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     

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     

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     

The enhanced contractility in phospholamban deficient mouse hearts is not associated with alterations in Ca2+-sensitivity or myosin ATPase-activity of the contractile proteins

Work performing heart preparations from hypercontractile, phospholamban deficient mouse hearts showed no change in parameters of contraction or relaxation in response to isoproterenol stimulation. Thus, the aim of the present study was to investigate whether or not changes at the level of the contractile apparatus occur in addition to the altered expression of Ca²⁺-regulating proteins observed in these mouse models, e. g., phospholamban, ryanodine receptors. Triton-X skinned fiber preparations from phospholamban deficient (n = 9) and wild-type (n = 10) mice were used and the Ca²⁺-activated force as well as the myosin ATPase-activity were simultaneously measured. The tension dependent ATPase-activity was unchanged in phospholamban deficient animals when compared to controls. The SERCA 2a-inhibitor cyclopiazonic acid did not affect myosin ATPase-activity in this system. The Ca²⁺-sensitivity of Ca²⁺-activated force and myosin ATPase were unchanged as well. Comparison of the concentrations needed to achieve half maximal activation of the myosin ATPase-activity and force demonstrated that the Ca²⁺-sensitivity of the myosin ATPase was higher compared to the Ca²⁺-sentivity of tension development. This holds true for phospholamban deficient mice (EC₅₀ ATPase: 0.9 ± 0.2 μmol/l; tension: 1.7 ± 0.3 μmol/l; p < 0.001) and wild-type controls (1.1 ± 0.01 μmol/l; 2.2 ± 0.4 μmol/l; p < 0.01). The myosin ATPase-activity and force were correlated to each other in both, phospholamban deficient mice and controls and did not change at submaximal Ca²⁺ concentrations. The ATPase/force-ratio, as a parameter of tension cost, was similar in either phospholamban deficient mice or controls. Thus, the present study provides evidence that at the level of the contractile proteins regulation of Ca²⁺-activated force and energy demand of force development are not altered in phospholamban deficient mice with enhanced myocardial performance. At the level of the regulation of crossbridge interaction, no adaptive or compensatory mechanisms have been initiated by ablation of phospholamban. Received: 22 March 1999, Returned for revision: 26 April 1999, Revision received: 1 July 1999, Accepted: 12 July 1999     

Reduction of oxygen delivery during post-ischemic reperfusion protects the isolated guinea pig heart

Objective: To further characterise the influence of oxygen delivery during early reperfusion (first 5 min) in the isolated guinea pig heart, three modes of coronary reperfusion were chosen, differing with respect to reperfusion flow and arterial PO₂. Methods: Isolated working guinea pig hearts underwent ischemia and reperfusion (15 min each). Reperfusion was at constant pressure (Group 1, 60 mmHg, n = 7) or at constant flow (Group 2, 5 ml/min, n = 7) with a PO₂ of 600 mmHg. Group 3 (n = 8) was reperfused at 5 ml/min with a PO₂ of 300 mmHg for 5 min and a PO₂ of 600 mmHg thereafter. Lactate release and oxygen consumption were determined during reperfusion. Glutathione release served to assess myocardial oxidative stress. Results: After ischemia, hearts in Group 1 (mean coronary flow 14.4±1.1 ml/min during the first 5 min of reperfusion) performed external heart work at 31 ± 2 % of the pre-ischemic level. Performance in Group 2 recovered to 50 ± 3 % and in Group 3 to 68 ± 3 %. Myocardial oxygen consumption during early reperfusion (2nd min) was lowest in Group 3 (1.9 μmol/min) and highest in Group 1 (8.3 μmol/min). No difference in lactate release was observed. Release of glutathione during the first 5 min of reperfusion was 43.8 ± 7.9 nmol in Group 1, but only 3.6 ± 0.7 in Group 2 (p < 0.05). Conclusions: In isolated guinea pig hearts, controlled oxygen delivery during post-ischemic reperfusion by both, reduction of coronary flow and PO₂, improves recovery of pump function. The effect is accompanied by less oxidative stress, as indicated by lowered rates of glutathione release. Received: 1 December 1998, Returned for 1. revision: 4 January 1999, 1. Revision received: 28 January 1999, Returned for 2. revision: 8 February 1999, 2. Revision received: 18 February 1999, Accepted: 2 March 1999     

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     

Analysis of mitochondrial DNA deletions in four chambers of failing human heart: hemodynamic stress, age, and disease are important factors

Mitochondrial DNA (mtDNA) mutations are not only responsible for organ dysfunction due to inefficient energy production but also indicators of metabolic and functional stresses in the organ. To analyze the significance of deletion mutation in human myocardium, we screened the presence of two common deletions (7.4 kb from 8637–16084 nt, 5.0kb from 8470–13477 nt) in four chambers using long PCR, and using serial-dilution PCR, measured the amount of deleted mtDNA in normal heart (NL) of brain-dead victims of road accidents (n = 9, age = 10–59) and failing hearts (CHF) of patients who underwent heart transplantation (n = 24, age = 17–63). Frequency of both deletions was higher in ventricles (Vt) than in atria (At) (Vt: At = 25/33 : 12/33 for 7.4 kb, 19/33 : 6/33 for 5 kb) (p < 0.05), whereas it was the same in the right and left chambers. In ventricles, both deletions were more frequent among older persons (> 35 yrs) than in younger persons /≤ 35 yrs) (older : younger = 16/20 : 9/13 for 7.4 kb, 15/20 : 4/13 for 5 kb) (p < 0.05). In ventricles of failing heart, the 5-kb deletion was more frequent than in those of normal heart (CHF : NL = 17/24 : 2/9) (p < 0.05), whereas the 7.4-kb deletion was frequent both in failing and normal hearts (CHF : NL = 19/24 : 6/9). The association of mutation with aging or disease process observed in ventricles was not found in the atria. Although the amount of mutant mtDNA in the left ventricle tended to increase according to a disease process, it was small, at most 1.56% or 0.012% of total mtDNA for a 7.4- or 5-kb deletion, respectively. No deletion was found, however, in lymphocytes from any patient who underwent transplantation. In conclusion, deletion mutation of mtDNA is frequently, but in a small amount, found in the ventricle of older failing heart than in the atrium of younger normal heart. This suggests that hemodynamic stress, age, and disease are factors to induce mtDNA mutation that represents the indicator of stresses on the heart and might turn into a contributor of progressive heart failure under extreme conditions. Received: 26 March 1999, Returned for 1. revision: 28 April 1999, 1. Revision received: 17 June 1999, Returned for 2. revision: 17 August 1999, 2. Revision received: 17 September 1999, Accepted: 6 October 1999     

Defective suppression of the aldosterone biosynthesis during stroke permissive diet in the stroke-prone phenotype of the spontaneously hypertensive rat

Previous studies have shown that short-term high salt intake unmasks blunted plasma aldosterone suppression in stroke-prone spontaneously hypertensive rats (SHRsp). The aim of this study was to evaluate the response of aldosterone biosynthesis and production to a sustained exposure to the stroke-permissive Japanese-style diet (JD) in young stroke-prone and stroke-resistant SHRs. For this purpose, 6-week old male rats from both strains were divided into 2 dietary groups and received regular diet (SHR = 37, SHRsp = 32) or the JD and 1% saline to drink (SHR = 34, SHRsp = 30) for 4 weeks. All measurements were carried out at the end of the dietary periods. After JD, plasma aldosterone levels were significantly decreased in SHR (from 357.8±57 to 163.3±31.5 pg/ml, p < 0.05) but markedly increased in SHRsp (from 442±56.5 to 739±125.7 pg/ml, p < 0.05). Consistently, the adrenal aldosterone synthase expression was reduced by JD in SHR (p < 0.05), whereas it was even slightly raised by JD in SHRsp so that, at the end of JD, aldosterone synthase mRNA was 5-fold higher in SHRsp than in SHR. Urinary sodium excretion (mEq/24h) achieved lower levels in SHRsp, so that fractional excretion of sodium was 80.2±9% in SHR and 40.3±8% in SHRsp (p < 0.05) in balance studies performed at the end of JD. These different responses of mineralocorticoid biosynthesis and urinary sodium excretion to JD were not accounted for by different adaptions of the renin-angiotensin and atrial natriuretic peptide systems, of serum potassium levels, or of adrenal 11β-hydroxylase expression in the two strains. Systolic blood pressure was comparable in both strains throughout the experiment. These results demonstrate enhanced aldosterone biosynthesis, associated with reduced urinary excretion of sodium in response to JD in SHRsp before the onset of stroke. This abnormality may play a role in the higher susceptibility of stroke of this model. Received: 1 March 1999, Returned for 1. revision: 22 March 1999, 1. Revision received: 7 June 1999, Returned for 2. revision: 28 June 1999, 2. Revision received: 4 August 1999, Returned for 3. revision: 10 September 1999, 3. Revision received: 28 October 1999, Accepted: 17 November 1999     

Density dependent expression of PDGF-A modulates the angiotensin II dependent proliferation of rat cardiac fibroblasts

The proliferative effect of angiotensin II (Ang-II) on primary cardiac fibroblasts is not well understood and controversially discussed. Results described here show that fibroblasts from adult rat hearts exhibit a cell density dependent Ang-II induced cell proliferation. Whereas we could not detect a proliferative effect of Ang-II on confluent cells, which are still able to divide as shown by stimulation with platelet derived growth factor (PDGF), we observed an Ang-II induced cell division of approximately 20% in non-confluent cells. At both densities, signal transduction molecules such as the mitogen activated protein kinases (MAPK) are activated. There has been substantial evidence that Ang-II may induce the expression and secretion of several growth factors. We demonstrated an approximately fivefold increase in platelet derived growth factor-A (PDGF-A) chain and transforming growth factor β 1 (TGF-β1) mRNA-expression within non-confluent cardiac fibroblasts by semiquantitative reverse polymerase chain reaction, TR-PCR. In contrast, the mRNA-expression of basic fibroblast growth factor (bFGF), insulin-like growth factor 1 (IGF-1), PDGF-A/-B chains, and TGF-β1 remained unchanged within confluent cardiac fibroblasts. Experiments with neutralizing antibodies showed that PDGF-AA is an important growth factor regulating cell proliferation, whereas TGF-β1 interferes with cell size regulation. In summary, we could show that the Ang-II induced cell proliferation in adult cardiac fibroblasts is mainly due to cell density dependent expression of growth factors like PDGF-AA. Received: 15 April 1999, Returned for revision: 11 May 1999, Revision received: 14 June 1999, Accepted: 12 July 1999     

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     

Hyperthyroidism is associated with preserved preconditioning capacity but intensified and accelerated ischaemic contracture in rat heart

Background: The present study was undertaken to define the effects of thyroxine administration on ischaemic preconditioning (PC) and the ischaemic contracture. Methods: Hyperthyroidism was induced by administration of L-thyroxine in rats (THYR) while normal animals served as controls (NORMa). Isolated rat hearts were perfused in a Langendorff preparation. NORMa control (n = 16) and THYR control (n = 9) hearts underwent 20 min of ischaemia and 45 min reperfusion while NORMa PC (n = 16) and THYR PC (n = 14) were subjected to PC before ischaemia. Additional normal hearts were subjected to 30 min of ischaemia with and without PC, NORMb control, n = 8 and NORMb PC, n = 6. Postischaemic recoveries of left ventricular (LV) developed pressure were expressed as % of the initial value (LVDP%). Severity of contracture was measured by the time (Tmax) and magnitude (Cmax) of peak contracture. Results: LVDP% was significantly higher after PC, both in NORMa and THYR rats. In NORMa control hearts, ischaemic contracture had not yet reached a plateau at 20 min of ischaemia. Contracture appeared earlier in THYR control and PC than in NORMa control and PC groups. Tmax was 22.1 (0.9) vs 16.8 (1.4) min for NORMb control and PC, p < 0.05 and 12.5 (1.0) vs 9.3 (1.1) min for THYR control and PC hearts, p < 0.05. Tmax was earlier in both THYR groups compared to NORMb groups, p < 0.05. Cmax was significantly higher in both THYR groups compared to both NORMb groups. Conclusion: Ischaemic contracture is both accelerated and accentuated in thyroxine treated hearts while preconditioning capacity is preserved. Preconditioning and thyroxine administration shorten Tmax in an additive way, whereas Cmax in hyperthyroid hearts did not further increase by preconditioning. Received: 7 January 1999, Returned for 1. revision: 1 February 1999, 1. Revision received: 4 March 1999, Returned for 2. revision: 31 March 1999, 2. Revision received: 19 April 1999, Accepted: 21 April 1999     

Ventricular arrhythmias following coronary artery occlusion in rats: is the diabetic heart less or more sensitive to ischaemia?

Rhythm disorders are common complications in diabetic patients, due to their enhanced sensitivity to ischaemia. However, experimental studies are inconsistent, and both higher and lower vulnerability to injury has been reported. Our objectives were to compare susceptibility to ventricular arrhythmias in rats with prolonged duration of diabetes induced hy streptozotocin (45 mg/kg, i. v.), utilising two different models. Following 8 weeks, either anaestetised open-chest rats in vivo or isolated Langendorff-perfused hearts were subjected to 30 min regional zero-flow ischaemia induced by occlusion of LAD coronary artery. In addition, cardiac glycogenolysis and lactate production were measured. In open-chest rats, 90% of the controls exhibited ventricular tachycardia (VT) which represented 55.4% of total arrhythmias, whereby only 19.9% of arrhythmias occurred as VT in 44% of the diabetic rats (P < 0.05 vs controls). Duration of VT and ventricular fibrillation (VF) was reduced from 35.5 ± 11.1 and 224.8 ± 153.9 s in the controls to 4.8 ± 2.5 and 2.2 ± 0.2 s in the diabetics, respectively (P < 0.05). Accordingly, severity of arrhythmias (arrhythmia score, AS) was also lower in the diabetics (2.0 ± 0.38 vs 3.3 ± 0.3 in the controls; P < 0.05). In the isolated hearts, high incidence of VF was decreased in the diabetic hearts, and although VT occurred in almost all of the diabetic hearts, the duration of VT and VF was substantially shorter (61.5 ± 14.5 and 5.5 ± 0.5 s vs 221.5 ± 37 and 398.5 ± 55 s in the controls, respectively; P < 0.05). AS was reduced to 2.9 ± 0.12 from 4.1 ± 0.3 in the controls (P < 0.05). Postischaemic accumulation of lactate was lower in the diabetic than in the non-diabetic myocardium (20.4 ± 1.9 vs 29.5 ± 2.9 μmol/l/g w.wt.; P < 0.05). These results suggest that rat hearts with chronic diabetes, despite some differences in the arrhythmia profiles between the in vivo model and isolated heart preparation, are less sensitive to ischaemic injury and exhibit lower susceptibility to ventricular arrhythmias and reduced accumulation of glycolytic metabolites. Received: 3 April 2000, Returned for 1. revision: 9 May 2000, 1. Revision received: 5 July 2000, Returned for 2. revision: 7 August 2000, 2. Revison received: 11 September 2000, Returned for 3. revision: 27 September 2000, 3. Revision received: 13 October 2000, Accepted: 16 October 2000     

Myosin heavy chain synthesis during the progression of chronic tachycardia induced heart failure in rabbits

Chronic tachycardia causes LV dilatation and dysfunction, with no hypertrophy. However, the contributing mechanisms responsible for the left ventricular (LV) remodeling in the absence of myocardial growth in this model of heart failure remain unclear. Therefore, the goal of the present study was to serially examine changes in LV function, steady state myosin heavy chain (MHC) mRNA levels, in vivo synthesis rates, and abundance with the progression of chronic tachycardia induced heart failure. Adult rabbits (3.5–4.5 kg) were studied after one, two, or three weeks of pacing ventricular tachycardia (VT; 400 bpm) and in controls (n=6 for all groups). LV fractional shortening was reduced by 30 % at week one and by over 50 % at week three of chronic VT. End‐diastolic dimension (EDD) increased at week two compared to controls (1.66 ± 0.10 vs 1.35 ± 0.11 cm, p < 0.05) and increased further at week three of VT (1.70 ± 0.06 cm, p < 0.05). The progressive changes in LV geometry and function with chronic VT were not associated with concomitant time dependent changes in LV mass or MHC mRNA levels. In contrast, MHC fractional synthesis rates increased and reached statistical significance at week three of VT compared to controls (8.3 ± 0.8 vs 5.5 ± 0.5 %/day, p < 0.05). Despite the stable or increased MHC protein synthesis rates, there was no change in MHC protein abundance at any point during the progression of VT induced heart failure, implicating enhanced MHC protein degradation. Thus, this study demonstrated that a contributory mechanism for the LV remodeling and lack of myocardial growth, which occurs with VT induced heart failure, is enhanced contractile protein degradative processes. Received: 17 July 1997 , Returned for revision: 20 August 1997, Revision received: 8 September 1997, Accepted: 7 October 1997     

Effects of different inotropic interventions on myocardial function in the developing rabbit heart

The development of the mammalian heart is characterized by substantial changes in myocardial performance. We studied the ontogeny of myocardial function with and without various inotropic interventions in the developing isolated, antegrade-perfused rabbit heart (2d, 8d, 14d, 28d, n = 96). Myocardial function was related to the protein expression of the sarcolemmal Na⁺-Ca²⁺ exchanger and to the sarcoplasmic Ca²⁺-ATPase. In neonatal hearts an age-dependent increase in maximal developed pressure velocity (dP/dt_max) by 45 % and peak negative pressure velocity (dP/dt_min) by 75 % within days 2 to 8 were observed. In response to inotropic intervention with isoproterenol, ouabain, calcium and the Na⁺-channel modulator BDF 9148, dP/dt_max and dP/dt_min increased in a concentration dependent manner. Significant differences between neonatal, juvenile and adult hearts could be demonstrated in a repeated measurement ANOVA model on the concentration-response curves for BDF 9148 (dP/dt_max and dP/dt_min), ouabain (dP/dt_min) and calcium (dP/dt_min), but not for isoproterenol. At the maximum isoproterenol concentration of 1 μmol/l, the increase in dP/dt_max and dP/dt_min was significantly higher in adult compared to neonatal hearts (t-test, p < 0.01). The significant decline of the Na⁺-Ca²⁺ exchanger protein expression from neonatal (1822 ± 171) to adult hearts (411 ± 96 S.E.M. [units per 20 μg protein], p < 0.01) was related to an increase in myocardial function (dP/dt_max r = 0.63, p < 0.01, dP/dt_min r = 0.62, p < 0.01). Contractility, relaxation and the observed positive inotropic effects were in general significantly lower in neonatal compared to adult hearts. In the individual heart an increase in contractility and relaxation was related to a decrease in Na⁺-Ca²⁺ exchanger expression. Received: 22 May 2000, Returned for 1. revision: 21 June 2000, 1. Revision received: 27 November 2000, Returned for 2. revision: 19 December 2000, 2. Revision received: 2 January 2001, Returned for 3. revision: 17 January 2001, 3. Revision received: 25 May 2001, Accepted: 11 June 2001     

Inhibition of apoptotic responses after ischemic stress in isolated hearts and cardiomyocytes

Recent findings on the induction of anti-apoptotic gene expression in ischemic/reperfused hearts encouraged us to investigate whether ischemic/reperfused hearts may be protected against apoptosis induction. To analyze this hypothesis we performed studies on isolated perfused hearts of rat. For apoptosis induction, hearts were perfused with the NO donor (±)-S-nitroso-N-acetylpenicillamine (SNAP, 10 μM) for 30 minutes. Four hours thereafter apoptosis was detected by DNA laddering and TUNEL assay. Under normoperfusion SNAP induced 5.5 ± 1.4 TUNEL-positive myocytes per tissue section (vs. 1.8 ± 0.5 in controls). But when hearts were subjected to 20 minutes of no flow ischemia, which was sufficient for energy depletion of the hearts without inducing severe necrotic or apoptotic cell death, reperfusion in the presence of SNAP did not induce apoptosis. To analyze if this mode of protection is a property of the cardiomyocytes, we performed corresponding experiments on ventricular cardiomyocytes of rat. Again, under normoxic conditions SNAP (100 (μM) increased the number of TUNEL-positive cells to 12.6 ± 4.9 % (vs. 5.4 ± 0.7 % in controls). But when SNAP was added after 3 h of simulated ischemia, which was sufficient for energy depletion of the cells without inducing apoptotic cell death, the number of apoptotic cells did not increase. The ischemia-induced protection of hearts and cardiomyocytes goes along with an increased expression of several anti-apoptotic genes, mainly of the bcl-2 family. This indicates that ischemic conditions induce an anti-apoptotic gene program in cardiomyocytes, which may also be responsible for the observed anti-apoptotic actions in the intact ischemic/reperfused myocardium. Received: 20 March 2002, Returned for 1. revision: 8 April 2002, 1. Revision received: 30 April 2002, Returned for 2. revision: 21 May 2002, 2. Revision received: 29 May 2002, Returned for 3. revision: 29 May 2002, 3. Revision received: 6 June 2002, Accepted: 12 June 2002 Correspondence to: Dr. G. Taimor     

Xanthine oxidase contributes to preconditioning's preservation of left ventricular developed pressure in isolated rat heart: developed pressure may not be an appropriate end-point for studies of preconditioning

Studies of preconditioning frequently use the isolated rat heart model in which recovery of post-ischemic function is the end-point. However, function following an episode of ischemia/reperfusion represents a composite of both stunning, which is related to free radical production and is not attenuated by preconditioning, and tissue salvage, the primary effect of preconditioning. Brief ischemia/reperfusion is also known to diminish adenosine release during subsequent ischemia by a mechanism independent of preconditioning's anti-infarct effect. Reduced purine release would diminish generation of free radicals by xanthine oxidase in rat heart and thus produce less stunning. In this paradigm preserved post-ischemic function in rat heart might look similar to salvage by preconditioning, but its mechanism would be quite different and not be relevant to the xanthine oxidase-deficient human heart. This hypothesis was tested in isolated rat hearts. Control or ischemically preconditioned hearts were subjected to 30 min of global ischemia and 60 min of reperfusion, either in the presence or absence of 25 μmol/l allopurinol, an inhibitor of xanthine oxidase. In non-preconditioned hearts allopurinol increased left ventricular developed pressure after 60 min of reperfusion from 26 ± 5 mmHg in control hearts to 47 ± 7 mmHg, whereas developed pressure in preconditioned hearts following reperfusion was 59 ± 5 mmHg and was unaffected by allopurinol. Developed pressure in non-preconditioned hearts treated with allopurinol was midway between that for untreated control and preconditioned hearts suggesting that at least 50 % of the recovery of developed pressure in preconditioned hearts may be related to free radical-induced stunning. In xanthine oxidase-deficient rabbit hearts, return of function was not different between non-preconditioned and preconditioned hearts. Therefore, post-ischemic developed pressure in the rat is significantly affected by purine-dependent stunning, and, hence, may be an unreliable marker of tissue salvage and also a poor index of what might be cardioprotective in man. Received: 2 May 2001, Returned for 1. revision: 25 May 2001, 1. Revision received: 13 June 2001, Returned for 2. revision: 18 June 2001, 2. Revision received: 11 July 2001, Accepted: 18 July 2001     

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     

Energy generation in hypertrophied postischemic myocardium Feasibility of prolonged inotropic stimulation with dopamine in hypertrophied reperfused left ventricles

Background Non-hypertrophied reversibly injured postischemic myocardium can be stimulated for a prolonged period without detrimental effects. Since no data on hypertrophied myocardium are available, our aim was to examine the effects of a prolonged postischemic positive inotropic stimulation on moderately hypertrophied left ventricles. Methods Using a Langendorrf-type isovolumically contracting isolated heart model, moderately hypertrophied (+50% of ventricular mass) hearts from spontaneously hypertensive rats (SHR) were investigated and compared to data from non-hypertrophied hearts of normotensive rats. A 30 minutes noflow ischemia was performed, and in the postischemic period dopamine was continuously administered for 20 minutes in order to stimulate the postischemic hearts to the control level of function. Data were compared to postischemic hearts without stimulation and to non-ischemic controls. After 50 minutes of reperfusion and cessation of the catecholamine steady state function, maximum contractile response, and high energy phosphates were determined. Results 30 minutes ischemia followed by 50 minutes reperfusion caused a significant reduction in developed LVP to 77.8±4.2% in SHR. Dp/dtmax was reduced to 67.0±2.3%. After cessation of dopamine stimulation developed LVP was 64.3±3.5% and dp/dtmax 69.3±3.7% in SHR. The double product was identically reduced in all postischemic groups. The contractile reserve was comparable in stimulated and non-stimulated postischemic SHR hearts. In hypertrophied myocardium, ATP was reduced to 1.1±0.1 μmol/gww (non-ischemic controls 2.5±0.3 μmol/gww) in unstimulated and to 1.0±0.1 μmol/gww in stimulated postischemic hearts. Comparably the ischemia-induced reduction in ATP in non-hypertrophied myocardium was 1.3 μmol/gww. Similar results were obtained for ADP and AMP. Creatine phosphate levels were normal in stimulated and non-stimulated postischemic myocardium of hypertrophied and non-hypertrophied hearts. Conclusion These results indicate that prolonged stimulation of stunned hypertrophied myocardium is feasible without detrimental effects on poststimulation contractile function. The energy generating apparatus is capable to deliver sufficient energy during stimulation of stunned hypertrophied hearts. Received: 28 July 1997, Returned for Revision: 4 September 1997, 1. Revision received: 20 October 1997, Accepted: 21 November 1997     

Endogenous adenosine suppresses norepinephrine-induced ventricular arrhythmias in rat heart

Adenosine is an antiarrhythmic substance particularly effective in catecholamine-dependent tachycardias. Although endogenous adenosine substantially accumulates in catecholamine-stimulated hearts, little is known about the antiarrhythmic potency of endogenous adenosine in this condition. Therefore, we sought to demonstrate a potential antifibrillatory effect of endogenous adenosine either by blockade of adenosine receptors with 8-phenyltheophylline (8-PT) or by suppression of endogenous adenosine release with nitrobenzyl-6-thioinosine (NBTI). The study was performed in spontaneously beating Langendorff-perfused rat hearts. Adenosine release into the effluent was determined by HPLC methods. Catecholamine stimulation was induced by perfusing the hearts with norepinephrine (1 μmol/l) for 30 min, which caused ventricular tachycardia (VT) in 31% and ventricular fibrillation (VF) in 25% of control hearts (n=35). When 8-PT (10 μmol/l) was added to the perfusion buffer prior to norepinephrine, the incidence of VT and VF increased to 79 and 68%, respectively. The addition of 8-PT did not affect the catecholamine-dependent formation of adenosine. Perfusion of the hearts with NBTI (10 μmol/l) prior to norepinephrine reduced adenosine release and increased the occurrence of both VT (65%) and VF (40%). In summary, the results indicate that adenosine is an endogenous antiarrhythmic substance, which accumulates in catecholamine-stimulated myocardium to a level, which effectively suppresses the occurrence of ventricular arrhythmias. Received: 26 November 1997, Returned for 1. revision: 18 December 1997, 1. Revision received: 15 January 1998, Returned for 2. revision: 29 January 1998, 2. Revision received: 18 February 1998, Accepted: 18 February 1998     

Evidence that cytosolic and ecto 5'-nucleotidases contribute equally to increased interstitial adenosine concentration during porcine myocardial ischemia

The purpose of this study was to determine the roles of cytosolic and ecto 5'-nucleotidase in myocardial ischemia-induced increases in interstitial fluid (ISF) adenosine. Pentobarbital anesthetized, open chest pigs were instrumented with two microdialysis fibers in the distally perfused bed of the left anterior descending (LAD) coronary artery to estimate ISF metabolites. Fibers in control hearts were perfused with standard Krebs buffer. In two additional groups, after collecting one dialysate sample with normal Krebs, fibers were perfused with buffer supplemented with either L-homocysteine thiolactone (5 mM) or the ecto 5'-nucleotidase inhibitor α, β-methylene adenosine 5'-diphosphate (AOPCP, 5 mM). Hearts were then submitted to 60 minutes LAD occlusion and two hours reperfusion. Dialysate nucleosides and AMP were measured by high performance liquid chromatography. The local delivery of homocysteine did not alter preischemic dialysate adenosine concentration (0.30 ± 0.04 μM) compared to pre-homocysteine infusion (0.39 ± 0.04 μM) or control hearts (0.36 ± 0.04 μM), but AOPCP significantly decreased preischemic dialysate adenosine levels (from 0.36 ± 0.02 to 0.14 ± 0.03 μM). During LAD occlusion both homocysteine and AOPCP reduced dialysate levels by approximately 50 %. At 30 minutes ischemia dialysate adenosine concentrations were 19.47 ± 2.72, 11.41 ± 2.44, and 7.93 ± 1.01 μ:M in control, homocysteine, and AOPCP hearts, respectively. AOPCP significantly increased dialysate AMP levels; at 60 minutes ischemia AMP levels were 6.22 ± 2.97 μM in control hearts and 38.60 ± 5.69 μM in AOPCP treated hearts. These results suggest that both cytosolic and ecto 5'-nucleotidase contribute to ischemia-induced increases in ISF adenosine in porcine myocardium. Received: 13 October 1998, Returned for revision: 5 November 1998, Revision received: 17 December 1998, Accepted: 4 January 1999