4 hr) likely result from direct effects of the ions themselves
on the channels or from posttranslational protein modifications. Although this
study does not address this type of posttranslational effect, we are currently
conducting experiments exploring the effects of Zn and V on protein phosphorylation
to gain mechanistic insight into the rapid effects these ions have on spontaneous
beat rate. In an effort to investigate potential molecular mechanisms requiring
transcriptional changes that may not be evident within 4 hr of exposure, we
chose to study alterations in gene expression after longer exposures to Zn
and V (6 and 24 hr), selecting representative genes encoding for several ionic
currents responsible for maintaining cellular transmembrane electrochemical
potential. A thorough review of these currents can be found in several references
(Nerbonne 2000; Nerbonne et al. 2001; Strauss and Brown 2001).
In the heart, potassium and calcium currents are principal determinants of
the time course of repolarization of cardiac myocytes; thus, their activity
is responsible for the duration of the action potential plateau. After depolarization
(a rapid rise in membrane potential primarily attributed to a rapid inflow
of sodium ions), voltage-gated potassium channels open, allowing potassium
to leave the cell. Simultaneously, calcium channels open to allow calcium ions
to enter the cell. The combined and opposing effects of the potassium and calcium
currents contribute to repolarization (i.e., a return of the cell to its negative
resting level) and provide the characteristic plateau seen in phase 2 of the
cardiac cell action potential. Hence, alterations in gene expression of cardiac
potassium and calcium channels may dramatically affect the capability of a
cardiac myocyte to repolarize.
The effects of potassium channel gene regulation on ion currents, protein
density, and the cardiac action potential have been demonstrated previously.
Investigators crossing dominate negative Kv4.2 mice with Kv1.4 knockout mice
essentially eliminated two important outward potassium currents, which produced
an increase in action potential duration resulting in QT prolongation and arrhythmia
(Guo et al. 2000). In streptozocin-induced diabetic rats, ventricular Kv4.2
mRNA levels were decreased 41%, whereas Kv1.4 mRNA levels were increased 179%
compared with nondiabetic controls. Western blot analysis showed a correlation
between corresponding changes in the mRNA levels to decreases in Kv4.2 protein
and increases in Kv1.4 protein (Nishiyama et al. 2001). A link between expression
of potassium channel genes and postmyocardial infarction-related arrhythmias
has been demonstrated, highlighting the importance of downregulated ion channels
after cardiac injury (Huang et al. 2000). Increases in action potential duration
and decreases in two potassium currents correlated to decreases in messenger
and protein levels of Kv4.2/4.3 and Kv2.1. Nerbonne et al. (2001) provide a
comprehensive summary of studies describing the consequences of genetic manipulation
of cardiac potassium channels and the resulting effects on ion currents and
cellular phenotype.
In addition to the role played by ion channels, cellular coupling of cardiac
myocytes is also vital for a coordinated propagation of electrical and chemical
impulses. Moreover, the strength of electrotonic interaction is primarily related
to the magnitude of cellular coupling as determined by gap junctional conductance.
Gap junctions are specialized membrane proteins that regulate the passage of
small molecules, ions, and electrical current between neighboring cells. In
adult ventricular myocytes, gap junctions are composed primarily of Cx43, whereas
Cx40 is present in the ventricle early in cardiac development (Kwak et al.
1999). Therefore, alterations in the expression of these proteins may well
modify both membrane potential and gap junctional conductance, leading to changes
in heart rate (beat rate in our cell culture model) and arrhythmia.
In this study we considered several representative genes involved in modulating
repolarization, several proteins that carry potassium and calcium currents,
and two proteins essential for gap junctional communication. We found both
time- and metal-dependent changes in the expression of several ion channels
and Cx43. Increased Cx43 gene expression was observed after both 6- and 24-hr
exposure. After 24-hr exposure, Zn also produced statistically significant
changes in gene expression of Kv4.2 (a fast-activating and -inactivating potassium
current), KvLQT1 (a very slow-activating and -inactivating potassium current),
and the 1 subunit of the L-type Ca2+ channel. We also
saw a large increase in Kv1 (a fast-activating and slow-inactivating potassium
current), although this change was not statistically significant. In contrast,
the effect of V exposure on these channels was strikingly different. V induced
a small increase in Cx43 gene expression but did not induce the expression
of any ion channel gene. The only ion channel gene affected by V, KvLQT1, was
actually downregulated. These findings are consistent with previous studies
and support the likelihood that Zn and V exert their effects via distinct mechanisms
(Evangelou and Kalfakakou 1993; Werdan et al. 1980).
Our data also show that transition metals commonly found on air pollution
particles can also stimulate the production of inflammatory cytokines in cultured
cardiomyoctes. Although still distinct, the changes noted in these transcripts
after Zn and V exposure are more similar than those noted for the ion channel
and gap junction proteins. After 6- and 24-hr exposure, both Zn and V induced
small but significant increases in the expression of IL-1 and IL-6 genes and
in HSP70, a protein shown to possess a protective role in inflammation and
ischemic disease, and which may well have a regulatory role in cytokine biosynthesis.
Whereas other cell types may produce a greater cytokine response, these data
are important in demonstrating that Zn and V are both capable of altering the
gene expression of cytokines in ventricular myocytes. This is potentially significant
because cytokines can contribute to pathways leading to myocardial infarction,
and in turn, myocardial infarction can further lead to hypertrophy of the myocardium,
resulting in heart failure and arrhythmia. In addition, various cytokines have
previously been shown to alter potassium currents (Diem et al. 2001, 2003),
gap junction connectivity (Chandross et al. 1996; Chanson et al. 2001), and
cardiac contractility (Finkel et al. 1992). Although our studies do not fully
support a conclusion that cytokine release results in modification of ion channel
gene regulation, we see a substantial cytokine response before major changes
in ion channel gene regulation is intriguing and deserves further investigation.
Although in vitro cell culture models are valuable in exploring mechanisms
by which PM components affect cardiac dysfunction, this work does have limitations.
In this study, we measured only changes in mRNA expression. Changes in mRNA
accumulation typically but not always result in modified protein production
(Nishiyama et al. 2001). If the mRNA alterations seen here are translated into
changes in protein assembly, we would expect to see changes in impulse formation,
the safety factor that protects against aberrant impulse propagation and conduction
patterns in myocardial tissue, as well as a significant inflammatory response.
Twenty-four-hour changes in beat rate may be reflective of the altered gene
regulation of Cx43 and the ion channels we chose to study. However, as a vast
number of proteins must interact harmoniously to contribute to the many repolarization
currents, it would be presumptuous to imply that the changes in spontaneous
beat rate observed in this study are due to the transcriptional changes in
the few ion channels we have considered. Because of the large number of potassium
channels found in the heart and because of the redundancy in the activity of
these channels, it is difficult to determine the effect that the message up-
or downregulation of a small number of channels would have on repolarization
and subsequent impulse formation. Nevertheless, it is evident that remodeling
of the ion channels is occurring to some degree, more so during Zn exposure,
and it is logical to assume that the transcription of ion channels other than
those we have measured are likely to be affected as well.
In summary, this study provides intriguing data suggesting that the chemical
composition of PM is very important in producing cardiac toxicity. Our data
demonstrate that Zn and V, metals commonly found in air pollution particles,
have very distinct effects on the spontaneous beat rate of cultured cardiac
myocytes, a measure that corresponds to heart rate in vivo. Although
we cannot provide a temporal account for rapid changes in spontaneous beat
rate, we do provide evidence that both metals, after a longer exposure, result
in alterations in ion channel and Cx gene expression that would be expected
to produce altered action potential characteristics and beat rate. Additionally,
Zn and V also produce changes in the transcription of cytokines, which in previous
studies have been linked to alterations in ion currents and gap junction connectivity.
Furthermore, although we cannot exclude the influence of other biological processes,
we provide stimulating hypothesis-generating evidence of a potential cellular
mechanism explaining the acute changes in heart rate observed in epidemiologic
and toxicologic studies of PM exposure. Further studies are being conducted
to determine the effects of Zn, V, and other PM constituents on the cardiac
action potential, the phosphorylation state of the connexins and functional
measures of cellular coupling after both short-term and long-term exposures.
These studies will help determine the precise mechanisms by which these metals
exert their distinct effects on the heart. |
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