Contractility and ischemic response of hearts from transgenic mice with altered sarcolemmal KATP...

Final Accepted VersionH-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 2002 by the American Physiological Society.

AJP-Heart Articles in PresS. Published on April 18, 2002 as DOI 10.1152/ajpheart.00107.2002

Final Accepted VersionH-00107-2002-R1

2.

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 non-

transgenics, 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


3.

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


4.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


5.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 α-

myosin 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


6.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


7.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 pre-

ischemic 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 Krebs-

Henseleit 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


8.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.


9.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


10.concentration to 100 nM lead to a similar increase in LVDP, 237 ± 38% for Non-

TG 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).


11.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,


12.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


13.(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 protein-

coupled 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


14.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


15.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.


16.

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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21.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 non-

TG 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.


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 2-

deoxyglucose (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


23.averaged / group and the values expressed as mean ± standard error. *p < 0.05 as

compared to non-TG hearts.


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*

Number of Hearts 27 28

Values are mean ± SE. *p < 0.05 as compared to paired non-transgenic hearts.


25.Figure 1.

0

10

20

30

40

50

60

70

Paced - 6 Hertz Unpaced

Non-Transgenic

Transgenic

Left

Ven

tric

ular

Dev

elop

edP

ress

ure

(mm

Hg)

*

*

n=27n=28

n=24n=22


26.Figure 2.

50

60

70

80

90

100

110

120

1.20 1.30 1.40 1.50 1.60 1.70 1.80

Non-Transgenic

Transgenic

Left

Ven

tric

ular

Dev

elop

ed P

ress

ure

(% R

elat

ive

to 1

.75

mM

Ca

2+)

[Ca2+] (mM)

*


27.Figure 3.

0

20

40

60

80

100


Control10 nM Iso

Pea

k LV

DP

(m

m H

g)

*

379 ± 23beats/min

383 ± 36beats/min

497 ± 38beats/min

491 ± 34beats/min


28.Figure 4.

150

200

250

300

350

400

450

500

550

0 100 200 300 400 500 0

Non-TransgenicTransgenic

Hea

rt R

ate

(bea

ts /

min

)

Carbachol (nM)

≈

postcontrol


29.Figure 5.

0

10

20

30

40

50

60

70

80

Non-Transgenic Transgenic, Line 4

No Preconditioning

Ischemic Preconditioning

Pos

t-Is

chem

ic L

VD

P(%

Rel

ativ

e to

Pre

-Isc

hem

ic)

*

n=15n=18

n=9 n=10

A.

0

10

20

30

40

50

60


Pos

t-Is

chem

ic I

ncre

ase

in

End

Dia

stol

ic P

ress

ure

(mm

Hg)

*

B.


30.Figure 6.

0

20

40

60

80

100

120

0 500 1000 1500 2000 2500 3000

Left

Ven

tric

ular

Dev

elop

ed

Pre

ssur

e (%

of

Pre

trea

tmen

t)

A.

2-DOG CN

Non-Transgenic

Transgenic

0

10

20

30

40

50

60

70

80

0 500 1000 1500 2000 2500 3000

End

Dia

stol

icP

ress

ure

(mm

Hg)

Time (sec)

B.

Non-Transgenic

Transgenic


31.Figure 7.

0

100

200

300

400

500

600

700

800

Cessation of Beating Rigor Contracture

Tim

e of

Ons

et (

sec)

*

*

A.

Non-TG TG Non-TG TG0

10

20

30

40

50

60

70

Pos

t-M

et In

hib

LVD

P (

%)

*

B.

Non-TG TG

Contractility and ischemic response of hearts from transgenic mice with altered sarcolemmal KATP...

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Transcript of Contractility and ischemic response of hearts from transgenic mice with altered sarcolemmal KATP...