Diminished posttetanic potentiation in failing human myocardium

In heart failure a decreased function of SERCA2 has been demonstrated. The present study aimed at investigating the relation between sarcoplasmic reticulum-Ca²⁺-load (SR-Ca²⁺-load) and the activity of the SERCA2. SR-Ca²⁺ load was evaluated by measuring posttetanic potentiation (PTP) in human nonfailing (NF, n=10) and endstage failing myocardium (DCM, n=11). In addition, the effect of cyclopiazonic acid (CPA), a specific inhibitor of SERCA2, on PTP was studied in both NF and DCM. In crude membrane preparations from the same hearts the maximal SERCA2 activity was determined and correlated with the PTP. In failing myocardium the PTP was significantly reduced compared to nonfailing myocardium (13.7±0.75 mN/mm² vs. 17.1±1.55 mN/mm², p<0.05, ±SEM). When PTP was studied in the presence of increased extracellular Ca²⁺-concentrations, the difference between NF and DCM was further pronounced. CPA decreased PTP in both nonfailing and failing human tissue. The maximal SERCA2 activity was significantly reduced in failing myocardium (NF 267±18.5 nmol ATP/mg protein · min⁻¹ vs. DCM 191±13.4 nmol ATP/mg protein · min⁻¹, p<0.05, ± SEM). Correlation of the PTP and maximal SERCA2 activity revealed a close correlation between both parameters in NF and DCM. In summary, the presented results suggest that reduced SERCA2 activity in DCM influences posttetanic force potentiation probably through a reduced SR-Ca²⁺-load. Received: 30 July 1999 Returned for 1. revision: 9 September 1999 1. Revision received: 24 November 1999 Returned for 2. revision: 26 January 2000 2. Revision received: 26 April 2000 Accepted: 9 May 2000     

Early rather than delayed administration of lisinopril protects the heart after myocardial infarction in rats

Background:ACE inhibitors have shown beneficial results in several studies after myocardial infarction (MI). However, this studies have shown conflicting results about the ideal starting time of the ACE inhibitors administration after MI and the importance of infarct size. Objectives: This study was designed to assess the long-term effects of lisinopril on mortality, cardiac function, and ventricular fibrosis after MI, in rats. Methods: Lisinopril (20 mg/kg/day) was given on day 1 or 21 days after coronary occlusion in small or large infarctions. Results: The mortality rate was reduced by 39% in early treatment and 30% in delayed treatment in comparison to the untreated rats. Early treatment reduced cardiac dysfunction in small MIs; however, delayed treatment did not. No statistical difference was observed among the groups for large MIs. No statistical difference was observed among the groups with large or small MIs on myocardial hydroxyproline concentration. Conclusions: Both early and delayed treatments with lisinopril increased survival. Treatment exerts no marked effects on fibrosis; early treatment has exerted beneficial influences on cardiac function whereas delayed treatment had no consistent effects. The protective effect of lisinopril is detectable only in small (< 40% of LV) MIs. Received: 6 May 1999, Returned for 1. revision: 28 May 1999, 1. Revision received: 20 July 1999, Returned for 2. revision: 26 August 1999, 2. Revision received: 28 September 1999, Accepted: 29 September 1999     

Chronic effects of ACE-inhibition (quinapril) and angiotensin-II type-1 receptor blockade (losartan) on atrial natriuretic peptide in brain nuclei of rats with experimental myocardial infarction

Alterations of the central nervous system may be important for imbalance of cardiovascular and fluid regulation in heart failure. The central renin-angiotensin and atrial natriuretic peptide (ANP) systems act as mutual antagonists. The effects of angiotensin converting enzyme (ACE) inhibition (quinapril, 6 mg/kg/day) and angiotensin II type 1 (AT₁) receptor blockade (losartan, 10 mg/kg/day) on ANP levels in 18 selected, microdissected brain nuclei were determined in sham-operated rats and rats with left ventricular dysfunction 8 weeks after myocardial infarction (MI). Plasma ANP tended to increase in MI rats and was further increased by quinapril. ANP was decreased in 12 brain areas of MI rats. ANP concentration was also significantly decreased by quinapril in six brain nuclei including subfornical organ and organum vasculosum laminae terminalis (areas lacking blood-brain barrier), and by losartan in 16 brain nuclei outside and within the blood-brain barrier in sham operated rats. However, both quinapril and losartan prevented a further reduction of central ANP as a result of myocardial infarction. These data suggest that there are effects on central ANP that result from chronic left ventricular dysfunction as well as an ACE-inhibitor and AT₁-antagonist. Mechanisms and consequences of central ANP depression remain unclear. They could, however, support systemic vasoconstriction and sodium and fluid retention. Received: 8 October 1999, Returned for 1. revision: 28 December 1999, 1. Revision received: 2 March 2000, Returned for 2. revision: 17 April 2000, 2. Revision received: 20 October 2000, Accepted: 9 November 2000     

Uncoupling between left ventricular contractility and relaxation after direct-current counter shocks

Left ventricular (LV) contractility and relaxation are physiologically coupled on the basis of intracellular calcium cycling. The relation has been reported to be unique. However, this may not be always true if relaxation is predominantly impaired. Direct current counter (DC) shocks develop myocardial interstitial edema, inducing diastolic heart failure. Thus, we hypothesized that LV contractility-relaxation coupling would be altered in an experimental model of diastolic dysfunction by DC shocks. The relation between Emax (LV contractility index) and the time constant of LV pressure decay (tau) was evaluated in isovolumic contraction of seven isolated, blood perfused dog hearts. There existed a hyperbolic relation between Emax and tau in control (= pre-DC) hearts. After the application of five consecutive 80 J DC shocks, Emax was unchanged (from 4.6 ± 1.0 to 5.2 ± 0.8 mmHg · ml⁻¹· 100 g) but tau was markedly prolonged (from 36 ± 12 to 74 ± 38 ms, P < 0.01). Thus, DC shocks induced a strikingly upward displacement of the hyperbolic curve compared with the control. The slope of the linear relation between Emax and the reciprocal of tau (= a relaxation velocity index normalized for contractility) significantly decreased after DC shocks. We conclude that the coupling between LV contractility and relaxation is not unique, but can be altered acutely by DC shocks. A dissociation of contractility-relaxation coupling may be of help for distinguishing diastolic heart failure and exploring its pathogenesis. Received: 2 November 1998, Returned for 1. revision: 2 December 1998, 1. Revision received: 15 February 1999, Returned for 2. revision: 2 March 1999, 2. Revision received: 14 April 1999, Accepted: 24 April 1999     

Left ventricular adaptation to chronic pressure overload induced by inhibition of nitric oxide synthase in rats

Summary Recent studies have indicated that chronic inhibition of nitric oxide synthase induces arterial hypertension without myocardial hypertrophy. We investigated the mechanisms of left ventricular (LV) adaptation to this condition, Also, we analyzed the effect of the angiotensin-converting enzyme inhibitor (ACEI), lisinopril, in this experimental model of ventricular pressure overload. Fiftyeight Wistar rats received eight weeks of treatment with either N^w-nitro-L-arginine-methyl ester (L-NAME group, n=19), lisinopril (LISINOPRIL group, n=19) or the combination of both drugs (LNAMELIS group, n=20). All results were compared to age and sex matched untreated rats (CONTROL group, n=18). Tail-cuff blood pressure rose significantly in L-NAME treated rats (195±29 mm Hg) compared to the CONTROL (141±12 mm Hg), LISINOPRIL (97±13 mm Hg), and LNAMELIS (113±16 mm Hg) groups. There was no myocardial hypertrophy in the chronically hypertensive rats. The ventricular unstressed volume was significantly reduced in the L-NAME group (0.119±0.027 mL) compared to the CONTROL (0.158±0.026 mL) indicating a disproportional reduction in ventricular volume related to the myocardial mass. The chamber size modification resulted in a systolic stress which was comparable to the CONTROL even though the isovolumetric systolic pressure was higher. The systolic functional data indicated preserved myocardial contractility in L-NAME. LV compliance was increased in the LISINOPRIL group and myocardial passive stiffness was lower in all treated rats compared to CONTROL. We conclude that LV adaptation to chronic pressure overload without hypertrophy involves changes in chamber geometry and myocardial diastolic mechanical properties. Also, ACEI fully prevents L-NAME induced hypertension, reduces myocyte cross-sectional area, and myocardial passive stiffness. The combination of L-NAME plus lisinopril decreases the load independent index of myocardial contractility. Received: 21 May 1997, Returned for 1. Revision: 18 June 1997, 1. Revision received: 22 August 1997, Returned for 2. Revision: 20 October 1997, 2. Revision received: 18 November 1997, Accepted: 10 December 1997     

Effects of selenium deficiency on the rat myocardial protein pattern – investigation by two-dimensional gel electrophoresis

Dietary selenium deficiency represents an etiological factor in “Keshan disease”, a distinct form of an endemic cardiomyopathy. The biochemical effects of selenium depletion in the myocardium are, however, not yet known. Therefore, we investigated the changes in the myocardial protein pattern in rats after long-term selenium deficiency. The myocardial proteins were analyzed in samples from five selenium-depleted rats (Se-deficient group) and five rats supplied with adequate amounts of the element (Se-adequate group). Isoelectric focusing (IEF) with carrier ampholytes on large 2-DE gels was used for the separation of proteins in the first dimension and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) for the second dimension. The protein patterns were evaluated by means of a computer-assisted gel analysis system. The biochemical identification of the proteins of interest was achieved by matrix-assisted laser desorption/ionization mass spectrometry (MALDI) or immunoblotting. On average, 588 ± 68 protein spots were found on the gels. No significant difference in spot numbers existed between the groups. A pattern of 270 spots with identical positions was found on every gel; 247 of these spots were not saturated and used for quantitative comparison. Thirty-five, i. e., 14%, differed significantly in their relative intensity in the two groups. Twenty-eight protein spots were decreased in the Se-deficient group and seven were increased. Sarcomeric creatine kinase M chain, α-myosin heavy chain (α-MHC) and myosin light chain 1 and 2 (MLC 1 and 2) were largely decreased in Se-deficiency. Three protein spots were increased by more than twofold or appeared only in the Se-deficient group. A mitochondrial creatine kinase was identified in this group. The results suggest that selenium deficiency affects myocardial energy metabolism and contractile proteins. These changes probably reflect non-specific alterations in heart failure. Received: 19 February 1997, Returned for 1. revision: 7 April 1997, 1. Revision received: 29 January 1999, Returned for 2. revision: 18 February 1999, 2. Revision received: 21 December 1999, Accepted: 6 January 2000     

Electrophysiological characterization of murine myocardial ischemia and infarction

Background Genetically altered mice will provide important insights into a wide variety of processes in cardiovascular physiology underlying myocardial infarction (MI). Comprehensive and accurate analyses of cardiac function in murine models require implementation of the most appropriate techniques and experimental protocols. Objective In this study we present in vivo, whole-animal techniques and experimental protocols for detailed electrophysiological characterization in a mouse model of myocardial ischemia and infarction. Methods FVB mice underwent open-chest surgery for ligation of the left anterior descending coronary artery or sham-operation. By means of echocardiographic imaging, electrocardiography, intracardiac electrophysiology study, and conscious telemetric ECG recording for heart rate variability (HRV) analysis, we evaluated ischemic and post-infarct cardiovascular morphology and function in mice. Results Coronary artery ligation resulted in antero-apical infarction of the left ventricular wall. MI mice showed decreased cardiac function by echocardiography, infarct-typical pattern on ECG, and increased arrhythmia vulnerability during electrophysiological study. Electrophysiological properties were determined comprehensively, but were not altered significantly as a consequence of MI. Autonomic nervous system function, measured by indices of HRV, did not appear altered in mice during ischemia or infarction. Conclusions Cardiac conduction, refractoriness, and heart rate variability appear to remain preserved in a murine model of myocardial ischemia and infarction. Myocardial infarction may increase vulnerability to inducible ventricular tachycardia and atrial fibrillation, similarly to EPS findings in humans. These data may be of value as a reference for comparison with mutant murine models necessitating ischemia or scar to elicit an identifiable phenotype. The limitations of directly extrapolating murine cardiac electrophysiology data to conditions in humans need to be considered. Received: 5 October 2000, Returned for 1. revision: 2 November 2000, 1. Revision received: 24 November 2000, Returned for 2. revision: 28 November 2000, 2. Revision received: 13 December 2000, Accepted: 14 December 2000     

Brief preconditioning ischemia alters diacylglycerol content and composition in rabbit heart

In order to give further insight into the potential role of PKC in beneficial effects of ischemic preconditioning, we have characterized the production of diacylglycerol, the endogenous activator of PKC, and its molecular species composition in ischemic control and preconditioned hearts. Preconditioning was induced by 1 cycle of 5 min of ischemia followed by 5 min of reperfusion. In control and preconditioned groups, hearts were harvested under deep anesthesia at baseline (preischemia) and at 2,5 and 10 min into the sustained coronary artery occlusion, i.e., preceding myocyte death. Diacylglycerol content and fatty acid composition were analysed by thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC), respectively. Myocardial diacylglycerol content was increased at 2 min into the sustained ischemia in the control group (481 ± 34 vs 292 ± 64 ng.mg⁻¹ at baseline; p < 0.05), but was comparable to the baseline value at 5 and 10 min. In the preconditioned group, diacylglycerol production remained unchanged throughout the 10-min test ischemia (317 ± 17 at 2 min vs 312 ± 38 ng.mg⁻¹ at baseline; p = NS). A detailed analysis of the molecular species composition at the time of 2 min revealed a reduced contribution of phosphatidy-linositol to diacylglycerol production in preconditioned myocardium (global correlation coefficient 0.57 vs 0.66 in control myocardium) with a trend toward an enrichment of diacylglycerol composition with some species originating from phosphatidylcholine. Thus, our study revealed that brief preconditioning ischemia: (1) prevents the increase of diacylglycerol content in the early minutes of the sustained ischemia, and (2) emphasizes the contribution of phosphatidylcholine in diacylglycerol formation to the detriment of that of phosphatidylinositol. Received: 12 October 1999, Returned for 1.revision: 3 November 1999, 1.Revision received: 6 January 2000, Returned for 2.revision: 14 February 2000, 2.Revision received: 21 April 2000, Accepted: 9 May 2000     

Effects of BIIB513 on ischemia-induced arrhythmias and myocardial infarction in anesthetized rats

Na⁺/H⁺ exchange (NHE) plays an important role in the regulation of the intracellular pH (pH_i) and in cardiac cell injury induced by ischemia and reperfusion. In the present study, we investigated the effects of BIIB513, a selective NHE-1 inhibitor on myocardial ischemia induced arrhythmias and myocardial infarction, provoked by 30 minutes of left main coronary artery occlusion followed by 2 hours of reperfusion in an anesthetized rat model. Intravenous administration of BIIB513 (0.01–3.0 mg/kg) did not induce changes in blood pressure or heart rate. BIIB513 (0.01, 0.1, 0.3, 1.0, 3.0 mg/kg) given prior to the coronary artery occlusion dose-dependently reduced ventricular premature beats, ventricular tachycardia, and a complete suppression of ventricular fibrillation down to the dose of 0.1 mg/kg. BIIB513 (0.01, 0.1, 0.3, 1.0, 3.0 mg/kg) given prior to the coronary artery occlusion dose-dependently reduced the infarct size with an ED50 value of 0.16 mg/kg. BIIB513 (1.0 mg/kg) given prior to reperfusion also reduced infarct size by 47.3 ± 13.1%. The reduction in infarct size was accompanied by a decrease in circulating levels of creatine phosphokinase (CPK). In conclusion, the present study demonstrates the cardioprotective ability of NHE-1 inhibition during myocardial ischemia and reperfusion by reducing serious ventricular arrhythmias and myocardial infarct size in anesthetized rats. Received: 18 November 1999, Returned for 1.revision: 9 December 1999, 1.Revision received: 2 May 2000, Returned for 2.revision: 24 May 2000, 2.Revision received: 5 June 2000, Accepted: 7 June 2000     

Effect of non-selective endothelin blockade, TAK-044, on the ischemic cellular injury of rat heart

The aim of this study is to evaluate the role of non-selective endothelin blockade (TAK-044) in ischemic myocardial injury. Forty anesthetized rats were separated into four groups: 1) TAK-I group, after preinjection of TAK-044 (3 mg/kg), LAD was ligated for 60 min and reperfused for 60 min; 2) Saline (SAL)-I group, LAD ligation and reperfusion without TAK-044; 3) TAK-C group, sham operated TAK group; 4) SAL-C group, sham-operated SAL group. Myocardium from each group was separated and analyzed by biochemical and ultrastructural procedures. Reperfusion arrhythmia (VT) was observed in 88 % of the SAL-I Group, in contrast to only 36% of the TAK-I group. At the end of reperfusion, hemodynamics indicated no significant differences between these two groups. The Ca⁺⁺-ATPase activity of sarcoplasmic reticulum (SR) was 3.9μmoles Pi/mg protein/h (39 % of SAL-C group) in the SAL-I group, while that in the TAK-I group was significantly higher at 6.1 (54%). The ratio of infarct/risk area was 58% in the SAL-I group and 36% in the TAK-I group. In the ultrastructural observations, irreversibly injured cells of the ischemic portion were reduced significantly from 35% (SAL-I group) to 14% (TAK-I group). Thus, our results indicated that endothelin blockade reduced ischemic cellular injury. The mechanism of this reduction was speculated to be a restistance to ischemic injury in the subcellular levels of the myocardium conferred by a reduction of vascular constriction and improvement of imbalance in the energy supply and demand. Received: 28 July 1998, Returned for 1. revision: 27 August 1998, 1.Revision received: 14 September 1998, Returned for 2. Revision: 1 October 1998, 2. Revision received: 13 October 1998, Returned for 3. Revision: 3 November 1998, 3. Revision received: 1 December 1998, Accepted: 7 December 1998     

Ventricular remodeling and transforming growth factor-beta 1 mRNA expression after nontransmural myocardial infarction in rats: effects of angiotensin converting enzyme inhibition and angiotensin II type 1 receptor blockade

With the application of early reperfusion therapy after acute MI, the incidence and importance of nontransmural infarction is increasing. In a rat model with nontransmural infarction, we evaluated 1) the changes of LV dimension, LV interstitial fibrosis and transforming growth factor-β1 (TGF-β1) mRNA expression and 2) the effects of angiotensin converting enzyme (ACE) inhibitor and angiotensin II type 1 (AT1) receptor antagonist treatment. Female Sprague-Dawley rats were subjected to 45 min of coronary occlusion followed by reperfusion, and five days after the operation the animals were randomized to untreated (n = 19), captopril-treated (n = 15) and losartan-treated (n = 14) groups. Twenty-six days after MI, echocardiographic examination revealed a remarkable dilatation of LV. Captopril or losartan treatment reduced the extent of LV cavity dilatation. Collagen volume fractions in noninfarcted septum as well as in peri-infarct area decreased with captopril or losartan treatment, compared to those of the untreated rats. In noninfarcted septum of untreated rats, TGF-β1 mRNA expression increased more than two fold (P < 0.05 vs. pre-MI) 5 and 10 days after MI. Captopril or losartan treatment suppressed the acute induction of TGF-β1 mRNA expressions. These results indicate that ACE inhibitor or AT1 receptor antagonist treatment after nontransmural infarction 1) attenuates LV remodeling as in transmural infarction and 2) decreases interstitial fibrosis at least partly by blocking the acute induction of TGF-β1 mRNA expression. Received: 10 December 1998, Returned for revision: 29 January 1999, Revision received: 15 March 1999, Accepted: 22 March 1999     

Arrhythmogenic potential of positive inotropic agents

The clinical use of positive inotropic agents has been associated with increased mortality, with proarrhythmia speculated to be a contributing factor. This study compares the arrhythmogenic potential of six positive inotropic agents representing different mechanistic classes: the β-adrenergic agonist dobutamine, the adenylyl cyclase activator forskolin, the phosphodiesterase-III inhibitor milrinone, the cardiac glycoside ouabain, and the sodium channel agonists DPI 201-106 and BDF 9148. These agents were studied in dogs with anterior myocardial infarction using lower and higher dose i. v. regimens targeted to elicit 20–40% and 70–90% increases in LV+dP/dt, respectively. Precipitation of new ventricular arrhythmia by programmed ventricular stimulation was observed in all treatment groups. Incidences of new arrhythmia were comparable in the lower dose regimens, ranging from 16.7% (3/18 animals with BDF 9148) to 31.6% (6/19 animals with DPI 201-106), and in the higher dose regimens, ranging from 10.0% (1/10 animals with milrinone) to 27.7% (5/18 animals with DPI 201-106). The overall incidence of new ventricular arrhythmia ranged from 27.3% (3/11 animals with ouabain) to 47.4% (9/19 animals with DPI 201-106). No differences were observed in underlying infarct size or time from infarction to electrophysiologic study between subgroups of animals in which new arrhythmias were precipitated vs. those remaining non-responsive in any treatment group. The positive inotropic agents tested displayed diverse total group effects on heart rate, electrocardiographic intervals including QTc and ventricular refractoriness. Within individual treatment comparisons revealed a general but not universal pattern of greater ventricular refractory period values in newly inducible vs. non-inducible subgroups in the DPI 201-106, BDF 9148 and ouabain (low and high dose); milrinone and dobutamine (high dose) treatment groups. These findings indicate that regardless of underlying cellular mechanisms of action, the six positive inotropic agents tested all displayed comparable proarrhythmic potentials unrelated to underlying infarct size and time from infarction. This observation suggests the general shared property of increased myocardial contractility, potentially adversely affecting myocardial oxygen balance, myocardial perfusion and electrical stability in the setting of previous myocardial infarction, to be a common underlying cause for arrhythmogenesis. Additionally, alterations in ventricular refractoriness and repolarization may contribute significantly to proarrhythmia with some positive inotropic interventions. Received: 20 July 1999, Returned for 1. revision: 16 September 1999, 1. Revision received: 26 October 1999, Returned for 2. revision: 24 November 1999, 2. Revision received: 22 December 1999, Accepted: 6 January 2000     

Alcohol and the risk of myocardial infarction

Epidemiological studies have repeatedly demonstrated a beneficial effect of moderate alcohol consumption on the incidence of coronary heart disease, myocardial infarction and overall mortality. The latter increases with excessive alcohol consumption. Although most epidemiological studies demonstrate a beneficial effect of alcohol consumption independent from the specific kind of alcoholic beverage, there is increasing evidence that wine and in particular red wine might contain pharmacological substances, which prevent atherosclerosis and myocardial infarction independent from the wine ethanol. Pathophysiological mechanisms mediating these beneficial effects include effects of wine phenols and tannins on LDL-cholesterol oxidation status, thrombocyte aggregation, endothelial function and smooth muscle cell proliferation. Identification and characterization of the pharmacologically active substances might provide the stage for the development of new substances to be used in the prevention of coronary artery disease and myocardial infarction. Received: 14 August 2000, Returned for 1. revision: 6 September 2000, 1. Revision received: 23 November 2000, Returned for 2. revision: 5 December 2000, 2. Revision received: 21 December 2000, Accepted: 8 January 2001     

A role of PKC in the improvement of energy metabolism in preconditioned heart

Objectives. A possible link between activation of PKC and improvement of energy metabolism during reperfusion in ischemic preconditioning hearts was examined. Methods. Isolated perfused rat hearts were preconditioned by 5-min ischemia and 5-min reperfusion in the presence and absence of a PKC inhibitor polymyxin B (50 μM) and then subjected to 40-min sustained ischemia and subsequent 30-min reperfusion. In another set of experiments, the hearts pretreated with and without a PKC activator PMA (15 pmol/5 min) were subjected to the sustained ischemia and reperfusion. Myocardial high-energy phosphates, glycolytic intermediates and mitochondrial oxygen consumption capacity were determined at appropriate experimental sequences. Results. Preconditioning enhanced the recovery of cardiac function such as left ventricular developed pressure, heart rate and rate-pressure product of the reperfused heart, suppressed the release of creatine kinase, enhanced the reperfusion-induced restoration of myocardial high-energy phosphates, attenuated the reperfusion-induced accumulation in glucose 6-phosphate and fructose 6-phosphate contents, abolished the ischemia-induced increase in tissue lactate content and prevented the ischemia-induced decrease in mitochondrial oxygen consumption capacity. Treatment of the perfused heart with PMA mimicked the effects of preconditioning on post-ischemic contractile function, enzyme release, levels of myocardial energy store, glycolytic intermediates and lactate, and mitochondrial function. Polymyxin B-treatment abolished the preconditioning-induced recovery of post-ischemic contractile function, the suppression of the release of CK, the restoration of myocardial energy store, and the preservation of mitochondrial function, whereas it did not cancel the improvement of glycolytic intermediate levels and the reduction in tissue lactate accumulation. Post-ischemic contractile function was closely related to restoration of high-energy phosphates and mitochondrial oxygen consumption capacity in all hearts subjected to ischemia/reperfusion. Conclusion. The results suggest that activation of PKC and preservation of mitochondrial function are closely linked with each other in the preconditioned heart, which may lead to the improvement of post-ischemic contractile function. Received: 29 January 1999, Returned for 1. revision: 26 February 1999, 1. Revision received: 27 April 1999, Returned for 2. revision: 18 May 1999, 2. Revision received: 12 July 1999, Returned for 3. revision: 26 July 1999, 3. Revision received: 25 October 1999, Accepted: 3 November 1999     

Cardiac isoform of α-2 macroglobin, a novel serum protein, may induce cardiac hypertrophy in rats

Earlier studies from this laboratory have identified a novel high molecular weight (182 kDa) serum protein suggested to be involved in the development of cardiac hypertrophy. In the present case the role of this novel serum protein in the development of pressure-induced cardiac hypertrophy and the molecular events associated with it in experimental rats has been investigated. Multiple injections of this purified protein intravenously (through tail vein) into the normal animals lead to the development of cardiac hypertrophy and this is accompanied by an induction of muscle specific genes such as that of MLC2 and β-MHC characteristics of pressure overloaded heart. Further, the hypertrophy-specific serum protein has been found to be identical to rat α-2 macroglobulin (α-2M) in molecular weight (182 kDa) and in its appearance in blood serum. α-2M is an acute phase serum protein that increases markedly after inflammatory stimuli in hepatocytes in liver and gets secreted into the blood. The studies at present suggest that the 182kDa serum protein that appeared during the early stage of development of cardiac hypertrophy in aorta constricted rats is a glycoprotein localized in the heart that showed immunological cross reactivity with α-2M and is expressed in the heart as evinced by Northern blot analysis. Further this protein showed certain differences from rat α-2M under denaturing conditions in isoelectric focusing and partial peptide mapping. Partial peptide sequencing of the internal peptides of tryptic digest of 182kDa showed 100% identity of the sequences with α-2M sequences. Rat α-2M does not, however, have any influence on the development of cardiac hypertrophy and its antibody does not cross react with the 182 kDa protein. These data suggest that the 182 kDa protein that may play an indispensable role in the development of cardiac hypertrophy in experimental rats in cardiac specific, and may be an isoform of liver α-2M belonging to macroglobulin family. Received: 10 December 1998, Returned for 1. revision: 15 January 1999, 1. Revision received: 2 September 1999, Returned for 2. revision: 29 September 1999, 2. Revision received: 15 February 2000, Returned for 3. revision: 20 April 2000, 3. Revision received: 30 June 2000, Accepted: 5 July 2000     

Beta-adrenergic receptors and intracellular signalling pathway in stunned and non-ischemic regions of pig myocardium

The β-adrenergic pathway may have a role in the pathophysiology of ischemic syndromes characterised by reversible left ventricular dysfunction, such as myocardial stunning and other clinical conditions of unstable angina or coronary spasms, or chronic reversible left ventricular dysfunction, which might be a consequence of repeated events of short-term ischemia (“repetitive stunning”). A partial-to-total occlusion of the left anterior descending coronary artery in pigs was used to induce short periods of ischemia (total ischemic time 12 ± 2 min). Hypokinesis and dyskinesis of the myocardium were considered signs of myocardial dysfunction. We found a maintained function of the β-adrenergic signalling system. Density and affinity of β-adrenergic receptors were not different in stunned and non-ischemic regions, nor were cyclic AMP and cyclic GMP intracellular contents and ratio, nor well as the ratio of stimulatory/inhibitory G protein a subunits. Our findings are in agreement with a maintained β-adrenergic signalling system in the pathophysiology of chronic reversible left ventricular dysfunction. Received: 25 September 2000 / Returned for 1. revision: 9 October 2000 / 1. Revision received: 22 November 2000 / Returned for 2. revision: 7 December 2000 / 2. Revision received: 8 January 2001 / Accepted: 11 January 2001     

Endocardial versus epicardial differences of sarcoplasmic reticulum Ca2+-ATPase gene expression in the canine failing myocardium

It is unknown whether the transmural heterogeneity of sarcoplasmic reticulum (SR) Ca²⁺-ATPase gene expression is present within the left ventricular (LV) wall. Moreover, the changes of transmural distribution have not been examined in the failing hearts. We thus quantified steady-state mRNA abundance of SR Ca²⁺ regulatory proteins by Northern blot analysis in both subendocardial and subepicardial LV layers from normal and rapid pacing-induced heart failure (HF) dog hearts. For normal LV, Ca²⁺-ATPase mRNA abundance (normalized to glyceraldehyde-3-phosphate dehydrogenase [GAPDH] mRNA) was significantly reduced in the subendocardium, whereas calsequestrin mRNA abundance was comparable between the two layers. For HF LV, Ca²⁺-ATPase mRNA abundance in the subendocardium was also reduced compared to the subepicardium. However, the endocardium to epicardium ratio was comparable between control and HF (0.62 ± 0.08 vs. 0.65 ± 0.07; p = NS). Therefore, the transmural gradient of this gene was constant in both control and HF. Even though the data on the transmural heterogeneity of protein level is not available, the subendocardium contained significantly less Ca²⁺-ATPase mRNA, which might contribute, at least in part, to the transmural gradients of biochemical and mechanical function. Received: 8 February 1999, Returned for revision: 8 March 1999, Revision received: 1 April 1999, Accepted: 23 April 1999     

Cyclic GMP attenuates cyclic AMP-stimulate inotropy and oxygen consumption in control and hypertrophic hearts

We tested the hypothesis that increasing myocardial cyclic GMP would attenuate cyclic AMP induced positive inotropy and O₂ consumption, in part, through changes in cyclic AMP and that renal hypertension-induced cardiac hypertrophy (HYP) would alter this relationship. Anesthetized, open chest rabbits (N = 48) were divided into four groups of control (CON) and HYP animals which received vehicle (VEH), isoproterenol 10⁻⁶M (ISO), 3-morpholinosyndnonimine 10⁻⁴M, (SIN-1), or a combination of ISO+SIN-1. Coronary blood flow (micro-spheres) and O₂ extraction (microspectrophotometry) were used to determine O₂ consumption in both subepicardium (EPI) and subendocardium (ENDO). Left ventricular change in wall thickness (%) was increased significantly by ISO in both CON (16 ± 4 to 31 ± 6) and HYP (17 ± 2 to 24 ±3). Percent change in wall thickness was similar in the CON, SIN-1, and ISO+SIN-1 groups. Myocardial O₂ consumption (ml O₂/min/100 g) was increased by ISO in CON (10.3 ± 1.0 to 13.6 ± 2.0 EPI; 10.9 ± 1.0 17.1 ±1.7 ENDO) and HYP (8.2 ± 1.4 to 12.3 ± 2.2 EPI; 6.6 ± 1.4 to 14.8 ± 1.8 ENDO). Oxygen consumption was unaffected by SIN-1 in CON and HYP animals. ISO+SIN-1 caused attenuated ISO-induced increases in O₂ consumption in CON in EPI and ENDO, and in EPI in HYP. Cyclic GMP (pmol/g) was unchanged by ISO in CON and HYP, and increased by SIN-1 in CON (8.1 ± 1.3 to 19.2 ± 2.3 EPI) and HYP (9.1 ± 1.5 to 12.8 ± 2.0 EPI). Cyclic GMP remained elevated with ISO+SIN-1 in both groups. Cyclic AMP (pmol/g) was increased significantly by ISO in CON (496 ± 43 to 725 ± 106 EPI; 534 ± 44 to 756 ± 148 ENDO) and insignificantly in HYP (435 ± 50 to 566 ± 35 EPI; 497 ± 51 to 583 ± 47 ENDO). Cyclic AMP levels were unaffected by SIN-1 in either group. Isoproterenol induced increases in cyclic AMP were blunted by ISO+SIN-1 in CON (496 ± 43 to 537 ± 59 EPI) and not affected in HYP. The current study demonstrated attenuation of cyclic AMP mediated increased inotropy and O₂ consumption by increasing cyclic GMP, which appeared, in part, related to cyclic GMP-induced reduction in cyclic AMP. This effect of cyclic GMP on cyclic AMP was not observed in myocardial hypertrophy. Received: 4 January 1999, Returned for 1. revision: 29 January 1999, 1. Revision received: 30 March 1999, Returned for 2. Revision: 3 May 1999, 2. Revision received: 3 May 1999, Returned for 3. Revision: 12 May 1999, 3. Revision received: 23 June 1999, Returned for final revision: 7 July 1999, Accepted: 22 July 1999     

Frequency-dependent changes in calcium cycling and contractile activation in SERCA2a transgenic mice

Objective: This study was undertaken to investigate the mechanism of altered contractility in hearts from transgenic mice overexpressing the sarcoplasmic reticulum (SR) Ca²⁺ ATPase (SERCA2a). In particular, we sought to determine whether the reported increase in contractility is freqnency-dependent, as might be expected if attributable to changes in SR Ca²⁺ loading. Methods: Intracellular [Ca²⁺] and contractile force were measured at room temperature (22 °C) simultaneously in fura-2-loaded isometrically-contracting trabeculae dissected from the hearts of FVB/N control (n=6) or SERCA2a transgenic (n=6) mice. Results: SERCA transgenics exhibit a positive force-frequency relationship, but this was flat in age- and strain-matched controls. SERCA transgenics exhibit a sizable increase in calcium transient amplitude relative to controls, with a concomitant increase in force generation at higher frequencies of stimulation. Amplitudes of Ca²⁺ transients (transgenics: 1.56 ± 0.09 μmol/l, controls: 1.21 ± 0.14) and twitches (transgenics: 21.71 ± 0.91 mN/mm², controls: 13.74 ± 1.67) were significantly different at 2.0 Hz stimulation (P < 0.05). Conclusion: An increase in SERCA expression increases the ability of the sarcoplasmic reticulum to store calcium, such that more calcium is available to be released during each heartbeat at higher stimulation rates. Received: 4 June 1999, Returned for revision: 1 July 1999, Revision received: 4 October 1999, Accepted: 14 October 1999     

Regulation by carnitine of myocardial fatty acid and carbohydrate metabolism under normal and pathological conditions

This review focuses on the regulation of myocardial fatty acids and glucose metabolism is physiological and pathological conditions, and the role of L-carnitine and of its derivative, propionyl-L-carnitine. Fatty acids are the major oxidation fuel for the heart, while glucose and lactate provide the remaining need. Fatty acids in cytoplasm are transformed to long-chain acyl-CoA and transferred into the mitochondrial matrix by the action of three carnitine dependent enzymes to produce acetyl-CoA through the β-oxidaton pathway. Another source of mitochondrial acetyl-CoA is from the oxidation of carbohydrates. The pyruvate dehydrogenase (PDH) complex, the key irreversible rate limiting step in carbohydrate oxidation, is modulated by the intra-mitochondrial ratio acetyl-CoA/CoA. An increased ratio results in the inhibition of PDH activity. A decreased ratio can relieve the inhibition of PDH as shown by the transfer of acetyl groups from acetyl-CoA to carnitine, forming acetylcarnitine, a reaction catalyzed by carnitine acetyl-transferase. This activity of L-carnitine in the modulation of the intramitochondrial acety-CoA/CoA ratio affects glucose oxidation. Myocardial substrate metabolism during ischemia is dependent upon the severity of ischemia. A very severe reduction of blood flow causes a decrease of substrate flux through PDH. When perfusion is only partially reduced there is an increase in the rate of glycolysis and a switch from lactate uptake to lactate production. Tissue levels of acyl-CoA and long-chain acylcarnitine increase with important functional consequences on cell membranes. During reperfusion fatty acids oxidation quickly recovers as the prevailing source of energy, while pyruvate oxidation is inhibited. A considerable body of experimental evidence suggests that L-carnitine exert a protective effect in in vitro and in vivo models of heart ischemia and hypertrophy. Clinical trials confirm these beneficial effects although controversial results are observed. The actions of L-carnitine and propionyl-L-carnitine cannot be explained as exclusively dependent on the stimulation of fatty acid oxidation but rather on a marked increase in glucose oxidation, via a relief of PDH inhibition caused by the elevated acetyl-CoA/CoA ratio. Enhanced pyruvate flux through PDH is beneficial for the cardial cells since less pyruvate is converted to lactate, a metabolic step resulting in the acidification of the intracellular compartment. In addition, L-carnitine decreases tissue levels of acyl moieties, a mechanism particularly important in the ischemic phase. Received: 25 January 1999, Returned for 1. revision: 18 February 1999, 1. Revision received: 10 May 1999, Returned for 2. revision: 7 June 1999, 2. Revision received: 21 December 1999, Accepted: 21 December 1999