INTRODUCTION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

THE ATRIOVENTRICULAR pressure gradient is the motive force behind left ventricular diastolic filling. The ventricular component of the atrioventricular pressure gradient depends on both the rate and duration of ventricular relaxation, an active process, and chamber elasticity, a passive process. Ischemia slows left ventricular filling (37, 48) at least in part because ischemia slows left ventricular relaxation (19, 26, 32, 45, 55). Ischemia also affects the passive elastic characteristics of the myocardium and left ventricle (36, 58). To study the role of the ventricular components of the atrioventricular pressure gradient, it is necessary to separate the effects of active relaxation and passive filling that occur simultaneously. There is also evidence from studies in whole hearts (30, 53) and isolated muscle (20, 21, 52) that muscle length or stretch affects relaxation. In addition, studies in isolated muscle demonstrate that ischemia modulates the effects of length or stretch on relaxation (31). Although there have been some detailed studies on the rate and duration of relaxation and the effects of early filling on relaxation in normal ventricles (35, 36), there have been no studies on the effects of ischemia on the time course of left ventricular relaxation or the effect of stretch due to filling on the rate, duration, and extent of relaxation in intact hearts.

To separate the active component of ventricular relaxation and the passive component due to the diastolic pressure-volume curve (i.e., filling), we implanted an artificial mitral valve that can be closed at end systole to completely prevent the ventricle from filling (39, 47). This end-systolic occlusion allows the ventricle to relax completely in the absence of filling and without a subsequent increase in pressure due to stretch of the passive myocardium. We also occluded the mitral valve at progressive times during left ventricular filling to investigate the relationship between ventricular filling (stretch of the myocardium) and relaxation. We studied the effect of regional ischemia on the rate and extent of left ventricular relaxation independent of the passive effects of filling and the effects of stretch (via increasing amounts of filling) on the rate of relaxation. This study shows that ventricular filling slows the rate of ventricular relaxation and that regional ischemia further slows the rate of relaxation and increases the sensitivity of relaxation rate to ventricular volume.

	METHODS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

The experiment was approved by the Committee on Animal Research at the University of California and conforms to the guiding principles of the American Physiological Society.

Surgical preparation. Juvenile pigs (32-43 kg) were premedicated with a subcutaneous injection of ketamine (20 mg/kg) mixed with xylazine (2 mg/kg) and atropine (0.5 mg), which was given in the neck, and then they were placed on the operating room table and anesthetized with -chloralose (100 mg/kg) via an ear vein. Each pig was intubated and mechanically ventilated with room air. A femoral vein was catheterized for the introduction of additional anesthetics, for blood gas sampling, and for a lidocaine drip (0.6 mg/min). Anesthesia was maintained with fentanyl (30 µg) and pancuronium (4 mg) every 20 min or as needed. The pigs were placed in the supine position, a midline sternotomy was performed, and the pericardium was opened to create a pericardial cradle. Blood gases were measured after each intervention to maintain the pH between 7.40 and 7.50, PCO₂ between 42 and 46 mmHg, and PO₂ between 95 and 100 mmHg by infusing sodium bicarbonate, adjusting oxygen flow, or adjusting the respirator.

Hemodynamic instrumentation. Two 5-Fr Millar micromanometers were inserted: one through the anterior wall of the left atrial appendage and the other through the apex of the left ventricle to measure left atrial and left ventricular pressures (Fig. 1). A third micromanometer was inserted into the right ventricle through the anterior wall of the right ventricle to measure right ventricular pressure. The micromanometers were warmed to 37°C overnight and then zeroed and calibrated against a mercury manometer. An ultrasonic flow transducer (Transonic Systems, Ithaca, NY) was placed around the left anterior descending coronary artery. Two pairs of endocardial segment-length crystals (Sonometrics, London, Ontario, Canada), 1-cm apart perpendicular to the long axis and parallel to the circumferential fibers of the ventricular wall, were used to measure regional myocardial function: one pair in the region perfused by the left anterior descending coronary artery and another pair in the region perfused by the circumflex coronary artery.

View larger version (22K): [in this window] [in a new window]   Fig. 1.   Typical hemodynamic pressures and volumes comparing normal, partial, and nonfilling beats at baseline (A) and during (B) ischemia. LV, left ventricular; RV, right ventricular; LA, left atrial; *, beginning of mitral occlusion.

A 7-Fr eight-electrode conductance catheter (Webster Laboratories, Baldwin Park, CA) was placed in the left ventricle to measure left ventricular volume via an apical approach and was connected to electronics (Leycom-Sigma 5, Leiden, The Netherlands) that convert the conductance signal into a volume. Proper positioning of the conductance catheter was checked echocardiographically and from the contours of the conductance catheter segment volume signals. A standard calibration function was used to correct the raw conductance catheter signal by adjusting for the parallel conductance volume and slope in each pig. This correction was estimated by comparing the conductance volumes (V_c) with volumes measured using two-dimensional echocardiography (V) during baseline and ischemia in each pig according to where 1/_c is the slope of the relation between conductance volumes and two-dimensional echocardiographically measured volumes (4, 8). The echocardiographic measurements were performed using a Hewlett-Packard ultrasound system (Sonos 2500) equipped with 2.5/3.5-MHz and 5.0-MHz transducers. An epicardial approach was used, positioning the transducer over the left ventricular apical dimple. Biplane recordings of the left ventricular cavity were obtained adjusting transducer angulation to maximize the left ventricular long axis. _c and V_c were determined by fitting this equation with a linear regression to the largest and smallest echocardiographic volumes determined using Simpson's rule during baseline and ischemia, which has been validated against cineangiography (7, 44) and magnetic resonance imaging (25). These echocardiographic volumes were compared with the largest and smallest volumes measured under the same steady-state hemodynamic conditions (but not necessarily the same beats) with the conductance catheter. The parallel conductance volume was 51.2 ± 8.4 (SD) ml, and _c was 0.79 ± 0.16.

A pair of pacing wires was placed on the right atrial appendage. The hearts were slowed by using zatebradine (ULFS-49), a bradycardic agent that acts on the sinoatrial node without hemodynamic effects (27), and the hearts were paced at 70 beats/min during the experiment.

A C-clamp left anterior descending constrictor was placed proximal to the ultrasonic flow transducer around the left anterior descending coronary artery to reduce coronary flow and, consequently, regional myocardial function (51). The inferior vena cava was dissected, and umbilical tape was placed around it to form a snare (IVC occluder), which could be constricted to define the diastolic pressure-volume curve over a wide range of end-diastolic volumes both before and after reduction of coronary flow.

Left ventricular volume clamp. The heart was instrumented with a remote-controlled mitral valve that can be closed to prevent filling at any time during the cardiac cycle (39, 47). A waterproof funnel made of polyester-cotton blend fabric was sutured around the left atrial appendage. A 1-0 braided polyester fiber (Ticron) suture with the point of the needle blunted was pushed into the right atrium at its junction to the inferior vena cava and pushed out from the superior-medial portion of the right atrium, close to the left atrium, and then anchored to the left atrial wall. After systemic heparinization (2,000 U/kg), the left atrium was opened and a modified 25-mm Bjork-Shiley valve was inserted through the funnel into the atrium and placed between the native mitral valve and the pulmonary veins. The 1-0 Ticron suture was then tightened into the groove of the prosthetic valve assembly and secured in place. The blood in the funnel was then squeezed back into the atrium and the excess funnel material tied off so the remaining funnel area was approximately the size of the native atrial chamber. The control cable for the mitral valve was attached to a solenoid system that alternately moves the control cable forward to occlude flow and backward to allow normal physiological flow.

In some beats, we activated the volume clamp during systole and held it throughout the subsequent diastole, preventing all filling. In others, we applied the clamp at various times through diastole, which permitted some filling before the occlusion of the mitral valve (Figs. 1 and 2). The first occlusion occurs during systole before the onset of filling to prevent any filling during diastole. The next occlusion occurs 40 ms later after the onset of filling, preventing filling from that point until the end of diastole. Then the next occlusion occurs 40 ms later during midearly filling, preventing the rest of filling and so on throughout diastole. The clamp was applied for one beat every fifth beat while data was recorded.

View larger version (23K): [in this window] [in a new window]   Fig. 2.   Passive diastolic pressure-volume relation at baseline (A-C) and during ischemia (D-F). A: LV volume. Numbers 1-5 mark times of mitral valve occlusions with number 1 reflecting a nonfilling beat and number 5 reflecting a normal filling beat. B: LV pressure. , Times of mitral valve occlusion; numbers 1-5 () show minimum pressure achieved following a mitral occlusion. C: pressure-volume relation determined from plotting A and B. , Passive pressure-volume points related to increasing volume from mitral occlusions in A. Solid line is a normally filling pressure-volume relation. D-F, volume, pressure, and passive pressure-volume relation during ischemia, respectively.

Hemodynamic measurements. Hemodynamic variables were measured from the left atrial and left ventricular pressure waveforms. Left ventricular end-diastolic pressure was taken at the rapid upstroke in the left ventricular pressure, which occurred at ~10% of the maximal rate of pressure development (dP/dt_max). Left atrial pressure crossover of the left ventricular pressure, which occurs when the mitral valve opens, was determined from the matched left atrial and left ventricular pressure traces. The pressures were matched by adjusting the left atrial and left ventricular pressure waveform during middiastole at the beginning of the experiment. The atrioventricular pressure gradient was quantified by calculating the time integral between left atrial and left ventricular pressure from mitral valve opening until the first reversal of pressure crossover and by the mean gradient during this time.

The extent of relaxation (P) is the pressure in the fully relaxed ventricle at any given volume. P was determined experimentally by performing a mitral occlusion at end systole, which prevents filling and allows the ventricle to relax completely isovolumically, and by measuring the minimum pressure.

The time constant of isovolumic relaxation () was determined by fitting the left ventricular pressure between the times of minimum rate of pressure development (dP/dt_min) and left atrial pressure crossover to P = (P₀  P)e^t/ + P (36, 58), where P₀ is the pressure at time 0, and P is the corresponding fully relaxed pressure on a nonfilling beat at the same left ventricular volume.  was estimated by linear regression of ln(P  P) against time t. When the rate of relaxation of the nonfilling beats during baseline and ischemia are compared,  of the occluded beat was calculated (Fig. 1). When the effect of filling on relaxation by occluding throughout diastole is calculated,  of the subsequent beat was calculated.

Determination of duration of relaxation. The duration of relaxation is calculated by occluding the mitral valve progressively later during filling to identify the point in time at which preventing additional filling is not associated with a pressure drop, indicating that the ventricular pressure is changing only because of moving up the passive diastolic pressure-volume curve and not because of active relaxation (36). The duration of relaxation was measured from dP/dt_min. If the ventricular pressure continues to fall after the mitral occlusion, then relaxation is not yet completed at the time of the occlusion. A mitral occlusion when the ventricular pressure does not fall marks the time of the end of relaxation.

Figure 2 shows left ventricular volumes and pressures during diastole for beats with mitral occlusions performed progressively throughout diastole. Figure 2A shows the volumes and Fig. 2B shows the left ventricular pressure decay after mitral occlusions were performed progressively throughout diastole. Figure 2C shows the corresponding data plotted in the pressure-volume plane, together with a pressure-volume curve for a normal filling beat. During baseline, points 1-3 fall below the normal filling pressure trace (solid line associated with point 5 in Fig. 2B) and points 4-5 fall on the pressure trace, whereas, during ischemia, points 1-2 fall below the normal filling pressure trace, point 3 falls slightly below the pressure trace, and points 4-5 fall on the pressure trace (Fig. 2E).

Characterization of passive left ventricular diastolic pressure-volume relation. The left ventricular diastolic pressure-volume relation was characterized using Nikolic's approach (36) where S_p is a parameter that describes the curvature of the diastolic pressure-volume relation, V₀ is the equilibrium volume, and V_m is the maximum attainable volume of the ventricular chamber. The parameters S_p, V_m, and V₀ were estimated from nonfilling beats using the Marquardt-Levenberg nonlinear regression in SigmaStat v2.03.

Characterization of passive myocardial stress-strain relation. To estimate the myocardial stress-strain relation, we used the ventricular pressure-volume relation based on a thick-walled version of the Laplace relation and the exponential stress-strain relation for the myocardium (18). The passive diastolic stress-strain relation that describes the nonlinear stiffness of the myocardium is (18) where  is stress (force/area in the myocardial wall);  is Lagrangian strain = (l  l₀)/l₀, where l  l₀ is the fractional extension from rest length (l₀), and  and  are parameters that describe muscle stiffness.  and  were calculated directly from the diastolic pressure-volume relation using (18) where The V₀ is taken as a fixed value obtained from fitting Nikolic's equation to the data. To characterize the changes in the passive elastic properties of the myocardium, this equation was used to fit the end-diastolic pressure-volume data points from the vena caval occlusion performed before and after regional ischemia. The ln was used as the parameter for estimation purposes to provide a better conditioned nonlinear parameter estimation using SigmaStat v2.03.

Systolic pressure-volume relation. The slope of the end-systolic pressure-volume relation (E_max), an index of contractility (13), was determined by fitting a straight line to the end-systolic pressure (P_es) and volume (V_es) data obtained during inferior vena caval occlusions using the method by Kono et al. (29) to where V_d is the volume-axis intercept of the end-systolic pressure-volume relation (49). (Note that the V_es defined from the pressure-volume loop is slightly larger than the minimum volume, which we used to calibrate the conductance catheter. V_es reported in the tables and discussed in the RESULTS is end-systolic volume defined from the pressure-volume loop.)

Protocol. We collected data at baseline and during ischemia. Ischemia was produced by reducing left anterior descending coronary artery flow until regional function, quantified by absolute systolic segment-length shortening, was reduced by at least 20%, based on the segment length at the time of left ventricular end diastole. (Ischemia was maintained for 45-75 min, depending on the ease with which we maintained a steady state.) Three interventions were performed during each state: 1) mitral occlusion at end systole (end-systolic occlusions) during steady state to determine the effect of ischemia on the rate of relaxation comparing the filling and nonfilling left ventricle; 2) mitral occlusions performed progressively throughout diastole to progressively increase the amount of ventricular filling to determine how myocardial length or stretch affected the rate of relaxation; and 3) mitral occlusions performed at end systole during an inferior vena caval occlusion in a nonfilling beat at a given end-diastolic volume to determine the effect of ischemia on the extent of left ventricular relaxation.

Statistical analysis. The end-systolic occlusion data were analyzed using two-way repeated-measures analysis of variance to determine the effects of ischemia and ventricular filling. (Because there are only two levels of each factor, we did not need to use a multiple comparison procedure.) We report the least square means from the ANOVA and associated standard errors. The means for treatment reflect the main effect of ischemia independent of ventricular filling, and the means for ventricular filling reflect the main effect of ventricular filling independent of the presence or absence of ischemia. Comparisons between two conditions (for example, when there were no occluded beats) were done with paired or unpaired t-tests, as appropriate.

The progressive diastolic occlusion data were analyzed by linear regression analysis to determine changes in the slope of the relations between left ventricular end-diastolic or filling volumes and the rate of relaxation of the subsequent beat at baseline and during ischemia. An overall test of coincidence was then performed to determine whether there was a difference between the two regression lines for baseline and ischemia for each pig. The slopes of these lines measured during baseline and ischemia were compared using a paired t-test.

To determine the number of pigs required, we used results from previous work in this laboratory on the effects of partial occlusion of the left anterior descending coronary artery. We would expect to see a change of 4 ± 4.5 (SD) mmHg in end-diastolic pressure in response to ischemia. To attain an 80% power to detect this difference with  = 0.05, a minimum of seven pigs was required. Computations were done with SigmaStat v2.03. We considered differences significant when P < 0.05.

	RESULTS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Hemodynamic effects of regional ischemia. Regional ischemia was quantified by measuring the change in segment-length shortening in the region perfused by the left anterior descending coronary artery (Table 1). Systolic segment-length shortening in the region perfused by the left anterior descending coronary artery decreased significantly (P < 0.001) from 4.2 ± 0.5 to 2.0 ± 0.5 mm during partial occlusion of this artery. Segment-length shortening in the circumflex region did not change during regional left anterior descending ischemia (P = 0.49).

The decrease in segment-length shortening was associated with a decrease in overall left ventricular contractility. Contractility, quantified by E_max, decreased during regional ischemia (3.2 ± 1.6 vs. 4.5 ± 1.7 mmHg/ml, P < 0.05). The decrease in contractility did not result in a change in the end-diastolic (P = 0.68) or end-systolic (P = 0.92) volumes. Regional ischemia did lead to an increase in the end-diastolic pressure (13.6 ± 1.4 vs. 9.9 ± 1.4 mmHg, P < 0.036) and a decrease in the atrioventricular pressure gradient (34.1 ± 6.4 vs. 43.6 ± 7.6 mmHg-ms, P < 0.026), measured as the time integral of difference between left atrial and left ventricular pressure from the time of pressure crossover (mitral valve opening) to the first reversal of pressure crossover. The mean atrioventricular pressure gradient during this same time interval was also decreased during ischemia (0.62 ± 0.07 vs. 0.78 ± 0.08 mmHg, P < 0.04). The decrease in the atrioventricular pressure gradient resulted in a decrease in filling volume (18.2 ± 3.3 vs. 21.9 ± 2.9 ml, P < 0.046).

Mean right ventricular pressure, right ventricular end-diastolic pressure, and peak right ventricular pressure, which can affect the atrioventricular pressure gradient, did not change with regional ischemia, and there were no signs of right ventricular failure. (Pigs are susceptible to right ventricular failure, which would reflect a more global form of ischemia outside of the design of the model of regional ischemia used in this study.)

Effect of filling on rate of relaxation during regional ischemia.  is faster in the nonfilling ventricle (39.6 ± 6.3 vs. 47.3 ± 5.4 ms, P < 0.03). The rate of relaxation is also faster in the nonfilling ventricle during ischemia (52.2 ± 5.5 vs. 59.4 ± 5.1 ms, P < 0.026). When the rate of relaxation in the nonfilling ventricle at baseline is compared with that during regional ischemia, active relaxation is impaired during regional ischemia (P < 0.001). This result shows that the active component of relaxation is impaired independently of the effects of filling during regional ischemia.

Effect of stretch on rate of relaxation during regional ischemia. The atrioventricular pressure gradient, which drives early filling, is in part, determined by . Figure 3 shows a typical example of the effect on  of changes in end-diastolic volume (A) and filling volume (B) produced by volume clamps at different volumes during the preceding diastole. The first data point (smallest volume) represents a nonfilling beat. At baseline, greater left ventricular volumes are associated with slower isovolumic relaxation (i.e., larger ) during the subsequent cycle. Ischemia slows the rate of relaxation at any given volume (i.e., the curves shift up). In addition, the slope of the relationship between  and volume is steeper during ischemia than baseline, indicating that the myocardium becomes more sensitive to the effects of length or stretch in the presence of regional ischemia.

View larger version (12K): [in this window] [in a new window]   Fig. 3.   Change in slope of relationships between isovolumic relaxation time constant () and volume increases during regional ischemia, whether volume is measured by end-diastolic volume (A) or filling volume (B). Each symbol represents an experiment.

Table 2 and Fig. 4 present the statistical analysis of these data. We first conducted an overall test of coincidence for the regression lines for baseline and ischemia in each pig individually. In all cases, the lines were significantly different. At baseline, the overall slope of the regression of  with increasing volume for all the pigs is 0.12 ± 0.06 (means ± SD) ms/ml (Table 2). During ischemia, the slope of the regression line of  against end-diastolic volume increases significantly to 0.17 ± 0.08 ms/ml (P < 0.028, by paired t-test), indicating that during ischemia ventricular filling slows  faster than at baseline. (One obtains the same result when using filling volume as the independent variable; Table 2 and Fig. 4.) These results suggest the active component of relaxation was slowed during regional ischemia and that regional ischemia increases the sensitivity of ventricular relaxation to stretching of the myocardium due to filling.

View this table: [in this window] [in a new window]   Table 2.   Slope of  vs. volume during progressive diastolic occlusions

View larger version (15K): [in this window] [in a new window]   Fig. 4.   Rate of  after occlusion of mitral valve progressively later in diastole at baseline and during ischemia as measured by end-diastolic volume (A) and filling volume (B). Slope of linear regression (solid line) reflects effect of volume on rate of relaxation.

Effect of regional ischemia on extent of relaxation. The extent of relaxation is the pressure in the fully relaxed ventricle (P) and is determined by measuring the minimum left ventricular pressure in the nonfilling left ventricle. We found P in the normal nonfilling left ventricle was 1.2 ± 0.7 mmHg (Table 3) compared with 1.3 ± 1.0 mmHg during ischemia (P < 0.002). The increase in P in the nonfilling ischemic ventricle could be due to either an increase in pressure due to an upward shift in the end-diastolic pressure-volume curve or an increase in pressure by moving further up the same passive end-diastolic pressure-volume curve.

Figure 5 shows the end-diastolic pressure-volume data points of nonfilling beats at baseline and during ischemia in the seven pigs. The increase in P during regional ischemia in three of the pigs (pigs 581, 582, and 585) was due to an upward shift in the end-diastolic pressure-volume curve during regional ischemia, whereas the increase in P in the other pigs (pigs 580, 583, 588, and 589) was due to moving up the same end-diastolic pressure-volume curve. We compared the shifts in the end-diastolic pressure-volume curves to the changes in contractility, quantified by E_max, in the two groups of pigs (Table 4). E_max was significantly greater (5.9 ± 1.0 vs. 3.5 ± 1.4 mmHg/ml, P < 0.05) in the pigs with an upward shift of the end-diastolic pressure-volume curve than in the pigs with a moved up single end-diastolic pressure-volume curve. Consistent with an earlier study in dogs (51), these findings suggest the upward shift in the end-diastolic pressure-volume curve during regional ischemia depends on contractile function at baseline, with upward shifts occurring in stronger ventricles. The hearts with stronger contractile function at baseline resulted in upward shifts during ischemia, whereas the weaker hearts moved up the same end-diastolic pressure volume curve. The upward shift in the end-diastolic pressure-volume curve suggests that the duration of relaxation in these pigs would be shortened, whereas in pigs in which the shift was up the same end-diastolic pressure-volume curve the duration of relaxation would be longer.

View larger version (13K): [in this window] [in a new window]   Fig. 5.   End-diastolic pressure-volume relation from end-systolic occlusions (nonfilling beats) during an inferior vena caval occlusion (decreasing preload) at baseline and during ischemia for each pig. Ischemia shifted end-diastolic pressure-volume curve up in pigs 581, 582, and 585 but not the other pigs.

Effect of ischemia on the duration of relaxation. Figure 2A shows left ventricular volume (solid line) during filling with the numbers (1-5) reflecting increasing times during diastole when the mitral valve was occluded. Number 1 represents an occlusion performed during systole (before the onset of filling) and 5 is the end-diastolic point (just before ventricular contraction). The corresponding pressure points (1-5), indicating the minimum pressure reached, for each of these occlusions is shown in Fig. 2B. The open circles in Fig. 2B show when the occlusions were performed, and the solid points show the minimum pressure points associated with each mitral occlusion.

In the absence of filling, the pressure in the ventricular chamber falls as long as the left ventricle is actively relaxing. When left ventricular relaxation is complete, preventing mitral inflow has no effect on ventricular pressure (36). Figure 2B illustrates that in this pig, the duration of relaxation (measured from the time of dP/dt_min) is shorter during ischemia. Because it is possible that the direction of the shift in the end-diastolic pressure-volume relationship could influence the duration of relaxation differently, we analyzed the data from the end-diastolic pressure-volume curve that shifted upward separately from the data that shifted up the same end-diastolic pressure-volume curve (Table 4). The upward shifted pigs showed a decrease in duration of relaxation during regional ischemia (210 ± 9 vs. 286 ± 10 ms), whereas the pigs whose end-diastolic pressure-volume points fell on the same curve during regional ischemia showed no significant change in the duration of relaxation (259 ± 13 vs. 247 ± 19 ms). These results show that the duration of relaxation decreased during regional ischemia in the first group of pigs due to the upward shift of the end-diastolic pressure-volume curve (P = 0.002 for ischemia × shift in a two-way repeated measures analysis of variance). Thus the direction of the end-diastolic pressure-volume curve suggests what changes occur in the duration of relaxation.

Effect of regional ischemia on myocardial and chamber stiffness. Myocardial stiffness decreased during regional ischemia, reflected by a decrease in  (3.4 ± 0.8 vs. 4.3 ± 0.7, P < 0.013) and ln (2.5 ± 0.2 vs. 2.9 ± 0.2, P < 0.05), parameters that describe muscle elasticity (Table 5). The left ventricular chamber, like the myocardium, was also less stiff during ischemia, as indicated by S_p, a parameter that describes the curvature of the pressure-volume relation (1.8 ± 0.5 vs. 3.4 ± 1.3, P < 0.018) (Table 5).

	DISCUSSION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

As the ventricle fills, the myocardium is stretched, acting as a constantly increasing load on the myocardial fibers throughout diastole. Previous studies using isolated muscles showed that isotonic lengthening depends on loading conditions (20). Isolated muscle experiments also showed that the rate of relaxation was slower at longer muscle lengths and suggested that this load dependence was a function of length-dependent Ca²⁺ sensitivity (50). Nikolic et al. (35, 36) showed in an intact heart model of a dog that the rate of relaxation was slowed in response to the stretch of the myocardium. Their study (36) was limited by the fact that they did not measure absolute volumes. Rather, they measured mitral flow and added the integrated flow to an unknown and assumed constant end-systolic volume. In contrast, our study is based on directly measured absolute chamber volumes. Using volume clamping progressively throughout diastole, we confirmed (in pigs) that stretching the normal myocardium also slows the rate of relaxation.

Regional ischemia has been shown to impair early diastolic filling (37, 48, 56). Impaired early diastolic filling is due, in part, to changes in left ventricular relaxation. Slowed left ventricular relaxation decreases the atrioventricular pressure gradient, resulting in reduced early diastolic filling (38). Quantifying changes in the full course of left ventricular relaxation is complicated by the fact that the active component of relaxation overlaps the passive component of filling, which begins before relaxation has ended. This study investigated the effect of regional ischemia on the active component of relaxation independent of the passive effects of filling and the effect of stretch on the rate, duration, and extent of relaxation. Consistent with earlier studies (2, 5, 6, 10, 11, 14, 16, 58), we found that the active component of left ventricular relaxation is impaired during regional ischemia. This slower relaxation is associated with a lower atrioventricular pressure gradient and less filling. This study adds to the information that regional ischemia increases the sensitivity of relaxation to the stretch of the myocardium due to ventricular filling.

Ischemia-induced impairment of active relaxation has been related to the inhibition of the reuptake of Ca²⁺ by the sarcoplasmic reticulum (34, 46) or increased cellular calcium inflow (24), resulting in impaired detachment of force-generating sites between actin and myosin (23). In experiments in which papillary muscle ischemia was induced in rats, the rate of relaxation did not change in response to increasing preload (59). These findings in isolated muscle were in contrast to the findings in this study showing that regional ischemia further slows the rate of relaxation reflected by an increase in  in response to stretch. At any given volume,  was larger in the ischemic heart compared with the normal heart. Significantly, we found the increase in  in response to stretch due to filling did not parallel the increase observed in the normal heart.  increased faster in the ischemic heart as a result of stretch. Nakamura et al. (33) had similar findings in isolated rat ventricular myocardium where the degree of slowed relaxation depended on the end-systolic length. This faster increase in  shows that relaxation is more sensitive to changes in stretch in response to increases in volume during regional ischemia.

Nikolic et al. (36) showed that during a lower inotropic state, relaxation is completed later than during a high inotropic state. This result suggests that during ischemia, which reduces ventricular contractility, the duration of relaxation would also be increased. We found that the duration of relaxation depends on the effect of regional ischemia on the passive end-diastolic pressure-volume relation. Regional ischemia resulted in two different shifts in the end-diastolic pressure-volume curve, an upward shift and a shift further up the end-diastolic pressure-volume relation of the same baseline curve (a rightward and upward shift on the same curve). In the ischemic pigs in which there was an upward shift in the end-diastolic pressure-volume relation, the duration of relaxation decreased, whereas in the ischemic pigs, in which the end-diastolic pressure-volume data points were further up the same end-diastolic pressure-volume curve, the duration of relaxation was unchanged.

The end-diastolic pressure-volume relation shifting in two different directions suggests different mechanisms. It has previously been suggested that so-called demand and supply ischemia produce different effects on the diastolic pressure-volume curve (3). In demand ischemia there is a critical stenosis that does not affect coronary perfusion at rest but which prevents adequate flow to the myocardium when the demand for oxygen is increased, for example by pacing. The upward shift in the end-diastolic pressure-volume relation has been attributed to persistent cross-bridge interaction during diastole (23) as a result of impaired calcium uptake by the sarcoplasmic reticulum, leading to an increase in diastolic myoplasmic calcium. In supply ischemia, when the coronary artery is constricted enough to compromise systolic function at rest (the model we used in this paper), the rightward shift in the end-diastolic pressure-volume relation is thought to result from a buildup of tissue metabolites (H⁺) due to decreased coronary washout. The buildup of metabolites results in intracellular acidosis and reduced myofilament calcium sensitivity subsequently reducing contractile activity. Because the myofilaments cannot interact during diastole if they do not interact during systole, the end-diastolic pressure-volume relation cannot shift upward (40). In contrast, using a model of demand ischemia, Takano and Glantz (51) showed that both of these shifts could occur, depending on the contractility of the ventricle before the induction of ischemia. The diastolic pressure-volume curve shifted upward in strong hearts and rightward in hearts with depressed contractility at baseline. In this study of supply ischemia, we also produced both upward and rightward shifts of the diastolic pressure-volume curve (albeit with a small sample size). As in the study by Takano and Glantz (51) of demand ischemia, we found that the stronger hearts shifted upward and the weaker ones shifted rightward. It appears that the level of baseline contractile function, not the type of ischemic intervention, is the best predictor of the direction of shift in the end-diastolic pressure-volume relation during ischemia.

In addition to the rate and duration of relaxation being impaired during regional ischemia, the extent of relaxation was also impaired. In the normal, fully relaxed nonfilling ventricle, the minimum pressure can be negative and has been referred to as elastic recoil. This negative pressure occurs when the ventricle contracts below its equilibrium volume, the volume at zero transmural pressure, and generates restoring forces similar to that of compressing a spring, which are released when the mitral valve opens (36, 54, 58). During ischemia, a decrease in elastic recoil has been shown, in part, to be due to a decrease in the magnitude of apical rotation during isovolumic relaxation and filling (17, 42), a consequence of decreased contractility consistent with the effects on contractility we observed. A decrease in elastic recoil is reflected by the inability of the fully relaxed, nonfilling left ventricle to generate a negative pressure during regional ischemia. In the upward shifted curves, the inability to generate a negative pressure may be due to persistent interaction of cross bridges in the ischemic myocardium throughout diastole (9, 22). This persistent interaction may be due to impaired calcium reuptake by the sarcoplasmic reticulum (34) and an increase in diastolic calcium concentration in myocytes during ischemia (28). Thus elastic recoil is not present in ischemic hearts (that showed an upward shift during regional ischemia) due to a decrease in contractility, resulting in a reduced extent of relaxation.

Myocardial and chamber stiffness decreased during regional ischemia. Although this result was unexpected, it is not without precedent. Forrester et al. (15) found a decrease in ventricular stiffness shortly after myocardial infarction. Hess et al. (26) showed that changes in stiffness were dependent on the degree of ischemia. An increase in the distensibility of the myocardium in the nonischemic zone has been shown to play a role in this decrease in stiffness (26). The activity of the cardiac nerves has also been shown to be involved in modulating myocardial stiffness during ischemia (1). Amano et al. (1) showed that cardiac nerves are responsible for increasing regional function in the nonischemic region of the heart while delaying increases in myocardial stiffness in the ischemic region. Other studies, which reported an increase in stiffness, were a result of severe ischemia following total coronary occlusion (12, 41, 43, 57). Thus the severity of ischemia plays a role in the changes in myocardial and chamber stiffness.

This study confirms the findings of Nikolic et al. (35, 36) about the effects of stretch due to filling on the rate, duration, and extent of relaxation and extends these results to investigate the effects of ischemia. Nikolic et al. (36) showed, as we do, that the rate of relaxation is slowed and the duration of relaxation is prolonged during filling. Their study was limited because it used derived ventricular volumes, whereas we used directly measured ventricular volumes. During regional ischemia, active relaxation was impaired in the nonfilling ventricle, which slowed the rate of relaxation. The slowed relaxation results in a decrease in the atrioventricular pressure gradient and slows early transmitral filling. An important new finding of this study shows that stretching the myocardium as the ventricle fills increases  faster during regional ischemia impairing early filling. The greater increase in  in response to regional ischemia reflects an increased sensitivity to stretch due to filling and an increased dependence of relaxation on volume. The duration of relaxation depends on the effect of regional ischemia on the end-diastolic pressure-volume relation, which is a reflection of baseline contractile function. A stronger baseline contractile function results in an upward shift in the end-diastolic pressure-volume relation during regional ischemia. In these curves that were shifted upward, we also found that the inability of the ventricle to develop a negative pressure during ischemia is, in part, due to a decrease in the extent of relaxation. All of these effects combine to reduce the atrioventricular pressure gradient and reduce left ventricular filling during regional ischemia.