(Circulation. 2000;102:III-365.)
© 2000 American Heart Association, Inc.
Myocardial Protection and Vascular Biology |
Vesnarinone Restores Contractility and Calcium Handling in Early Endotoxemia
From the Departments of Anesthesiology and Cardiac Surgery and the Anesthesia/Critical Care Medicine Laboratory, Children’s Hospital and Harvard Medical School, Boston, Mass.
Correspondence to Francis X. McGowan, Jr, Cardiac Anesthesia Service, Children’s Hospital, 300 Longwood Ave, Boston, MA 02115. E-mail mcgowan_f{at}a1.tch.harvard.edu
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Abstract |
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Background—Endotoxin (lipopolysaccharide, LPS) is a trigger of the systemic inflammatory response. We have previously found that vesnarinone and amrinone, when given before LPS, prevented cytokine production and LPS-related cardiac dysfunction. We tested the hypothesis that vesnarinone would improve intracellular Ca2+ handling and calcium-activated contractile force after the onset of endotoxemia.
Methods and Results—Adult rabbits received a bolus injection of
LPS or vehicle. Vesnarinone (3 mg/kg) was given
intravenously 90 minutes later. Two hours after LPS
administration, hearts were perfused in the isolated Langendorff mode.
Peak left ventricular developed pressure, ±dp/dt, oxygen
consumption (M
O2), and ratexpressure
product were evaluated in conjunction with fluorescent
spectroscopic determinations of intracellular calcium concentrations
(Cai) and the rate of Cai transient decline
during diastole (
Ca). Peak left ventricular
developed pressure and ±dp/dt were significantly lower in the LPS
group. These were completely restored by vesnarinone. There was
significantly slower diastolic calcium removal (increased
Ca) in LPS hearts that was also corrected by vesnarinone; however,
the cytosolic calcium overload characteristic of LPS hearts was only
partially improved. Reduced mechanical inefficiency (the ratio of
rate-pressure product to MO2) and
myofilament sensitivity to Cai were also significantly
improved by vesnarinone.
Conclusions—Acute endotoxemia caused contractile protein calcium insensitivity, oxygen wastage, and abnormal calcium cycling. Vesnarinone, given in the rescue mode, normalized LPS-induced myocardial dysfunction and partially restored abnormal calcium cycling. Although the mechanisms responsible for these effects require further clarification, it appears that agents such as vesnarinone may be useful to treat inflammatory-induced myocardial dysfunction.
Key Words: calcium • contractility • proteins • inflammation
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Introduction |
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Endotoxin (lipopolysaccharide, LPS) is a prime trigger of the systemic inflammatory response that produces multiple organ dysfunction during sepsis.1 LPS release during cardiopulmonary bypass has also been linked to cardiac dysfunction.2 In experimental endotoxemia models, systolic dysfunction and reduced contractile reserve can be observed within 1 to 4 hours; these occur in the absence of systemic acidosis, hypotension, or decreased coronary perfusion, and before significant increases in circulating proinflammatory cytokines.3 4 5 More recently, we have shown that myocardial intracellular calcium (Ca2+i) handling and contractile protein sensitivity to Ca2+i are rapidly impaired (within 2 hours) by endotoxemia.6 These rapid effects of endotoxin on myocardial function and calcium cycling were not improved by ß-agonist administration. We have also found that vesnarinone, which has phosphodiesterase-inhibiting, ion channel, and immunomodulating activity, significantly reduced cytokine production and myocardial contractile dysfunction when given before LPS administration.7 The purpose of the present study was to test the hypothesis that vesnarinone would improve intracellular Ca2+ handling and calcium-activated contractile force after the onset of LPS-induced contractile dysfunction.
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Methods |
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Reagents
Vesnarinone was a gift from Otsuka Pharmaceutical Co. All other chemicals were purchased from Sigma Chemical Company, unless otherwise noted, and were of the highest grade available. LPS (lot 126H4099) was also purchased from Sigma. The calcium-sensitive, cell-permeable fluorescent dye Rhod-2AM was from Molecular Probes.
Animal Model
Animal procedures received institutional approval and were
conducted in conformity with the Guiding Principles in the Care and Use
of Animals of the American Physiology Society and the Guide for the
Care and Use of Laboratory Animals published by the National Institutes
of Health (NIH publication No. 85-23, revised, 1985). New Zealand White
rabbits (2.5 to 3.0 kg) received a bolus injection of 0.2 mg/kg
Salmonella typhimurium LPS dissolved in 1 mL/kg sterile
saline (LPS group, n=7) or sterile saline alone (1 mL/kg, control
group, n=6). Ninety minutes after LPS administration or saline
administration (n=6 each), animals received 3 mg/kg vesnarinone as an
intravenous infusion. Two hours after LPS or saline
administration, animals received ketamine (100 mg/kg) and
heparin (1000 U) intravenously. Their hearts were then
rapidly excised, placed in ice-cold Krebs-Henseleit (K-H) buffer, and
rapidly perfused retrograde through the aorta in the Langendorff mode
with the use of modified K-H crystalloid perfusate, exactly as
described previously.6 A latex fluid-filled balloon
connected to a micromanometry catheter (Miller Instruments) was used
for isovolumic left ventricular function measurements.
Hearts were paced at 150 bpm by atrioventricular pacing
wires sutured to the right atrium and the free wall of the right
ventricle. Peak developed left ventricular (LV) pressures
were measured at an LV end-diastolic pressure of 5
mm Hg. Coronary flow was measured by timed collection of the
coronary effluent. Oxygen content was calculated from the
measured oxygen tension (ABL-3 Acid-Base Laboratory, Radiometer) of
simultaneous aortic perfusate and coronary
venous effluent samples; myocardial oxygen consumption
(MO2) was calculated as the
measured arteriovenous oxygen difference multiplied by coronary
flow and divided by dry heart weight. Relative mechanical efficiency
was determined by the ratio of work, quantified by the ratexpressure
product (RPP), to myocardial oxygen consumption
(RPP/MO2).
Calcium Measurements
Measurement of beat-to-beat intracellular calcium transients was
performed as we have previously described and validated in
detail.6 8 Because Rhod-2 has no spectral shift when
it binds calcium, it is necessary to account for changes in tissue
Rhod-2 concentration over time (eg, leakage or photobleaching) as well
as differences in dye loading. To do this,
F589 was corrected for tissue absorbance
as follows. Excitation light was scanned at wavelengths between 524 nm
(peak Rhod-2 absorbance in cardiac tissue) and 589 nm (reference
wavelength at which there is insignificant Rhod-2 absorbance) and the
ratio of reflected light at 524:589 used to estimate tissue dye
concentration. The calcium fluorescence signal was also
corrected for tissue autofluorescence by subtracting the
fluorescence spectra of the heart before Rhod-2 loading from
that of the Rhod-2–loaded heart. Instantaneous ratioing of the
fluorescent intensity (F) at 589 nm to reflected excitation
light at 524 nm was done electronically to correct for heart motion.
Calcium transient values were expressed as peak fluorescent
intensity corrected for autofluorescence divided by the
absorbance correction for dye concentration (F/A).
After stabilization of dye loading and baseline signal acquisition,
beating hearts were perfused in a stepwise fashion for 2 minutes each
with K-H buffer containing 0.75, 1.25, and 2.25 mmol/L calcium. In
this way, the relation of extracellular calcium
(Cao) to intracellular calcium
(Cai) and Cai to
contractile force could be demonstrated. LV pressure and Rhod-2
fluorescence data were recorded simultaneously.
All data analysis was performed with commercially available
software (Sigma Plot, Jandel Scientific). The rate of the
Cai transient decline during
diastole, Ca was assessed by curve-fitting to a
monoexponential function as described
previously,6 using the following equation:
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Ca was calculated with this equation between
20% and 70% decline of transient amplitude.
Statistical Analysis
All values are reported as mean±SEM. Data are from 6
experiments each. Multiple group comparisons were made using ANOVA
followed by the Bonferroni procedure. Differences were considered
significant at P<0.05 with adjustment made for multiple
comparisons.
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Results |
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Effects of LPS and Vesnarinone
One of 7 animals that received LPS alone died (at
90
minutes after LPS administration); there were no deaths in any of the
other groups. No animal, regardless of treatment group, was hypotensive
(mean arterial pressure was 
50 mm Hg in all
animals) before cardiac harvesting. Peak developed LV pressure was
significantly lower in LPS hearts than in normal hearts (91±2 versus
110±5 mm Hg, P=0.05). This was restored by
vesnarinone administration to a supranormal level (138± 8 mm Hg,
P=0.01 versus LPS alone) that was not significantly
different from the response of normal hearts to vesnarinone
(142±5 mm Hg). Positive dp/dt (+dp/dt) and negative dp/dt
(-dp/dt) were also significantly lower in LPS hearts than in control
hearts; these parameters were also restored by vesnarinone
treatment (Figure 1
).
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Typical Rhod-2 fluorescence tracings are shown in Figure 2. Both systolic and
diastolic calcium concentrations were significantly higher
in LPS-treated hearts than in normal hearts. The rate of
diastolic calcium removal (Ca) was significantly lower
in LPS hearts than in normal hearts. Although vesnarinone corrected
Ca (Figure 3), it only incompletely
restored cytosolic calcium levels toward normal (Figure 4). As can be seen in Figure 4, LPS hearts developed significantly less pressure despite higher
cytosolic calcium concentrations; vesnarinone treatment of LPS hearts
was associated with significantly increased contractile force, but with
only a modest reduction in Cai. This apparent
improvement in myofibrillar calcium sensitivity (increased force at the
same or somewhat lower calcium concentration) was also seen in control
hearts exposed to vesnarinone (Figure 4).
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) developed significantly
less contractile force compared with controls (
). Vesnarinone
treatment of both LPS (
) and normal (•) hearts caused
significantly increased contractile force at somewhat reduced
systolic calcium levels. Data are mean±SEM, n=6 each.
Response to Calcium-Induced Inotropy
The ability to increase contractility in response
to increasing extracellular calcium (calcium-induced inotropy) from
0.75 mmol/L to 2.25 mmol/L was assessed. The ratio of
increase in pressure to the increase in intracellular calcium produced
by increasing pefusate calcium was (arbitrary units) 11±4 in control
hearts, 1±0.5 in LPS hearts (P=0.01 versus LPS) and 6±2 in
vesnarinone-treated LPS hearts (P=0.05 versus LPS alone and
control). In other words, contractility in normal
hearts was very responsive to the small changes in
Cai produced by increasing extracellular calcium.
In contrast, increasing perfusate calcium in LPS hearts led to
much larger changes in Cai, which were
nevertheless accompanied by minimal increases in
contractility. Although contractile force was
normalized in LPS-vesnarinone hearts, there was still increased calcium
cycling (see Figure 4
) leading to a lower value for the
efficiency of calcium-induced inotropy.
Overall mechanical efficiency was estimated from the ratio of the LV
rate-pressure product to oxygen consumption
(RPP/MO2) in response to
calcium-induced inotropy. As can be seen in Figure 5, LPS hearts developed significantly
less force despite higher oxygen consumption, and this effect was
corrected by vesnarinone.
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Discussion |
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The major finding of this study was that vesnarinone, given in the "rescue mode" 90 minutes after the onset of endotoxemia, improved LPS-stimulated abnormalities in myocardial function. Our data do not allow us to precisely define the mechanism(s) of action of vesnarinone in this setting. However, it is clear that vesnarinone led to substantial improvements in contractility, contractile responsiveness to calcium, diastolic calcium removal, and mechanical efficiency.
One or more intracellular sites may be responsible for the
observed calcium handling abnormalities during endotoxemia. In the
adult rabbit (as in the adult human) heart, cytosolic calcium is
primarily (75% of calcium influx) determined by the sarcoplasmic
reticulum (SR).9 The rate of
[Ca2+]i transient decline
is thus mainly a function of the SR Ca2+-ATPase
that transports calcium from the cytosol into SR; other, quantitatively
smaller sites include the sarcolemmal
Na+/Ca2+ exchanger that
transports Ca2+ from the cytosol into the
extracellular space and buffering of Ca2+ by
intracellular proteins. We therefore speculate that vesnarinone
improved SR Ca-ATPase function as part of its effect to speed
diastolic calcium removal. Increased production of
cAMP increases SR calcium uptake by stimulation of the SR Ca-ATPase and
enhances diastolic relaxation and increases inotropic
state; thus, phosphodiesterase inhibition may explain several of the
observed effects of vesnarinone. However, in a similar model,
dobutamine did not improve contractility or
calcium handling when given 2 hours after LPS despite evidence of
preserved ß-adrenergic signaling.7 It is therefore
unlikely that phosphodiesterase-mediated increases in cAMP are entirely
responsible for the effects observed in the present study. It is
also interesting that vesnarinone did not completely normalize
cytosolic calcium levels despite correcting the rate of
diastolic calcium removal. After vesnarinone, LPS hearts
still appeared "leaky" to calcium, as demonstrated by higher
calcium levels and greater increases in Cai when
exposed to higher perfusate calcium. Whether this was due to
decreased control of transmembrane calcium flux or to abnormal opening
of the SR calcium release channel, as may be produced by oxygen and
nitrogen radical species, requires further study.10
There are several advantages to the use of Rhod-2 to measure intracellular calcium transients. It excites in the visible range and thus tissue absorbance, autofluorescence, interference from other endogenous fluorophores, and photobleaching are low. It efficiently loads into the cell at 37°C with minimal uptake into nonmyocytes or intracellular organelles, displays high quantum yield on binding calcium, and has a favorable Kd for calcium relative to the contractile proteins (710 nmol/L in the presence of 0.5 mmol/L myoglobin).8 Overall, these properties give good specificity, depth of tissue penetration, large dynamic range, and detailed transient morphology. However, one disadvantage is that Rhod-2 does not exhibit a shift in excitation or emission wavelength when binding calcium; one must measure and correct for changes in dye concentration (see Methods section). We also corrected for motion artifact by gently constraining the heart against an optical window and using a reflected light reference signal correction; thus, motion artifact accounts for <2% to 3% of signal.
The cause of reduced myofibrillar calcium sensitivity in LPS
hearts, particularly after only 2 hours of exposure, is uncertain. The
number of potential mediators released by LPS is large and includes
tumor necrosis factor-
, interleukin-1(ß), platelet activating
factor, endothelin, and nitric oxide.1 2 3 4 5 11 12 13 14 These can
stimulate numerous intracellular signaling pathways, including
phospholipases, protein kinase C, and sphingomyelinase, which can then
modify calcium regulatory sites in the contractile proteins as well as
in the sarcolemma, sarcoplasmic reticulum, and mitochondria.
Contractile protein sensitivity to calcium may be directly affected by
phosphorylation of sites that regulate calcium binding
(eg, troponin I) by production of cGMP, activation of protein
kinase C, and so forth. Metabolic factors such as ATP,
pHi, Mg2+,
Pi, and the free energy of ATP hydrolysis are
also known to alter contractile protein calcium sensitivity. Results
from a previous study suggest that LPS increased
Pi 2-fold and that this increase could account
for much of the contractile dysfunction.6 15 16
We speculate that the primary effect of vesnarinone is through
alterations of one or more of the aforementioned signaling pathways.
Additional pharmacological effects of vesnarinone include sodium
channel opening, decreased inward and outward potassium currents, and
prolonged action potential duration. It has been shown to have various
"immunomodulatory" effects that include inhibition of
cytokine production and viral replication. We have
previously demonstrated that vesnarinone and amrinone prevented or
reduced many features of the acute endotoxemic response, such as
LPS-induced death, fever, acidosis, cardiac dysfunction, and elevated
plasma cytokine concentrations.7 More recently, we
found that these compounds prevented initiation of inflammatory
signaling by preventing activation of NF
B, a transcription factor
that leads to expression of a wide array of cytokine and other
stress genes.17 The mechanisms of these effects are
uncertain but may in part be due to increased cAMP production,
leading to altered gene expression and to inhibition of
"stress-induced" activation of specific membrane receptors and the
signaling pathways linked to them, including those leading to the
production of phosphatidic acid and
ceramide.7 18 19 20 21 22 An ability to specifically inhibit
stress or cellular activation signals may be a potential advantage
compared with other agents with anti-inflammatory properties such as
corticosteroids.
We conclude that relatively brief exposure (2 hours) to LPS in vivo caused contractile dysfunction, abnormal calcium cycling, and oxygen wastage. Similar abnormalities of calcium handling and contractile protein calcium sensitivity have been found in failing hearts and after ischemia-reperfusion.15 23 24 25 The ability to completely restore contractile function and mechanical efficiency, improve abnormal calcium cycling, and inhibit inflammatory signaling suggests that agents such as vesnarinone may have a role in the treatment of myocardial dysfunction in settings such as cardiopulmonary bypass, sepsis, transplant rejection, and myocarditis.
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Acknowledgments |
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This work was supported in part by National Institutes of Health grants HL-52589 (to Dr McGowan) and HL-42607 (to Dr del Nido).
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