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AJP-Heart Articles in PresS. Published on April 18, 20 as DOI 10.152/ajpheart.0017.20
Final Accepted Version
H-00107-2002.R1
Contractility and ischemic response of hearts from transgenic
mice with altered sarcolemmal KATP channels.
R. Rajashree1, J.C. Koster2, K.P. Markova2, C.G. Nichols2, and P.A. Hofmann1
Running title: Contractile function in KATP transgenic hearts.
From the 1Department of Physiology, University of Tennessee School of Medicine, 894
Union Avenue, Memphis, Tennessee 38163 and the 2Department of Cell Biology and
Physiology, Washington University School of Medicine, 660 South Euclid Avenue, St.
Louis, Missouri 63110
Address all correspondence and reprint requests to:
Polly Hofmann, Ph.D.
Department of Physiology,
University of Tennessee School of Medicine,
894 Union Avenue, Memphis, Tennessee 38163
Ph: (901) 448-7348
FAX: (901) 448-7126,
e-mail: [email protected]
Copyright 20 by the American Physiological Society.
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Abstract
The functional significance of KATP channels is controversial. In the present study
transgenic mice expressing a mutant Kir6.2, with reduced ATP sensitivity, were used to
examine the role of sarcolemmal KATP in normal cardiac function, and after an ischemic
or metabolic challenge. We found left ventricular developed pressure (LVDP) is 15-20%
higher in hearts from transgenics in the absence of cardiac hypertrophy. β-adrenergic
stimulation causes a positive inotropic response from non-transgenics that is not observed
in transgenic hearts. Decreasing extracellular Ca2+ decreases LVDP in hearts from nontransgenics, but not in transgenics. These data suggest an increase in intracellular [Ca2+]
in transgenic hearts. Additional studies demonstrated hearts from non-transgenic and
transgenics have a similar post-ischemic LVDP. However, ischemic preconditioning
does not improve post-ischemic recovery in transgenics. Transgenic hearts also
demonstrate a poor recovery following metabolic inhibition. These data are consistent
with the hypothesis that sarcolemmal KATP channels are required for development of
normal myocardial function, and perturbations of KATP channels leads to hearts which
respond poorly to ischemic or metabolic challenges.
Keywords: KATP, transgenic, ischemic preconditioning, metabolic inhibition, left
ventricular developed pressure, heart rate, diastolic pressure, Kir6.2
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Introduction
ATP-sensitive K+ (KATP) channels are formed through the association of inwardly
rectifying K (Kir6.x) and sulfonylurea receptor (SURx) subunits (reviewed in 1, 21, 23).
Four Kir subunits assemble to create the pore of the KATP channel that is surrounded by
four SUR subunits. Tissue and membrane specific isoform combinations of Kir-SUR
contribute, in part, to the functional diversity of KATP channels. Myocardial sarcolemmal
KATP channels are thought to be composed of Kir6.2-SUR2A (3, 10) while mitochondrial
KATP channels may be a complex of Kir6.1-SUR1 (16, 24).
Native KATP channels are closed at physiologic concentrations of ATP, but open
as ATP decreases (17). ATP responsiveness may be significant in allowing the coupling
of metabolic state to membrane potential and hence myocardial excitability. Supporting
the theory that KATP channels are typically inactive at physiologic ATP concentrations,
basal myocardial contractility is unaffected by knockout of the murine Kir6.2 (25).
However, acute opening of sarcolemmal KATP channels due to metabolic inhibition
shortens action potential duration, and decreases left ventricular developed pressure
(LVDP; 25). To gain further insight into the effect(s) of activation of KATP channels on
cardiac function, we have generated transgenic mice expressing mutant sarcolemmal KATP
channels that have greatly reduced ATP sensitivity and are open under physiological
conditions. The first goal of the present study was therefore to characterize the
contractile function of hearts from these transgenic animals.
Improved myocardial post-ischemic recovery brought about by pre-ischemic
conditioning with brief, transient ischemia (ischemic preconditioning) may involve
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opening of KATP channels (reviewed in 4, 9, 18, 20). Gross and Auchampach were
the first to show that KATP channel blockers inhibit the protective effects of ischemic
preconditioning, and pre-ischemic treatment with KATP channel openers mimics the
cardioprotection afforded by ischemic preconditioning (8). It was initially hypothesized
that during ischemic preconditioning the resultant fall in ATP would allow sarcolemmal
KATP channels to open, reduce action potential duration, decrease Ca2+ influx, and protect
the heart from subsequent ischemic damage due to Ca2+ overload. However, it was
subsequently determined that a decrease in action potential duration may not be necessary
to observe the reduction in post-ischemic infarct size associated with ischemic
preconditioning (27). More recent studies using KATP inhibitors and activators
purportedly specific for mitochondrial KATP channels implicate mitochondrial rather than
sarcolemmal KATP channels as responsible for the cardioprotective effects of ischemic
preconditioning (4, 9, 18, 20). However, the interpretation of these studies may not be
straightforward since the specificity of KATP openers/blockers are condition specific. For
example, diazoxide a KATP opener considered specific for mitochondrial KATP channels
has no effect on sarcolemmal KATP channels in the absence of MgADP, but activates
sarcolemmal KATP at in vivo levels of MgADP (6). Thus, the second goal of the present
study was to determine if hearts from transgenic mice with sarcolemmal KATP channels
having decreased ATP sensitivity respond differently than non-transgenic hearts to (a)
global ischemia with and without ischemic preconditioning, and (b) metabolic inhibition
using cyanide and 2-deoxyglucose pretreatment.
For these studies we examined the contractility of hearts expressing mutant
Kir6.2 (12). In excised patch clamp experiments, sarcolemmal KATP channels
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from transgenic mice were nearly 100-fold less sensitive to inhibition by ATP than
non-transgenic controls (12). Somewhat counterintuitively, the maximal KATP current
density was also decreased 4-fold in myocytes from transgenic mice (12). Therefore,
when [ATP]i falls (e.g. during the onset of ischemia), KATP channels in transgenic hearts
will be expected to open earlier than in non-transgenic controls. Similarly, transgenic
KATP channels will stay open longer as [ATP]i rises (e.g during the onset of reperfusion),
even though the maximal KATP conductance is lower than in control. Thus the present
study will provide insight into the role the sarcolemmal KATP plays in normal and
pathological myocardial function using transgenic mice that have a greatly decreased
ATP sensitivity and a reduced number of functioning KATP channels at the sarcolemma.
Materials and Methods
Transgenic Mice. Previously we have demonstrated COSm6 cells co-transfected with
SUR2A and Kir6.2 containing an N-terminal truncation of 30 amino acids (∆N) in
combination with a point mutation, K185Q, has a pronounced ATP-insensitivity of their
KATP channels (13). Thus, transgenic constructs were made using the cardiac specific αmyosin heavy chain promoter with Kir6.2[∆N,K185Q] and a green fluorescent protein
tag on the C-terminus (Kir6.2[∆N,K185Q]-GFP; 12). Transgenic mice were identified
by PCR on mouse-tail DNA using GFP specific oligonucleotide primers. Four founder
mice expressing the Kir6.2[∆N,K185Q]-GFP transgene were isolated, and bred to
isogenicity. Myocytes from hearts of line 4 mice had the highest levels of fluorescence
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with all myocytes fluorescing in a punctate, cross-striated pattern (12). Hearts
from line 4 transgenic mice (TG) were examined in the present study.
Langendorff Perfused Heart Preparations and Assessment of Ventricular Function.
Hearts were removed from male or female mice anaesthetized with methoxyflurane
inhalation or sodium pentobarbital. Sodium pentobarbital was used after methoxyflurane
became unavailable in the United States. No difference in ventricular pressures were
observed between hearts isolated with the two anesthetics. The aorta was cannulated
with a 20G stainless steel blunt needle filled with a modified Krebs-Henseleit buffer with
the heart completely immersed in ice cold buffer. Krebs-Henseleit solution contained 4.7
mM KCl, 118 mM NaCl, 1.2 mM MgSO4, 1.75 mM CaCl2, 17 mM NaHCO3, 11 mM
glucose, 1.2 mM KH2PO4, 0.05 mM EDTA, 2 mM lactic acid, pH 7.4. Following
cannulation the heart was retrograde perfused with Krebs-Henseleit solution at a constant
pressure of 65 mm Hg without recirculation. The Krebs-Henseleit solution was
continually gassed with 95% O2 - 5% CO2 at 37°C. The heart was immersed in a 37°C
organ bath filled with Krebs-Henseleit solution and, where indicated, paced at 6 Hz.
External pacing was discontinued during prolonged ischemia and recommenced after 3-5
minutes of reperfusion.
A left atriotomy was performed and an open-ended beveled polyethylene cannula
with outer diameter of 1.27 mm (PE90) was passed into the left ventricle. This cannula
was rigid and maintained the left ventricle at a constant volume throughout the
experiment. PE90 tubing was selected since the specific outer diameter fixes ventricular
volume such that maximum LVDP is obtained in mouse hearts. LVDP was better
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maintained over a 2-3 hour period (typical duration of experimental protocol) when
ventricular volume was fixed by PE90 tubing as compared to a balloon inflated to a
similar diameter in the ventricle. The fluid filled tubing was connected to a pressure
transducer (BLPR, World Precision Instruments, Sarasota, FL). Fluid was continually
retained in the tubing since the tube with transducer forms a vacuum. Pressures from the
transducer were digitized using an AD/DA conversion board (NB-MIO-16XL-18 µsec,
National Instruments, Austin, TX) and stored in a Macintosh computer. Acquisition and
data analysis was carried out using computer programs generated in LABVIEW software
(National Instruments, Austin, TX).
LVDP was calculated as the difference between peak systolic pressure and end
diastolic pressure (EDP). Averages of LVDP and EDP from the final 3-5 minutes under
a given experimental conditioning are reported. Post-ischemic increases in end diastolic
pressure (EDP) were calculated as the difference between post-ischemic EDP and preischemic EDP.
Experimental Protocols.
Dose-response effects of isoproterenol (Iso) and carbachol
were obtained in the same set of hearts. Hearts were instrumented but not paced, and
baseline contractility was obtained over 30 minutes. Hearts were then exposed to KrebsHenseleit containing 10 nM Iso for 5 minutes, washed with Krebs-Henseleit solution
without Iso for 25 minutes, and exposed to Krebs-Henseleit containing 100 nM Iso for 5
minutes. Average heart rate and peak increase in LVDP at each concentration of Iso was
determined. Immediately following the Iso dose-response determination, carbachol was
added to the 100 nM Iso solution. Carbachol concentration was sequentially increased
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every 5 minutes without washing in the maintained presence of 100 nM Iso. Heart
rate was reported as the average heart rate over the entire period of carbachol exposure at
a given concentration. Following exposure to 500 nM carbachol (highest dose) hearts
were exposed to a Krebs-Henseleit solution containing 100 nM Iso without carbachol for
10 minutes.
For ischemia - reperfusion protocols, hearts were instrumented and paced at 6 Hz
for a 20 minute equilibration period. Following the equilibration period either no change
for an additional 40 minutes (no preconditioning), or 4 cycles of 5 minutes of ischemia 5 minutes of reperfusion were carried out (ischemic preconditioning). This was followed
by a prolonged ischemia of 22 minutes, and reperfusion for 60 minutes.
Effects of decreasing extracellular [Ca2+] and metabolic inhibition were obtained
in the same set of hearts. Hearts were instrumented, allowed to recover for 15 minutes,
paced, and sequentially exposed to Krebs-Henseleit solution containing 1.75 mM Ca2+,
1.50 mM Ca2+, and 1.25 mM Ca2+. Data was averaged and reported for the final 3
minutes of a 10 minute exposure time / Ca2+ concentration. Hearts were then returned to
a Krebs-Henseleit solution containing 1.75 mM Ca2+ for 10 minutes to re-establish
baseline pressure values. Subsequently, glycolysis was reduced by perfusion with a
modified Krebs-Henseleit solution containing 1 mM 2-deoxyglucose (2-DOG) without
glucose or lactate for 5 minutes (2). Oxidative phosphorylation was blocked by
perfusion with a modified Krebs-Henseleit solution containing 2 mM NaCN without
glucose, lactate or 2-DOG for 10 minutes (2). Hearts were then perfused for 60 minutes
with a Krebs-Henseleit solution with glucose and lactate but without 2-DOG or CN.
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Statistics. Analysis of variance (ANOVA) and either a Fisher's LSD post hoc test
or Students t-test were applied. A p < 0.05 was considered statistically significant.
Results
Baseline Characterization of Contractility. Heart-to-body weight ratios did not change
in the KATP TG mice as compare to gender matched non-TG littermates (Table 1).
Systolic pressure (Table 1) and LVDP (Figure 1) were significantly higher in TG hearts
than in paired non-TG hearts both in the presence and absence of external pacing.
Intrinsic heart rates of the excised, Langendorff perfused hearts were not different
between hearts from KATP TG mice as compare to gender matched non-TG littermates,
383 ± 35 versus 380 ± 23 beats / min respectively (mean ± SEM, n = 5).
Calcium dependence on contractility was determined in hearts from TG and
paired non-TG mice (Figure 2). Decreasing extracellular [Ca2+] from 1.75 to 1.50 to 1.25
mM significantly decreased LVDP in hearts from non-TG mice while there was no
systematic decrease in LVDP in TG hearts.
Inotropic Reserve in Control and KATP Transgenic Hearts. The inotropic effects of
β−adrenergic and muscarinic receptor activation were determined in hearts from TG and
paired non-TG mice. The β−adrenergic receptor agonist Iso increased heart rate to a
similar extent in TG and non-TG hearts (Figure 3). Concomitantly, an incremental,
positive inotropic effect was observed in hearts of non-TG mice, while in TG hearts
LVDP did not change upon exposure to 10 nM Iso (Figure 3). Increasing the Iso
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concentration to 100 nM lead to a similar increase in LVDP, 237 ± 38% for NonTG and 183 ± 42% for TG (mean ± SEM, n=5), and heart rate. It should be noted that
Control hearts from TG mice demonstrated a trend towards higher LVDP as compared to
Control Non-TG mice, but this did not reach statistical significance (p of 0.20 for 5 hearts
/ group). Statistical analysis using data from all Control hearts in this study (Figure 1)
indicates LVDP is significantly higher in hearts of TG mice.
The negative chronotropic effect of muscarinic action was determined in the
presence of 100 nM Iso. No statistically significant difference exists in the carbachol
concentration - heart rate relationship between hearts from TG and non-TG mice (Figure
4). Following application of the highest concentration of carbachol, hearts were washed
free of carbachol and the effect of 100 nM Iso alone was redetermined to identify the
extent of run down and receptor desensitization in the preparations. Similar heart rates
were observed in hearts stimulated with isoproterenol prior to and following the
carbachol dose-response measurements (Figure 4).
Functional response to global ischemia and reperfusion in the presence and absence of
ischemic preconditioning. Figure 5 presents the post-ischemic recovery of contractile
function of hearts which underwent 22 minutes of ischemia followed by 60 minutes of
reperfusion with and without ischemic preconditioning. Similar post-ischemic recovery
of LVDP and increase in EDP was obtained in non-preconditioned hearts from both TG
and non-TG mice. Post-ischemic recovery of LVDP and EDP improved in ischemic
preconditioned hearts from non-TG mice, but not in hearts from TG mice (Figure 5).
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Functional response to metabolic inhibition. Figure 6 presents the results from
representative experiments to examine the response of metabolic inhibition in hearts of
gender matched TG and non-TG. Both hearts underwent metabolic inhibition by
replacement of glucose with 2-deoxyglucose, and exposure to NaCN (see Methods).
Recovery of the non-TG heart from metabolic inhibition was significantly better as
determined by a higher final LVDP (Figure 6A) and lower EDP (Figure 6B). During
metabolic inhibition, the decrease in LVDP was delayed but the development of rigor
was accelerated in TG hearts (Figures 6). Cumulative results of hearts which underwent
metabolic inhibition (Figure 7) are consistent with this individual paired observation in
that an earlier onset of rigor contracture and a lower recovery of LVDP was observed in
TG hearts, yet spontaneous beating ceased at a significantly later time in hearts from TG
mice (Figure 7).
Discussion
KATP transgenic hearts have increased myocardial developed pressure, and are
insensitive to changes in extracellular [Ca2+] and submaximum β−adrenergic
stimulation. Knockout of Kir6.2, the pore forming subunit of the KATP channel, abolishes
KATP channel activity in mouse ventricle and the effects of KATP-channel openers on
action potential duration (25). Expression of the Kir6.2[DN,K185Q] in mouse heart
leads to profound reduction of ATP sensitivity, yet decreased KATP channel density (12).
Counter to predictions from previous studies, the reduction of ATP sensitivity does not
lead to marked action potential shortening or excitation failure under normal conditions,
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and heart rate is reduced by about 15% in conscious animals (12). Why the
phenotype is so mild, and what are the broader consequences of altered KATP channel
activity on cardiac function remain open questions. The present study begins to address
these questions by examination of contractile function in Kir6.2[DN,K185Q] TG hearts.
Left ventricular developed pressure was higher in TG hearts as compared to hearts from
paired non-TG animals. This increase was not due to hypertrophy. In addition, there was
no decrease in LVDP of transgenic hearts when extracellular Ca2+ was decreased from
1.75 to 1.25 mM, and β−adrenergic responsiveness decreased in transgenic hearts. This
decrease in sensitivity of LVDP to extracellular Ca2+ could be explained by either (i) an
increase in myofilament Ca2+ sensitivity of force production, or (ii) altered Ca2+ handling.
Loss of β−adrenergic responsiveness in other transgenic mouse models has been
correlated with an increased intracellular Ca2+ and resultant Ca2+-induced inhibition of
adenylate cyclase (22). Thus our data is most consistent with the hypothesis that there is
an increase in intracellular [Ca2+] in transgenic hearts that accounts for the increase in
LVDP, maintained contractility at lower [Ca2+], and reduced inotropic reserves as
assessed by 10 nM isoproterenol stimulation. Future cellular studies will definitively
establish if intracellular Ca2+ and/or myofilament Ca2+ sensitivity are increased in
transgenic hearts with a high expression of Kir6.2[∆N,K185Q]-GFP.
At least two possibilities exist as to how transgene overexpression can lead to the
apparent elevation in contractile state in transgenic hearts under control conditions. First,
the altered KATP channel activity in the sarcolemma may lead to compensatory increases
in Ca2+ current, or decreases in voltage-gated K current, either of which might explain the
variable prolongation of action potential duration seen in isolated transgenic myocytes
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(12). Second, KATP channels may be present in sarcoplasmic reticular membranes,
and affect trans-sarcoplasmic reticular membrane Ca2+ distributions. At the present time,
these possibilities are speculation, but may be resolved by future studies at the cellular
level.
Transgenic hearts and muscarinic activation. Muscarinic modulation of heart rate is an
important physiologic index of heart function. The negative chronotropic effect of
muscarinics is due to activation of muscarinic K channels (KAch) in the sinoatrial node
and atrium. The channel is activated by G protein-coupled receptors and is thought to be
a heterotetrameric complex with equal numbers of Kir3.1and Kir3.4 subunits (5). In the
present study hearts from transgenic and non-transgenic mice responded in a similar
fashion to increasing concentrations of a muscarinic agonist. This suggests G proteincoupled KAch channels are not altered in hearts of transgenic mice with
Kir6.2[∆N,K185Q]-GFP. In addition it is consistent with the idea that mutagenesis, in
and of itself, does not lead to non-specific changes in cardiac ion channels.
KATP transgenic hearts have a differential recovery from ischemia and metabolic
inhibition. Myocardial functional recovery following 22 minutes of global ischemia was
similar in hearts from paired TG and non-TG mice. The rapid contractile failure
observed in ischemia results from decreasing pH, accumulation of phosphate, and
subsequent inhibition of the myofilaments (7). Consistent with ischemia-induced cardiac
failure being independent of KATP channels, recovery from ischemia was similar in KATP
TG and non-TG hearts. By contrast metabolic inhibition leads to a delayed contractile
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failure (cessation of beating occurred at 600 rather than 400 seconds), but an
earlier onset of rigor contracture and a very poor functional recovery in TG as compared
to non-TG hearts. The contractile failure normally observed in metabolic inhibition is
likely due to KATP activation and subsequent action potential shortening (7, 14). We have
previously demonstrated that at greater than 200 seconds into metabolic inhibition the
current density in KATP TG myocytes is 4 fold less than in non-TG myocytes (12). Thus,
in KATP TG hearts a lower absolute level of KATP current during metabolic inhibition in
combination with a higher contractile state may delay the cessation of contraction. This
continued contraction during metabolic inhibition and the higher baseline inotropic state
would result in increased ATP consumption that would cause an earlier onset of rigor and
concomitant worsening of contractile recovery following removal of metabolic blockers.
Hence, any protective effects brought about by an intial, more rapid activation of the KATP
in TG hearts is counteracted.
Acute and/or chronic changes in sarcolemmal KATP impedes myocardial functional
recovery from ischemic preconditioning. In hearts from TG mice ischemic
preconditioning failed to improve post-ischemic myocardial functional recovery. This
result seems contradictory to the observations that implicate the mitrochondrial KATP
channel as the primary transducer of cardioprotection afforded by preconditioning (4, 9,
18, 20), and is consistent with studies indicating a role for the sarcolemmal KATP
channels in cardioprotection (11, 15, 19). However, it should be noted there may be
indirect consequences of the KATP transgene expression on preconditioning. First, chronic
reduction in KATP channel density may allow for an increase in cardiac electrical
abnormalities during development and aging, and subsequent damage to the heart. Our
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observations of an increase in baseline LVDP of the transgenic hearts and a similar
post-ischemic recovery in LVDP in transgenics and non-transgenics that were not
preconditioned suggest the transgenic hearts are not failing in general. Second,
experimental conditions such as duration and timing of preconditioning, heart rate and
inotropic state of the heart can have a significant impact on the benefits brought about by
ischemic preconditioning. Since the inotropic state appears to be affected by the
transgene, it is conceivable that the necessary conditions for effective preconditioning
may be altered in the transgenic hearts. Nevertheless, the recent demonstration that
preconditioning is also abolished in hearts from Kir6.2 knockout animals (26) indicates
further consideration of the role of sarcolemmal KATP in this phenomena is warranted.
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Acknowledgements
This study was supported by National Institute of Health grants HL-48839 (PAH) and
HL45742 (CGN), and an American Heart Association Established Investigatorship
(PAH).
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Figure Legends
Figure 1. Cumulative left ventricular developed pressure under baseline conditions in
hearts from KATP TG mice, and hearts from gender matched non-TG littermates. Hearts
were either unpaced or paced at 6 Hz. Values are expressed as the mean ± standard error.
*p < 0.05 as compared to hearts from paired non-TG mice.
Figure 2. Cumulative left ventricular developed pressure (LVDP) as a function of
extracellular Ca2+ in hearts from KATP TG and hearts from paired non-TG mice. LVDP
was normalized to the LVDP observed when extracellular Ca2+ was 1.75 mM for each
heart. Data from 8 hearts was averaged / group and the values expressed as mean ±
standard error. *p < 0.05 as compared to LVDP at an extracellular Ca2+ of 1.75 mM.
Figure 3. Effect of 10 nM isoproterenol (Iso) on heart rate and peak increase in left
ventricular developed pressure (LVDP) in hearts from KATP TG and paired non-TG mice.
Data from 5 hearts was averaged / group and the values expressed as mean ± standard
error. *p < 0.05 as compared to Control hearts from the same group.
Figure 4. Cumulative heart rate as a function of increasing concentrations of carbachol in
the presence of 100 nM isoproterenol in hearts from KATP TG and hearts from paired nonTG mice. Following the highest dose of carbachol, heart rate was re-determined for 100
nM isoproterenol alone (0 nM Carbachol). Data from 5 hearts was averaged / group and
the values expressed as mean ± standard error.
Final Accepted Version
H-00107-2002-R1
22.
Figure 5. Cumulative post-ischemic recovery of left ventricular developed pressures
(LVDP; A) and increase in end diastolic pressure (B) in Non-TG and TG hearts that
underwent 22 minutes of global ischemia followed by 60 minutes of reperfusion in the
presence and absence of ischemic preconditioning. Values are expressed as the mean ±
standard error. *p < 0.05 as compared to hearts from the same group that did not undergo
ischemic preconditioning.
Figure 6. Representative examples of left ventricular developed pressure (A) and end
diastolic pressure (B) in hearts that underwent metabolic inhibition. Metabolic inhibition
consisted of a 5 minute exposure to a Krebs-Henseleit solution containing 1 mM 2deoxyglucose (2-DOG) in the absence of glucose, followed by a 10 minute exposure to 2
mM NaCN (CN) in the absence of 2-DOG and glucose, and a final perfusion with a
normal Krebs-Henseleit solution. Hearts were from a gender and littermate pair of mice
expressing the KATP transgene, and a non-transgenic.
Figure 7. Cumulative data of time of cessation of beating and onset of rigor contracture
(A) in metabolically inhibited hearts, and post-metabolic inhibition (Post-Met Inhib)
recovery of left ventricular developed pressure (LVDP; B). Hearts are from KATP TG
mice, and littermates that were non-TG. Time of onset for rigor and cessation of beating
was measured from the start of 2-deoxyglucose perfusion. Rigor contracture was defined
as the point at which end diastolic pressure increased by 20 mm Hg over baseline. LVDP
is expressed as relative to pre-metabolic inhibition LVDP. Data from 6 hearts was
Final Accepted Version
H-00107-2002-R1
23.
averaged / group and the values expressed as mean ± standard error. *p < 0.05 as
compared to non-TG hearts.
Final Accepted Version
H-00107-2002-R1
24.
Table I. Characterization of Hearts
Non-Transgenic
Transgenic
Heart Weight (mg)
122.1 ± 3.2
120.7 ± 3.3
Body Weight (g)
25.9 ± 0.9
26.9 ± 0.8
Heart / Body Weight (mg/g)
4.8 ± 0.1
4.5 ± 0.1
Diastolic Pressure (mm Hg)
10.4 ± 0.9
9.8 ± 2.2
Systolic Pressure (mm Hg)
65.9 ± 3.3
74.8 ± 3.6*
27
28
Number of Hearts
Values are mean ± SE. *p < 0.05 as compared to paired non-transgenic hearts.
Final Accepted Version
H-00107-2002-R1
25.
Figure 1.
Non-Transgenic
Left Ventricular Developed
Pressure (mm Hg)
Transgenic
*
70
60
*
50
40
30
20
n=28
n=27
n=22
n=24
10
0
Paced - 6 Hertz
Unpaced
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H-00107-2002-R1
26.
Left Ventricular Developed Pressure
(% Relative to 1.75 mM Ca 2+ )
Figure 2.
120
Non-Transgenic
110
Transgenic
100
90
80
*
70
60
50
1.20
1.30
1.40
1.50
[Ca2+] (mM)
1.60
1.70
1.80
Final Accepted Version
H-00107-2002-R1
27.
Figure 3.
Peak LVDP (mm Hg)
Control
10 nM Iso
*
100
80
60
497 ± 38
beats/min
40
20
379 ± 23
beats/min
491 ± 34
beats/min
383 ± 36
beats/min
0
Non-Transgenic
Transgenic
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H-00107-2002-R1
28.
Figure 4.
Non-Transgenic
Transgenic
500
450
400
350
300
250
200
150
≈
Heart Rate (beats / min)
550
0
100
200
300
Carbachol (nM)
400
500
0
post
control
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H-00107-2002-R1
29.
Figure 5.
Post-Ischemic LVDP
(% Relative to Pre-Ischemic)
A.
80
No Preconditioning
*
70
Ischemic Preconditioning
60
50
40
30
20
10
n=18
n=9
n=15
n=10
0
Non-Transgenic
Transgenic, Line 4
Post-Ischemic Increase in
End Diastolic Pressure (mm Hg)
B.
60
50
40
30
20
*
10
0
Non-Transgenic
Transgenic
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H-00107-2002-R1
30.
Figure 6.
2-DOG
CN
Left Ventricular Developed
Pressure (% of Pretreatment)
A.
120
100
80
60
Non-Transgenic
40
20
Transgenic
0
0
500
1000
1500
2000
2500
3000
B.
80
Transgenic
End Diastolic
Pressure (mm Hg)
70
60
50
40
Non-Transgenic
30
20
10
0
0
500
1000
1500
Time (sec)
2000
2500
3000
Final Accepted Version
H-00107-2002-R1
31.
Figure 7.
A.
Time of Onset (sec)
800
700
600
*
500
*
400
300
200
100
0
Non-TG
TG
Cessation of Beating
Non-TG
TG
Rigor Contracture
Post-Met Inhib LVDP (%)
B.
70
60
50
40
30
20
*
10
0
Non-TG
TG