Coronary blood flow control is impaired at rest and during exercise in conscious diabetic dogs

This study tested whether diabetes mellitus impairs coronary blood flow control sufficiently to alter the balance between myocardial oxygen delivery and metabolism. Dogs (n = 7) were instrumented with catheters in the aorta and coronary sinus, and with a flow transducer on the circumflex coronary artery. Coronary blood flow, myocardial oxygen consumption (MVO₂), heart rate and aortic pressure were measured at rest and during treadmill exercise before and after induction of diabetes with alloxan monohydrate (40 – 60 mg/kg). Arterial plasma glucose concentration increased from 4.6 ± 0.2 mM in non-diabetic, control dogs to 20.2 ± 2.3 mM one week after alloxan injection. In non-diabetic control dogs, exercise increased MVO₂ 3.1-fold, coronary blood flow 2.7-fold, and heart rate 2.4-fold. Coronary venous PO₂ decreased from 19.4 ± 0.6 mmHg at rest to 14.7 ± 0.7 mmHg during exercise. Diabetes significantly attenuated exercise coronary hyperemia and reduced coronary venous PO₂ at rest (15.6 ± 0.5 mmHg) and during exercise (12.6 ± 0.8 mmHg). Diabetes also significantly reduced myocardial oxygen delivery at each level of exercise. Acute hyperglycemia alone did not alter exercise-induced coronary vasodilation or reduce coronary venous PO₂. These findings demonstrate that experimental diabetes attenuates functional coronary hyperemia and impairs the balance between coronary blood flow and myocardial metabolism. However, this deleterious effect is not related to acute hyperglycemia but to the chronic disease process of diabetes mellitus. Received: 19 July 2001, Returned for 1. revision: 20 August 2001, 1. Revision received: 17 October 2001, Returned for 2. revision: 19 October 2001, 2. Revision received: 2 November 2001, Accepted: 5 November 2001     

Endogenous nitric oxide regulates right coronary blood flow during acute pulmonary hypertension in conscious dogs

This study investigated the role of nitric oxide (NO) in the control of right coronary (RC) blood flow at rest and during acute pulmonary hypertension. Experiments were performed in seven chronically instrumented, conscious dogs. NO synthesis was inhibited by systemic administration of N^ω-nitro-L-arginine (LNA, 35 mg/kg). Inflation of a balloon in the main pulmonary artery raised right ventricular (RV) peak systolic pressure from 34 ± 2 to 47 ± 3 mmHg before LNA and from 37 ± 2 to 47 ± 3 mmHg after LNA, but did not affect mean systemic arterial pressure. RV O₂ consumption (MVO₂) increased from 4.4 ± 0.7 to 6.1 ± 0.7 ml/min/100 g. 82 % of the elevated RV MVO₂ was provided by RC blood flow, which increased from 46 ± 7 to 61 ± 8 ml/min/100 g. After LNA, resting RV MVO₂ and RC flow fell. RC venous PO₂ fell, but RV lactate uptake was not altered. During pulmonary hypertension, the increase in RC blood flow was blunted by LNA, so that only 66 % of the elevated RV MVO₂ was supplied by increased RC flow. Analysis of O₂ supply variables as functions of RV MVO₂ further demonstrated a significant role of NO in regulating RC flow at rest and during moderate pulmonary hypertension. Conclusions NO is required for the RC hyperemic response to acute pulmonary hypertension as well as for normal resting RC blood flow. After blockade of NO synthesis, RV O₂ supply at rest and during pulmonary hypertension was sustained by increased RV O₂ extraction. Received: 2 April 2002, Returned for 1. revision: 17 April 2002, 1. Revision received: 13 May 2002, Returned for 2. revision: 29 May 2002, 2. Revision received: 5 June 2002, Accepted: 10 June 2002     

The inotropic response of the isolated,perfused, working rat heart to norepinephrine is attenuated by inhibition of nitric oxide

Experiments on isolated, perfused, working left ventricular (LV) hearts of 66 female Wistar rats were done to examine whether nitric oxide (NO) influences the effects of norepinephrine (NE) on coronary flow as well as on contraction and relaxation. Functional parameters were monitored before and after application of NE at a concentration of 3 × 10⁻⁸ M in the absence and presence of the nitric oxide synthase (NOS) inhibitor L-nitro-arginine (L-NA) at a concentration of 1 × 10⁻⁴ M and of the spontaneous NO donor sodium (Z)-1-(N,N-diethylamino) diazen-1-ium-1,2-diolat (DEA/NO) at a concentration of 1 × 10⁻⁷ M. In control experiments, heart rate was varied by electrical stimulation between 200 and 400 beats/min. Within this range of heart rates, coronary flow and cardiac output remained constant, while stroke volume, LV peak pressure and LV dP/dt_max decreased with increasing heart rate. NE increased coronary flow from 7.6 ± 0.4 to 9.8 ± 0.7 ml/min and induced the well-known positive chronotropic and inotropic effects. DEA/NO increased coronary flow; however, the inotropic and lusitropic parameters were not affected. Simultaneous infusion of NE with DEA/NO further increased coronary flow from 9.8 ± 0.7 to 12.1 ± 0.8 ml/min without a significant effect on any other functional parameter. When NOS was inhibited by L-NA, the positive inotropic effect of NE was attenuated. Cardiac output, however, was increased, while coronary flow did not change significantly. Under these conditions, NE increased dP/dt_max by 65.5 ± 5.8% (from 2999 ± 97 to 4929 ± 230 mmHg/s) compared with an increase by 92.8 ± 6.7% (from 3770 ± 82 to 7234 ± 211 mmHg/s) under control conditions. Application of DEA/NO reversed the attenuated inotropic response, but relaxation remained partially impaired. Thus, the presence of NO seems to be necessary for the inotropic effect of NE to become manifest. Received: 9 February 2001, Returned for 1. revision: 22 February 2001, 1. Revision received: 25 May 2001, Returned for 2. revision: 12 June 2001, 2. Revision received: 20 July 2001, Returned for 3. revision: 2 August 2001, Accepted: 20 August 2001     

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     

Acute coronary vascular and myocardial perfusion effects of conjugated equine estrogen in the anesthetized dog

Women generally exhibit angina rather than myocardial infarction as the first manifestation of heart disease. Postmenopausal use of hormone replacement therapy, specifically estrogens, is associated with reduced incidence of major cardiac events suggesting estrogen may protect the heart during ischemia. We recently showed that acute administration of conjugated equine estrogens prior to ischemia attenuated the ventricular arrhythmias of ischemia as well as those of reperfusion. This study looks at basal effects of estrogen on coronary blood flow and the effects of estrogen on regional blood flow during ischemia to determine if estrogen exerts its antiarrhythmic effects during ischemia by altering blood flow. Under conditions of natural blood flow, estrogen caused cyclic changes in blood flow. When coronary blood flow was controlled and limited, estrogen increased coronary perfusion pressure (118±8 mmHg vs. 85±10 mmHg in non-treated dogs, P<0.05) demonstrating an overall vasoconstrictor effect. Coronary blood flow and regional myocardial perfusion were determined before and during ischemia in anesthetized dogs with and without acutely-administered estrogen. Colored microspheres were injected at steady state prior to ischemia, and during steady state myocardial ischemia. Conjugated equine estrogen (10 μg/kg), administered about 6 min before ischemia, had no effect on regional perfusion under steady state conditions, nor in the non-ischemic zone during ischemia. Perfusion in the subepicardial and subendocardial ischemic zones in estrogen-treated dogs was significantly lower than in non-treated dogs [0.14±0.01 ml/min/g vs. 0.23±0.02 ml/min/g (P<0.05) in the epicardial ischemic zone; and, 0.15±0.02 ml/min/g vs. 0.22±0.03 ml/min/g (P<0.05) in the endocardial ischemic zone]. We conclude that the acute, systemic administration of estrogen in the anesthetized dog decreases regional perfusion in the ischemic myocardium and causes significant coronary vasoconstriction when flow is controlled and limited. Received: 3 March 1998, Returned for 1. revision: 28 April 1998, 1. Revision received: 29 May 1998, Accepted: 18 June 1998     

Effect of atrial dilatation on electrophysiologic properties and inducibility of atrial fibrillation

Introduction: Atrial dilatation may play an important role in the occurrence of atrial fibrillation (AF) in clinical situations. However, the electrophysiologic characteristics of dilated atria are still unclear. Methods and results: In 18 isolated Langendorff-perfused canine hearts (14.6 ± 2.2 kg), we measured atrial effective refractory periods (ERPs) at four different sites, conduction velocity and percentage of slow conduction on the right atrium (using a high-density electrode plaque), and assessed the inducibility of AF at the baseline (0 cm H₂O) and high (15 cm H₂O) atrial pressure. The atrial ERPs did not change significantly, but the dispersion of ERP increased significantly (40 ± 18 vs 25 ± 9 vs ms, p = 0.01) during high atrial pressure. The percentage of slow conduction (< 25 cm/s) over the mapping area, and the inducibility of AF increased during high atrial pressure (23.7 ± 10.2 % vs 32.1 ± 12.5 %, p = 0.02). The AF inducibility significantly correlated with the ERP dispersion (R = 0.75, p < 0.001) and maximal percentage of slow conduction (R = 0.88, p < 0.001). Furthermore, ERPs were significantly shorter in the induced AF group than those without induced AF (68 ± 17 vs 84 ± 16 ms, P < 0.05). Conclusions: The increased inhomogenity in atrial electrophysiological properties during atrial dilatation contributed to the inducibility of AF. Received: 26 November 2001?Returned for 1. revision: 2 January 2002?1. Revision received: 11 February 2002?Returned for 2. revision: 25 March 2002?2. Revision received: 6 May 2002?Returned for 3. revision: 10 June 2002?3. Revision received: 21 August 2002?Accepted: 11 September 2002     

Nifedipine limits infarct size via NO-dependent mechanisms in dogs

Objectives Amlodipine increases NO levels in coronary vessels and aorta via bradykinin-dependent mechanisms in vitro. We have previously reported that nifedipine increases cardiac NO levels in the ischemic canine hearts, suggesting that nifedipine may also have protective effects against ischemia and reperfusion injury, because the enhancement of NO production limits infarct size. We tested whether nifedipine limits infarct size via NO-dependent mechanisms. Methods In open chest dogs, the left anterior descending coronary artery was perfused with blood through a bypass tube and occluded for 90 min followed by 6 hours of reperfusion. Infarct size was assessed at 6 hours of reperfusion. Nifedipine of 3 or 6 μg/kg/min was infused into the bypass tube between 10 min prior to the onset of ischemia and 60 min of reperfusion. Results Neither systemic blood pressure nor heart rate changed during infusion of nifedipine. Infarct size was reduced by the administration of nifedipine (3 or 6 μg/kg/min) compared with the untreated condition (25.6 plusmn; 2.6 and 19.1 ± 3.5 vs. 43.4 ± 5.6 %, respectively), which was completely blunted by L-NAME (45.0 ± 3.6 and 45.4 ± 4.2 vs. 47.9 ± 3.9 % in the nifedipine (3 or 6 μg/kg/min) with L-NAME groups vs. the L-NAME group). Myeloperoxidase activity of the myocardium increased after 6 hours of reperfusion, which was attenuated by nifedipine. The limitation of infarct size and the attenuation in myeloperoxidase activity were completely blunted by L-NAME. There were no significant differences in collateral blood flow at 45 min of ischemia between each group. Conclusions We conclude that the Ca channel blocker, nifedipine, limits infarct size via NO-dependent mechanisms. Received: 21 September 2000, Returned for 1. revision: 9 October 2000, 1. Revision received: 17 January 2001, Returned for 2. revision: 5 February 2001, 2. Revision received: 13 February 2001, Accepted: 14 February 2001     

A1 adenosine receptor overexpression decreases stunning from anoxia-reoxygenation: role of the mitochondrial K(ATP) channel

Myocardial A₁ adenosine receptor (A₁AR) overexpression protects hearts from ischemia-reperfusion injury; however, the effects during anoxia are unknown. We evaluated responses to anoxia-reoxygenation in wild-type (WT) and transgenic (Trans) hearts with ∼200-fold overexpression of A₁ARs. Langendorff perfused hearts underwent 20 min anoxia followed by 30 min reoxygenation. In WT hearts peak diastolic contracture during anoxia was 45 ± 3 mmHg, diastolic pressure remained elevated at 18 ± 3 mmHg after reoxygenation, and developed pressure recovered to 52 ± 4 % of pre-anoxia. A₁AR overexpression reduced hypoxic contracture to 29 ± 4 mmHg, and improved recovery of diastolic pressure to 8 ± 1 mmHg and developed pressure to 76 ± 3 % of pre-anoxia. Mitochondrial K_ATP blockade with 100 μM 5-hydroxydecanoate (5-HD) increased hypoxic contracture to 73 ± 6 mmHg in WT hearts, reduced post-hypoxic recoveries of both diastolic (40 ± 5 mmHg) and developed pressures (33 ± 3 %). In contrast, 5-HD had no effect on hypoxic contracture (24 ± 8 mmHg), or post-hypoxic diastolic (10 ± 2 mmHg) and developed pressures (74 ± 3 %) in Trans hearts. In summary, (i) A₁AR overexpression improves myocardial tolerance to anoxia-reoxygenation, (ii) intrinsic mitochondrial K_ATP channel activation decreases hypoxic contracture and improves functional recovery in wild-type hearts, and (iii) mitochondrial K_ATP channels do not appear to play a major role in the functional protection from anoxia afforded by A₁AR overexpression. Received: 5 February 2001, Returned for 1. revision: 21 February 2001, 1. Revision received: 20 August 2001, Returned for 2. revision: 3 September 2001, 2. Revision received: 24 October 2001, Accepted: 25 October 2001     

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     

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     

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     

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     

Skeletal muscle abnormalities in rats with experimentally induced heart hypertrophy and failure

Background: In congestive heart failure (CHF), function and metabolism of skeletal muscles are abnormal. Aim: To evaluate whether the reduced oxidative capacity of skeletal muscles in CHF is due to impaired O₂ utilisation. Methods: CHF was induced in rats by injecting 50 mg/Kg monocrotaline. Several animals received the same dose of monocrotaline but only compensated right ventricular hypertrophy and no sign of congestion resulted. Two age- and diet-matched groups of control animals were also studied. In soleus and extensor digitorum longus (EDL) muscles, we studied skeletal muscle blood flow, oxidative capacity and respiratory function of skinned muscle fibres. Results: In CHF, we observed a decrease of muscle blood flow (statistically significant in the soleus, p < 0.05 vs. controls). In compensated rats, a similar trend in blood flow was observed. In both soleus and EDL, a significant reduction of high energy phosphate and a shift of the redox potential towards accumulation of reducing equivalents were observed. The reduction of energy charge was not correlated to the decrease of blood flow. In skinned myofibres, the ratio of O₂ utilised in the presence and in absence of ADP (an index of phoshorilating efficiency) was reduced from 8.9 ± 1.9 to 2.7 ± 0.2 (p < 0.001) and from 5.7 ± 1.0 to 2.0 ± 0.3 (p < 0.01) in soleus and EDL, respectively. Activity of the different complexes of respiratory chain was investigated by means of specific inhibitors, showing major abnormalities at the level of complex I. In fact, inhibition of VO₂ by rotenone was decreased from 83.5 ± 3.2 to 36.4 ± 9.6 % (p < 0.005) and from 81.8 ± 6.1 to 38.2 ± 7.4 % (p < 0.005) in soleus and EDL, respectively. Conclusions: In rats with CHF, abnormalities of oxidative phosphorylation of muscles occur and complex I of the respiratory chain seem to be primarily affected. The metabolic alterations of skeletal muscles in CHF may be explained, at least in part, by an impaired O₂ utilisation. Received: 22 February 2002, Returned for 1. revision: 14 March 2002, 1. Revision received: 5 June 2002, Returned for 2. revision: 21 June 2002, 2. Revision received: 23 August 2002, Accepted: 12 September 2002 Correspondence to: Dr. C. Ceconi     

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     

Differential G protein receptor kinase 2 expression in compensated hypertrophy and heart failure after myocardial infarction in the rat

The onset of heart failure is associated with characteristic changes in myocardial expression of G protein receptor kinase 2 (GRK2). Although, GRK2 significantly contributes to the regulation of myocardial function in the failing heart, the GRK2 expression during cardiac hypertrophy without heart failure remains to be explored. We here report a differential expression of GRK2 in cardiac hypertrophy with or without heart failure in response to a myocardial infarction in the rat. Postmyocardial infarction animals were divided into two groups depending on the absence or presence of pulmonary edema, which is a manifestation of heart failure. Remarkably, cardiac GRK2 expression and activity were inhibited in animals with cardiac hypertrophy without heart failure, whereas animals with heart failure had elevated GRK2. Thus, three weeks after the infarction cardiac GRK2 expression in animals with hypertrophy alone was decreased to 0.34 of control, whereas in the group of animals with heart failure GRK2 expression was 1.89-fold higher than in sham-operated animals. GRK2 activity was affected in a similar way, three and nine weeks after the infarction cardiac GRK2 activity was reduced to 0.58 and 0.62 in animals with hypertrophy without heart failure when compared to sham operated animals. By contrast, GRK2 activity was increased by 1.32- and 1.21-fold three and nine weeks postinfarction in animals with heart failure when compared to sham animals. These data suggest that GRK2 expression is differentially regulated in hypertrophic, non-failing and hypertrophic, failing hearts. Received: 26 August 2002, Returned for 1. revision: 9 September 2002, 1. Revision received: 25 September 2002, Returned for 2. revision: 24 October 2002, 2. Revision received: 3 November 2002, Accepted: 9 November 2002 Correspondence to: S. P. Sheikh     

Critical timing of mitochondrial K(ATP) channel opening for enhancement of myocardial tolerance against infarction

Objective The present study was designed to assess the relationship between the timing of a mitoK_ATP channel opener, diazoxide, and its infarct size-limiting effect. Methods In isolated rabbit hearts, infarction was induced by 30 min of global ischemia and 2 h of reperfusion, and infarct size was determined by tetrazolium staining and expressed as a percentage of the left ventricle (%IS/LV). Diazoxide, a mitoK_ATP channel selective opener, and/or 5-hydroxydecanoate (5-HD), a mitoKATP channel blocker, were infused before or after the onset of ischemia. When these agents were infused during the ischemic period, they were dissolved in a hypoxic buffer at concentrations 10-fold higher than those in the pre-ischemic period, and the infusion rate was set at 2 % of the pre-ischemic coronary flow. Results In untreated controls, %IS/LV was 53.2 ± 4.1 (SE). Pretreatment with diazoxide (100 μM) with a 10-min washout period reduced %IS/LV to 7.8 ± 2.4 and this protection was abolished by co-infusion of 5-HD (50 μM). Pre-ischemic infusion of diazoxide without a washout period reduced %IS/LV to 7.3 ± 1.4, and infusion of diazoxide from 10 min after the onset of ischemia also limited %IS/LV to 14.9 ± 4.6. However, diazoxide infusion from 25 min after the onset of ischemia failed to reduce infarct size (%IS/LV = 54.5 ± 7.2). Furthermore, pretreatment with 5-HD (50 μM) also completely abolished the protection afforded by early post-ischemic diazoxide infusion (%IS/LV = 48.3 ± 6.5). Neither infusion of 5-HD nor the anoxic vehicle alone during ischemia modified %IS/LV. Conclusion These findings suggest that opening of mitoK_ATP channels before ischemia and during early ischemia, but not that upon reperfusion, is important for enhancement of myocardial tolerance against infarction. Received: 6 November 2000, Returned for revision: 21 November 2000, Revision received: 24 January 2001, Accepted: 25 January 2001     

Antioxidant properties of acetaminophen and cardioprotection

The acute administration of acetaminophen to isolated, perfused guinea pig hearts appears to have cardioprotective effects against the injury/mechanical dysfunction caused by global, low-flow, myocardial ischemia and reperfusion. In the current study we selected ischemia/reperfusion and administration of sodium pentobarbital as perturbations of the electrical stability of the myocardium. We investigated their ability to induce ventricular arrhythmias and changes in the characteristics of monophasic action potentials in the absence and presence of acetaminophen (0.35 mmol/l). The numbers of ventricular premature beats and ventricular salvos encountered in the presence of pentobarbital were significantly (P < 0.05) reduced by acetaminophen. The combined frequency of these arrhythmias was 0.14 ± 0.06/min vs 0.03 ± 0.01/min (P < 0.05) in the absence and presence of acetaminophen, respectively. The incidence of ventricular salvos increased steadily in vehicle-treated hearts after administration of pentobarbital. No such trend was seen with acetaminophen. After 10 min of global, low-flow myocardial ischemia, MAP50 and MAP90 (monophasic action potentials at 50 and 90 % repolarization, respectively) decreased without acetaminophen (e.g. MAP50, 31 ± 4 ms) but did not change during the same time interval with acetaminophen (e.g. MAP50, 57 ± 6 ms)(P < 0.05). During ischemia and reperfusion, acetaminophen attenuated the release of hydroxyl radicals and peroxynitrite. Collectively these data reveal cardioprotective, antioxidant behavior of acetaminophen. Under selected conditions (e.g. those causing release of free radicals and other oxidants) such behavior might also prevent ventricular arrhythmias. Received: 30 October 2000, Returned for 1. revision: 20 November 2000, 1. Revision received: 12 December 2000, Returned for 2. revision: 2 January 2001, 2. Revision received: 14 January 2001, Accepted: 31 January 2001     

Central hypercapnic chemoreflex modulation of renal sympathetic nerve activity in experimental heart failure

Activation of the sympathetic nervous system plays an important role in the pathophysiology and progression of congestive heart failure (CHF). The precise mechanisms responsible for sympathetic activation in CHF are not yet clearly established. An altered central hypercapnic chemoreflex modulation of sympathetic nerve activity (SNA) might be an explanation. Therefore, the response of postganglionic renal SNA to elevation of CO₂ concentration in the inspiratory air to 2, 4, and 6% was determined in anesthetized, artificially ventilated rats after denervation of peripheral baro- and chemoreceptors 2 weeks (group A; n=8) or 6 weeks (group B; n = 11) after induction of an aorto-caval shunt, or 4 weeks after aortic banding (group C; n = 7). In all CHF models, left ventricular enddiastolic pressure was increased (A 8 ± 1, B 8 ± 1, C 10 ± 2 mmHg) as compared to sham operated controls (A 3 ± 1, B 4 ± 1, C 5 ± 1 mmHg). Indicative of left ventricular hypertrophy and pulmonary congestion, wet weight of heart (A + 60%, B + 93%, C + 49%) and lungs (A + 15%, B + 36%, C + 12%) were also enhanced as compared to controls. Elevation of inspiratory CO₂ concentration to 2, 4, and 6% increased renal SNA by approximately 10, 20, and 30% from resting activity in all groups. The maximum SNA responses at 6% CO₂ in the groups with CHF (A + 390 ± 95, B + 425 ± 133, C + 368 ± 158 μVs) did not differ from those in the respective controls (A + 510 ± 130, B + 570 ± 180, C + 275 ± 25 μVs). It is concluded that under these experimental conditions the central hypercapnic chemoreflex sensitivity is not altered in either of the employed models of CHF and therefore may not play a major role for the well-known elevation of SNA in CHF. Received: 16 July 2001, Returned for revision: 25 July 2001, Revision received: 27 August 2001, Accepted: 11 September 2001     

The expression of heat shock protein 60 in myocardium of patients with chronic atrial fibrillation

Objective Cardiomyocytes respond to stress with the expression of different heat shock proteins (HSP). HSP60 is induced by various stress factors. The aim of this study was to investigate the expression of HSP60 in human atrial fibrillation (AF). Method Right atrial samples from 14 patients undergoing elective cardiac surgery were excised and immediately frozen in liquid nitrogen. Eight patients had chronic AF and six patients were in sinus rhythm. The HSP60 protein level was determined by SDS-PAGE, Western blot and quantified by optical densitometry according to the immunoreactive bands of actin. Results In myocardial samples from patients with chronic AF, we found a more than 2.5-fold increase in HSP60 expression compared to atrial myocardium of patients in sinus rhythm. Conclusion This result indicates an up regulation of HSP60 in response to chronic atrial fibrillation Received: 31 October 2001, Returned for 1. revision: 20 Novemver 2001, 1. Revision received: 12 December 2001, Returned for 2. revision: 3 January 2002, 2. Revision received: 25 January 2002, Accepted: 6 February 2002     

Myocardial blood flow in awake dogs with chronic tricuspid regurgitation

The primary purpose of this study was to define regional blood flow in dogs with chronic tricuspid regurgitation (TR) in order to determine if the marked hypertrophy of the right atria resulted in compromised myocardial perfusion. Myocardial blood flow (ml/min/gm) was measured with radiolabeled microspheres in eight dogs with TR during rest, moderate exercise (5 dogs), and infusion of adenosine (1 mg/kg/min), an index of minimal vascular resistance. Similar measurements were obtained in eight normal dogs. In TR, the ratio of right atrium (RA) and right ventricle (RV) to body weight was greater than in normal dogs, 77 % and 30 %, respectively. During rest, flow in RA appendage was less than in nonappendage region in the normal dogs; no differences were noted in TR dogs, indicating an augmented hemodynamic role of the appendage in TR. Both RA and RV blood flow was greater in TR during rest but no other differences in flow were found between the two groups. Minimum vascular resistance in RV but not RA was slightly increased in TR versus normal. During marked myocyte hypertrophy, the vasculature of RA develops sufficiently to provide the same flow capacity as in the normal heart. Received: 11 June 1997, Returned for revision: 15 July 1997, Revision received: 11 August 1997, Accepted: 7 September 1997