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Negative inotropic drugs alter indexes of cytosolic [Ca²⁺]-left ventricular pressure relationships after ischemia

Anesthesiology Research Laboratories, ¹Department of Anesthesiology and ²Department of Physiology, and ³Cardiovascular Research Center, The Medical College of Wisconsin, Milwaukee 53226, ⁴Research Service, Veterans Affairs Medical Center, Milwaukee 53295, ⁵Department of Biomedical Engineering, Marquette University, Milwaukee, Wisconsin 53223; and ⁶Department of Anesthesiology and Intensive Care Medicine, University Hospital Münster, 48129 Münster, Germany

Submitted 2 December 2003 ; accepted in final form 29 March 2004

	ABSTRACT

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Negative inotropic agents may differentially modulate indexes of cytosolic [Ca²⁺]-left ventricular (LV) pressure (LVP) relationships when given before and after ischemia. We measured and calculated [Ca²⁺], LVP, velocity ratios {[(d[Ca²⁺]/dt_max)/(dLVP/dt_max); VR_max] and [(d[Ca²⁺]/dt_min)/(dLVP/dt_min); VR_min]}, and area ratio (AR; area [Ca²⁺]/area LVP per beat) before and after global ischemia in guinea pig isolated hearts. Ca²⁺ transients were recorded by indo 1-AM fluorescence via a fiberoptic probe placed at the LV free wall. [Ca²⁺]-LVP loops were acquired by plotting LVP as a function of [Ca²⁺] at multiple time points during the cardiac cycle. Hearts were perfused with bimakalim, 2,3-butanedione monoxime (BDM), nifedipine, or lidocaine before and after 30 min of ischemia. Before ischemia, each drug depressed LVP, but only nifedipine decreased both LVP and [Ca²⁺] with a downward and leftward shift of the [Ca²⁺]-LVP loop. After ischemia, each drug depressed LVP and [Ca²⁺] with a downward and leftward shift of the [Ca²⁺]-LVP loop. Each drug except BDM decreased d[Ca²⁺]/dt_max; nifedipine decreased d[Ca²⁺]/dt_min, whereas lidocaine increased it, and bimakalim and BDM had no effect on d[Ca²⁺]/dt_min. Each drug except bimakalim increased VR_max and VR_min before ischemia; after ischemia, only BDM and nifedipine increased VR_max and VR_min. Before and after ischemia, BDM and nifedipine increased AR, whereas lidocaine and bimakalim had no effect. At 30 min of reperfusion, control hearts exhibited marked Ca²⁺ overload and depressed LVP. In each drug-pretreated group Ca²⁺ overload was reduced on reperfusion, but only the group pretreated with nifedipine exhibited both higher LVP and lower [Ca²⁺]. These results show that negative inotropic drugs are less capable of reducing [Ca²⁺] after ischemia so that there is a relatively larger Ca²⁺ expenditure for contraction/relaxation after ischemia than before ischemia. Moreover, the differential effects of pretreatment with negative inotropic drugs on [Ca²⁺]-LVP relationships after ischemia suggest that these drugs, especially nifedipine, can elicit cardiac preconditioning.

bimakalim; butanedione monoxime; nifedipine; lidocaine; indo 1; Ca²⁺ transients; Ca²⁺ concentration

Ischemia and cardiotonic drugs alter the relationships between cytosolic Ca²⁺ and contractility and relaxation. We have analyzed the cyclic relationship between LVP and cytosolic [Ca²⁺] in isolated hearts to better understand how positive inotropic drugs alter the dynamics of Ca²⁺ flux and its relationship to mechanical function (11, 36). This is especially important in hearts compromised by ischemia and reperfusion (I/R) injury (26, 28, 56, 60) and congestive heart failure (23, 31), where abnormal cytosolic Ca²⁺ homeostasis is associated with depressed myocardial contractility and relaxation. Cardiac I/R causes long-lasting bursts of Na⁺ channel opening and prolongs Na⁺ channel inactivation that leads to Na⁺ loading and Ca²⁺ overload by slowed or reverse Na⁺/Ca²⁺ exchange.

The aims of this study were, first, to observe how negative inotropic drugs that work on different cardiac receptors or ion channels alter several indexes of the Ca²⁺-LVP relationship; second, to assess whether these drugs are equally effective after ischemia as before ischemia; and, finally, to determine whether these drugs protect the heart against I/R injury. We used the fluorescent probe indo 1-AM to simultaneously measure transient cytosolic [Ca²⁺] and isovolumetric LVP during the cardiac cycle and analyzed several indexes of this relationship to better understand how different negative inotropic drugs alter Ca²⁺-LVP relationships before and after ischemia.

Four drugs were selected. Bimakalim (Bim), an opener of ATP-sensitive K⁺ (K_ATP) channels (4, 20), shortens the cardiac AP, which in turn retards Ca²⁺ influx per contraction. Lower concentrations of K_ATP channel openers are known to elicit cardioprotection without altering AP duration (4), although the mechanism for this is unclear (19). In the normoxic heart, the K_ATP channel is thought to be inactive; during I/R, however, the decline in ATP results in K_ATP channel activation, which in turn reduces postischemic Ca²⁺ loading (42). K_ATP channel opening may modulate cytosolic Ca²⁺-controlling mechanisms, possibly through altered mitochondrial bioenergetics (13, 19, 43). The K_ATP channel located within the sarcolemmal membrane is also thought to be located in the mitochondrial membrane, but its existence in mitochondria has not been verified. These channels appear to be key elements in effecting cardiac preconditioning (20).

2,3-Butanedione monoxime (BDM) has broad effects. It reversibly uncouples excitation from contraction to produce a negative inotropic effect in all contractile elements including those in skeletal and cardiac muscle cells. BDM retards cross-bridge function by inhibiting actinomyosin ATPase, by decreasing myofilament Ca²⁺ sensitivity (17, 21, 22, 45), by depressing transsarcolemmal Ca²⁺ fluxes, by reducing SR Ca²⁺ uptake and release, and by shorting the AP (21). Nifedipine (Nif), a dihydropyridine, blocks voltage-gated L-type Ca²⁺ channels and is used clinically to treat angina, hypertension, and ventricular tachycardia (47, 61). Lidocaine (Lid) depresses phase 0 of the cardiac AP by blocking the fast Na⁺ channel (10) and is used primarily as an antidysrhythmic and nerve blocking agent. Lid also depresses contractility by decreasing cytosolic [Ca²⁺] secondary to enhanced forward-mode Na⁺/Ca²⁺ exchange with a reduction in Na⁺ influx (41).

	METHODS

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Isolated heart preparation and measurements. The investigation conformed to the National Institutes of Health Guide for the Care and Use of Laboratory Animals (NIH Pub. No. 85-23, Revised 1996). Prior approval was obtained from the Medical College of Wisconsin Animal Studies Committee. Our preparation and measurements have been described in detail (3, 11, 36, 49, 56). In brief, hearts were isolated from anesthetized guinea pigs (250–300 g) and perfused by the Langendorff method at a constant pressure of 55 mmHg and at 37°C with a modified Krebs-Ringer (KR) solution equilibrated with 95% O₂ and 5% CO₂ and containing (in mM) 137 Na⁺, 4.5 K⁺, 2.4 Mg²⁺, 1.25 Ca²⁺, 134 Cl^–, 15.5 HCO₃^–, 1.2 H₂PO₄^–, 11.5 glucose, 2 pyruvate, 16 mannitol, 1.05 EDTA, and 0.1 probenecid with 5 U/l insulin.

LVP was measured with a transducer connected to a thin, saline-filled latex balloon inserted into the LV through a cut in the left atrium. Baseline systolic LVP was 50–55 mmHg; this was due to half normal CaCl₂ in the perfusate, the cardiodepressant effect of probenecid, and the cytosolic buffering of [Ca²⁺] by the fluorescence dye indo 1. The first derivative of LVP, dLVP/dt, was derived on-line, and maximum (dLVP/dt_max) and minimum (dLVP/dt_min) values represent the maximum and minimum time derivatives of LVP. Balloon volume was adjusted to maintain a diastolic LVP of 0 mmHg during the initial control period so that any increase in diastolic LVP indicated an increase in LV wall stiffness or diastolic contracture. Two pairs of bipolar electrodes were placed in each heart to monitor intracardiac electrograms, from which spontaneous atrial heart rate was determined from the right atrial beat-to-beat interval.

Coronary flow (CF) was measured at constant temperature (37°C) with a self-calibrating in-line, ultrasonic flowmeter. Coronary effluent Na⁺, K⁺, Ca²⁺, PO₂, PCO₂, and pH were measured off-line with an intermittently self-calibrating analyzer system (Radiometer Copenhagen ABL 505; Copenhagen, Denmark). Coronary sinus effluent was collected through a cannula inserted into the right ventricle through the pulmonary artery after the venae cavae were ligated. Coronary outflow (coronary sinus) O₂ was also measured continuously on-line with a Clark-type O₂ electrode placed in the outflow. Because myocardial metabolism is altered by negative inotropic drugs and by I/R (3, 24, 59), we measured myocardial O₂ consumption (MO₂) and %O₂ extraction (%O₂E). MO₂ was calculated as (CF/heart weight) x (arterial PO₂ – venous PO₂) x 24 µl O₂/ml, and %O₂E was calculated as 100 x [(arterial PO₂ – venous PO₂)/arterial PO₂] at 760 mmHg and 37°C.

Measurement of cytosolic and noncytosolic free Ca²⁺ in isolated hearts. We have described details of our method to monitor, calibrate, and assess indo 1 fluorescence signals as a measure of cytosolic [Ca²⁺] in the LV of isolated hearts (3, 11, 36, 49, 56). All experiments were conducted in a light-blocking Faraday cage. Briefly, the heart was suspended via the aortic cannula in the perfusate bath at 37°C, and the distal end of a trifurcated fiberoptic cable (surface area 3.85 mm²) was placed gently against the LV epicardial surface through a hole in the bath to excite the tissue with light filtered at 350 nm and recorded at 385 and 456 nm. A rubber O-ring was placed over the fiberoptic tip to seal the hole, and netting was applied around the heart for optimal contact with the fiberoptic tip. Background autofluorescence was determined for each heart after initial perfusion and equilibration for 30 min.

Each heart was then loaded with indo 1-AM for 20–30 min with the recirculated KR solution at a final indo 1-AM concentration of 6 µM. Residual interstitial indo 1-AM was washed out by perfusing the heart with standard KR solution for 20 min. Additional experiments (3 hearts for each of the 5 groups) were conducted to assess changes in tissue autofluorescence due to changes in the redox state (primarily a measure of NADH) and drug autofluorescence. None of the drugs exhibited a significant change in autofluorescence. I/R, as noted previously (37), caused an initial increase and then a decrease in NADH; these autofluorescence values were subtracted from the fluorescence signal obtained with indo 1.

The fluorescence emissions at 385 and 456 nm (F₃₈₅ and F₄₅₆) were recorded using a modified luminescence spectrophotometer (SLM Aminco-Bowman II, Spectronic Instruments; Urbana, IL). The arc lamp shutter was opened only for 2.5-s recording intervals to prevent photobleaching. The F₃₈₅-to-F₄₅₆ ratio remained stable during the 3-h course of these studies, indicating no change in effective measured [Ca²⁺]. After indo 1 was loaded, systolic-diastolic LVP was slightly altered in nonischemic hearts over this time period. Cytosolic [Ca²⁺] was distinguished from total cell [Ca²⁺] after the mitochondrial-derived fluorescence was quenched at the end of each experiment with MnCl₂ (3, 11, 36, 49, 56). Pilot experiments using fresh sections of non-dye-loaded LV free wall (2–3 mm thick) placed between the probe tip and the LV wall from an intact dye-loaded heart showed an attenuation of 80–85% in emission signal strength from the epicardial to endocardial surface. Although tissue underlying the fiberoptic probe in the middle myocardial band becomes infarcted in this model, the nonviable cells do not contribute to the signal because they do not have intact cell membranes.

Simultaneous [Ca²⁺] and LVP recordings were obtained at designated time points. Customized software was developed in MATLAB (Mathworks, Natick, MA) for off-line signal processing of recorded data. LVP and fluorescence data were digitally lowpass filtered using a fourth-order bidirectional Butterworth filter at 25 Hz. Data were analyzed for peak systolic, peak diastolic, and systolic-diastolic LVP (mmHg) and [Ca²⁺] (nM). First derivatives of [Ca²⁺] (d[Ca²⁺]/dt) and LVP (dLVP/dt) were derived on-line, and values for d[Ca²⁺]/dt_max and dLVP/dt_max (peak rate of total free Ca²⁺ inflow and contractility) as well as d[Ca²⁺]/dt_min and dLVP/dt_max (peak rate of Ca²⁺ outflow and relaxation) were determined. Area [Ca²⁺] and area LVP (systolic-diastolic LVP time integral), i.e., total LVP (potential work) and total cytosolic [Ca²⁺] during one beat, were computed.

Concentration-response curves for each drug were not obtained so direct comparisons among drugs for a given variable would not be considered valid. However, velocity ratios (VR) and area ratios (AR) were utilized to compare responses to drugs because these ratios normalized the individual values for Ca²⁺ and LVP for each drug. The index of (d[Ca²⁺]/dt_max)/(dLVP/dt_max), i.e., the maximal VR (VR_max), assessed the ratio of the maximal rate of change of cytosolic Ca²⁺ influx, with or without drug treatment, to the maximal rate of change in contractility. Inversely, the index of (d[Ca²⁺]/dt_min)/(dLVP/dt_min), or minimal VR (VR_min), assessed the ratio of the maximal rate of change in cytosolic Ca²⁺ outflow, with or without drug treatment, to the maximal rate of change in relaxation.

The index of area [Ca²⁺]/area LVP, or AR, was used to assess the net amount of free Ca²⁺ moved in and out of the cytosol to effect cardiac (potential) work assessed over one beat. Area [Ca²⁺] was defined as the integral of the mean systolic minus diastolic free [Ca²⁺] during a mean cardiac cycle, i.e., the total amount of [Ca²⁺] available during one heartbeat to generate contraction and relaxation. Area LVP was defined as the integral of systolic-diastolic LVP (contractile and relaxation) during a mean cardiac cycle. AR normalizes the two area indexes under all experimental conditions, i.e., links total available cytosolic [Ca²⁺] for contraction and relaxation in the presence and absence of drugs. The three ratios were used to compare responses among the four drugs, before as well as after reperfusion, irrespective of drug concentration. For example, a doubling of AR would indicate a 50% reduction in the amount of LVP work for a given amount of Ca²⁺ available.

Protocol. Forty hearts were divided randomly and equally among five groups: no-drug ischemia controls, Bim, BDM, Nif, and Lid groups. Each experiment lasted 210 min (Fig. 1). Initial background (before indo 1 loading) measurements were obtained after 30 min of stabilization. On-line recordings were sampled and stored every 1–5 min throughout each experiment. Data shown are limited to four distinct periods: at the end of the washout period (baseline, at 75 min), during drug perfusion before 30 min of ischemia (at 82 min), at 30 min of reperfusion (at 165 min), and during repeat drug perfusion (at 172 min). In this model, LV infarct size is 50% of total ventricular weight after 30 min of ischemia and 60–120 min of reperfusion (37–39). CF was stopped for 30 min to cause global no-flow ischemia. Drugs were perfused at concentrations that submaximally lowered LVP as established from pilot studies. Bim (1 µM), BDM (4 mM), Nif (125 nM), or Lid (150 µM) was perfused for 2 min, 30 min before ischemia (at 80 min) in each of the drug groups, and again for 2 min at the same concentration beginning after 30 min of reperfusion (at 170 min). Each drug caused a small reversible depression of LVP within 3–7 min of perfusion. All measured variables returned to control values after their washout. All analog signals were digitized (PowerLab/8 SP, ADInstruments; Castle Hills, Australia) and recorded at 125 Hz (Chart & Scope v3.63, ADInstruments) on Power Macintosh G4 computers (Apple; Cupertino, CA) for later analysis using MATLAB and Microsoft Excel (Microsoft; Redmond, WA) software. Prior frequency spectral analysis showed no peaks above 50 Hz for [Ca²⁺] as measured by indo 1.

View larger version (15K): [in this window] [in a new window]   Fig. 1. Experimental protocol. Hearts were perfused and stabilized with Krebs-Ringer (KR) solution for 30 min, loaded with indo 1-AM for 30 min, and washed out of residual indo 1-AM for 20 min. At 80 min, drug [bimakalim (Bim; 1 µM), 2,3-butanedione monoxime (BDM; 4 mM), nifedipine (Nif; 125 nM), or lidocaine (Lid; 150 µM)] was perfused for 2 min before ischemia (time 80 min) and after 30 min of reperfusion (time 170 min). Control hearts were perfused only with KR solution at the same time points. Hearts were subjected to 30 min of global ischemia at 37°C (time 110 to 140 min) and reperfused for 60 min (time 140 to 200 min). MnCl2 (50 mM) was perfused at the end (time 220–210 min) to quench noncytosolic Ca2+.

	RESULTS

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Figure 2 shows representative tracings of simultaneously obtained LVP and myoplasmic [Ca²⁺] (converted from raw Ca²⁺ transients) before ischemia in the absence and presence of Nif. The indexes of cytosolic [Ca²⁺] and LVP were derived from such traces; Nif reduced both [Ca²⁺] and LVP.

View larger version (24K): [in this window] [in a new window]   Fig. 2. Plot of simultaneously recorded cytosolic [Ca2+] (broken lines) and isovolumic left ventricular pressure (LVP; solid line) before and during Nif exposure. Nif decreased both cytosolic [Ca2+] transient and LVP. This effect of Nif was reversible upon its washout (data not shown).

View larger version (32K): [in this window] [in a new window]   Fig. 3. A: averaged cytosolic [Ca2+]-LVP loops (6–10 cardiac cycles/heart in 8 hearts) during baseline conditions for four negative inotropic drug groups before drug perfusion, and control (no drug), before ischemia. Loop area was acquired by integrating [Ca2+]-LVP over 6–10 beats for 8 hearts/group. Mean loop area (103 nM·mmHg) before and after ischemia for each treatment is shown next to the drug name; there were no differences in loop area. B: averaged [Ca2+]-LVP loops at 30 min of reperfusion (no drugs) after hearts were treated with Bim, BDM, Nif, or Lid before ischemia. Loop areas after ischemia were similar in the control and Nif groups, lower and similar in the Lid, Bim, and BDM groups, and not different between the Nif and Lid groups (P < 0.05). See Table 1 for significance.

View larger version (21K): [in this window] [in a new window]   Fig. 4. Averaged cytosolic [Ca2+]-LVP loops obtained during perfusion of 1 µM Bim (A) and 4 mM BDM (B) 30 min before (preischemia) and 30 min after reperfusion (postischemia). Pre- and postischemia loop areas were not different for each drug (P < 0.05). See Table 1 for significance.

View larger version (22K): [in this window] [in a new window]   Fig. 5. Averaged cytosolic [Ca2+]-LVP loops obtained during perfusion of 125 nM Nif (A) and 150 µM Lid (B) 30 min before (preischemia) and 30 min after reperfusion (postischemia). The pre- and postischemia loop area was greater for Nif (P < 0.05) after ischemia and not different for Lid. See Table 1 for significance.

View larger version (52K): [in this window] [in a new window]   Fig. 6. d[Ca2+]/dtmax (A) and dLVP/dtmax (B) at baseline, during peak response of drugs (drug 1) before ischemia, at 30 min of reperfusion (RP 30 min), and during peak response of drugs (drug 2) after ischemia. d[Ca2+]/dtmax increased significantly in all groups after ischemia (RP 30 min). dLVP/dtmax was reduced significantly by all drugs (drug 1) before ischemia and depressed by all drugs except for Nif after ischemia (drug 2).

View larger version (43K): [in this window] [in a new window]   Fig. 7. d[Ca2+]/dtmin (A) and dLVP/dtmin (B) at baseline, during peak response of drugs before ischemia, at 30 min of reperfusion, and during peak response of drugs after ischemia. Reperfusion increased d[Ca2+]/dtmin before and after ischemia but increased dLVP/dtmin less after ischemia than before.

View larger version (37K): [in this window] [in a new window]   Fig. 8. A: ratio of (d[Ca2+]/dtmax)/(dLVP/dtmax), VRmax, at baseline, during peak response of drugs before ischemia (drug 1), at 30 min of reperfusion, and during the peak response of drugs after ischemia (drug 2). Reperfusion at 30 min increased VRmax in all groups except Nif. Before ischemia (drug 1) all drugs except BIM increased VRmax; after ischemia (drug 2), Nif and BDM, but not Bim or Lid, increased VRmax. B: ratio of (d[Ca2+]/dtmin)/(dLVP/dtmin), VRmin, at baseline, during peak response of drugs before ischemia, at 30 min of reperfusion, and during the peak response of drugs after ischemia. The effect of the drugs before (drug 1) and after (drug 2) ischemia were qualitatively similar to Fig. 6A.

View larger version (29K): [in this window] [in a new window]   Fig. 9. Ratio of area Ca2+ to area LVP, AR, at baseline, during the peak response of drugs before ischemia (drug 1), at 30 min of reperfusion, and during the peak response of drugs after ischemia (drug 2). Reperfusion at 30 min increased AR in all groups.

Figure 9 shows that the AR was increased by BDM and Nif before (drug 1) and after ischemia (drug 2) compared with the baseline and 30 min of reperfusion. There was no significant change in AR by Bim or Lid before ischemia or over that caused by ischemia alone. At 30 min of reperfusion, AR was increased significantly above baseline in all groups; only Nif had a lower AR compared with the control group. Postischemia drug (drug 2) treatments showed greater increases in AR for each group compared with the preischemia drug treatment (drug 1).

	DISCUSSION

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This study shows that, first, reperfusion after 30 min of ischemia in control hearts (no drug) shifts the cytosolic [Ca²⁺]-LVP loop downward, i.e., decreased LVP, and rightward, i.e., increased cytosolic [Ca²⁺]. Second, at 30 min of reperfusion in drug-pretreated hearts, the [Ca²⁺]-LVP loop is shifted leftward, i.e., decreased systolic [Ca²⁺], and upward, i.e., increased LVP, compared with nontreated hearts; this indicates drug-induced preconditioning. These shifts are most pronounced in Nif-treated hearts. Third, before ischemia, each drug reduces the loop area with respect to baseline by reducing LVP and for Nif by also lowering systolic [Ca²⁺]. Fourth, after ischemia, each drug differently shifts the [Ca²⁺]-LVP loop by reducing LVP more than systolic [Ca²⁺]. Fifth, in the control hearts, I/R injury results in decreased contractile and relaxation responses, i.e., increased VR_max and VR_min. Each drug treatment before ischemia, except for Bim, reduces the contractile response, i.e., increased VR_max and VR_min. Sixth, at 30 min of reperfusion, AR increases in all groups; however, Nif-treated hearts exhibit the least increase. This suggests that, by comparison, reduced total [Ca²⁺] (Table 1, i.e., less area [Ca²⁺]) is required to effect contraction and relaxation (Table 1, larger area LVP) in the Nif-treated hearts. Overall, our study shows that negative inotropic drugs are less effective in reducing [Ca²⁺] than LVP after ischemia. Finally, and importantly, each drug, especially Nif, given before ischemia induces a preconditioning effect as shown by improved LVP and lower [Ca²⁺] on reperfusion after ischemia.

Selection and comparison of negative inotropic drugs.

This study follows up our recent work (11) in which we examined the differential alteration of Ca²⁺-LVP relationships before and after I/R by four positive inotropic agents. Here, we selected four negative inotropic drugs that either 1) directly block sarcolemmal Ca²⁺ channels (Nif) or depress myofilament sensitivity (BDM) among other effects; 2) indirectly alter Ca²⁺ uptake by depressing Na⁺ channel activation to enhance Na⁺/Ca²⁺exchange activity (Lid); or 3) hasten repolarization to reduce transient Ca²⁺ influx and reduce [Ca²⁺] loading (Bim).

As in our recent studies (11, 36), we used a lower [CaCl₂] (1.25 mM) to better distinguish maximal effects on contractility and cytosolic [Ca²⁺] by the positive inotropic agents and selected only one concentration of each drug that provided submaximal depression of LVP. It was not our intent to conduct concentration-response curves to compare the efficacy of these drugs, so we could not directly compare cardiac effects of different drugs. For much of the results (Tables 1 and 2 and Figs. 6–9), we compared changes in responses only within a drug group.

We chose the isolated heart model to minimize influence of cardiac preload and afterload, blood-borne factors, and autonomic nervous system function. Nif, BDM, and Lid, but not Bim, decreased heart rate by 35%. This indicates that these drugs have direct effects on pacemaker cells and Purkinje fibers possibly via Ca²⁺ and Na⁺ channels. Lid, for example, may reduce sinoatrial node activity by depressing L-type Ca²⁺ channels (30) or may depress spontaneous activity of Purkinje fibers by inhibiting the fast Na⁺ channel (1).

Three ratios (VR_max, VR_min, and AR) furnished normalized values for indexes of LVP and Ca²⁺ and so allowed comparison among drugs. We derived these ratios as they each yield information on the cost of performing work, i.e., the net amount of Ca²⁺ available to produce a comparable decrease in contractility and relaxation over the cardiac cycle. Therefore, an increase in any ratio meant that contractile responsiveness was decreased, and vice versa. Unlike the other three drugs, Bim perfusion before and after ischemia did not increase VR_max, and VR_min, and Bim and Lid did not alter AR. These findings indicate that these drugs improved the cost-effective use of available [Ca²⁺] for the contraction and relaxation responses compared with baseline or reperfusion. I/R injury, assessed at 30 min of reperfusion, attenuated this responsiveness as shown by increased VR_max and VR_min in the control and drug-pretreated groups except for the Nif group, which showed no change in VR_max, VR_min, and AR.

It should be noted that [Ca²⁺]-LVP loops and the above analyses of various indexes of Ca²⁺ plotted against LVP do not furnish any temporal information. This, however, can be extracted from our study in which we compared effects of two positive and two negative inotropic drugs on several additional static and dynamic indexes of the [Ca²⁺]-LVP relationship in the absence of ischemia (36). For those drugs that might reduce heart rate to increase cardiac cycle length, the rates of cytosolic Ca²⁺ inflow and outflow and the rates of LVP development and relaxation are slowed. The heart rate effect is normalized to some degree by the ratios of an index of Ca²⁺ to the same index of LVP.

Ischemia-reperfusion and cytosolic [Ca²⁺]-to-LVP relationships.

We reported that I/R injury increases cytosolic [Ca²⁺] and decreases contractility in the isolated heart model (3, 11, 56). We found that [Ca²⁺]-LVP response curves generated by changing extracellular CaCl₂ concentration before and after ischemia showed a reduced maximal contractile response and a rightward shift of the normalized [Ca²⁺]-LVP relationship. This indicated that ischemia causes a reduced maximal activated contractile force and reduced sensitivity to Ca²⁺ (3, 56). In this model, I/R injury causes not only cardiac dysfunction but also infarction. In other studies under similar conditions, we found 50–60% ventricular infarction and 40% recovery of systolic-diastolic LVP after 1–2 h of reperfusion in control hearts (3, 56). We (11) showed recently that the [Ca²⁺]-LVP loop relationship is altered by ischemia by shifting the loop base upward (increased diastolic LVP) and to the right (increased systolic [Ca²⁺]) on reperfusion. In the present study, we found that I/R injury significantly increased VR_max, VR_min, and AR. Although the negative inotropic drugs given before ischemia variably preconditioned hearts as evidenced by loop characteristics, we did not observe any difference in contraction (dLVP/dt_max) or relaxation (dLVP/dt_min) effects between control and drug-treated groups at 30 min of reperfusion. However, reperfusion-induced systolic Ca²⁺ overloading was attenuated in all pretreated hearts after ischemia. These results confirm and extend our previous results that the decrease in myocardial Ca²⁺ responsiveness and increase in Ca²⁺ loading can be ameliorated by preconditioning by negative inotropic drugs.

Preconditioning effects of negative inotropic drugs.

Ca²⁺ channel blockers are well known to be cardioprotective. Nif protects when given after I/R injury (12, 25, 34, 35, 41), but little is known about a possible preconditioning effect before ischemia. Cain et al. (7) conducted in vitro experiments on human atrial trabecular tissue removed from patients receiving Ca²⁺ channel blockers and found that ischemic preconditioning was prevented; moreover, exogenous CaCl₂ added to the bath elicited preconditioning (8). We found that Nif reduced both cytosolic [Ca²⁺] and LVP before ischemia and preserved [Ca²⁺]-LVP loop characteristics better than the other drugs that reduced LVP but not systolic [Ca²⁺]. At 30 min of reperfusion, the Nif-treated hearts were more efficient in utilizing Ca²⁺ (smaller area Ca²⁺) for LVP (area LVP) generation and relaxation (reduced VR_max).

The preconditioning effect of Nif may be indirectly related to its Ca²⁺ channel-blocking action. For example, adenosine preconditioning may be mediated in part by reduced transsarcolemmal Ca²⁺ uptake. A₂ agonist stimulation of Ca²⁺ uptake attenuated adenosine preconditioning, and Nif abolished the A₂ agonist-induced attenuation of preconditioning (50). It was suggested that Nif blocked the increase in Ca²⁺ uptake by A₂ agonists as well as their effect to attenuate preconditioning by adenosine. Thus Nif may precondition the heart in part by reducing [Ca²⁺], which then may trigger intracellular events (memory effect) leading to preconditioning.

K_ATP channel openers like Bim are well known to elicit preconditioning (9, 13, 20). BDM given just before the onset of ischemia is cardioprotective (5), possibly by virtue of its effects to inhibit actinomyosin ATPase and reduce ATP consumption (45) or to decrease myofilament Ca²⁺ sensitivity (17, 21, 22). Overall, Bim, like Nif, may be effective therapeutically in the postischemic heart as K_ATP channel-induced preconditioning is a potential design for clinical trials, especially in patients subjected to short periods of ischemia, e.g., in angioplasty.

Lid given before or early during ischemia protects against I/R injury (16, 32, 52, 54, 55), but it appears not to be protective when given after ischemia (16). Possible mechanisms for Lid preconditioning are its effects on blocking Na⁺ channels, its antioxidant activity (14), its energy-sparing effect due to reduced Na,K-ATPase activity, and its indirect effect to reduce Ca²⁺ loading secondary to reduced Na⁺/Ca²⁺ exchange. Any of these potential effects of negative inotropic drugs must only initiate, but not mediate, the process of preconditioning because the drug is washed out before ischemia.

Effects of negative inotropic drugs on [Ca²⁺]-LVP relationships.

Cardiac K_ATP channels are believed to play a role in modulating cardiac function, particularly under conditions of metabolic stress, as in I/R injury (9). K_ATP channel opening shortens the AP by enhancing phase 3 repolarization; this inhibits the L-type Ca²⁺ channel and slows reverse-mode Na⁺/Ca²⁺ exchanger. These actions may lead to a decrease in cytosolic [Ca²⁺] and a subsequent reduction in contractility (40). Bim had a small negative inotropic and vasodilatory effects when given before ischemia, as shown previously (42). In our study, Bim reduced cytosolic Ca²⁺ loading on reperfusion compared with the control group and depressed both LVP and [Ca²⁺] compared with 30 min of reperfusion; this suggests that after ischemia the myocardium may become sensitized to the K_ATP channel agonist, perhaps as a result of altered ATP level in the vicinity of K_ATP channels (42). This notion is supported by findings that the stunned myocardium may have a reduced amount of ATP for an extended period on reperfusion (58). Postischemia Bim also improved contractile responsiveness, VR_max and AR, for the [Ca²⁺] available. This may be due in part to an increase in CF after ischemia (Table 2).

BDM exerts its negative inotropic actions by reducing the Ca²⁺ current to shorten the AP, by inhibiting cross-bridge function (45), and by decreasing myofilament sensitivity to Ca²⁺ (21, 22). BDM may inhibit Ca²⁺ entry by dephosphorylation of L-type Ca²⁺ channels (2, 21, 44). These findings are consistent with our results in that 4 mM BDM given before ischemia depressed LVP without altering [Ca²⁺] (Table 1). After reperfusion, depression of LVP by BDM was associated with reduced [Ca²⁺], as evidenced by a downward and leftward shift in the [Ca²⁺]-LVP curve (Table 1). This suggests that structural changes due to I/R injury could alter cell membrane properties (17) so that cells become more susceptible to modulation of Ca²⁺ homeostasis by BDM. Indeed, it is reported that 10 mM BDM added to the perfusate after ischemia attenuated a reperfusion-induced rise in resting tension and reduced [Ca²⁺] (17).

Nif, unlike Bim, BDM, or Lid, reduced both [Ca²⁺] and LVP before and after ischemia to cause a downward and slight leftward shift in the [Ca²⁺]-LVP loop (Table 1 and Figs. 4 and 5), which was not altered by ischemia, as it was with BDM, Bim, and Lid. However, Nif perfusion after 30 min of reperfusion increased AR less than did Bim. Nif pretreatment preserved the [Ca²⁺]-LVP loop at 30 min of reperfusion better than did Bim pretreatment, but Nif treatment, per se, was less effective in depressing both LVP and [Ca²⁺] as shown by the loops (Figs. 4 and 5), by the smaller AR after reperfusion, and by the increased VR_max and VR_min compared with Bim. In our recent study (36), we showed that Nif exhibited less loop hysteresis compared with Lid; this suggested a more efficient linkage of [Ca²⁺] to elicit contraction with Lid than with Nif.

Lid exerts its negative inotropic action primarily by suppressing the fast Na⁺ channel (32, 52–54, 57) and possibly also by disrupting active Ca²⁺ transport by the SR (57). In our study, hearts briefly exposed to Lid and washed out before ischemia were protected against Ca²⁺ overload (Table 1). Lid depressed CF and MO₂ in the preischemic heart but not in the postischemic heart, which suggests that depression of LVP before ischemia could be due in part to decreased metabolic function. Lid affinity for the Na⁺ channel increases during I/R injury due to reduced intracellular pH and inactivation of the channel (53). Reduced [Ca²⁺] on reperfusion in Lid-treated hearts may be attributed to a decrease in Na⁺ influx during I/R (55). Lid may also reduce cytosolic [Ca²⁺] (27, 48), especially at higher concentrations, by inhibiting the L-type Ca²⁺ channel (27, 51). Decreases in both [Ca²⁺] and LVP by Lid in the postischemic hearts, but not in preischemic hearts, may also be attributed in part to changes in the affinity of Lid for Na⁺ channels. Na⁺ channel blockade can lead to decreased Na⁺ influx and subsequently to decrease [Ca²⁺] and contractility. The shift in the [Ca²⁺]-LVP loop (Fig. 5B) downward and leftward after ischemia, but not before ischemia, illustrates differential modulation of [Ca²⁺]-LVP loop characteristics.

Summary and conclusions.

This study first provides a better understanding of the mechanisms and differences among negative inotropic drugs on several [Ca²⁺]-LVP indexes during the cardiac cycle. We showed that differently acting negative inotropic agents given before and after ischemia modulate [Ca²⁺]-LVP relationships differently both within and across treatment comparisons. Second, using these same indexes, we showed that these drugs differently precondition hearts. In in vivo blood-perfused and innervated hearts, the effects of these drugs given over a period of time would likely be different from our observations, but the overall effect of cytosolic [Ca²⁺] on beat-to-beat contractility and relaxation is probably similar. Thus, if these isolated heart results were to be applied to the clinical setting, they would suggest that diverse classes of drugs like Na⁺ and Ca²⁺ channel blockers, K_ATP channel openers, and actinomyosin ATPase inhibitors remain effective in reducing contractility after ischemia, albeit at higher levels of cytosolic [Ca²⁺]. Moreover, in a clinical setting, these drugs may elicit cardiac preconditioning as a defense against I/R injury.

	GRANTS

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This research was supported in part by National Institutes of Health Grants R01-HL-58691, KO1 HL-073246-01, and R01–5T32 GM-08377, by American Heart Association Grant 0355608Z, and by the Innovative Medizinische Forschung Grant Ri610005, Westfälische Wilhelms-Universität Münster, Germany.

	DISCLOSURES

TOP ABSTRACT METHODS RESULTS DISCUSSION GRANTS DISCLOSURES REFERENCES

 
Portions of this work have appeared in abstract form in Biophys J 82: 66A, 653A, and 654A, 2002; FASEB J 16: A857, 2002; and Anesthesiology 95: A622, 2001.

	ACKNOWLEDGMENTS

 
The authors thank Dr. Srinivasan G. Varadarajan, Dr. Ming Tao Jiang, Jim Heisner, and Anita Tredeau for valuable contributions.

	FOOTNOTES

 

Address for reprint requests and other correspondence: D. F. Stowe, M4280, 8701 Watertown Plank Rd., Medical College of Wisconsin, Milwaukee Regional Medical Center, Milwaukee, WI 53226 (E-mail: dfstowe{at}mcw.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.

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