AJP - Heart Calcium Transients and Cell-Sarcomere
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Am J Physiol Heart Circ Physiol 284: H758-H771, 2003. First published October 31, 2002; doi:10.1152/ajpheart.00286.2002
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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


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Increased diastolic chamber stiffness (up-arrow 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 up-arrow DCS. Second, quick length changes reversed up-arrow 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 up-arrow DCS, hearts were exposed to 10 mmol/l calcium. Calcium doubled contractility and accelerated relaxation velocity, but without affecting up-arrow DCS. Thus up-arrow 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 up-arrow DCS.

stiffness; left ventricular end-diastolic pressure; quick length change; heterogeneity


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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.


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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 approx 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 (tau ) 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 tau , 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 tau  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. tau  was calculated by the variable asymptote method (P = P0e-t/tau  + 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.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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.

                              
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  		Table 1.   Characteristics of groups before interventions


                              
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  		Table 2.   Effect of interventions

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 approx 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. up-arrow  and down-arrow , 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.

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.

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.

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 tau  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 3.   Characteristics of isovolumic contraction and relaxation at baseline and during ischemia


                              
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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 (tau ) 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.

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: tau  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. tau  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 (tau  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 tau ). Calcium increased developed pressure, indicating increased intracellular calcium concentration, and reduced tau , but failed to affect LVEDP.

During continued ischemia, contractile function (developed pressure, 22 ± 3 mmHg) and tau  (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 (tau  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 (tau  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 (tau  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 (tau  increased). During continued ischemia, contractile function and tau  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 tau ) without reducing increased diastolic stiffness.

Hence, although rate of relaxation was significantly reduced by ischemia, it was corrected by deliberate calcium loading (tau  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.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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 tau ) 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 tau  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 tau  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 tau  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 tau  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 tau  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 tau  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.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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