INTRODUCTION

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THE STRIKINGLY RAPID DECLINE of cardiac contraction at the onset of myocardial ischemia was described by Harvey in 1628 (7), but the mechanism responsible for this rapid fall in contractile force has still not been identified. Bristow et al. (2) proposed that the rapid fall of contractile force at the initiation of ischemia is, in fact, an active downregulation of contractile function. Thus myocardial energy demand may be reduced sufficiently to reestablish a new balance between oxygen supply and demand, so that irreversible injury or necrosis is avoided (2, 12). The crucial question remains to be answered, i.e., how this contractile downregulation is initiated and modulated to match the reduction in flow.

Energetically mediated (4, 8, 9) and vascularly mediated (10) mechanisms have been proposed to account for the rapid contractile downregulation. Because of the rapidity of the downregulation at the onset of ischemia, some investigators have postulated that energetic changes would not be rapid enough to account for the decline of contraction. With a 0.5-s resolution, we reported the first data showing that cytosolic P_i increases before and more rapidly than the decline of developed left ventricular pressure (LVP) after the onset of ischemia in an isolated rat heart preparation (8). However, the coronary perfusion pressure (CPP) also fell before the decline in contractile force, so "vascular collapse" as proposed by Koretsune et al. (10) might also initiate contractile downregulation.

To determine whether vascular collapse produced by the rapid drop of CPP initiates contractile downregulation at the onset of ischemia, this investigation examined LVP following abrupt, varying reductions of coronary flow. Experiments were conducted in isolated rat hearts in which LVP declines very rapidly with no change in heart rate (HR) during abrupt, brief coronary flow reductions (8); therefore, changes in LVP could index the changes in cardiac contractile force. Abrupt reductions in coronary flow produced decreases in LVP, which always correlated well with changes in flow but not with changes in CPP. Our findings of similar LVP at different CPP are not compatible with the vascular collapse theory.

	METHODS

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Experimental preparation. Six rats (350-400 g body wt) were anesthetized with pentobarbital sodium (50 mg/kg ip). Their hearts were rapidly excised and perfused in the Langendorff mode with low-phosphate physiological salt solution containing the following (in mM): 127 NaCl, 5.8 KCl, 0.2 KH₂PO₄, 1.1 MgSO₄, 25 NaHCO₃, 2.5 CaCl₂, 5.5 glucose, and 2 pyruvate; and equilibrated with a gas mixture of 95% O₂-5% CO₂ at 37°C. A digital perfusion pump (Ismatec Instruments) was used to precisely control coronary flow.

CPP was measured with a transducer connected to the inflow line close to the heart and set at 60 mmHg for the baseline condition. A latex balloon was placed in the left ventricle through an incision in the left atrium and the mitral orifice for continuous measurement of LVP and HR. The end-diastolic pressure was set at 6-8 mmHg by partial inflation of the balloon.

Protocol. The relationships between LVP and CPP were determined following the onset of four different degrees of ischemia induced by abrupt reduction of coronary flow by 1) 50% of baseline for 27 s followed by 3 min of reperfusion; 2) 70% of baseline for 27 s followed by 3 min of reperfusion; 3) 85% of baseline for 18 s followed by 5 min of reperfusion; and 4) 0% of baseline for 8 s followed by 5 min of reperfusion.

To avoid possible deterioration or/and preconditioning of the preparation, which might have been induced by multiple episodes of severe ischemia (reduction of coronary flow by 85 or 100%), the durations of these ischemias were restricted to 18 or 8 s, respectively. Each degree of ischemia and reperfusion was repeated 10 times successively followed by a 10-min normal perfusion period before we started the next series of more severe ischemic episodes. LVP, HR, and CPP were continuously recorded with a Grass polygraph at a paper speed of 1 cm/s.

Statistical analyses. Data are presented as means ± SE. LVP and CPP values measured at selected times following abrupt, varying reductions of coronary flow were evaluated using one-way analysis of variance followed by Student-Newman-Keuls test for multiple comparisons. LVP values measured at selected CPP resulting from abrupt, varying reductions of coronary flow were also evaluated using one-way analysis of variance followed by Student-Newman-Keuls test for multiple comparisons. Differences with P < 0.05 are described as significant.

	RESULTS

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Stability of preparations. Coronary flow during baseline was 8.9 ± 0.4 ml · min¹ · g¹. The stability of the preparation was verified by unchanged LVP, HR, and CPP during normal perfusion periods throughout the experiments, i.e., before ischemia and after every 10 episodes of ischemia. During these normal perfusion periods, LVP averaged 152 ± 9, 154 ± 9, 153 ± 6, 155 ± 6, and 154 ± 11 mmHg; HR averaged 187 ± 9, 185 ± 10, 187 ± 9, 186 ± 8, and 178 ± 6 beats/min; and CPP averaged 64 ± 1, 64 ± 1, 65 ± 2, 66 ± 1, and 67 ± 3 mmHg.

Changes in LVP and CPP following abrupt reduction of coronary flow. Changes in LVP and CPP following the onset of varying degrees of ischemia are shown in Fig. 1. Figure 1A shows all data, and Fig. 1B with an expanded time scale shows data collected during the first 5 s of the protocol. After abrupt reductions of coronary flow, the fall in LVP lagged behind the decrease in CPP. At 1.5 s after flow reductions ranging from 50 to 100%, decreases in contractile function did not differ significantly, although CPP varied significantly from 45 ± 1 to 20 ± 2 mmHg. By 5 s (dashed line in Fig. 1A) after the onset of ischemia, CPP had dropped to near its lowest value, whereas LVP was still falling steeply. The more severe the ischemia, the greater was the decrease in CPP. However, declines in LVP were not significantly different even by 5 s.

View larger version (28K): [in this window] [in a new window]   Fig. 1.   A: time course of left ventricular pressure (LVP) and coronary perfusion pressure (CPP) after abrupt reductions of coronary flow by 50, 70, 85, and 100% of baseline. By 5 s after onset of ischemia (verticle dashed line), CPP had dropped to near its lowest value for each respective flow reduction, whereas LVP was still falling steeply. B: changes in LVP and CPP during first 5 s of protocol with an expanded time scale. By 1.5 s after onset of ischemia, CPP had decreased to significantly different values, whereas LVP values did not differ significantly. By 5 s after onset of ischemia, CPP had essentially reached new steady states, whereas LVP was still falling steeply.

Relationship of decline in LVP to decrease in CPP at onset of ischemia. The relationships between the rapid decline in LVP and in CPP after the abrupt reductions of coronary flow to different degrees are shown in Fig. 2. These data demonstrate that LVP fell in response to decreases in flow and not directly in response to decreases in CPP. For LVP values <135 mmHg, CPP values for respective flow reductions differed significantly. With the most moderate flow reduction (50%), CPP was still above ~35 mmHg when LVP had fallen to ~80 mmHg. With the most severe flow reduction (100%), CPP had fallen to ~2 mmHg when LVP reached ~80 mmHg. For contractile function equal to 50% of baseline, CPP ranged from 35 ± 0.5 to 2.5 ± 1 mmHg for flow reductions ranging from 50 to 100%. Also, it is clear from Fig. 2 that the LVP-CPP relationships begin to diverge according to the degree of ischemia at relatively high CPP values. Similar values of LVP and widely varying values of CPP are incompatible with the vascular collapse theory.

View larger version (25K): [in this window] [in a new window]   Fig. 2.   Relationships between LVP and CPP after abrupt reductions of coronary flow by 50, 70, 85, and 100% of baseline. For LVP values <135 mmHg, CPP values for respective flow reductions differed significantly. Although decline of LVP following each respective flow reduction was accompanied by rapid decrease in CPP, LVP values were not associated with unique values of CPP.

	DISCUSSION

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The major finding of this investigation is that ventricular contractile function varied directly with coronary flow but not with CCP following abrupt, varying reductions of coronary flow. Therefore, the fall in CCP is not the mediator of contractile downregulation at the onset of ischemia in this model.

These observations were obtained by summarizing the responses of the isolated heart to multiple ischemic episodes. It was crucial that the preparation remained stable, i.e., no deterioration and preconditioning were induced by multiple episodes of ischemia. The stability of the preparations was verified by the unchanged hemodynamic indexes during the periods of normal perfusion throughout the experiments.

Our results do not support the hypothesis of Koretsune et al. (10) that coronary vascular collapse induces a rapid fall in myocardial contractile function at the onset of ischemia. They reported that LVP of perfused ferret hearts fell by >50% at 30 s after the onset of ischemia, with no significant changes in energy metabolites. To gauge the role of intravascular pressure in this fall of LVP, they compared ischemia induced by massive coronary microembolization at the level of the precapillary arterioles to ischemia induced by stopping coronary flow. They found that contractile depression developed significantly slower in the microembolized hearts, despite similar accumulation of P_i and H⁺ at 30 s in both models. Thus they concluded that coronary vascular collapse is responsible for the fall of contractile function during early ischemia. A major concern is that embolization with microspheres made it difficult to determine precisely the onset of ischemia. The embolization process might have induced a progressive and diffuse ischemia and, thus, a more gradual fall in contractile force. The failure of Koretsune et al. (10) to detect energetic changes at 30 s of ischemia most likely resulted from the necessity of pooling data from four hearts to obtain satisfactory ³¹P nuclear magnetic resonance spectra.

Evidence opposing the vascular collapse hypothesis was also reported by Galinanes et al. (6). They compared the decrease of contractile function during slow (stopping the perfusion pump) and fast (reversing the perfusion pump for 5 s to create a negative pressure) vascular collapse in isolated rat hearts. They found that the rate and extent of the decline of contractile function during early ischemia were not altered by changing the rate of vascular collapse. Our results are consistent with their observation that faster decreases in CPP did not produce correspondingly greater or faster declines in LVP. However, our protocol of reducing coronary flow from 50 to 100% of normal baseline is more physiologically and clinically relevant than the imposition of a negative CPP performed by Galinanes et al. (6) Furthermore, negative CPP may immediately collapse epicardial vessels, thus sparing the downstream circulation from the intended faster reduction in distending pressure. If that had happened, no decrement of contractile function would be expected. Our protocol and results more conclusively rule out a significant role for vascular collapse in the downregulation of contractile function during early ischemia.

In the current investigation, coronary flow was the controlled variable, and observations of contractile function and perfusion pressure were analyzed. When coronary pressure was reduced independently in relevant earlier experiments, contractile function (1, 3) and myocardial O₂ consumption (1) did not decline as long as pressure-flow autoregulation prevented a fall in coronary flow. These results support the current findings that coronary pressure does not directly regulate contractile function. The current investigation, however, addressed more severe conditions in which both coronary flow and pressure were compromised.

Rather than hemodynamic, the mediator responsible for the initiation and modulation of contractile downregulation at the onset of ischemia is most likely energetic. As we reported previously, P_i increases before and is closely correlated with the decline in contractile force following abrupt reductions of coronary flow (8). This rapid rise in P_i may function as a metabolic signal that rapidly downregulates contractile force, either at the contractile proteins (5, 11, 13) or the sarcoplasmic reticulum (14), or, indirectly, by lowering the phosphorylation potential of ATP (4, 9).