Vol. 284, Issue 3, H758-H771, March 2003
TRANSLATIONAL PHYSIOLOGY
Mechanisms underlying ischemic diastolic dysfunction:
relation between rigor, calcium homeostasis, and relaxation
rate
Niraj
Varma1,2,
James P.
Morgan2, and
Carl S.
Apstein1
1 Boston University School of Medicine, Boston
02118; and 2 Cardiovascular Division, Beth Israel
Hospital, Harvard Medical School, Boston, Massachusetts 02215
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ABSTRACT |
Increased
diastolic chamber stiffness (
DCS) during ischemia may result
from increased diastolic calcium, rigor, or reduced velocity of
relaxation. We tested these potential mechanisms during severe
ischemia in isolated red blood cell-perfused isovolumic rabbit
hearts. Ischemia (coronary flow reduced 83%) reduced left ventricular (LV) contractility by 70%, which then remained stable. DCS
progressively increased. When LV end-diastolic pressure had increased 5 mmHg, myofilament calcium responsiveness was altered with 50 mmol/l
NH4Cl or 10 mmol/l butanedione monoxime. These affected
contractility (i.e., a calcium-mediated force) but not
DCS.
Second, quick length changes reversed
DCS, supporting a rigor
mechanism. Third, ischemia increased the time constant of isovolumic pressure decline from 47 ± 3 to 58 ± 3 ms
(P < 0.02) but concomitantly abbreviated the
contraction-relaxation cycle, i.e., pressure dissipation occurred
earlier without diastolic tetanization. Finally, to assess any link
between rate of relaxation and
DCS, hearts were exposed to 10 mmol/l
calcium. Calcium doubled contractility and accelerated relaxation
velocity, but without affecting
DCS. Thus
DCS developed during
ischemia despite severely reduced contractility via a rigor
(and not calcium mediated) mechanism. Calcium resequestration capacity
was preserved, and reduced relaxation velocity was not linked to
DCS.
stiffness; left ventricular end-diastolic pressure; quick length
change; heterogeneity
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INTRODUCTION |
ACUTE
DIASTOLIC heart failure, characterized by reduced rate of
relaxation and increased diastolic chamber stiffness, occurs in angina,
left ventricular (LV) hypertrophy, and hypertension (where
subendocardial ischemia may be important) (12, 15) with pulmonary sequelae (27) that may ultimately produce
edema (12, 25). The underlying etiology remains unclear,
but persistently increased diastolic calcium (6, 18),
disturbed high-energy phosphate metabolism (16, 36), and
reduced relaxation velocity (7) have all been implicated.
However, the precise nature of the ischemic insult, i.e.,
"supply" or "demand," may determine the mechanisms underlying
diastolic dysfunction (3, 5, 15, 26, 35). Demand
ischemia, where tachycardia occurs during moderately reduced
coronary flow, characteristically results in an upward shift of the
pressure-volume loop, i.e., increased diastolic stiffness, whereas
contractile function remains preserved. Intracellular calcium has not
been measured during demand ischemia, but we (37) recently reported a rigor-bond mechanism underlying increased diastolic
stiffness in an experimental model. In contrast, supply ischemia, e.g., during acute coronary thrombosis or
experimental ligation, results in contractile failure and an initial
increase in diastolic distensibility. Increased diastolic calcium has
been reported to occur experimentally (6, 7, 18) but is
postulated to be unable to express effects on diastolic tone because of
accumulated intracellular metabolites, e.g., protons and inorganic
phosphate, which reduce calcium sensitivity (3, 5, 26,
35).
We characterized the mechanisms responsible for diastolic dysfunction
in the important clinical condition of supply ischemia. In
isolated hearts, we reproduced physiological stable ischemia, simulating perfusion in an acute infarcted region with low coronary flow (without tachycardia). We observed that an initial increase in
diastolic compliance was followed by a later increase in stiffness. To
identify the underlying mechanism for increased stiffness, we tested
the role of diastolic calcium by deliberately altering intracellular
calcium concentration or myofilament calcium responsiveness, and we
used quick length changes to assess the role of rigor-bond formation.
Finally, we assessed the relation of reduced velocity of pressure
decline to end-diastolic pressure.
The results indicated that severe supply ischemia did not
"protect" against diastolic failure (15, 26, 35) and
that the negative lusitropic effects of ischemia, i.e., reduced
relaxation velocity and increased diastolic stiffness, could be
separated into calcium-sensitive and calcium-insensitive components, respectively.
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METHODS |
The experimental preparation of isolated, balloon in LV rabbit
hearts utilizing an erythrocyte perfusate at 37°C has been described
in detail previously (11). At baseline, the LV balloon volume was adjusted to achieve a stable LV end-diastolic pressure (LVEDP) of 10 mmHg. Thereafter, the volume was not altered for the
duration of each experiment, i.e., each heart contracted
isovolumically. Because the pericardium was removed and the right
ventricle decompressed, passive chamber stiffness was determined by
myocardial stiffness. Hence, LV diastolic chamber stiffness was indexed
by isovolumic LVEDP, measured after systolic pressure dissipation on
the "flat" portion of the pressure tracing. LV contractility was
indexed by isovolumic LV systolic, or developed pressure (i.e.,
systolic minus diastolic), and by the peak positive derivative of LV
pressure (+dP/dt). Hearts were paced constantly throughout
each experiment at 2.7 Hz, replicating resting sinus rates. During an
initial stabilization period of 20 min, hearts were perfused at
coronary flows eliciting coronary artery perfusion pressures of 80 mmHg (normoxia). Ischemia was then imposed by reducing coronary flow to a constant low rate eliciting a coronary artery perfusion pressure (CPP) of
15 mmHg, simulating perfusion conditions during acute myocardial infarction (22). Thereafter, the coronary flow
rate remained unchanged during ischemic perfusion. LVEDP
initially decreased but then progressively increased, indicating
increasing diastolic chamber stiffness. To determine the etiology of
this diastolic dysfunction, a series of interventions was performed with the use of intracoronary infusions. Agents (diluted in saline) were delivered directly into the coronary circulation via aortic infusion (infusion rates never exceeded 5% of coronary flow). Concentrations are expressed below as the final arterial concentrations delivered. After the interventions, ischemic coronary flow
rates were continued for a further 5 min. In groups undergoing
reperfusion, coronary flow rates were returned to individual baseline
values in each heart. Thus the effect of coronary vascular turgor on diastolic stiffness remained constant, and diastolic chamber stiffness during reperfusion could be compared with preischemic values.
Assessment of increased diastolic stiffness.
The effect of deliberately altered myofilament calcium responsiveness
on increased diastolic tension was assessed by agents that do not
affect intracellular calcium concentration but which alter myofilament
calcium sensitivity at both systolic and diastolic levels of calcium
(21). Changes in isovolumic LV systolic pressure (dependent on calcium-activated cross-bridge cycling) in response to
these interventions represented effects on contractility at the
myofilament level and provided an "internal control" to compare with effects (if any) occurring on increased isovolumic LVEDP.
Myofilament calcium sensitivity is affected by intracellular pH. We
used ammonium chloride (NH4Cl), which produces
intracellular alkalanization, followed by "washout" acidification
(21), to test the influence of altered myofilament calcium
sensitivity on ischemia-induced increased diastolic stiffness.
We hypothesized that if increased diastolic stiffness was calcium
driven, then NH4Cl should exert a biphasic effect on
isovolumic LVEDP. Hearts received a 50 mmol/l infusion for 1 min,
commencing when LVEDP had increased ~5 mmHg (NH4Cl,
n = 6).
Butanedione monoxime (BDM) reduces calcium-activated cross-bridge
cycling without altering the calcium transient (at concentrations
10
mmol/l), i.e., results in excitation-contraction uncoupling. However,
it is incapable of breaking formed rigor bonds (17, 21).
We reasoned that if increased diastolic stiffness due to prolonged
ischemia resulted from persistent calcium-activated tension,
then this should be reduced by BDM. To test this, hearts received a 10 mmol/l infusion for 5 min after LVEDP had increased ~5 mmHg (BDM,
n = 8).
Controls (n = 7) received saline for 5 min, after LVEDP
had increased ~5 mmHg. Hearts were reperfused 5 min after saline infusion.
Quick stretch release (QSR) discriminated calcium- from rigor-driven
increased diastolic stiffness in isolated contracting hearts
(37). Quick alterations in LV balloon volume were
performed by a moving piston, driven by compressed air, that could
rapid deliver and withdraw a constant fluid volume in 0.5 s. The
volume was varied precisely in each individual heart to equal 25% of the baseline intraventricular volume, resulting in a 3%
circumferential fiber length change. The small magnitude of this
stretch had no deleterious effect on baseline systolic or diastolic
hemodynamics, i.e., it did not disrupt myocyte function or connective
tissue cytoarchitecture (37).
In the current study, hearts (n = 6) received QSR when
LVEDP had increased ~5 mmHg during ischemia. Pacing was
terminated immediately before QSR, but resumed immediately thereafter.
Thus QSR was delivered during the diastolic period. After QSR, constant low-flow ischemia was continued for a further 5 min. To observe recovery of function, hearts were then reperfused.
Assessment of relaxation rate.
Ischemia reduces contractile function and rate of relaxation
(7). Increased diastolic stiffness then follows. We tested whether an ischemia-related reduction in relaxation rate
sufficed to affect LVEDP (i.e., "incomplete relaxation" producing
increased LVEDP) by quantifying changes occurring in the time constant
of isovolumic exponential pressure decline (
) and also the total relaxation period relative to the entire contraction-relaxation cycle.
Low-flow ischemia was imposed as above, and hearts were paced
constantly at 2.7 Hz (n = 6). When function had
stabilized (3-5 min), LV pressure was analyzed for the following
parameters: peak LV systolic pressure, LVEDP, LV developed pressure,
time from beginning of contraction to peak systolic pressure (TP), and
time from peak systolic pressure to 90% pressure decline
(T90), i.e., a measure approximating the duration of the
relaxation period. Curve fitting was performed to derive
, the time
constant of exponential decay (7, 13).
We then tested whether reduced rate of relaxation reflected a
diminished ability for diastolic calcium clearance during
ischemia. We reasoned that if this was the case, then further
calcium loading should saturate the calcium-handling ability, slow the
rate of pressure decline, and result in increased residual diastolic
calcium, which would simultaneously drive an increase in diastolic
stiffness. In contrast, if increased diastolic stiffness during
ischemia was determined by rigor, then the rate of relaxation
(calcium driven) should be dissociated from diastolic stiffness,
because these two parameters would not share common determinants
(24). To test this, we assessed responses to calcium
during ischemia both before and after diastolic chamber
stiffness had increased.
Hearts received a brief (1 min) infusion of 10 mmol/l calcium chloride
at 5 min of ischemia. A subsequent increase in diastolic stiffness occurred in all hearts during sustained low-flow perfusion. When LVEDP had increased 10 mmHg, hearts received a further, brief (1 min) infusion of 10 mmol/l calcium chloride.
Data acquisition.
LV pressure measurements were recorded continuously. In individual
hearts, data are reported at baseline, 5 min after imposition of
ischemia, and during intervention (means ± SE). Pressure
was measured with the use of a fluid-filled system with the use of a
rigid, short length of polyethylene tubing. Initially, we compared simultaneous results with those derived from a high-fidelity
micromanometer Millar catheter placed within the balloon in the LV
cavity. Similar to other investigators (7, 24), we found
LV systolic pressure, diastolic pressure, and
to be identical with
the use of both solid-state and fluid-filled systems. For the current
experiments, all measurements were made from fluid-filled systems.
LV pressure was digitized by a 12-bit analog-to-digital converting
board at a sampling rate of 1 kHz (model DAP 800/3, Microstar;
Bellevue, WA) and stored on a Gateway 2000 personal computer. The
digital signal was analyzed with custom-designed software.
was
calculated by the variable asymptote method (P = P0e
t/
+ Pa, where P is LV pressure, Pa is asymptotic
pressure, t is time, and P0 is LV pressure at
minimum dP/dt). Pa was allowed to vary
because this provides the most accurate description of LV relaxation
(13). Only a correlation coefficient >0.98 was accepted.
At each recording time point during experiments, ~20 beats were
averaged to derive hemodynamic data.
Statistical comparisons between groups were performed by two-way ANOVA.
If overall ANOVA indicated a significant difference of groups, trials,
or interaction, values at specific time points were examined by the
method of least-significant differences. A value of P < 0.05 was considered significant.
All animal handling and procedures strictly complied with the
regulations of Boston University Animal Care and the National Society
for Medical Research.
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RESULTS |
Coronary flows, duration of ischemia, and
hemodynamics at baseline and before intervention were similar
in all groups (Tables 1 and 2). In each
heart, coronary flow and CPP remained constant during ischemia
and were unaffected by interventions. Hence, in our experiments,
altered isovolumic LVEDP in response to interventions indicated an
effect on diastolic stiffness without any confounding contribution from
altered coronary vascular turgor.
Ischemia.
Overall, low-flow ischemia (reduction of CPP from 80 to 15 mmHg) was accompanied by an 83% reduction of coronary flow from 1.3 ± 0.06 to 0.22 ± 0.01 ml · min
1 · g
LV wet wt
1 (P < 0.001). In all hearts,
LVEDP initially decreased with ischemia and then, after
5-10 min, started to increase gradually, representing increasing
diastolic stiffness. Isovolumic LVEDP increased
5 mmHg during
21 ± 4 min of ischemia (P < 0.001).
Ischemia initially reduced LV contractile function (developed
pressure and +dP/dt) by ~70%, but this then remained
constant despite progressively increasing diastolic stiffness.
Increased LV diastolic stiffness.
Individual groups were similar before interventions performed during
ischemic diastolic dysfunction (Table
2).
NH4Cl elicited an initially positive, followed by a
negative inotropic effect (Fig.
1A). The peak positive effect,
where developed pressure increased from 27 ± 5 to 34 ± 6 mmHg (P < 0.005) (Fig. 1B) and
+dP/dt increased, was consistent with an initial
alkalanization and increased myofilament calcium sensitivity, but was
unaccompanied by any further increase in increased diastolic chamber
stiffness. During the following negative inotropic effect, developed
pressure decreased from 34 ± 6 to 22 ± 3 mmHg
(P < 0.001), indicating diminished myofilament calcium
sensitivity (during "washout acidification"), but increased
diastolic stiffness was not concomitantly reduced. Thus the
intracellular pH-induced changes in systolic active tension generation
were dissociated from increased diastolic tension, which remained
unaffected. Function in controls remained unchanged. Five minutes after
NH4Cl washout, function was similar in control and
NH4Cl groups.

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Fig. 1.
A: representative tracing of ammonium
chloride (NH4Cl). Ischemia [decreasing coronary
perfusion pressure (CPP) to 15 mmHg] reduced left ventricular (LV)
systolic pressure (LVSP) to 49 mmHg and LV end-diastolic pressure
(LVEDP) to 7 mmHg. Diastolic stiffness increased gradually.
NH4Cl (50 mM), imposed when LVEDP had increased to 11 mmHg,
initially increased LVSP from 56 to 69 mmHg and then reduced it to 41 mmHg, indicating a positive, followed by a negative, inotropic effect,
consistent with its known effects on intracellular pH (pHi)
and hence myofilament calcium sensitivity. However, elevated LVEDP did
not change in the same direction, implying that it was not increased by
calcium-driven cross-bridge cycling. and , increased and
decreased pHi, respectively. B: group data.
During ischemia, LV developed pressure (LVDP) remained constant
in controls, but demonstrated a biphasic response to NH4Cl.
Diastolic stiffness increased and was unaffected by NH4Cl.
dP/dt, peak derivative of LV pressure.
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BDM progressively reduced contractile function, consistent with its
property of reducing cross-bridge cycling (Fig.
2A). LV +dP/dt and
developed pressure were significantly reduced (developed pressure BDM
vs. control = 15 ± 2 vs. 25 ± 3 mmHg,
P < 0.001) (Fig. 2B). However, BDM failed
to reduce increased LVEDP. On the contrary, diastolic tension continued
to increase during BDM exposure, i.e., moved in a direction opposite to
that expected of a calcium-mediated tension, suggesting that this was
mediated by a calcium-independent process. When BDM was discontinued,
developed pressure recovered slightly from 15 ± 2 to 17 ± 2 mmHg, 5 min post-BDM (P < 0.05), indicating that
calcium-mediated cross-bridge cycling partially resumed. However,
increased LVEDP was unaltered and continued to increase, and was no
different to control. This again contrasted the calcium sensitivity of
contractile function with the calcium insensitivity of increased
diastolic stiffness during ischemia.

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Fig. 2.
A: representative tracing of butanedione
monoxime (BDM). Ischemia reduced LVSP to 34 mmHg and LVEDP to
9.5 mmHg, which then gradually increased. BDM progressively reduced
LVSP from 43 to 39 mmHg (developed pressure from 28 to 19 mmHg),
indicating inhibition of calcium-activated myofilament cross-bridge
cycling. However, elevated LVEDP was not simultaneously reduced (and
continued to increase), indicating that it was not calcium driven. Five
minutes after BDM was discontinued, contractile function recovered
partially (developed pressure increased from 19 to 23 mmHg), but LVEDP
continued to rise. B: group data demonstrating the peak
effects of BDM. During ischemia, BDM reduced LVDP but failed to
reduce increased LVEDP, which continued to rise indistinguishably from
controls. These opposite effects of BDM on contractile and lusitropic
function implied that increased diastolic stiffness was not calcium
mediated.
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Hence, interventions (NH4Cl and BDM) affecting
calcium responsiveness at the myofilament level failed to alter
increased diastolic stiffness resulting from ischemia in the
direction expected of a calcium-dependent tension (in contrast to their
effects on contractility).
QSR delivered after isovolumic LVEDP had increased during prolonged
ischemia, immediately lysed increased diastolic tension (LVEDP
post- vs. pre-QSR = 8 ± 0.4 vs. 15 ± 0.4 mmHg,
P < 0.001). Thus chamber stiffness returned to
precontracture values [LVEDP post-QSR vs. precontracture = 8 ± 0.4 vs. 8 ± 0.5 mmHg, P = not significant
(NS)] with no immediate tension recovery (Fig. 3, A and
B). The decrement of LVEDP
produced by QSR was identical in magnitude to the "upward shift" of
isovolumic LVEDP sustained during ischemia. Hence, QSR elicited
a characteristic rigor-lysis response without a calcium-mediated
component (37). Contractile function remained constant,
both pre- and post-QSR.

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Fig. 3.
A: representative tracing of the effect of quick stretch
release (QSR). Ischemia reduced LVSP to 34 mmHg and LVEDP to 8 mmHg, which then gradually increased. QSR, imposed when LVEDP had
increased to 14 mmHg, reduced LVEDP to precontracture values, with no
tension redevelopment, i.e., a typical response for rigor-bond-mediated
increase in tension (37). During continued
ischemia post-QSR, diastolic stiffness gradually increased
again. Reperfusion (i.e., return of coronary flow to baseline rates)
resulted in resolution of diastolic dysfunction because isovolumic
LVEDP returned to its baseline value of 10 mmHg in 5 min. Contractile
function recovered partially. B: group data depicting
hemodynamic function pre-QSR and immediately post-QSR. QSR reversed
ischemia-induced increased diastolic stiffness without
affecting contractility.
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After QSR, isovolumic LVEDP gradually increased again from 8 ± 0.4 to 14 ± 0.2 mmHg (P < 0.01) during 5 min of
continued ischemia. This was unaccompanied by any change in
contractile function. This recurrent increase in LV diastolic stiffness
suggested a redevelopment of rigor bonds. Reperfusion reversed this
process because diastolic stiffness was restored to baseline values
(isovolumic LVEDP decreased to 11.5 ± 1 mmHg in 5-min reperfusion
vs. 10 mmHg preischemia, P = NS) (Fig.
3A). In controls, increased stiffness was only partially
reversed by reperfusion (LVEDP decreased from 20 ± 3 at end
ischemia to 18 ± 2 mmHg at 5-min reperfusion). Lesser degrees of contracture sustained during ischemia, in response to stretch, or pharmacological intervention (15, 32) may
be associated with improved diastolic function during reperfusion. Contractile function recovered to 67% of preischemic values
with reperfusion in both QSR and controls developed pressure at 5-min reperfusion in QSR = 62 ± 5 mmHg vs. control = 67 ± 2 mmHg, P = NS). Peak positive and negative
derivatives of LV pressure (±dP/dt) were also similar in
QSR and controls (QSR vs. control at 5-min reperfusion:
+dP/dt = 774 ± 136 vs. 865 ± 38 mmmHg/s,
P = NS;
dP/dt = 609 ± 92 vs.
750 ± 44 mmHg/s, P = NS).
Relaxation rate.
Ischemia initially decreased LVEDP and increased
from
47 ± 3 to 58 ± 3 ms (P < 0.02), reflecting
increased chamber distensibility and reduced velocity of relaxation,
respectively (Tables 3 and 4). However, ischemia abbreviated
the duration of both contraction (TP) and relaxation (T90),
thus diminishing the isovolumic contraction-relaxation cycle (TP + T90) from 251 ± 8 to 216 ± 6 ms
(P < 0.001) (Fig. 4).
Reduced T90 reflected earlier total pressure dissipation, despite reduced rate of relaxation (because during ischemia,
the decline in pressure commenced earlier, from a lower peak systolic pressure, compared with baseline). Thus the diastolic period prolonged (at constant heart rate). Hence, during ischemia, the reduced rate of relaxation became less likely to shift the end-diastolic pressure-volume relation.
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Table 4.
Effects of calcium on isovolumic contraction and relaxation at 5 and 30 min of ischemia, i.e., before and during ischemic
diastolic dysfunction
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Fig. 4.
Isovolumic LV pressure: baseline compared with
ischemia at constant heart rate (n = 6).
Although the time period of pressure decline ( ) increased during
ischemia (from 47 ± 3 to 58 ± 3 ms), it was
associated with an abbreviated mechanical transient (TP + T90 was reduced from 251 ± 8 to 216 ± 6 ms),
and complete relaxation was achieved earlier (i.e., the "flat"
diastolic period, after pressure decline had been completed, was
prolonged). TP, time to peak systolic pressure; T90, time
to 90% decline in pressure from TP.
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Calcium.
After 5 min of ischemia, during stable function, calcium
increased developed pressure (reflecting increased intracellular calcium; see Table 4) (1) and accelerated relaxation rate:
shortened to 43 ± 2 ms (P < 0.01), i.e., to
preischemic values, but without affecting isovolumic LVEDP
(Fig. 5, A and B).

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Fig. 5.
Effect of calcium. A: representative tracing.
Ischemia (reducing CPP to 10 mmHg) reduced LVSP to 27 mmHg and
LVEDP to 8 mmHg. increased from 47 ± 0.4 to 53 ± 0.4 ms. Calcium increased contractility (LVSP to 52 mmHg, +dP/dt
to 700 mmHg/s), indicating increased intracellular calcium
concentration, but did not simultaneously increase LVEDP, indicating
preserved calcium resequestration. Calcium accelerated rate of
relaxation ( diminished to 37 ± 0.5 ms, dP/dt
increased to 600 mmHg/s), but isovolumic LVEDP was not reduced. Thus
rate of relaxation was not linked to diastolic chamber stiffness
(indexed by isovolumic LVEDP, measured on the flat portion of the
diastolic tracing when dP/dt = 0). B:
group data. Ischemia reduced LV developed pressure, LVEDP, and
velocity of relaxation (increased ). Calcium increased developed
pressure, indicating increased intracellular calcium concentration, and
reduced , but failed to affect LVEDP.
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During continued ischemia, contractile function (developed
pressure, 22 ± 3 mmHg) and
(58 ± 5 ms) remained stable,
but diastolic stiffness progressively increased (Fig.
6A). When LVEDP had increased by 9.7 ± 0.5 mmHg (P < 0.001, after 31 ± 1 min ischemia), calcium more than doubled the developed pressure
to 45 ± 8 mmHg (P < 0.01) and simultaneously
accelerated relaxation velocity (
pre- vs. postintervention = 58 ± 5 vs. 47 ± 5 ms, P < 0.01), but
failed to affect increased diastolic stiffness (LVEDP pre- vs.
postintervention = 19 ± 0.5 vs. 23 ± 3, NS) (Fig.
6B).

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Fig. 6.
Effect of calcium during ischemic diastolic dysfunction.
A: representative tracing. Ischemia (coronary artery
perfusion pressure reduced to 18 mmHg) reduced systolic pressure, to 23 mmHg, and relaxation rate ( increased to 52 ± 2 ms). Diastolic
stiffness gradually increased. When LVEDP had increased to 19 mmHg (28 min), calcium (10 mM Ca2+) increased contractility
(systolic pressure from 35 to 58 mmHg, +dP/dt from 180 to
650 mmHg/s), indicating increased intracellular calcium concentration.
However, increased LVEDP did not further increase, indicating preserved
calcium resequestration ability. Calcium accelerated rate of relaxation
( diminished to 36 ± 1 ms, dP/dt increased to
580 mmHg/s) without reducing LVEDP, suggesting that rate of
relaxation was not linked to diastolic chamber stiffness (indexed by
LVEDP, measured on the flat portion of the diastolic tracing when
dP/dt equaled zero). B: group data.
Ischemia reduced LV developed pressure, isovolumic LVEDP, and
rate of relaxation ( increased). During continued ischemia,
contractile function and remained constant, but diastolic stiffness
gradually increased. Calcium increased LV developed pressure, but did
not increase diastolic stiffness further, indicating preserved calcium
resequestration ability during ischemic diastolic dysfunction.
Calcium increased velocity of relaxation (reduced ) without reducing
increased diastolic stiffness.
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Hence, although rate of relaxation was significantly reduced by
ischemia, it was corrected by deliberate calcium loading (
reversed to baseline values) during initial ischemia, when
diastolic stiffness was reduced, and also after prolonged low-flow
perfusion, when diastolic stiffness had increased. In each case,
the calcium-induced acceleration in velocity of relaxation failed
to affect diastolic stiffness while preserving the flat diastolic
period between systolic contractions (Figs. 5A and
6A). These results suggest that the reduced rate of
relaxation observed during ischemia does not equate with a
limited calcium-handling capacity and that changes in the rate can
occur independently of diastolic stiffness, i.e., the two are not
linked during ischemia.
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DISCUSSION |
The principal findings in this study were that increased diastolic
stiffness could develop during severe contractile dysfunction and
resulted from rigor and not diastolic calcium-driven cross-bridge cycling. The ischemia-induced reduction in relaxation rate was insufficient to affect the end-diastolic pressure-volume relation and
could be reversed (by calcium) without affecting increased diastolic
stiffness, suggesting that these were not coupled. Thus ischemia did not result in diminished calcium-handling capacity and its effects on relaxation rate and diastolic stiffness resulted from separate mechanisms.
Hemodynamic function.
An 85% reduction of coronary flow simulates acute infarction/coronary
ligation regions, in which extremely low perfusion continues via
collateral circulation (22). We recreated this globally, i.e., in the whole LV simulated an underperfused area. Ischemia decreased contractile function immediately, which then remained stable.
Diastolic distensibility initially increased (LVEDP decreased from 10 to 8 ± 0.5 mmHg, P < 0.005). This phenomenon has
been postulated to result from either vascular decompression, i.e., a
turgor effect, and/or intracellular metabolite accumulation (3,
5, 6, 26, 35). In a previous study (9) in isolated
hearts, the degree of underperfusion used here resulted in
intracellular acidosis (pH reduction to 6.2) and a 300% increase in
inorganic phosphate. These effects, by reducing myofilament calcium
sensitivity, may prevent increased diastolic calcium expressing effects
on diastolic tone and protect against the development of increased
diastolic tension. In the current model, the initial decrease in
diastolic stiffness was followed by a progressive increase, but without
any further alteration in contractile function (developed pressure and
+dP/dt remained stable), i.e., increased diastolic stiffness
developed despite, and independent of severely diminished
contractility. In our model, metabolite washout, although reduced,
continues during continued low, constant-rate perfusion (4,
11), and intracellular pH remains stable (9).
Hence, the temporal dissociation between contractile (initially
reduced, then stable) and diastolic (initially increased, followed by a progressive decrease in distensibility) function did not support a
mechanism of metabolite-mediated changing myofilament calcium sensitivity, accounting for the changes in diastolic function occurring
during prolonged low-flow ischemia. In such a case, diastolic
and contractile responses should have been paired. Similarly, contractile function remained constant both pre-QSR, when diastolic stiffness was increasing, and post-QSR, when it was reversed (Fig. 3B). Responses to NH4Cl-mediated intracellular
pH changes suggested that calcium sensitivity during ischemia
was preserved, to some extent, at calcium concentrations able to
generate force. Our model of low-flow ischemia illustrated the
separate effects of ischemia on contractile and diastolic
function, in contrast to experiments with zero-flow ischemia
resulting in complete contractile failure.
Increased diastolic stiffness: calcium versus ATP.
Ischemia-induced ATP depletion may cause sustained diastolic
actin-myosin interaction by the following: 1) diastolic
persistence of increased intracellular calcium concentration (due to
energy-limited ion pumps), 2) directly, resulting in failure
of the rigor complex to dissociate in the final stage of the
cross-bridge cycle, or 3) a combination of these mechanisms.
A calcium-mediated mechanism, widely favored, seems to be supported by
the observation of increased diastolic calcium, which occurs with
hypoxia, zero-flow ischemia, or metabolic inhibition (6,
7, 18). However, the relation of these experimental models to
clinical ischemic conditions is unclear. Furthermore, attempts
to correlate observations of increasing myocyte calcium concentration
to development of contracture do not necessarily confirm cause-effect
relationships. Thus some studies (2, 20) reported no
correlation, and increased diastolic calcium, assessed by calcium
indicators, may not reflect troponin C-bound calcium (10).
In contrast to these previous studies, here we assessed responses to
interventions designed to differentiate calcium- versus
rigor-mediated mechanisms, after stiffness had increased.
NH4Cl and BDM failed to affect increased diastolic stiffness but, in striking contrast, markedly influenced contractile function, illustrating that these interventions achieved their intended
intracellular action on calcium-activated cross-bridge cycling and
hence calcium-activated tension (Figs. 1 and 2). These results
therefore did not support a causative role for diastolic calcium-driven
cross-bridge cycling for increased diastolic stiffness, despite the
reported increase in diastolic calcium concentration during
ischemia. This conclusion was supported by quick length changes, which perfectly lysed ischemia-induced increased
stiffness, i.e., a response typical for a rigor mechanism, without any
calcium-mediated component (37) (Fig. 3).
The results support a mechanism of ATP depletion directly affecting
cross-bridge detachment underlying increased diastolic chamber
stiffness during ischemia. However, previous studies, including
ours (10, 23, 37), have failed to demonstrate a lower
average tissue [ATP] in hearts subjected to either supply or demand
ischemia, in which an increase in diastolic chamber stiffness
occurred, compared with hearts subjected to similar ischemia,
in which no increase in diastolic tension occurred. Thus the degree of
diastolic dysfunction sustained failed to correlate with the level of
depletion of high-energy phosphates during ischemia (32). To explain our results, we hypothesize that locked
cross bridges may develop during ischemia in only a few, more
vulnerable myocytes. Total tissue ATP estimations may then not reveal a
critical reduction in this minority. Additionally, tissue ATP
concentrations do not reflect turnover rates. Thus improved mechanical
function during ischemia in response to glycolytic substrate,
though markedly enhancing glycolytic ATP flux did not dramatically
alter total ATP content (9). Rigor development may be
modulated by other metabolites, e.g., increased [ADP] facilitated
rigor tension in the presence of only modest reductions in [ATP]
(39), and correlated with increased diastolic stiffness
(36).
The emergence of two distinct populations of myocytes in response
ischemia may also explain the dichotomous hemodynamic effects observed in this study, where diastolic stiffness increased but contractile function remained stable. One group, containing a relatively small number of cardiomyocytes, developed rigor, becoming inexcitable and incapable of developing contractile force (6, 17). These increased diastolic pressure in proportion to their number, which progressively increased during continued ischemia (but recovered function when normal perfusion conditions were restored
early). The second group comprised the majority of myocytes, which
continued to contract actively and generate phasic LV pressures, responsive to perturbations of calcium availability or sensitivity (e.g., BDM and NH4Cl), manifest as altered contractile
function. The contractility of these ischemic, contracting
cells may have been increased by stretch exerted by adjacent myocytes
in rigor (by a Frank-Starling effect and enhanced calcium sensitivity), acting to preserve overall contractile function. When ischemic contracting myocytes relengthened, they then contributed to relaxation dynamics in the whole heart. Thus a minority of myocytes developing rigor may have exerted sufficient effect to increase isovolumic LVEDP,
but remained insufficient to significantly reduce developed pressure.
This process may only be manifest during the delicate supply-demand
imbalance of the low-flow ischemic condition imposed in this
study, which is unlike experimental states of either zero-flow ischemia or prolonged metabolic inhibition, in which
irreversible contracture, calcium release, and cell death ensues
relatively rapidly in all myocytes. However, more prolonged
ischemia may eventually have resulted in sufficient myocyte
loss to diminish contractile function concomitantly with increased
diastolic tension.
Evidence for the development of two such populations of mycoytes during
low-flow ischemia was not directly demonstrated in the current
study. However, heterogenous responses occurring at both myocardial and
myocyte levels in response to conditions of energy limitation have been
reported previously. An examination of tissue oxygen gradients in
isolated hearts with the use of NADH fluorescence (33, 34)
revealed that inadequate oxygen delivery resulted in relatively large,
well-defined anoxic areas, developing within minutes of
imposition of global low-flow perfusion. Flow into these areas was
significantly reduced. The size of these areas remained stable, but
could be increased by increasing myocardial work by higher paced heart
rates, i.e., enhancing the supply-demand imbalance, and by acidosis.
[Such conditions are likely to have been reproduced in the current
study with the use of steady-state low-flow ischemia, with
intact beating hearts, under perfusion conditions demonstrated to
result in intracellular acidosis (9)]. The anoxic areas
could be reversed by restoring normal perfusion conditions, and
reproducibly recreated. The border zones between aerobic and anoxic
tissue were characteristically sharply defined, indicating a negligible
volume of only partially anaerobic myocardium. The results indicated
the development of two distinct populations of mitochondria: those
completely anerobic, juxtaposed with others with completely aerobic
function, i.e., regions where oxygen supply was sufficient to maintain
normal oxidative phosphorylation. The effect of this process may be the
development of mycoytes in rigor sited adjacently to others that
continue to contract. This is supported by ultrastructural studies in
isolated hearts during global low-flow ischemia, illustrating
some myocytes in contracture juxtaposed to cells with near-normal
ultrastructure (4). The condition may be exaggerated in
the subendocardium, which is more vulnerable to ischemia
(23, 41), particularly in hypertrophy (13,
29). Additionally, isolated myocytes exposed to metabolic inhibition demonstrated a variable time to onset of contracture (19). Thus some myocytes appear to be more susceptible to
energy deprivation, illustrating intermyocyte heterogeneity. Our
results therefore may demonstrate the hemodynamic sequelae of these
heterogenous responses to ischemia, where a fraction of
myocytes in severely ischemic tissue develop contracture,
whereas others in adjacent tissue with preserved flow and oxygen supply
(at a reduced level) continue to support contractile activity.
Relaxation rate.
Pressure decay closely followed calcium transient decay in myocytes,
reflecting the kinetics of calcium resequestration (21). In isovolumically contracting hearts, pressure and calcium transient decay increased in parallel with progressively reduced coronary (crystalloid) flow (7). This apparent "coupling" was
interpreted to reflect increasingly compromised calcium-handling
ability (due to energy-limited ion pumps) with increasing severity of
"ischemia," resulting in increased diastolic stiffness
(incomplete diastolic calcium clearance resulting in residual
cross-bridge cycling and thus persistent diastolic tension). However,
this presumed that the magnitude of reduction in relaxation rate was
sufficient to shift the diastolic pressure-volume relation and that
factors determining diastolic stiffness were identical to those
determining relaxation rate during ischemia (i.e., changes
occurring in diastolic stiffness should be accompanied by changes in
relaxation rate in the same direction) (24). Because our
results demonstrated a rigor- and not calcium-mediated mechanism for
increased diastolic stiffness during ischemia, we reasoned that
decreased relaxation rate (which is calcium related) should be
insufficient to affect end-diastolic pressure and be independent of
diastolic stiffness because these would be driven by different
mechanisms (24). Furthermore, if reduced relaxation rate
during ischemia implied impaired calcium reseqestration
ability, then an additional calcium load should be unable to accelerate
relaxation rate, but instead drive a further increase in diastolic stiffness.
Global low-flow ischemia reduced relaxation velocity (increased
) and prolonged the relaxation phase relative to the entire contraction-relaxation cycle (T90/TP + T90) (Fig. 4). Despite this, overall relaxation time
(T90) decreased because the mechanical transient was
abbreviated. Hence, counterintuitively, earlier pressure dissipation
occurred during low-flow perfusion, i.e., slowed relaxation became less
likely to influence end-diastolic pressure. In these experiments, we
assured complete relaxation, during constant baseline heart rates,
evidenced by a flat diastolic pressure, during which maximal negative
LV pressure derivative remained at zero (Figs. 4, 5A, and
6A). This flat diastolic period, during which diastolic
function is determined by passive chamber properties, actually
prolonged during ischemia, whether diastolic stiffness
decreased or increased, and remained flat even during calcium loading
(Figs. 5A and 6A). Therefore, systole did not commence before the end of the previous diastole, i.e., there was no
evidence for diastolic tetanization. Previously, incomplete relaxation
has been postulated to occur when
increases by 3.5 times
(13), but an increase of this magnitude did not occur here. This calculation assumes that pressure decays from the same point
in time (i.e., TP) and the same peak systolic pressure. However, during
ischemia, this decay commences earlier, and from a lower peak
pressure, and hence
has to increase >3.5 times to be able to
affect end-diastolic pressure (at constant heart rates).
Relaxation rate was independent of stiffness. Thus
increased with ischemia, but then remained constant, whether
diastolic stiffness decreased (e.g., with ischemia onset) or
increased. Calcium-driven acceleration of relaxation failed to reduce
diastolic stiffness (Fig. 5), even when this had increased (Fig. 6).
Thus reduced rate of relaxation during ischemia could be
corrected without altering diastolic stiffness in the same direction,
i.e., they were not linked. Similarly, exercise in patients with
ischemia shortened
but simultaneously increased diastolic
stiffness (8). The separation of the negative lusitropic
effects of ischemia namely reduced relaxation velocity and
increased stiffness, implies that they are not determined by the same
mechanism (24). Thus rate of relaxation reflects calcium
transient decline (21) and is necessarily calcium
dependent, contrasting with ischemia-related increased
diastolic stiffness, which is calcium independent, supporting our
earlier conclusion that this results from an actin-myosin interaction
from rigor. The failure of a deliberate calcium load to drive a further
increase in diastolic stiffness implies intact resequestration ability
during ischemia and hence provides further evidence against a
calcium-driven mechanism for increased diastolic tension. The
interesting observation of calcium-mediated acceleration of relaxation
rate may indicate an acceleration in calcium resequestration rate via a
sarcoplasmic reticulum responsive to an increased load or,
alternatively, an effect of increased contractility [although
is
reported to be a load-insensitive index of relaxation-velocity (13)].
Sarcoplasmic reticulum.
The results suggest that ischemia does not result in
sarcoplasmic reticulum dysfunction, despite its high metabolic
requirement. Constant contractility was maintained during a progressive
increase in diastolic stiffness suggesting a constant, reduced level of excitation-contraction coupling without progressive sarcoplasmic reticulum emptying. This conclusion in intact beating hearts is supported by the observation that isolated myocytes subjected to
metabolic stress maintained intact and responsive sarcoplasmic reticulum function, which was unaccompanied by depletion of calcium stores (14). This contrasts with a state where the
sarcoplasmic reticulum was deliberately disabled by caffeine
(21). The contraction-relaxation cycle was prolonged.
Relaxation velocity slowed to an extent where systole commenced before
the completion of the previous diastole (thus no flat diastolic
period), and increased diastolic tension resulted because active cross
bridges remained at end diastole ("tetanization"). Under these
conditions, reduced rate of relaxation was linked to increased
stiffness (24), which was unaffected by QSR
(37).
Supply versus demand ischemia.
Our results suggest that increased diastolic stiffness occurring in
both supply and demand ischemia share a common subcellular mechanism (37, 38), i.e., these states may not be
qualitatively distinct but represent variations in the imbalance of
myocardial oxygen supply relative to demand. This explains the clinical
and experimental observation that the distinction between supply
(predominant contractile dysfunction) and demand (predominant diastolic
dysfunction) may not always be clear because either may result in mixed
effects (3, 26, 35). The relative degrees of contractile
and diastolic dysfunction elicited may be determined by the outcome of
the balance among perfusion level, metabolic demand, and time. Reduced
perfusion determines contractile dysfunction ("perfusion-contraction
matching"), which may be set by nonenergetic factors (14,
28). In contrast, diastolic dysfunction does seem to be related
to reduced ATP. In demand ischemia, where perfusion is usually
maintained but metabolic demand exceeds supply, contractile dysfunction
is minimal and diastolic dysfunction predominates and occurs early
because ATP may be relatively rapidly driven down by repetitive
depolarizations during tachycardia. Prolonged underperfusion, during
constant low heart rate, represented a supply-demand imbalance less
severe than that associated with a superimposed tachycardia, resulting in a slower, and later, increase in stiffness. The duration, extent, and severity of ischemia relative to demand underlie the
differences. Species differences may also contribute, e.g., diastolic
stiffness increases earlier in rodent hearts compared with larger
mammalian hearts (5).
Ramifications of study.
This study offers a novel insight into diastolic response of the
myocytes to ischemia. We infer that cross-bridge detachment of
the actin-myosin "rigor complex" formed during the cross-bridge cycle is more sensitive to disturbed high-energy phosphate
concentrations resulting from ischemia than ionic pump activity
governing calcium homeostasis (which remain intact). Although
rigor-bond formation is regarded as the final result of prolonged
zero-flow ischemia (i.e., "stone heart") (16),
our results suggest that it appears as the earliest diastolic response
to ischemia (supply or demand), develops independent of changes
in contractility, and lesser degrees of which may be reversible with
early reperfusion (e.g., post-QSR; Fig. 3A), when the
supply-demand mismatch is corrected (37) or when the ATP
supply is increased by glycolytic substrate (9, 38). The
results appear to support a heterogenous myocyte response to conditions
of energy limitation. Ischemia illustrates a condition where
relaxation rate is determined by a separate mechanism to extent (i.e.,
diastolic stiffness) (13, 24). The abbreviated contraction-relaxation cycle (Fig. 4) may be the corollary of an
abbreviated action potential due to intracellular ATP-sensitive K+ channel activation also resulting from
ischemia-related reduced cytosolic ATP-to-ADP ratios
(30).
Advantages of model.
Our unique model permitted observation of the salient effects of
ischemia. A red blood cell colloid perfusate at normal
hematocrit and temperature is critical for imposition of a true, stable
low-flow state. In contrast, ischemia surrogates, e.g., hearts
subjected to hypoxia, or isolated muscle strips or myocytes subjected
to metabolic inhibition, do not reproduce true ischemia and may
introduce artifacts. High (nonphysiological) flow rates during
crystalloid perfusion contribute independent effects to preserve
contraction (via sarcomere stretch) and increase diastolic stiffness
(via vascular engorgement), and may not evince the results of this study. Thus "ischemia" (20% of baseline crystalloid flow)
reduced the relaxation rate, without abbreviating the mechanical
transient (7). Reduction to even 10% of baseline
perfusion levels failed to approach true ischemic coronary flow
rates. Hypoxic crystalloid perfusion is paradoxically associated with
even higher flow rates, with no concomitant intracellular pH reduction
(9), and does not shorten the duration of
contraction-relaxation (18). Significantly, in a previous
study (37), the diastolic pathophysiology of demand ischemia could not be reproduced during crystalloid perfusion, but was successfully recreated with blood perfusate. Perfusate temperature <37°C alter cellular functions and may result in
prolonged mechanical transients with a reduced rate of relaxation
(18). Here, meticulously controlled constant flow rates
eliminated vascular engorgement effects (13, 40), and
37°C ensured physiological ion pump function, e.g., of the
sarcoplasmic reticulum. Interpretation of relaxation rate was
facilitated in this isovolumic model that eliminated confounding
effects of viscoelastic factors, volume loading, filling
(13), and lengthening loads of early diastole (31), and homogenous global ischemia
eliminated segmental dysynchrony (13). The right
ventricle was decompressed and the pericardium freed, eliminating
potential effects of ventricular interaction (13).
Study limitations.
We did not measure calcium concentrations or sensitivity in response to
perfused agents. Exposure to calcium, NH4Cl, and BDM, in
the concentrations we used, translates into the intended intracellular actions at the myofilament level, which has been well characterized by
others (1, 14, 21). Because only reduced perfusion was imposed on hearts, preserving active contraction, we inferred intracellular actions from changes in LV contractile pressure, i.e., a
known calcium-dependent force, as an "internal control," to compare
with diastolic pressure. We did not measure high-energy phosphates or
intracellular pH, but have done so previously during comparable
low-flow ischemia (9).
The isovolumic model is widely used for studying diastolic dysfunction.
Although this facilitates experimental study, results should be
carefully extrapolated to the in vivo condition, where isovolumic
relaxation represents a shorter phase of diastole. The reduced
relaxation rate observed during clinical angina, by upward displacement
of the early part of the diastolic pressure-volume relation, may have
the clinical sequelae of affecting early diastolic filling, mean left
atrial pressure, and coronary artery flow (13, 15, 31).
In conclusion, although conditions of energy deprivation have been
postulated to impair calcium homeostasis and thereby increase diastolic
tension, in this study true ischemia had a different effect,
where disturbed high-energy phosphate metabolism directly affected the
cross-bridge cycle to create rigor bonds (and thus increase diastolic
stiffness) unrelated to either diminished rate of relaxation or
impaired calcium reuptake.
 |
ACKNOWLEDGEMENTS |
We thank Dr. Hinrik Stromer for performing calculations of
and
Soeun Ngoy for excellent technical assistance.
 |
FOOTNOTES |
This study was supported by National Heart, Lung, and Blood Institute
Grant HL-48175. N. Varma received the Physician-Investigator Fellowship
of the American Heart Association, Massachusetts Affiliate, 13-614-923.
Address for reprint requests and other correspondence: N. Varma, Dept. of Cardiology, Univ. Hospital of Cleveland, Lakeside 3085, Case Western Reserve Univ., 11100 Euclid Ave., Cleveland OH 44106 (E-mail: nxv11{at}po.cwru.edu).
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
First published October 31, 2002;10.1152/ajpheart.00286.2002
Received 3 April 2002; accepted in final form 12 October 2002.
 |
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