Reversible effects of isoproterenol-induced hypertrophy on in situ left ventricular function in rat hearts

The aim of the present study was to evaluate specifically left ventricular (LV) function in rat hearts as they transition from the normal to hypertrophic state and back to normal. Either isoproterenol (1.2 and 2.4 mg.kg(-1).day(-1) for 3 days; Iso group) or vehicle (saline 24 microl.day(-1) for 3 days; Sa group) was infused by subcutaneous implantation of an osmotic minipump. After verifying the development of cardiac hypertrophy, we recorded continuous LV pressure-volume (P-V) loops of in situ ejecting hypertrophied rat hearts. The curved LV end-systolic P-V relation (ESPVR) and systolic P-V area (PVA) were obtained from a series of LV P-V loops in the Sa and Iso groups 1 h or 2 days after the removal of the osmotic minipump. PVA at midrange LV volume (PVA(mLVV)) was taken as a good index for LV work capability (13, 15, 20, 21). However, in rat hearts during remodeling, whether PVA(mLVV) is a good index for LV work capability has not been determined yet. In the present study, in contrast to unchanged end-systolic pressure at midrange LV volume, PVA(mLVV) was significantly decreased by isoproterenol treatment relative to saline; however, these measurements were the same 2 days after pump removal. Simultaneous treatment with a beta(1)-blocker, metoprolol (24 mg.kg(-1).day(-1)), blocked the formation of cardiac hypertrophy and thus PVA(mLVV) did not decrease. The reversible changes in PVA(mLVV) reflect precisely the changes in LV work capability in isoproterenol-induced hypertrophied rat hearts mediated by beta(1)-receptors. These results indicate that the present approach may be an appropriate strategy for evaluating the effects of antihypertrophic and antifibrotic modalities.     

Na+/Ca2+ exchange inhibition protects the rat heart from ischemia-reperfusion injury by blocking energy-wasting processes

We have recently reported that exposure of rat hearts to high Ca(2+) produces a Ca(2+) overload-induced contractile failure in rat hearts, which was associated with proteolysis of alpha-fodrin. We hypothesized that contractile failure after ischemia-reperfusion (I/R) is similar to that after high Ca(2+) infusion. To test this hypothesis, we investigated left ventricular (LV) mechanical work and energetics in the cross-circulated rat hearts, which were subjected to 15 min global ischemia and 60 min reperfusion. Sixty minutes after I/R, mean systolic pressure-volume area (PVA; a total mechanical energy per beat) at midrange LV volume (mLVV) (PVA(mLVV)) was significantly decreased from 5.89 +/- 1.55 to 3.83 +/- 1.16 mmHg.ml.beat(-1).g(-1) (n = 6). Mean myocardial oxygen consumption per beat (Vo(2)) intercept of (Vo(2)-PVA linear relation was significantly decreased from 0.21 +/- 0.05 to 0.15 +/- 0.03 microl O(2).beat(-1).g(-1) without change in its slope. Initial 30-min reperfusion with a Na(+)/Ca(2+) exchanger (NCX) inhibitor KB-R7943 (KBR; 10 micromol/l) significantly reduced the decrease in mean PVA(mLVV) and Vo(2) intercept (n = 6). Although Vo(2) for the Ca(2+) handling was finally decreased, it transiently but significantly increased from the control for 10-15 min after I/R. This increase in Vo(2) for the Ca(2+) handling was completely blocked by KBR, suggesting an inhibition of reverse-mode NCX by KBR. alpha-Fodrin proteolysis, which was significantly increased after I/R, was also significantly reduced by KBR. Our study shows that the contractile failure after I/R is similar to that after high Ca(2+) infusion, although the contribution of reverse-mode NCX to the contractile failure is different. An inhibition of reverse-mode NCX during initial reperfusion protects the heart against reperfusion injury.     

Altered LV inotropic reserve and mechanoenergetics early in the development of heart failure

To test the hypothesis that alterations in left ventricular (LV) mechanoenergetics and the LV inotropic response to afterload manifest early in the evolution of heart failure, we examined six anesthetized dogs instrumented with LV micromanometers, piezoelectric crystals, and coronary sinus catheters before and after 24 h of rapid ventricular pacing (RVP). After autonomic blockade, the end-systolic pressure-volume relation (ESPVR), myocardial O(2) consumption (MVO(2)), and LV pressure-volume area (PVA) were defined at several different afterloads produced by graded infusions of phenylephrine. Short-term RVP resulted in reduced preload with proportionate reductions in stroke work and the maximum first derivative of LV pressure but with no significant reduction in baseline LV contractile state. In response to increased afterload, the baseline ESPVR shifted to the left with maintained end-systolic elastance (E(es)). In contrast, after short-term RVP, in response to comparable increases in afterload, the ESPVR displayed reduced E(es) (P < 0.05) and significantly less leftward shift compared with control (P < 0.05). Compared with the control MVO(2)-PVA relation, short-term RVP significantly increased the MVO(2) intercept (P < 0.05) with no change in slope. These results indicate that short-term RVP produces attenuation of afterload-induced enhancement of LV performance and increases energy consumption for nonmechanical processes with maintenance of contractile efficiency, suggesting that early in the development of tachycardia heart failure, there is blunting of length-dependent activation and increased O(2) requirements for excitation-contraction coupling, basal metabolism, or both. Rather than being adaptive mechanisms, these abnormalities may be primary defects involved in the progression of the heart failure phenotype.     

Calpain inhibitor-1 protects the rat heart from ischemia-reperfusion injury: analysis by mechanical work and energetics

We hypothesized that calpain inhibitor-1 protected left ventricular (LV) function from ischemia-reperfusion injury by inhibiting the proteolysis of alpha-fodrin. To test this hypothesis, we investigated the effect of calpain inhibitor-1 on LV mechanical work and energetics in the cross-circulated rat hearts that underwent 15-min global ischemia and 60-min reperfusion (n = 9). After ischemia-reperfusion with calpain inhibitor-1, mean end-systolic pressure at midrange LV volume and systolic pressure-volume area (PVA) at midrange LV volume (total mechanical energy per beat) were hardly changed, although they were significantly (P < 0.01) decreased after ischemia-reperfusion without calpain inhibitor-1. Mean myocardial oxygen consumption per beat (Vo(2)) intercepts (PVA-independent Vo(2); Vo(2) for the total Ca(2+) handling in excitation-contraction coupling and basal metabolism) of Vo(2)-PVA linear relations were also unchanged after ischemia-reperfusion with calpain inhibitor-1, although they were significantly (P < 0.01) decreased after ischemia-reperfusion without calpain inhibitor-1. There were no significant differences in O(2) costs of LV PVA and contractility among the hearts in control (or normal) postischemia-reperfusion and postischemia-reperfusion with calpain inhibitor-1. Western blot analysis of alpha-fodrin and the immunostaining of 150-kDa products of alpha-fodrin confirmed that calpain inhibitor-1 almost completely protected the proteolysis of alpha-fodrin. Our results indicate that calpain inhibitor-1 prevents the heart from ischemia-reperfusion injury associated with the impairment of total Ca(2+) handling by directly inhibiting the proteolysis of alpha-fodrin.     

Energetics of ventricular contraction as traced in the pressure-volume diagram

The pressure-volume (P-V) relationship of the canine left ventricle can reasonably be simulated by a time-varying elastance model. In this model the total mechanical energy generated by a contraction can be determined theoretically from the change in the elastance. Applying this theory to the actual left ventricle, we have found that the area in the P-V diagram circumscribed by the end-systolic P-V relation line, the end-diastolic P-V relation curve, and the systolic segment of the P-V trajectory is equivalent to the total mechanical energy generated by ventricular contraction. We call this area the systolic P-V area (PVA). We have studied experimentally the correlation between the PVA and myocardial oxygen consumption (VO2) in the canine left ventricle. VO2 was linearly correlated with PVA regardless of the contraction mode and loading conditions in a given left ventricle. The VO2-PVA relation parallel shifted upward with positive inotropic agents. This shift comprised a significant increase in VO2 component for the unloaded contraction. We therefore consider that further analyses of the VO2-PVA relationship will greatly promote our understanding of cardiac energetics.     

Dynamic effects of positive-pressure ventilation on canine left ventricular pressure-volume relations.

Positive-pressure ventilation (PPV) may affect left ventricular (LV) performance by altering both LV diastolic compliance and pericardial pressure (Ppc). We measured the effect of PPV on LV intraluminal pressure, Ppc, LV volume, and LV cross-sectional area in 17 acute anesthetized dogs. To account for changes in lung volume independent of changes in Ppc and differences in contractility, measures were made during both open- and closed-chest conditions, during closed chest with and without chest wall binding, and after propranolol-induced acute ventricular failure (AVF). Apneic end-systolic pressure-volume relations (ESPVR) were generated by inferior vena caval occlusions. With the open chest, PPV had no effects. With the chest closed, PPV inspiration decreased LV end-diastolic volume (EDV) along its diastolic compliance curve and decreased end-systolic volume (ESV) such that the end-systolic pressure-volume domain was shifted to a point left of the LV ESPVR, even when referenced to Ppc. The decrease in EDV was greater in control than in AVF conditions, whereas the shift of the ESV to the left of the ESPVR was greater with AVF than in control conditions. We conclude that the hemodynamic effects of PPV inspiration are due primarily to changes in intrathoracic pressure and that the inspiration-induced decreases of LV EDV reflect direct effects of intrathoracic pressure on LV filling. The decreases in LV ESV exceed the amount explained solely by a reduction in LV ejection pressure.     

Effects of changes in left ventricular contractility on indexes of contractility in mice

Measurement of left ventricular (LV) function is often overlooked in murine studies, which have been used to analyze the effects of genetic manipulation on cardiac phenotype. The goal of this study was to address the effects of changes in LV contractility on indexes of contractility in mice. LV function was assessed in vivo in closed-chest mice by echocardiography and by LV catheterization using a conductance pressure-volume (P-V) catheter with three different interventions that alter contractility by 1) atrial pacing to increase inotropy by augmentation of the force-frequency relation (modest increment of inotropy), 2) dobutamine to maximize inotropy, and 3) esmolol infusion to decrease contractility. Load-independent parameters derived from P-V relations, such as slope of end-systolic P-V relations (ESPVR) and slope of the first maximal pressure derivative over time (dP/dt(max))-end-diastolic volume relation (dP/dt-EDV), and standard echocardiographic parameters were measured. The dP/dt-EDV changed the most among parameters after atrial pacing and dobutamine infusion (percent change, 162.8 +/- 95.9% and 271.0 +/- 44.0%, respectively). ESPVR was the most affected by a decrease in LV contractility during esmolol infusion (percent change, -49.8 +/- 8.3%). However, fractional shortening failed to detect changes in contractility during atrial pacing and esmolol infusion and its percent change was <20%. This study demonstrated that contractile parameters derived from P-V relations change the most during a change in LV contractility and should therefore best detect a small change in contractility in mice. Heart rate has a modest but significant effect on P-V relationship-derived indexes and must be considered in the evaluation of murine cardiac physiology.     

Normal distribution of ventricular pressure-volume area of arrhythmic beats under atrial fibrillation in canine heart

We previously found the frequency distribution of the left ventricular (LV) effective afterload elastance (E(a)) of arrhythmic beats to be nonnormal or non-Gaussian in contrast to the normal distribution of the LV end-systolic elastance (E(max)) in canine in situ LVs during electrically induced atrial fibrillation (AF). These two mechanical variables determine the total mechanical energy [systolic pressure-volume area (PVA)] generated by LV contraction when the LV end-diastolic volume is given on a per-beat basis. PVA and E(max) are the two key determinants of the LV O(2) consumption per beat. In the present study, we analyzed the frequency distribution of PVA during AF by its chi(2), significance level, skewness, and kurtosis and compared them with those of other major cardiodynamic variables including E(a) and E(max). We assumed the volume intercept (V(0)) of the end-systolic pressure-volume relation needed for E(max) determination to be stable during arrhythmia. We found that PVA distributed much more normally than E(a) and slightly more so than E(max) during AF. We compared the chi(2), significance level, skewness, and kurtosis of all the complex terms of the PVA formula. We found that the complexity of the PVA formula attenuated the effect of the considerably nonnormal distribution of E(a) on the distribution of PVA along the central limit theorem. We conclude that mean (SD) of PVA can reliably characterize the distribution of PVA of arrhythmic beats during AF, at least in canine hearts.     

Induction of cardioplegic arrest immediately activates the myocardial apoptosis signal pathway

Myocardial ischemia-reperfusion, including cardioplegic arrest (CA), has been associated with cardiac apoptosis induction. However, the time course of apoptosis activation and the trigger mechanisms are still unclear. Because apoptosis inhibition may represent a novel therapeutic strategy for long-term myocardial preservation, we sought to investigate the time course of apoptosis signal-pathway induction during CA. As to method, Sprague-Dawley rats (300-350 g) were anesthetized, intubated, and mechanically ventilated. CA was initiated by infusion of ice-cold crystalloid solution (Custodiol, 10 ml/kg) into the aortic root, and hearts were rapidly excised and stored for 0, 30, 60, and 120 min in 0.9% sodium chloride solution (28 degrees C). In controls, no CA was initiated before removal and storage at 28 degrees C. In another group, calcium-rich cardioplegia was used, and an additional group received a caspase-8 inhibitor before CA induction. Left ventricular cytosolic extracts were isolated and investigated for the activity of caspase-3 and -6 (effector caspases) and caspase-8 and -9 (involved in extrinsic and intrinsic pathways of apoptosis induction). Fluorometric activity assays were performed by using specific substrates. As a result, activities of all tested caspases were significantly increased immediately after CA induction compared with controls. Administration of the caspase-8 inhibitor significantly reduced activities of all caspases. With calcium-rich cardioplegia, caspase activities were significantly lower compared with low-calcium CA. Control hearts also showed an increase of caspase activities during cold-storage ischemia without CA but had significantly different time courses compared with hearts with CA. In conclusion, our data show rapid apoptosis signal-pathway induction immediately following CA exposure. Thus apoptosis signal-pathway inhibition as a potential strategy for improved myocardial preservation would have the greatest effect when applied before CA exposure.     

Activation of PKC decreases myocardial O2 consumption and increases contractile efficiency in rats

The effect of protein kinase C (PKC) activation on cardiac mechanoenergetics is not fully understood. To address this issue, we determined the effects of the PKC activator phorbol 12-myristate 13-acetate (PMA) on isolated rat hearts. Hearts were exposed to PMA with or without pretreatment with the PKC inhibitor chelerythrine. Contractile efficiency was assessed as the reciprocal of the slope of the linear myocardial O2 consumption (VO2) pressure-volume area (PVA) relation. PMA decreased contractility (Emax; -30 +/- 8%; P < 0.05) and increased coronary perfusion pressure (+58 +/- 11%; P < 0.01) without altering left ventricular end-diastolic pressure. Concomitantly, PMA decreased PVA-independent VO2 [nonmechanical energy expenditure for excitation-contraction (E-C) coupling and basal metabolism] by 28 +/- 8% (P < 0.05) and markedly increased contractile efficiency (+41 +/- 8%; P < 0.05) in a manner independent of the coronary vascular resistance. Basal metabolism was not affected by PMA. Chelerythrine abolished the PMA-induced vasoconstriction, negative inotropy, decreased PVA-independent VO2, and increased contractile efficiency. We conclude that PKC-mediated phosphorylation of regulatory proteins reduces VO2 via effects on both the contractile machinery and the E-C coupling.     

Pressor effect of electroacupuncture on hemorrhagic hypotension

Neiguan (PC-6) is a traditional acupoint in each forearm and overlies the trunk of the median nerve. Previous studies show that electroacupuncture (EA) at the Neiguan acupoint could improve not only myocardial ischemic dysfunction by inducing a depressor response but also recover hemorrhagic hypotension by inducing a pressor response. However, their physiological mechanisms are not yet elucidated. We investigated the pressor effect of Neiguan EA and its mechanism by focusing on left ventricular (LV) performance in a canine hemorrhagic hypotension model. We hemorrhaged 36 anesthetized and thoracotomized mongrel dogs and decreased LV end-systolic pressure (ESP) to approximately 70 mmHg (35% decrease). We obtained LV pressure-volume (P-V) data with a micromanometer catheter and a conductance catheter. One-hour Neiguan EA significantly recovered the decreased ESP, end-diastolic volume, and stroke volume by 32 +/- 13%, 27 +/- 13%, and 39 +/- 17%, respectively (P < 0.05), without changing heart rate and the slope of the end-systolic P-V relation. Neiguan EA inhibited a hemorrhage-induced increase in plasma catecholamines. However, vecuronium (neuromuscular blocking agent) administration abolished the antihypotension effect of Neiguan EA. Furthermore, Neiguan EA was much more effective than a nonacupoint thigh EA. We conclude that Neiguan EA achieved the antihypotension effect by improving LV filling of the hemorrhage-depressed LV performance despite the inhibition of the hemorrhage-increased plasma catecholamines. This pressor effect seemed to accompany an increased venous return by Neiguan EA-increased vasomotor tone and muscle pump. This study demonstrated a scientific basis for the therapeutic efficacy of acupuncture in the treatment of hemorrhagic hypotension and shock.     

Characterization of right ventricular function after monocrotaline-induced pulmonary hypertension in the intact rat

We characterized hemodynamics and systolic and diastolic right ventricular (RV) function in relation to structural changes in the rat model of monocrotaline (MCT)-induced pulmonary hypertension. Rats were treated with MCT at 30 mg/kg body wt (MCT30, n = 15) and 80 mg/kg body wt (MCT80, n = 16) to induce compensated RV hypertrophy and RV failure, respectively. Saline-treated rats served as control (Cont, n = 13). After 4 wk, a pressure-conductance catheter was introduced into the RV to assess pressure-volume relations. Subsequently, rats were killed, hearts and lungs were rapidly dissected, and RV, left ventricle (LV), and interventricular septum (IVS) were weighed and analyzed histochemically. RV-to-(LV + IVS) weight ratio was 0.29 +/- 0.05 in Cont, 0.35 +/- 0.05 in MCT30, and 0.49 +/- 0.10 in MCT80 (P < 0.001 vs. Cont and MCT30) rats, confirming MCT-induced RV hypertrophy. RV ejection fraction was 49 +/- 6% in Cont, 40 +/- 12% in MCT30 (P < 0.05 vs. Cont), and 26 +/- 6% in MCT80 (P < 0.05 vs. Cont and MCT30) rats. In MCT30 rats, cardiac output was maintained, but RV volumes and filling pressures were significantly increased compared with Cont (all P < 0.05), indicating RV remodeling. In MCT80 rats, RV systolic pressure, volumes, and peak wall stress were further increased, and cardiac output was significantly decreased (all P < 0.05). However, RV end-systolic and end-diastolic stiffness were unchanged, consistent with the absence of interstitial fibrosis. MCT-induced pressure overload was associated with a dose-dependent development of RV hypertrophy. The most pronounced response to MCT was an overload-dependent increase of RV end-systolic and end-diastolic volumes, even under nonfailing conditions.     

Myocardial interstitial glucose and lactate before,during, and after cardioplegic heart arrest

The interstitial fluid of the human myocardium was monitored in 13 patients undergoing aortic valve and/or bypass surgery before, during, and after hypothermic potassium cardioplegia. The regulation of glucose and lactate was studied after sampling with microdialysis. The following questions were addressed. 1). Is the rate of transcapillary diffusion the limiting step for myocardial uptake of glucose before or after cardioplegia? 2). Does cold potassium cardioplegia induce a critical deprivation of glucose and/or accumulation of lactate in the myocardium? Before cardioplegia, interstitial glucose was approximately 50% of the plasma level (P < 0.001). Interstitial glucose decreased significantly immediately after induction of cardioplegia and remained low (1.25 +/- 0.25 mM) throughout cardioplegia. It was restored to precardioplegic levels 1 h after release of the aortic clamp. Interstitial glucose then decreased again at 25 and 35 h postoperatively to the levels observed during cardioplegia. Interstitial lactate decreased immediately after induction of cardioplegia but returned to basal level during the clamping period. At 25 and 35 h, interstitial lactate was significantly lower than before and during cardioplegia. Glucose transport over the capillary endothelium is considered rate limiting for its uptake in the working heart but not during cold potassium cardioplegia despite the glucose deprivation following perfusion of glucose-free cardioplegic solution. Lactate accumulated during cardioplegia but never reached exceedingly high interstitial levels. We conclude that microdialysis provides information that may be relevant for myocardial protection during open-heart surgery.     

Negative inotropism of hyperthermia increases oxygen cost of contractility in canine hearts

Heart temperature affects left ventricular (LV) function and myocardial metabolism. However, how and whether increasing heart temperature affects LV mechanoenergetics remain unclear. We designed the present study to investigate effects of increased temperature by 5 degrees C from 36 degrees C on LV contractility and energetics. We analyzed the LV contractility index (E(max)) and the relation between the myocardial oxygen consumption (MVO(2)) and the pressure-volume area (PVA; a measure of LV total mechanical energy) in isovolumically contracting isolated canine hearts during normothermia (NT) and hyperthermia (HT). HT reduced E(max) by 38% (P < 0.01) and shortened time to E(max) by 20% (P < 0.05). HT, however, altered neither the slope nor the unloaded MVO(2) of the MVO(2)-PVA relation. HT increased the oxygen cost of contractility (the incremental ratio of unloaded MVO(2) to E(max)) by 49%. When Ca(2+) infusion restored the reduced LV contractility during HT to the NT baseline level, the unloaded MVO(2) in HT exceeded the NT value by 36%. We conclude that HT-induced negative inotropism accompanies an increase in the oxygen cost of contractility.     

Deficiency of TIMP-1 exacerbates LV remodeling after myocardial infarction in mice

Recent studies have been directed at modulating the heart failure process through inhibition of activated matrix metalloproteinases (MMPs). We hypothesized that a loss of MMP inhibitory control by tissue inhibitor of MMP (TIMP)-1 deficiency alters the course of postinfarction chamber remodeling and induced chronic myocardial infarction (MI) in wild-type (WT) and TIMP-1(-/-) mice. Left ventricular (LV) pressure-volume loops obtained from WT and TIMP-1(-/-) mice demonstrated that LV end-diastolic volume [52 +/- 4 (WT) vs. 71 +/- 6 (TIMP-1(-/-)) microl] and LV end-diastolic pressure [9.0 +/- 1.2 (WT) vs. 12.7 +/- 1.4 (TIMP-1(-/-)) mmHg] were significantly increased in the TIMP-1(-/-) mice 2 wk after MI. LV contractility was reduced to a similar degree in the WT and TIMP-1(-/-) groups after MI, as indicated by a significant fall in the LV end-systolic pressure-volume relationship. Ventricular weight and cross-sectional areas of LV myocytes were significantly increased in TIMP-1(-/-) mice, indicating that the hypertrophic response was more pronounced. The observed significant loss of fibrillar collagen in the TIMP-1(-/-) controls may have been an important contributory factor for the observed LV alterations in the TIMP-1(-/-) mice after MI. These findings demonstrate that TIMP-1 deficiency amplifies adverse LV remodeling after MI in mice and emphasizes the importance of local endogenous control of cardiac MMP activity by TIMP-1.     

Single-beat estimation of right ventricular end-systolic pressure-volume relationship

Assessment of right ventricular (RV) contractility from end-systolic pressure-volume relationships (ESPVR) is difficult due to problems in measuring RV instantaneous volume and to effects of changes in RV preload or afterload. We therefore investigated in anesthetized dogs whether RV ESPVR and contractility can be determined without measuring RV volume and without changing RV preload or afterload. The maximal RV pressure of isovolumic beats (P(max)) was predicted from isovolumic portions of RV pressure during ejecting beats and compared with P(max) measured during the first beat after pulmonary artery clamping. In RV pressure-volume loops obtained from RV pressure and integrated pulmonary arterial flow, end-systolic elastance (E(es)) was assessed as the slope of P(max)-derived ESPVR, pulmonary artery effective elastance (E(a)) as the slope of end-diastolic to end-systolic relation, and coupling efficiency as the E(es)-to-E(a) ratio (E(es)/E(a)). Predicted P(max) correlated with observed P(max) (r = 0.98 +/- 0.02). Dobutamine increased E(es) from 1.07 to 2.00 mmHg/ml and E(es)/E(a) from 1.64 to 2.49, and propranolol decreased E(es)/E(a) from 1.64 to 0.91 (all P < 0.05). After adrenergic blockade, preload reduction did not affect E(es), whereas hypoxia and arterial constriction markedly increased E(a) and somewhat increased E(es) due to the Anrep effect. Low preload did not affect E(es)/E(a) and high afterload decreased E(es)/E(a). In conclusion, in the right ventricle 1) P(max) can be calculated from normal beats, 2) P(max) can be used to determine ESPVR without change in load, and 3) P(max)-derived ESPVR can be used to assess ventricular contractility and ventricular-arterial coupling efficiency.     

Evaluation of BM-573, a novel TXA2 synthase inhibitor and receptor antagonist, in a porcine model of myocardial ischemia-reperfusion

AIMS: To investigate whether BM-573 (N-tert-butyl-N'-[2-(4'-methylphenylamino)-5-nitro-benzenesulfonyl]urea), an original combined thromboxane A2 synthase inhibitor and receptor antagonist, prevents reperfusion injury in acutely ischemic pigs. METHODS: Twelve animals were randomly divided in two groups: a control group (n = 6) intravenously infused with vehicle, and a BM-573-treated group (n = 6) infused with BM-573 (10 mg kg(-1) h(-1)). In both groups, the left anterior descending (LAD) coronary artery was occluded for 60 min and reperfused for 240 min. Either vehicle or BM-573 was infused 30 min before LAD occlusion and throughout the experiment. Platelet aggregation induced by arachidonic acid ex vivo measured was prevented by BM-573. RESULTS: In both groups, LAD occlusion decreased cardiac output, ejection fraction, slope of stroke work--end-diastolic volume relationship, and induced end-systolic pressure-volume relationship (ESPVR) rightward shift, while left ventricular afterload increased. Ventriculo-arterial coupling and mechanical efficiency decreased. In both groups, reperfusion further decreased cardiac output and ejection fraction, while ESPVR displayed a further rightward shift. Ventriculo-arterial coupling and mechanical efficiency remained impaired. Area at risk, evidenced with Evans blue, was 33.2+/-3.4% of the LV mass (LVM) in both groups, and mean infarct size, revealed by triphenyltetrazolium chloride (TTC), was 27.3+/-2.6% of the LVM in the BM-573-treated group (NS). Histological examination and immunohistochemical identification of desmin revealed necrosis in the anteroseptal region similar in both groups, while myocardial ATP dosages and electron microscopy also showed that BM-573 had no cardioprotective effect. CONCLUSIONS: These data suggest that BM-573 failed to prevent reperfusion injury in acutely ischemic pigs.     

Separation of fluorescence signals from Ca2+ and NADH during cardioplegic arrest and cardiac ischemia

Determinations of intracellular [Ca(2+)](i) during ischemia using fluorescent indicators are hampered by overlapping cellular autofluorescence (AF), which largely depends on NADH. If Ca(2+) is to be determined under different kinds of ischemia, signal separation merits special attention. We used triple wavelength excitation fluorescence to separate autofluorescence from [Ca(2+)]-dependent fura-2 fluorescence. Excitation at 360 nm served as third, Ca(2+)-insensitive wavelength. Using an appropriate evaluation procedure, we separated Ca(2+)-dependent signals from autofluorescence which is semiquantitatively associated with NADH, an indicator of the cellular redox state. We compared changes of [Ca(2+)](i) in isolated hearts during ischemia following cardioplegic arrest with those after transient stop of nutritive perfusion. We observed [Ca(2+)] transients in spontaneously beating hearts, persisting during ischemic episodes, and an increase of mean [Ca(2+)](i). In contrast, cardioplegic arrest stopped periodical [Ca(2+)](i) transients and heart beats simultaneously. [Ca(2+)](i) remained at diastolic values, tended to decrease during the first minutes of cardioplegic arrest and then increased slowly. Autofluorescence increased under both conditions. During ischemia, this increase was faster than in cardioplegia experiments. It started after the last heart beat despite persisting perfusion. Our measurements demonstrate that rhythmical heart beat is essential for sufficient perfusion. Reduced [Ca(2+)](i) under cardioplegic arrest may influence metabolism.     

Sustained postischemic cardiodepression following magnesium-diltiazem cardioplegia

Magnesium-diltiazem cardioplegia was evaluated in the intact, perfused rat heart to determine whether the joint administration of these agents would adversely affect myocardial contractile and high-energy phosphate recovery following intermittent, normothermic global ischemic arrest. Sequential metabolic and functional analyses were performed on isolated perfused rat hearts during each phase of the experimental protocol: control (10 min), normoxic cardioplegia (10 min), intermittent global ischemic arrest (two 15-min periods separated by 2 min infusion of the normoxic cardioplegic perfusate), and normoxic postischemic control reperfusion (60 min). Four different cardioplegic solutions were evaluated: 30 mM KCl, 30 mM KCl with 2 mg diltiazem/liter, 20 mM MgCl2, and 20 mM MgCl2 with 2 mg diltiazem/liter. Myocardial phosphatic metabolite levels and intracellular pH were analyzed nondestructively in the intact hearts by phosphorus-31 NMR spectroscopy. Corresponding measurements of peak left intraventricular pressure, rate of peak pressure development (dP/dt), and contraction frequency were performed at the midpoint during each 5-min interval of 31P NMR signal averaging. Magnesium plus diltiazem-treated hearts were distinguished from all other groups by a marked delay in postischemic functional recovery consisting of a prolonged depression in contractility (34% of control, P less than 0.01) that persisted throughout the first 50 min of postischemic reperfusion. Diltiazem in combination with magnesium cardioplegia was detrimental to postischemic functional recovery, despite a rapid restoration of high-energy phosphate stores. The apparent adverse interactive effects of excess magnesium and diltiazem suggest that elective ischemic arrest with magnesium cardioplegia in combination with diltiazem may be contraindicated clinically. The mechanistic basis and drug specificity of this response require further clarification. The present findings appear to exclude ATP and PCr production, and structural causes as the basis for the observed aberrant functional recovery from global ischemia of magnesium plus diltiazem-arrested hearts.     

Global cardiac function: mechano-energetico-informatics

This review on the global cardiac function covers cardiac mechanics, energetics, and informatics that I have developed with my collaborators over the last 30 years in Japan and USA. We first established E(max) (end-systolic maximum elastance or pressure/volume ratio) as a new index of ventricular contractility using canine hearts. We then expanded the E(max) concept to PVA (systolic pressure-volume area consisting of external mechanical work and mechanical potential energy) as an innovative measure of total mechanical energy of ventricular contraction and discovered it to be a reliable determinant of ventricular energetics or O(2) consumption (V(O(2))). We have discovered that E(max) shifts the V(O(2))-PVA relation and the E(max) dependency (O(2) cost of E(max)) varies among different pathophysiological hearts. We also searched for the basis of E(max) in crossbridge behavior information contained in an X-ray diffraction of papillary muscle. Recently, we established a new integrative analysis to estimate total Ca(2+) recruited for excitation-contraction coupling in a beating heart using the E(max)-PVA-V(O(2)) information. These global, mechano-energetico-informatic approaches seem to facilitate better understanding of cardiac function, as required in the present post-genomic era when more physiomic knowledge is required not only in cardiac function but also in all other physiologic functions.