Download Role of Glycolytic Products in Damage to Ischemic Myocardium

816
Role of Glycolytic Products in Damage to Ischemic
Myocardium
Dissociation of Adenosine Triphosphate Levels and Recovery of
Function of Reperfused Ischemic Hearts
James R. Neely and Lee W. Grotyohann
From the Department of Physiology, The Milton S. Hershey Medical Center, The Pennsylvania State University, Hershey, Pennsylvania
Downloaded from http://circres.ahajournals.org/ by guest on June 16, 2017
SUMMARY. The mechanism of irreversible damage to ischemic myocardium was investigated in
the perfused rat heart. The time of transition from reversible to irreversible damage to contractile
function was accelerated by accumulation of glycolytic products and increases in extracellular
calcium. Both of these effects were largely independent of adenine nucleotide levels in the tissue.
With zero coronary flow and 1.25 nut calcium the decrease in ability of the heart to recover
ventricular function with reperfusion after 30 minutes of ischemia was directly correlated with
accumulation of glycolytic products (as estimated by tissue lactate) during ischemia. The extent
of lactate accumulation during ischemia was varied by preperfusing the hearts for 0, 10, or 15
minutes under anoxic, high coronary flow conditions to deplete tissue glycogen prior to ischemia,
and by adding lactate back to the perfusate of these hearts during the ischemic period. Recovery
of ventricular function was inversely related to tissue lactate during ischemia and varied from 28
to 92%, even though there was little or no change in tissue levels of residual adenosine
triphosphate. Increasing extracellular calcium accelerated the time of onset of irreversible damage
with little or no change in residual adenosine triphosphate levels. At any given calcium concentration, the time-dependent declines in the ability of the heart to recover ventricular function was
also largely independent of adenosine triphosphate levels. These studies suggest a major role of
anaerobic glycolytic products (lactate, hydrogen ion, or NADH) in ischemic damage to the heart
that is unrelated to loss of tissue adenine nucleotides. With zero or low flow ischemia, this effect
may result in irreversible damage to the myocardium before adenine nucleotides are reduced to
critically low levels. (Circ Res 55: 816-824, 1984)
THE ischemic myocardium progresses from a reversible to an irreversible state of damage within
several minutes to one or more hours, depending on
the severity and conditions of the ischemic insult.
The mechanisms responsible for this transition are
not known with certainty. It is widely accepted that
loss of adenine nucleotides and failure to restore
adenosine triphosphate (ATP) levels with reperfusion is a critical factor in the onset of irreversibility
(Kiibler and Spieckermann, 1970; Gudbjarnason et
al., 1970; Vary et al., 1970; Jennings et al., 1977;
Reibel and Rovetto, 1978; Watts et al., 1980). However, many other factors have also been implicated,
such as overloading the cells with Ca++ (Shen and
Jennings, 1972; Nayler, 1981), accumulation of metabolic products (Neely et al., 1973; Katz and Messineo, 1981; Neely and Feuvray, 1981) and structural
damage to membranes caused secondarily either by
low ATP and cell swelling (Jennings et al., 1977;
Jennings and Reimer, 1981) or accumulation of metabolic products with subsequent activation or inhibition of key enzymes (Katz and Messineo, 1981;
Neely and Feuvray, 1981).
Although both loss of adenine nucleotides and
accumulation of metabolic products may directly
and indirectly result in cellular damage, the relative
importance of and rate at which damage occurs from
these two processes is not known. The present study
provides data which disassociates irreversible damage from residual adenine nucleotide levels. Data
are presented which implicates glycogenolysis and
high tissue lactate (or associated metabolite changes)
as important contributing factors to the damage
process. Tissue levels of lactate during ischemia were
altered by preperfusing the hearts under anoxic
conditions to deplete tissue glycogen prior to ischemia and by adding lactate to the perfusate of
glycogen-depleted hearts during exposure to ischemia. Ischemic coronary flows of zero or 1 ml/
min were also used to study the effect of washout
of metabolic products. Recovery of ventricular function with reperfusion was negatively correlated with
tissue levels of lactate during ischemia. Functional
recovery was poorly correlated with residual levels
of ATP during reperfusion.
Methods
Isolated perfused hearts from 250- to 350-g male, Sprague-Dawley rats, were used. Hearts were perfused by the
Neely and Grotyohann/Protection of Ischemic Myocardium
Downloaded from http://circres.ahajournals.org/ by guest on June 16, 2017
Langendorff procedure as described earlier (Neely et al.,
1967), except that ventricular pressure was monitored by
placing a plastic catheter with a small perforated ball tip
into the left ventricle via the mitral valve. The catheter
was filled with perfusate and connected to a Statham
pressure transducer. The perfusate was Krebs-Henseleit
bicarbonate buffer containing 11 mM glucose and the free
Ca++ concentration shown in the tables and figures. The
hearts received a 10-minute washout perfusion with oxygenated buffer at 60 mm Hg aortic perfusion pressure.
The hearts then were switched to a perfusion with buffer
gassed with a 95:5 mixture of either O2:CO2 or N2:CO2
for 10-15 minutes before inducing global ischemia by
cross-clamping the aortic perfusion tube and reducing
coronary flow to zero or reducing coronary flow to 1 ml/
min with a constant flow rotary pump connected to the
aortic cannula. These ischemic hearts were maintained at
37°C for various times. The hearts then were reperfused
at a constant aortic pressure of 60 mm Hg with oxygenated
buffer, and recovery of ventricular function was followed
for 30 minutes.
For tissue analysis of metabolites, hearts were frozen
with Wollenberger clamps cooled in liquid nitrogen.
Groups of hearts were frozen after the 10- or 15-minute
preperfusion just prior to inducing ischemia. Other groups
were frozen at the end of the ischemic period and after
the 30 minutes of reperfusion. Tissue levels of ATP, adenosine diphosphate (ADP), adenosine monophosphate
(AMP), creatine phosphate (CP), glycogen, and lactate
817
were determined using neutralized perchloric acid extracts
as described earlier (Neely et al., 1973) by standard enzymatic procedures (Bergmeyer, 1963). Ventricular function was assessed by measuring developed pressure (i.e.,
the difference between systolic and diastolic pressures)
and heart rate. The percent recovery of ventricular function was calculated as the product of developed pressure
and heart rate after 30 minutes of reperfusion divided by
the same product measured before starting the experimental perfusion. In some groups of hearts, various concentrations of Na+ lactate were added to the recirculating perfusate during the anoxic and ischemic perfusions.
Results
The characteristic response of tissue adenine nucleotides during zero coronary flow, whole heart
ischemia is shown in Table 1. Tissue ATP decreased
rapidly during the first 25 minutes of ischemia, but
declined only from 6 to 3.4 /imol/g dry weight
between 25 and 45 minutes. With reperfusion after
each of the ischemic periods, ATP levels increased
slightly. This restoration of ATP was greater when
reperfusion was started after short periods of ischemia, with little recovery occurring after from 35
to 80 minutes of ischemia. The small recovery of
ATP was due to rephosphorylation of ADP and
AMP, and the difference between levels of ATP in
TABLE 1
Effects of Whole Heart Ischemia on Tissue Adenine Nucleotides
Tissue metabolites
(jimol/g dry)
Time of
ischemia
(min)
Reperfused
30 min
ATP
ADP
AMP
Total
0
+
24.1 ±0.6
3.3 ± 0.4
0.3 ± 0.05
27.6 ±0.9
+
9.6 ±0.8
16.5 ±0.3
7.4 ±0.14
2.4 ± 0.08
3.3 ± 0.5
0.16 ± 0.03
20.5 ± 0.19
19.1 ±0.36
+
6.1 ±0.4
9.6 ± 0.4
4.9 ± 0.6
2.6 ± 0.2
7.3 ± 0.6
0.9 ± 0.1
18.3 ± 0.2
13.1 ±0.4
+
4.0 ± 0.4
8.2 ± 0.3
4.2 ±0.5
2.9 ± 0.08
8.1 ± 0.06
1.5 ±0.12
16.3 ± 1
12.7 ± 0.3
+
4.3 ± 0.5
6.5 ± 0.4
4.3 ± 0.3
2.9 ± 0.2
7.4 ± 0.4
2.2 ± 0.2
16.6 ±0.4
11.6 ±0.1
+
3.7 ±0.2
5.1 ±0.3
4.4 ± 0.3
3.3 ±0.16
7.6 + 0.2
2.8 ± 0.2
15.7 ± 0.3
11.2 ±0.2
+
3.4 ± 0.2
4.4 ±0.14
3.8 ±0.4
3.3 ± 0.2
7.9 ± 0.6
2.8 + 0.15
15.1 ±0.3
10.5 ±0.2
50
+
4.0 ±0.1
4.0 ±0.3
3.2 + 0.05
11.1 ±0.4
60
+
3.4 ±0.09
2.7 ±0.06
3.2 + 0.02
9.3 ± 0.13
20
25
30
35
40
45
Hearts were perfused with oxygenated buffer containing glucose (11 mM) and 2.5 mM Ca++ for 10
minutes prior to inducing ischemia. The aortic perfusion tube was then clamped and the hearts
maintained at 37°C with zero coronary flow for the ischemic times shown. A group of four to six
hearts was frozen after each of the ischemic periods. Another group of six to ten hearts were reperfused
at 60 mm Hg perfusion pressure for 30 minutes after each of the ischemic periods and then frozen.
The data are means ± SE.
Circulation Research/Vol. 55, No. 6, December 1984
818
These changes in ATP are shown graphically in
Figure 1. In addition, the changes in tissue levels of
CP during ischemia and with reperfusion are shown,
along with recovery of ventricular function. Levels
of CP declined rapidly during ischemia with little
additional loss after 20 minutes. With reperfusion,
i
_
t
i
35
I"
i
t
•D 30
-? 25
3.
a
,'^Reperfused
s
Ischemic
0
10 20 30 40 50 60
^Reperfused
20
ol
15
u
10
70
60
i 5040
O
E
30
\
|
5
0
Ischemic
0 10 20 30 40 50 60
10
15
ATP (jumollg dry)
20
10
/diastolic
0
Time of Ischemia (min)
S
To determine the effect of Ca++ concentration on
the time course of ischemic damage and the relation
between recovery of function and residual ATP
levels with reperfusion, we perfused the hearts with
various concentrations of Ca++ at a constant coro-
O)
X
ires
45 40
Ul)
Downloaded from http://circres.ahajournals.org/ by guest on June 16, 2017
CP levels returned to above normal after 20 minutes
of ischemia, but did not fully recover after longer
periods. Likewise, ventricular pressures recovered to
about normal with reperfusion after 20 minutes of
ischemia, but recovery declined progressively after
longer periods of ischemia. Decreased function was
reflected in both a large rise in diastolic and a large
decrease in systolic pressures. Recovery of developed pressure was essentially zero after periods of
35-60 minutes of ischemia. Although heart rate was
decreased by 20-30% during reperfusion (data not
shown), the major effect on function was decreased
developed pressure, as shown in Figure 1. The relation between recovery of developed ventricular
pressure and residual ATP at the end of the 30minute reperfusion (Fig. 1) shows a fairly good
correlation at ATP levels below 10 /*mol/g dry.
These data are very comparable to those reported
earlier by others (Reibel and Rovetto, 1979) and
suggest that the tissue becomes progressively more
damaged with continued exposure to ischemia in
association with loss of total adenine nucleotides.
The hearts used for the data shown in Table 1 and
Figure 1 were perfused with buffer containing 2.5
mM free Ca++. Since this is higher than the physiological Ca++ concentration, and Ca++ overloading is
known to cause damage to ischemic myocardium,
the effects of Ca++ concentration on adenine nucleotides and recovery of ventricular function were determined.
control and reperfused hearts reflects loss of total
adenine nucleotides. It is interesting that, during the
first 20 minutes of ischemia, ADP was high, but its
concentration decreased with continued exposure to
ischemia. On the other hand, the concentration of
AMP continued to increase up to 25 minutes of
ischemia and then stayed at these very high levels
for at least 45 minutes. With reperfusion, ADP levels
returned to near normal at all time periods, whereas
levels of AMP returned to normal after 20 minutes
of ischemia, but remained higher than normal with
reperfusion after longer periods of ischemia. Loss of
total adenine nucleotide was associated with high
levels of AMP both during ischemia and with reperfusion. In this model of zero flow ischemia, reperfusion caused an additional loss of adenine nucleotides, and the magnitude of the loss increased
between 20 and 25 minutes, in association with the
large increase in AMP. Thus, with reperfusion after
20 minutes, most of the ADP and AMP was converted back to ATP with little additional loss of total
nucleotides. After 25 minutes of ischemia, however,
only 3.5 /*mol were rephosphorylated to ATP, and
5.2 /imol were lost from the cells with reperfusion.
With longer exposure to ischemia, about the same
amount of nucleotides was lost with reperfusion,
and most of this loss came from the AMP present
at the end of ischemia and was associated with
continued high levels of AMP during reperfusion.
20
10 20 30 40 50 60
FIGURE 1. Recovery of high energy phosphates
and ventricular function during reperfusion.
Hearts are the same as those for Table 1. ATP
and creatine phosphate levels at the end of the
ischemic period are shown by solid lines and
after reperfusion by dashed lines. Ventricular
pressures were determined at the end of the 30minute reperfusion period for each group. The
relationship between developed ventricular
pressure (systolic-diastolic) and residual ATP
after reperfusion is also shown.
Neely and Grotyohann /Protection of Ischemic Myocardium
TABLE 2
Recovery of ATP, CP, Total Creatine, and Ventricular
Function during 30 Minutes of Reperfusion after Exposure of
Hearts to Low Flow Anoxia at Various Ca*+ Concentrations
Perfusate
Ca + +
(mM)
2.5
Time
of low
flow
(min)
0
Tissue metabolites
(/jmol/g dry)
CP
ATP
Recovery of
ventricular
function
Total
(%)
creatine
24 ± 0.7 37 ± 1.7 63 ± 2.3
+
+
±
+
1.9
1.4
0.6
0.4
37
34
30
31
±
±
+
±
2.7
1.6
1.8
2.9
62
56
54
53
± 1.2 100 + 3
+ 2.3 92 ± 1 4
± 1 . 7 91 ± 11
± 1.2 77 ±7
9.2+
8.6 +
7.9 +
4.7 +
1.1
0.8
0.5
0.4
36
38
30
23
+
+
±
+
2.6
2.1
2.7
2.7
59
61
53
47
±
±
±
±
Downloaded from http://circres.ahajournals.org/ by guest on June 16, 2017
0.75
45
60
75
120
1.25
45
60
75
120
1.75
45
60
75
7.6 + 0.3 31 + 1.8 56 ± 1.8
6.5 + 0.4 29 + 3.2 52 ± 2.8
7.7 + 0.52 32 + 2.0 59 ± 2.4
2.5
20
30
45
60
75
9.4
6.1
6.6
4.6
3.6
11.8
9.8
8.2
6.8
±
±
+
±
+
0.26
0.16
0.49
0.73
0.53
32
22
23
17
12
+
±
±
+
+
0.9
0.7
0.5
3.0
2.1
56
39
48
43
39
+
±
±
±
±
92 ±10
3
101 + 9
3
2.7 88 ± 5
1.6 47 ± 10
0.5
1
2.6
2.3
2.3
83 ± 7.5
74 ± 8
85 ±12
79 ± 10
54 ± 5
35 ± 7
13 ± 6
7 ± 2.6
Hearts were perfused for 10 minutes under control conditions
with a coronary flow of 12 ml/min and a perfusate containing
glucose (11 mM), pyruvate (5 mM), and 2.5 miu Ca++. They were
then switched to a constant flow perfusion at 1 ml/min coronary
flow with perfusate gassed with 95:5, N2:CO2 and containing 11
mM glucose and the free Ca++ concentration shown in this table.
This low flow perfusion was continued for the times shown in
the table. The hearts were then reperfused for 30 minutes with
oxygenated perfusate containing glucose (11 mM), pyruvate (5
mM), and 2.5 mM Ca++ at coronary flows of about 12 ml/min
before freezing for measurement of metabolite levels. Because
decreased ventricular function involved both a decrease in developed pressure and heart rate, function was calculated as the
product of developed pressure and heart rate after 30 minutes of
reperfusion and is expressed as the percent of the preischemic
function for each heart. The data are means ± SE for four to six
hearts in each group.
nary flow of 1 ml/min to ensure exposure of the
heart to constant levels of extracellular Ca++ during
ischemia. However, to make the oxygen supply
comparable to the zero flow hearts shown in Figure
1, the perfusate was gassed with N2:CC>2 (95:5)
during the ischemic perfusions. The data from these
experiments show several things (Table 2). First at
any level of perfusate Ca++, recovery of function
with reperfusion declined with duration of exposure
to low flow anoxic conditions. As expected, increasing the Ca++ concentration accelerated the onset of
ventricular failure. For example, at Ca ++ concentrations of 0.75 and 1.25 mM, essentially 100% recovery
of function was obtained with reperfusion after 60
minutes, whereas only 74 and 13% of function was
recovered with reperfusion after 60 minutes of ischemia with 1.75 and 2.5 mM Ca++, respectively. At
a physiological Ca++ of 1.25 mM, 88% of function
819
was recovered after 75 minutes compared to only
7% at the same time in heart receiving 2.5 rr\M Ca++.
The second major observation from these data is
that, although residual ATP declined with time of
exposure to ischemia and with increased Ca++ concentration, the differences in ATP concentrations
were small, even though the ability of the hearts to
recover function varied widely. The largest decrease
in ATP with ischemic perfusion time occurred at 2.5
mM Ca++. Under this condition of high Ca++, a fairly
good correlation existed between residual ATP and
recovery of function similar to that obtained in
Figure 1 with zero coronary flow. Overall, however,
there was a very poor correlation between recovery
of ventricular function and residual ATP (Fig. 2).
Essentially full recovery was obtained with ATP
levels between 7 and 10 /umol/g dry and recovery
of function decreased from 80% to 7% over a narrow
range of ATP from 6 to 4 fimol/g dry. These data
suggest that cellular damage caused by overloading
the cells with extracellular Ca++ was not due to a
proportionally greater depletion of adenine nucleotides except perhaps under extreme conditions of
high Ca++. Also, the time-related deterioration observed at any Ca++ concentration occurred over a
very narrow range of residual ATP levels. Even at
2.5 mM Ca++, recovery of function decreased from
54% after 30 minutes to 13% after 60 minutes when
ATP decreased only from 6.1 to 4.6 /tmol/g dry.
Thus, the ability to recover mechanical function was
poorly related to residual ATP in this model of
ischemia. In fact, one could just as easily correlate
the decline in functional recovery with loss of tissue
total creatine (Fig. 2) or failure to recover creatine
phosphate (Table 2). These data strongly suggest
that a more general type of cellular damage occurred
that is independent of the loss of adenine nucleotides.
A major difference between the experiments of
Figure 1 and those of Table 2 was the maintenance
of low coronary flow for the hearts shown in Table
100
100
90
90
I
r
1*
80
70
70
sii
SO
7c
/.
/*
50
o
40
I
30
20
10
5
10
15
ATP (/jmol/g dry)
20
*
10
*
20 30 40
50 60
70
Total Creatine
(umol/g dry)
FIGURE 2. Relation of ventricular function and residual ATP and
total creatine with reperfusion following exposure to low flow anoxia.
These data are the same as presented in Table 2. Control hearts are
shown as • and the low flow anoxic hearts exposed to Ca** concentrations of 0.75, 2.25, 1.75, and 2.5 mMare indicated by • , O, A, and
+, respectively.
Circulation Research/Vo/. 55, No. 6, December 1984
820
Downloaded from http://circres.ahajournals.org/ by guest on June 16, 2017
2. Thus, with low flow, the ability to recover ventricular function was maintained for a longer exposure to ischemia and occurred at much lower levels
of ATP (Fig. 2) than with zero coronary flow (Fig.
1). These observations suggested a role of coronary
flow and accumulated metabolic products in the
onset of tissue damage that is independent of O2
supply and ATP levels.
A major metabolic product that accumulates during ischemia is lactate, and associated with this, H +
and NADH. Therefore, it was of interest to determine the role of glycolytic products in cellular damage during ischemia. Since the lactate that accumulates during zero flow ischemia is derived largely
from glycogenolysis, groups of hearts were exposed
to an anoxic preperfusion with high coronary flow
to deplete glycogen levels and wash out the lactate
produced prior to exposure to zero flow ischemia
(Table 3). In those hearts receiving a 10-minute
oxygenated perfusion prior to ischemia, levels of
ATP, CP, and glycogen were high and lactate was
low at the beginning of the 30-minute ischemic
period. At the end of ischemia, levels of ATP, CP
and glycogen were low, and lactate had increased
42-fold. After 30 minutes of reperfusion, ATP and
CP levels were partially restored, glycogen remained
low, and lactate, although greatly reduced from the
ischemic level, remained higher than the pre-
ischemic level. Functional recovery in these hearts
was only 28% of the preischemic level. This is a
much lower level of recovery than observed in the
hearts shown in Table 2 that were exposed to 1.25
rrtM Ca++, but where coronary flow was 1 ml/min
(100% recovery after 60 minutes), again emphasizing the role of washout of metabolic products.
In hearts exposed to 10 minutes of anoxic preperfusion, ATP was reduced by about 50% and CP was
almost depleted prior to ischemia (Table 3). Glycogen levels were decreased from 120 to 20 ^mol
glucose/g dry by the anoxic preperfusion. However,
conversion of this 100 /*mol of glycogen glucose to
lactate resulted in only 20 jtmol lactate accumulating
in the tissue because high coronary flow was available during anoxia to wash out the lactate. When
these hearts were subsequently exposed to 30 minutes of zero flow ischemia, ATP and CP levels
declined to amost nondetectable levels, much more
than in the oxygenated hearts, and glycogen and
lactate levels were much lower at the end of ischemia. With reperfusion, these hearts restored ATP
to about the same level as the oxygenated hearts,
CP was higher, and glycogen remained low. Lactate
decreased more than in the oxygenated hearts, and
recovery of ventricular function was increased to
68%.
When the period of anoxic preperfusion was ex-
TABLE 3
Tissue Metabolites and Recovery of Ventricular Function in Hearts Exposed to Various
Conditions of Oxygen Supply Prior to Ischemia
Condition
ATP
CP
Gly
Lac
Recovery of
ventricular
function
(%)
O2 (10 min) (10)
+ ischemia (10)
+ reperfusion (10)
18.7 ±0.34
3.7 ±0.22
7.2 ±0.31
21.9 ±0.51
5.4 ± 0.26
15.4 ±0.76
120 ± 4.6
49 ± 0.6
41 ± 2.6
3.9 ± 0.40
166 ±6.5
19.1 ± 2.7
28 ± 4.2
N2 (10 min) (8)
+ ischemia (8)
+ reperfusion (16)
10.5 ± 0.22
0.46 ± 0.05
8.5 ± 0.28
1.8 ±0.13
0.39 ± 0.07
30.0 ±1.2
20 ± 2.0
1.9 ±0.18
8.8 ± 1.3
19.8 ± 0.80
104 ±3.3
3.33 ± 0.23
68 ± 4.6
N2 (15 min) (4)
+ ischemia (4)
+ reperfusion (7)
8.8 ±0.18
0.52 ± 0.03
9.2 ± 0.45
2.2 ± 0.07
0.59 ±0.11
32.7 + 2.1
5.8 ± 1.0
1.9 ± 0.25
7.9 ± 1.3
22.8 ± 0.89
72.5 ± 1.9
3.6 ±0.61
92 ± 2.9
N2 (10 min)
+ O2 (10 min) (8)
+ ischemia (6)
+ reperfusion (10)
19.4 ± 0.5
0.45 ± 0.03
10.8 ± 0.33
37 ± 1.5
0.43 ± 0.09
32 ± 0.91
39 ± 3
1.7 ±0.1
15 ±0.8
4 ±0.6
108 ± 3
2.9 ± 4
75 ± 5
Tissue metabolites (/imol/g dry)
++
The perfusate contained glucose (11 nw) and 1.25 mM Ca in each case, and all hearts were
perfused under oxygenated conditions for 10 minutes before the experimental perfusions were begun.
The ischemic period was 30 minutes of zero coronary flow and reperfusion was for 30 minutes with
oxygenated buffer containing glucose (11 mM) and 1.25 mM Ca++ in each case. The preischemic
treatment was either 10 minutes of oxygenated perfusion, 10 or 15 minutes of anoxic perfusion or 10
minutes anoxic perfusion followed by 10 minutes of oxygenated perfusion. Coronary flows were about
13 ml/min in each case during the preischemic perfusion. Groups of hearts were frozen after the
preischemic perfusion, after 30 minutes of ischemia, and after 30 minutes of reperfusion for analysis
of tissue metabolites. Recovery of ventricular function was determined as the product of developed
ventricular pressure and heart rate and is expressed as the percent of the pretreatment function. The
data are means ± SE for the number of hearts shown in parentheses. Gly = glycogen. Lac = lactate.
Neely and Grotyohann/Protection of Ischemic Myocardium
/ery
100 r
80
80
60
60
821
B
o
40
40
\
20
20
0
10
20
30
40
0
Downloaded from http://circres.ahajournals.org/ by guest on June 16, 2017
>
5
10
15
20
ATP (^mol/g dry)
Tissue Lactate (mM)
100
100
80
80
FIGURE 3. Relation between recovery of ventricular function
and tissue lactate at the end of ischemia and tissue ATP after
reperfusion. The data in panels A and B are the same as shown
in Table 2 for oxygenated and 10- or 15- minute anoxic
preperfused hearts, and in panels C and D, the data are from
Table 4 with lactate added to the anoxic and ischemic perfusate.
60
o
40
20
10
20
30
40
Added Perfusate Lactate
(mM)
0
10
15
20
ATP (jimol/g dry)
tended to 15 minutes, the level of glycogen was
lower at the beginning of ischemia, and correspondingly less lactate was accumulated during ischemia.
Recovery of function with reperfusion was further
improved to 92%. This very large improvement in
ventricular function occurred in spite of much lower
levels of ATP and CP at the beginning of reperfusion
in the anoxic preperfused than in the oxygenated
preperfused hearts. There was no correlation between recovery of function and residual ATP levels
during reperfusion (Fig. 3, panel B), but a good
negative correlation was observed between tissue
levels of lactate during ischemia and recovery of
function with reperfusion (Fig. 3, panel A).
It is possible that hydrolysis of the large stores of
ATP and CP present in oxygenated hearts at the
time ischemia is introduced might contribute to cellular damage by production of H+. Also, the high
content of tissue oxygen at the beginning of ischemia
might contribute to free radical production and accelerate ischemic damage. Neither of these possibilities seemed to be important factors in the accelerated damage that occurred to hearts oxygenated
prior to ischemia. When hearts were perfused under
anoxic conditions for 10 minutes and then reoxygenation for 10 minutes prior to ischemia, the tissue
levels of ATP and CP were fully restored during the
10 minutes of reoxygenation but glycogen levels
remained low (Table 3). When these anoxic-reoxygenated hearts were subsequently exposed to ischemia, levels of ATP, CP, and glycogen were depleted much the same as in the anoxic preperfused
hearts that had not been reoxygenated. However,
lactate production from glycogenolysis remained
lower than in the oxygenated hearts, and functional
recovery with reperfusion was much better. Thus,
reoxygenation between the anoxic and ischemic perfusions restored tissue ATP and CP, but glycogen
remained low and the protective effect of anoxic
preperfusion on recovery of function was still observed.
Since low glycogen at the beginning of ischemia
and less lactate accumulation during ischemia appeared to be the only metabolic event that correlated
with improved recovery of function during reperfusion, the effect of adding Na+ lactate to the perfusate during the anoxic preperfusion and ischemic
period was determined (Table 4). Lactate was not
added to the perfusate during reperfusion. Addition
of lactate at 0, 10, 20, 30, and 40 mM had no effect
on the level of ATP and CP either at the end of
ischemia or with reperfusion. Likewise, glycogen
levels were similarly low at the end of ischemia and
reperfusion in all groups. However, the recovery of
Circulation Research/Vof. 55, No. 6, December 1984
822
TABLE 4
Effect of Perfusate Lactate on Tissue Metabolites and Recovery of Ventricular Function in
Glycogen-Depleted Hearts Exposed to Ischemia
ATP
CP
Gly
Lac
Recovery of
ventricular
function
(%)
(8)
(16)
0.46 ± 0.05
8.5 ±0.28
0.39 ±0.07
30 ± 1.2
1.9 ±0.18
8.8 ±1.3
104 ± 3.3
3.3 ± 0.23
68 ± 4.3
(6)
(6)
0.71 ±0.14
9.3 ± 0.33
0.44 ±0.06
33 ±0.16
1.3 ±0.17
8.6 ±.83
134 ±4.1
4.0 ± 0.44
51 ± 6.2
(6)
(6)
0.53 ±0.14
7.4 ± 0.65
0.96 ±0.34
24 ±2.3
2.1 ±0.27
6.8 ± 0.70
165 ±5.2
5.0 ± 0.96
43 ± 9.3
(6)
(6)
0.50 ± 0.05
7.9 ±0.16
1.0 ±0.22
28 ± 1.0
1.9 ±0.23
9.4 ±0.81
238 ± 10
6.7 ±0.9
25 ± 5
(6)
(6)
0.44 ± 0.05
7.4 ± 0.39
0.55 ±0.08
27 ±1.9
2.0 ±0.61
11.6 ± 1.2
263 ± 9
7.8 ±2.1
16 ± 5
Tissue metabolites (fimol/g dry)
Perfusate
Iactate
Condition
(mM)
Downloaded from http://circres.ahajournals.org/ by guest on June 16, 2017
Ischemia
Reperfused
0
Ischemia
Reperfused
10
Ischemia
Reperfused
20
Ischemia
Reperfused
30
Ischemia
Reperfused
40
++
The perfusate contained 11 mM glucose and 1.25 mM Ca throughout, and all hearts received a 10minute oxygenated initial perfusion. They were then switched to 10 minutes of anoxic perfusion
(coronary flows of 13 ml/min) with perfusate containing the lactate concentration shown prior to
exposure to 30 minutes of zero coronary flow ischemia followed by 30 minutes of oxygenated
reperfusion. When Na+ lactate was added, perfusate NaCl was reduced to compensate for the additional
ions. Groups of hearts were frozen after the ischemic and reperfusion periods for analysis of tissue
metabolites. Recovery of ventricular function was determined as the product of developed pressure
and heart rate and is expressed as the percent of the pre-anoxic function for each group. The data are
means ± SE for the number of hearts shown in parentheses. Gly = glycogen, Lac = lactate.
ventricular function with reperfusion was inversely
related to tissue lactate during ischemia. A good
negative correlation between added lactate and recovery of function was obtained (Fig. 3, panel C)
with no correlation existing between residual ATP
and functional recovery (Fig. 3, panel D). Thus, these
data provide more direct evidence that high levels
of lactate during ischemia are associated with accelerated cellular damage independent of ATP levels.
To determine whether this effect of lactate occurred during the ischemic period or with continued
high levels of lactate during reperfusion, groups of
hearts were made ischemic with oxygenated buffer
to allow lactate levels to increase, and then the rates
of lactate washout during reperfusion were determined. Tissue levels of lactate were 171 ± 5.7, 124
± 11, 78 ± 9, 25 ± 3.8, 21 ± 1.5, and 15 ± 4 ^mol/
g after 0, 0.5, 1.0, 2.0, 5.0, and 30 minutes of
reperfusion, respectively. Thus, 85% of the very
high levels of lactate at the end of ischemia was
washed out of the tissue within 2 minutes of reperfusion. However, the level of lactate still remained
higher than normal after 30 minutes of reperfusion
(15 ± 4 compared to control levels of 2.3 ± .6 ^mol/
g). Thus, the major effect of lactate probably occurred by exposure of the tissue to high levels during
ischemia. This conclusion is supported by the observation that addition of lactate to the perfusate during
reperfusion of ischemic hearts did not influence
function. Recovery of function in hearts preperfused
under anoxic conditions prior to ischemia was 68 ±
6% without lactate and 65 ± 7% when 30 mM lactate
was added only during the reperfusion period.
Discussion
During the transition of ischemic myocardium
from viable to necrotic tissue, every biochemical and
mechanical function of the heart will probably be
affected. However, before the cells become necrotic,
they pass through a transition phase from reversible
to irreversible damage. The time at which this transition occurs depends very much on the ischemic
model being used and on the cellular process being
investigated that becomes irreversibly damaged. In
the present study, recovery of mechanical function
was used as an estimate of cellular damage. Although this is a gross and indirect assessment of cell
viability, it nonetheless reflects damage to the contractile function of the cells. Irrespective of the cellular process being investigated, two primary factors
result in the time-dependent development of irreversible damage. These are loss of or decreased
oxidative production of ATP and accumulation of
metabolic products that cannot be oxidized to CO2
and H2O or removed from the tissue by washout of
the vascular space.
Both of these events may result in a very heterogeneous cascade of metabolic and structural alterations that collectively cause irreversible damage. The
decrease in oxidative metabolism, for example, not
only lowers production of and levels of high energy
Neely and Grotyohann/Protection of Ischemic Myocardium
Downloaded from http://circres.ahajournals.org/ by guest on June 16, 2017
phosphates, but results in net loss of total adenine
nucleotides. In the present study, this loss of total
nucleotides was associated with accumulation of
high levels of AMP. Accumulation of other metabolic products may cause damage through a variety
of mechanisms associated with their inhibition of
certain enzymes and perhaps activation of others
(Katz and Messineo, 1981; Neely and Feuvray,
1981). It seems clear that decreased oxidative production of ATP and the associated loss of adenine
nucleotides, if allowed to progress until adenine
nucleotides are critically low, can in itself result in
irreversibility. Certainly, when no adenine nucleotides are available for phosphorylation to ATP, the
cells are probably irreversibly damaged. Even when
ATP levels are not totally depleted, a good negative
correlation between residual ATP and cellular function has been reported for several models of ischemia (Gudbjarnason et al., 1970; Jennings et al.,
1977; Reibel and Rovetto, 1978; Vary et al., 1979;
Watts et al., 1980) and confirmed in the present
study for hearts perfused with 2.5 mM Ca in which
high levels of lactate accumulated. However, a
cause-and-effect relationship between low adenine
nucleotides and irreversible cellular damage has not
been clearly established. One reason for this is that
it has not been possible to restore ATP levels rapidly,
and to determine whether restoration of ATP results
in reversal of cellular damage (Reibel and Rovetto,
1979; Reimer et al., 1981).
The role of metabolic products in development of
ischemic damage has been appreciated for several
years. In 1935, Tennant and Wiggers (1935) demonstrated that a reduction in coronary flow had a
more profound effect on myocardial contractility
than did hypoxia. The early decrease in contractile
force of ischemic hearts was associated with increased tissue lactate (Neely et al., 1973) and H+
(Cobbe and Poole-Wilson, 1980; Jacobus et al., 1982)
with little change in tissue ATP (Katz, 1969;
Gudbjarnason et al., 1970; Neely et al., 1973;
Hearse, 1979). The onset of irreversible damage was
also related to the continued presence of high lactate
levels (Neely et al., 1973). Increased lactate and the
associated rise in cytosolic NADH was shown to
inhibit glycolysis and reduce anaerobic ATP production (Rovetto et al., 1973, 1975). Incubation of thin
slices of dog myocardium with 50 mM lactate resulted in mitochondrial changes after 10 minutes
that were similar to those found after 1 hour in
ischemic myocardium (Armiger et al., 1974). Thus,
high tissue lactate has been implicated as a factor
directly or indirectly causing cellular damage during
ischemia.
It seems clear from the data presented in the
present study that inability of the heart to recover
mechanical function was not due solely to the loss
of adenine nucleotides and, consequently, to low
levels of residual ATP during reperfusion. Recovery
of function varied from 28 to 100% of the preischemic function with little or no change in adenine
823
nucleotides. The two most important factors determining the ability of the hearts to recover ventricular
function were the levels of extracellular Ca++ and
accumulation of tissue lactate during ischemia. In
the first 75 minutes of low flow anoxia, Ca++ concentrations ranging from 0.75 to 1.75 mM had little
effect on the ability of the heart to recover function,
but with longer exposure to ischemia or at higher
Ca++, the effects of Ca++ were apparent. With 0.75
mM Ca++ present during ischemia, 77% of the preischemic function was recovered after 120 minutes
of ischemia, even though ATP was only 28% of
normal. At physiological Ca++, recovery of function
after 120 minutes was reduced to 47% with little
additional decrease in ATP. In contrast, when the
Ca++ concentration was increased to 2.5 mM, only
35% of preischemic function was recovered after 45
minutes of ischemia, and residual ATP was still 28%
of normal. Thus, the effects of Ca on ischemic
damage were largely independent of residual ATP
levels during reperfusion.
At physiological concentration of free Ca++ (1.25
mM), the ability to recover function was greatly
dependent on the amount of glycolytic products
present in the tissue during ischemia. Thus, when
hearts were preperfused for 0, 10, or 15 minutes
under high coronary flow anoxic conditions to deplete the tissue of glycogen prior to ischemia, recovery of function increased from 28 to 68 and 92%,
respectively, even though residual ATP during reperfusion ranged only between 7.2 and 9.2 jtmol/g
dry. This improved recovery of function was associated with less tissue glycogen at the beginning of
ischemia and less accumulation of lactate during
ischemia. Addition of lactate to the perfusate prior
to ischemia in the anoxic preperfused hearts reversed the beneficial effects of glycogen depletion
and lowering tissue lactate. In this case, recovery of
function was negatively correlated with the concentration of lactate added to the perfusate and decreased from 68 to 16% of preischemic function
with addition of 0 and 40 mM lactate. This change
in function occurred even though residual ATP during reperfusion varied only from 8.5 to 7.4 jtmol/g
dry and CP levels varied only from 30 to 27 /*mol/
g dry. Thus, the reverse effects of reduced lactate
during ischemia and rapid restoration of high lactate
during ischemia suggest a critical role of this glycolytic product in ischemic damage.
The mechanism of these beneficial effects of glycogen depletion or the harmful effects of lactate
accumulation are not known. The effects do appear
to depend on the exposure of the heart to high
lactate levels during ischemia and not during reperfusion. However, there was a continued higher than
normal level of lactate during reperfusion in those
hearts that recovered function poorly. This continued higher lactate level may simply be associated
with, rather than the cause of, poorer functional
recovery. The tissue damage associated with high
tissue levels of lactate during ischemia may be me-
Circulation Research/Vo/. 55, No. 6, December 1984
824
Downloaded from http://circres.ahajournals.org/ by guest on June 16, 2017
diated by changes in cellular pH both when lactate
is produced from endogeneous glycogen or when
added to the perfusate. Transport of weak acids
such as lactic into the cell probably occurs as the
protonated acid which dissociates in the intracellular
space releasing H+. Thus the addition of extracellular
lactate causes an increased concentration gradient
of lactate from extra- to intracellular spaces which
could result in an inward H+ pump.
The protective effect of decreased glycolysis during ischemia is contrary to the conclusions of previous publications (Hearse and Chain, 1972; Bricknell et al., 1981). These studies reported a special
protective role of glycolysis in ischemia. There may
be a beneficial role of glycolytic ATP under special
conditions, such as the high flow anoxia used by
Hearse and Chain (1972), where lactate accumulation would be low, and in the K+-arrested hearts
with maintained coronary flow as studied by Bricknell et al. (1981). Nonetheless, in the beating heart
with very low or zero coronary flow as used in the
present study, and with coronary flows that might
be expected to pertain clinically, accumulation of
lactate and/or other products of glycolysis can be
expected to accelerate tissue damage.
A reduction of glycolysis and maintenance of low
tissue lactate may be just as important in the protection of the heart by hypothermic cardioplegia as is
the preservation of adenine nucleotides. At the end
of 20 minutes of hypothermic (10°C) ischemia, tissue glycogen was still high (90% of normal) and
lactate was low (about 2 times normal) compared to
that present after 20 minutes of normothermic ischemia, glycogen (10% of normal) and lactate (13
times normal) (Ichihara et al., 1981).
This work was supported by National Institutes of Health Grant
HL-18206.
Address for reprints: Dr. fames R. Neely, Department of Physiology, The Milton S. Hershey Medical Center, The Pennsylvania State
University, P.O. Box 850, Hershey, Pennsylvania 17033.
Received May 4, 1984; accepted for publication September 13,
1984.
References
Armiger LC, Gavin JB, Herdson PB (1974) Mitochondrial changes
in dog myocardium induced by neutral lactate in vitro. Lab
Invest 31: 29-33
Bergmeyer HU (1963) Methods in Enzymatic Analysis. New York,
Academic Press
Brickneli OL, Daries PS, Opie LH (1981) A relationship between
adenosine triphosphate, glycolysis and ischemic contracture in
the isolated rat heart. J Mol Cell Cardiol 13: 941-945
Cobbe SM, Poole-Wilson PA (1980) The time of onset and
severity of acidosis in myocardial ischemia. J Mol Cell Cardiol
12: 745-760
Gudbjarnason S, Mathes P, Ravens KG (1970) Functional compartmentation of ATP and creatine phosphate in heart muscle.
J Mol Cell Cardiol 1: 325-339
Hearse DJ (1979) Oxygen deprivation and early myocardial contractile failure: A reassessment of the possible role of adenosine
triphosphates. Am J Cardiol 44: 1115-1121
Hearse DJ, Chain EB (1972) The role of glucose in the survival
and recovery of the anoxic isolated perfused rat heart. Biochem
J128: 1125-1133
Ichihara K, Robishaw JD, Vary TC, Neely JR (1981) Protection of
ischemic myocardium from metabolic products. Acta Med
Scand 210: 13-18
Jacobus WE, Pores IH, Lucas SK, Weisfeldt ML, Flaherty JT (1982)
Intracellular acidosis and contractility in normal and ischemic
heart examined by 3IP NMR. J Mol Cell Cardiol 14 (suppl 3):
13-20
Jennings RB, Reimer KA (1981) Lethal myocardial ischemia injury.
Am JPathol 102: 241-255
Jennings RB, Hawkins HK, Lowe JE, Hill ML, Klotman S, Reimer
KA (1977) Relation between high energy phosphate and lethal
injury in myocardial ischemia in the dog. Am J Pathol 92:187214
Katz AM (1969) The early "pump" failure of the ischemic heart.
Am J Med 47: 497-502
Katz AM, Messineo FC (1981) Lipid-membrane interactions and
the pathogenesis of ischemic damage in the myocardium. Cir
Res 48: 1-16
Kiibler W, Spieckermann PG (1970) Regulation of glycolysis in
the ischemic and the anoxic myocardium. J Mol Cell Cardiol 1:
351-377
Nayler WG (1981) The role of calcium in the ischemic myocardium. Am J Pathol 102: 262-270
Neely JR, Feuvray D (1981) Metabolic products and myocardial
ischemia. Am J Pathol 102: 282-291
Neely JR, Liebermeister H, Battersby EJ, Morgan HE (1967) Effect
of pressure development on oxygen consumption by isolated
rat heart. Am J Physiol 212: 804-814
Neely JR, Rovetto MJ, Whitmer JT, Morgan HE (1973) Effects of
ischemia on function and metabolism of the isolated working
rat heart. Am J Physiol 225: 651-658
Reibel DK, Rovetto MJ (1978) Myocardial ATP synthesis and
mechanical function following oxygen deficiency. Am J Physiol
234: H620-H624
Reibel DK, Rovetto MJ (1979) Myocardial adenosine salvage rates
and restoration of ATP content following ischemia. Am J
Physiol 237: H247-H252
Reimer KA, Hill ML, Jennings RB (1981) Prolonged depletion of
ATP and of the adenine nucleotide pool due to delayed resynthesis of adenine nucleotides following reversible myocardial
ischemic injury in dogs. J Mol Cell Cardiol 13: 229-239
Rovetto MJ, Whitmer JT, Neely JR (1973) Comparison of the
effects of anoxia and whole heart ischemia on carbohydrate
utilization in isolated, working rat heart. Circ Res 32: 699-711
Rovetto MJ, Lamberton WF, Neely JR (1975) Mechanisms of
glycolytic inhibition is ischemic rat hearts. Circ Res 37: 742751
Shen AC, Jennings RB (1972) Kinetics of calcium accumulation
in acute myocardial ischemic injury. Am J Pathol 67: 441-452
Tennant R, Wiggers CJ (1935) Effects of coronary occlusion on
myocardial contraction. Am J Physiol 112: 351-361
Vary TC, Angelakos ET, Schaffer SW (1979) Relationship between adenine nucleotide metabolism and irreversible tissue
damage in isolated perfused rat heart. Circ Res 45: 218-224
Watts JA, Koch CD, LaNoue KF (1980) Effect of Ca++ on heart
function after ischemia. Am J Physiol 238: H909-H916
INDEX TERMS: Metabolic products and ischemic damage • Lactate and ischemic damage • Mechanism of ischemic damage •
Glycolysis and ischemic damage
Role of glycolytic products in damage to ischemic myocardium. Dissociation of adenosine
triphosphate levels and recovery of function of reperfused ischemic hearts.
J R Neely and L W Grotyohann
Downloaded from http://circres.ahajournals.org/ by guest on June 16, 2017
Circ Res. 1984;55:816-824
doi: 10.1161/01.RES.55.6.816
Circulation Research is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX 75231
Copyright © 1984 American Heart Association, Inc. All rights reserved.
Print ISSN: 0009-7330. Online ISSN: 1524-4571
The online version of this article, along with updated information and services, is located on the
World Wide Web at:
http://circres.ahajournals.org/content/55/6/816
Permissions: Requests for permissions to reproduce figures, tables, or portions of articles originally published in
Circulation Research can be obtained via RightsLink, a service of the Copyright Clearance Center, not the
Editorial Office. Once the online version of the published article for which permission is being requested is
located, click Request Permissions in the middle column of the Web page under Services. Further information
about this process is available in the Permissions and Rights Question and Answer document.
Reprints: Information about reprints can be found online at:
http://www.lww.com/reprints
Subscriptions: Information about subscribing to Circulation Research is online at:
http://circres.ahajournals.org//subscriptions/