Decreased expression of Na+/H+ exchanger isoform 1 (NHE1) in non-infarcted myocardium after acute myocardial infarction

Although cardiac NHE1 is activated during myocardial ischemia and reperfusion injury, little is known about changes in expression in non-infarcted myocardium after acute myocardial infarction (AMI). The purpose of this study was to examine left ventricular function and region dependent NHE1 expression after myocardial infarction. Therefore, we produced two AMI models in rats, a small infarction model which was continuously ligated at the branches of the left coronary artery, and an extensive infarction model continuously ligated at the root of the artery. We examined NHE1 mRNA expression using RNase protection assay and protein levels using Western blot analysis in non-infarcted myocardium during the 24 hour period after AMI. The level of NHE1 mRNA and protein expression in the whole heart including the infarcted myocardium did not change after a small infarction. On the other hand, in the case of an extensive infarction, the levels of NHE1 mRNA and protein expression decreased significantly by 21.5% (P<0.05) and by 22.0% (P<0.05), respectively, in non-infarcted myocardium. Left ventricular systolic pressure (LVSP) decreased significantly by 13% and 38% with the branch and root ligation, respectively. However, left ventricular end-diastolic pressure (LVEDP) only increased with the root ligation. These results indicate that NHE1 expression decreased in response to extensive myocardial infarction only in non-infarcted myocardium. The present study may be important in furthering the understanding of NHE1 in myocardial infarction and suggests that decreased expression of NHE1 in non-infarcted myocardium may decrease the extent of cardiac cell injury.     

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     

Influence of mild hypothermia on myocardial contractility and circulatory function

Myocardial contractility depends on temperature. We investigated the influence of mild hypothermia (37–31°) on isometric twitch force, sarcoplasmic reticulum (SR) Ca²⁺-content and intracellular Ca²⁺-transients in ventricular muscle strips from human and porcine myocardium, and on in vivo hemodynamic parameters in pigs. In vitro experiments: muscle strips from 5 nonfailing human and 8 pig hearts. Electrical stimulation (1 Hz), simultaneous recording of isometric force and rapid cooling contractures (RCCs) as an indicator of SR Ca²⁺-content, or intracellular Ca²⁺-transients (aequorin method). In vivo experiments: 8 pigs were monitored with Millar-Tip (left ventricular) and Swan-Ganz catheter (pulmonary artery). Hemodynamics parameters were assessed at baseline conditions (37°), and after stepwise cooling on cardiopulmonary bypass to 35, 33, and 31°C. Hypothermia increase isometric twitch force significantly by 91 ± 16% in human and by 50 ± 9% in pig myocardium (31 vs. 37°C; p < 0.05, respectively). RCCs or aequorin light emission did not change significantly. In anesthetized pigs, mild hypothermia resulted in an increase in hemodynamic paramters of myocardial contractility. While heart rate decreased from 111 ± 3 to 73 ± 1 min⁻¹, cardiac output increased from 2.4 ± 0.1 to 3.1 ± 0.3 l/min, and stroke volume increased from 21 ± 1 to 41 ± 3 ml. +dP/dt_max increased by 25 ± 8% (37 vs. 31°C; p < 0.05 for all values). Systemic and pulmonary vascular resistance did not change significantly during cooling. Mild hypothermia exerts significant positive inotropic effects in human and porcine myocardium without increasing intracellular Ca²⁺-transients or SR Ca²⁺-content. These effects translate into improved hemodynamics parameters of left ventricular function. Received: 26 June 2000, Returned for revision: 20 July 2000, Revision received: 11 October 2000, Accepted: 17 October 2000     

Preconditioning one myocardial region does not neccessarily precondition the whole rabbit heart

A previous study in dogs indicated that preconditioning (PC) of a specific myocardial region not only evoked a local cardioprotective effect but also rendered remote myocardium resistant to infarction. In the present study we devised a method to test for remote PC in the rabbit which it is not possible to ligate two separate coronary branches on the same heart. In situ hearts were subjected to PC with two cycles of 5-min regional ischemia/5-min reperfusion. Following this in vivo PC protocol, the hearts were removed and perfused on a Langendorff apparatus with crystalloid buffer. They then underwent 30 min of global ischemia with the entire left ventricle at risk followed by 2 h of reperfusion. At the end of the experiment the myocardium previously subjected to the in vivo PC protocol (preconditioned region) was identified as the tissue without fluorescence after fluorescent particles had been injected into the aortic root following reocclusion of the snared branch of the left coronary artery. Infarcted myocardium was identified by triphenyltetrazolium chloride staining. Tissue salvage was observed only in the preconditioned region where 13.2 ± 3.6 % of the myocardium infarcted as opposed to 44.6 ± 1.3 % in the remaining non-preconditioned left ventricular tissue (p < 0.05, n = 6). In sham-operated hearts (snare but no PC), infarction was similar in both the snared vessel's perfusion territory and the rest of the left ventricular myocardium (49.2 ± 6.5 % vs. 43.7 ± 3.7 %, n = 5). Hence PC of one myocardial region does not necessarily confer PC protection to all regions of the heart. Because remote PC could not be demonstrated in rabbits, this phenomenon may be species or protocol-specific, and should not be assumed to occur in man. Received: 11 April 2001, Returned for revision: 22 April 2001, Revision received: 15 May 2001, Accepted: 17 May 2001     

Altered force-frequency relation in hypertrophic obstructive cardiomyopathy

The present study was designed to test the hypothesis that in hypertrophied myocardium of patients with hypertrophic obstructive cardiomyopathy (HOCM) a reduced contractile reserve provided by frequency dependent potentiation of force of contraction contributes to the myocardial dysfunction. Myectomy was performed in 8 HOCM patients with normal systolic left ventricular function at rest. Nonfailing myocardium from the hearts of three multiorgan donors was investigated for comparison. In thin myocardial strips we measured the inotropic effects of different stimulation frequencies (0,5–3.0Hz) at different extracellular Ca²⁺ concentrations (1.8–16.2 mmol/l). At 1.8 mmol/l extracellular Ca²⁺ concentration, increasing stimulation rates had no positive inotropic effect in HOCM myocardium, whereas in nonfailing myocardium force of contraction increased up to 3 Hz. Increasing extracellular Ca²⁺ concentrations induced a positive force-frequency relation in HOCM with a maximum at 5.4 mmol/l Ca²⁺. A further increase to 16.2 mmol/l Ca²⁺ resulted in a negative force-frequency relation in these specimens. The time to peak tension and the time to relaxation decreased at increasing stimulation frequencies at all Ca²⁺ concentrations investigated. In conclusion, in hypertrophied myocardium of HOCM patients increasing stimulation frequencies failed to have a positive inotropic effect at physiological extracellular Ca²⁺ concentrations. The induction of a positive force-frequency relation by higher Ca²⁺ concentrations suggests that an abnormal cellular Ca²⁺ handling may play an important pathophysiological role. Received: 27 December 1996, Returned for revision: 3 March 1997, Revision received: 1 September 1998, Accepted: 30 September 1998     

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     

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     

Calcium-sensitivity of the SR calcium release channel in failing and nonfailing human myocardium

Altered Ca²⁺ metabolism of the sarcoplasmic reticulum results in changes of the contractile behavior in failing human myocardium. The ryanodine-sensitive Ca²⁺ release channel of the sarcoplasmic reticulum plays a key role in the intracellular Ca²⁺ handling in cardiac myocytes. Recently, we showed that the density of ³H-ryanodine binding sites which correspond to the SR Ca²⁺ release channel in human myocardial homogenates is unchanged in failing human myocardium. However, the sensitivity of the channel towards Ca²⁺, which acts as the trigger signal of channel activation and thereby initiates contraction, has not yet been investigated in failing and nonfailing myocardium. Methods: Homogenates (100 μg protein) from hearts with dilated (DCM, n = 10) or ischemic (ICM, n = 9) cardiomyopathy were incubated with a saturating concentration of ³H-ryanodine (12 nM) in the presence of different Ca²⁺ concentrations ranging from 1 nM to 10 mM. For comparison, myocardium of 8 nonfailing hearts which could not be transplanted for technical reasons was investigated. Nonspecific binding was determined in the presence of a high concentration (10 μM) of unlabeled ryanodine. Results: ³H-ryanodine binding to the Ca²⁺ release channel showed a bell-shaped pattern with an increase in specific binding at submicromolar Ca²⁺ concentrations and a decrease at higher Ca²⁺ concentrations than 0.5 mM, whereas nonspecific binding was not influenced by different Ca²⁺ concentrations. In nonfailing myocardium, maximal ³H-ryanodine binding (Bmax) was 85.2 ± 3.1 fmol/mg protein and half-maximal binding was reached at a free Ca²⁺ concentration of 0.25 (0.22 – 0.30)μM (EC₅₀). Neither EC₅₀ values nor maximal specific ³H-ryanodine binding differed between nonfailing and failing myocardium of both etiologies. EC₅₀ values were 0.24 (0.23 – 0.26)μM (DCM, n = 10) or 0.28 (0.25 – 0.31)μM (ICM, n = 9), respectively. Caffeine (2 mM) and the ATP-analogon AMP-PCP (1 mM) led to a shift towards lower Ca²⁺ concentrations consistent with an activation of the channel by these compounds, whereas Mg²⁺ (0.7 mM) shifted the Ca²⁺-dependence of ³H-ryanodine binding towards higher Ca²⁺ concentrations indicating inhibition of channel opening. After activation of the Ca²⁺ release channel by caffeine or AMP-PCP as well as after the inhibition with MG²⁺ EC₅₀ values were the same in failing and nonfailing myocardium. Conclusion: Caffeine and AMP-PCP sensitize, whereas Mg²⁺ desensitizes the myocardial Ca²⁺ release channel to Ca²⁺. The determination of Ca²⁺-dependent ³H-ryanodine binding to the human myocardial Ca²⁺ release channel is a useful tool to investigate its open probability. Furthermore, the Ca²⁺-sensitivity and the pharmacological behavior of the human SR Ca²⁺ release channel are similar in failing and nonfailing myocardium. Received: 7 October 1997, Returned for revision: 9 December 1997, 1.Revision received: 1 December 1998, Accepted: 5 January 1999     

Intracellular mechanisms of cGMP-mediated regulation of myocardial contraction

The intracellular mechanisms of cGMP, a major intracellular mediator of nitric oxide that regulates the contractility of cardiac muscle, are still to some extent unknown. To investigate these mechanisms, we observed the effects of 8-bromo-cyclic GMP (8br-cGMP) on myofibrillar Ca²⁺ sensitivity and Ca²⁺ handling of the sarcoplasmic reticulum (SR) using β-escin-skinned preparations from Wistar rat hearts. Both low (1 μM) and high doses (100 μM) of 8br-cGMP significantly decreased the myofibrillar Ca²⁺ sensitivity obtained from pCa-tension relationships to a similar extent (pCa₅₀; from 6.04 to 5.95 by 1 μM 8br-cGMP and 6.00 to 5.89 by 100 μM 8br-cGMP, respectively, n = 9 each). Whereas this Ca²⁺ desensitization induced by 100 μM 8br-cGMP was blocked by 1 μM KT5823, a specific inhibitor of cGMP-dependent protein kinase (PKG), not induced by 1 μM 8br-cGMP was not effected by KT5823. When the amount of Ca²⁺ released from the SR was estimated by the peak amplitude of 25 mM caffeine-induced contractions after constant Ca²⁺-loading by pCa 6, both doses of 8br-cGMP significantly augmented the caffeine-induced peak force to a similar extent (125 ± 5.8 % by 1 μM 8br-cGMP and 116 ± 5.1 % by 100 μM 8br-cGMP, respectively, n = 6 each). The two observed effects of cGMP (a decrease in myofibrillar Ca²⁺ sensitivity and an increase in Ca²⁺ uptake by the SR) may participate in regulating myocardial contraction via nitric oxide. Low and high doses of cGMP seem to work mainly via PKG-independent and PKG-dependent pathways, respectively. Received: 8 January 2001, Returned for 1. revision: 24 January 2001, 1. Revision received: 16 April 2001, Returned for 2. revision: 2 May 2001, 2. Revision received: 10 May 2001, Accepted: 15 May 2001     

Correlation between heterogeneous myocardial flow and oxidative metabolism in normoxic and stunned myocardium

Myocardial blood flow exhibits considerable heterogeneity. Consequently, oxygen supply to the myocardium is also heterogeneous, as is myocardial metabolism. Many lines of evidence show a close correlation between local flow and local metabolism in the normoxic myocardium. So far, myocardial metabolism has pre-dominantly been assessed indirectly by using labeled substrates. We used the ¹⁸O isotope, permitting analytical separation of H₂ ¹⁸O from the ¹⁸O isotope, as well as quantification of regional oxidative metabolism by measuring the tissue residue of oxidation water in the rabbit myocardium. Correlation of local flow with oxidative metabolism was significant in the normoxic myocardium. This correlation was lost in the postischemic/reperfused myocardium. Apart from the established mechanisms underlying myocardial stunning, a mismatch between local flow and oxidative metabolism might thus also contribute to the postischemic dysfunction. In the normoxic myocardium, function should correlate with metabolism and blood flow. For technical reasons, function has not been assessed on a very local scale. Nevertheless, some considerations are presented on the heterogeneity of function as well as on the scale on which heterogeneity should be investigated to convey physiologically meaningful information on regulatory cardiac mechanisms. Received: 17 May 2001, Returned for revision: 23 July 2001, Revision received: 20 August 2001, Accepted: 12 September 2001     

Altered excitation-contraction coupling in myocytes from remodeled myocardium after chronic myocardial infarction.

Following myocardial infarction (MI), the left ventricle undergoes progressive dilatation and eccentric hypertrophy, i.e., remodeling, which is greater in the adjacent than the remote region. The cellular mechanisms underlying these regional differences were studied. One (n=5) and 8 weeks (n=8) after anteroapical MI in sheep, cardiac myocytes were isolated from the adjacent and remote regions. At 8 weeks after MI, myocyte function in the remote region was not different from values either in sham controls (n=3) or animals 1 week after MI. At 8 weeks after MI, myocyte contractile function (% contraction) was decreased, P<0.01, in the adjacent region (6.4+/-0.4%), as compared with the remote region (8.8+/-0.5%) and was associated with decreased amplitude of Ca(2+)transients (adjacent, 0.69+/-0.09 v remote, 1.08+/-0.20, P<0.05) and L-type Ca(2+)current density (adjacent, 3.6+/-0.2 v remote, 4.8+/-0.2 pA/pF, P<0.05). Relaxation was also impaired significantly in myocytes from the adjacent region, associated with decreased protein levels of SERCA2a. The myocytes were hypertrophied more in the adjacent region than the remote region. Furthermore, focal areas of central myofibrillar lysis and increased glycogen deposition were observed in the adjacent region. These results indicate that impaired excitation-contraction coupling underlies dysfunction of myocytes from the adjacent non-infarcted myocardium after chronic MI, even in the absence of heart failure. Hypertrophy is implicated as the mechanism, since these changes were noted at 8 weeks, but not at 1 week after MI.     

The effects of levosimendan on the left ventricular function and protein phosphorylation in post-isschemic guinea pig hearts

The widely accepted theories for the decreased function in the stunned myocardium relate to Ca²⁺ desensitization and free radical-mediated tissue damage of the myofilaments. The aim of the present study was to examine whether the depressed contractile function and Ca²⁺ responsiveness of the stunned myocardium may be restored by a new Ca²⁺ sensitizer (levosimendan), which has been shown to improve the Ca²⁺ response of the myofilaments. The effects of levosimendan on the left ventricular function and the in vivo protein phosphorylation were examined in both the non-ischemic and the stunned myocardium. Myocardial stunning was induced in Langendorff-perfused guinea pig hearts by suspending the circulation for 8 min, followed by a 20-min reperfusion period. Perfusion of post-ischemic guinea pig hearts with levosimendan (0.03–0.48 μM, 6 min) was associated with dose- and time-dependent increases in both dP/dt_max (contractility) and dP/dt_min (speed of relaxation). When the effectiveness of levosimendan was compared in non-ischemic and post-ischemic hearts, no significant differences were noted in the relative stimulatory effects on contractility and relaxation, at any given time point (time-response curve) or concentration (dose-response curve). Perfusion of the guinea pig hearts with a high (0.3 μM) levosimendan concentration did not reveal any qualitative or quantitative difference in the phosphodiesterase inhibitory potential of the compound (elevation of tissue cyclic AMP levels and characteristics of protein phosphorylation) between the non-ischemic and the post-ischemic myocardium. However, when isoproterenol was adminstered to induce maximal in vivo phosphorylation of cardiac phosphorproteins, an attenuation of the ³²P-incorporation into troponin I was noted in the post-ischemic hearts. The decrease in isoproterenol-induced ³²P-incorporation into troponin I was associated with similar alterations in the tissue level of this protein. We conclude that the Ca²⁺ sensitizer levosimendan exerts dose- and time-dependent positive inotropic and lusitropic effects on the postischemic myocardium, lending support to the hypothesis tha Ca²⁺ desensitization of the myofibrils is involved in myocardial stunning. Received: 20 July 1998, Returned for 1. revision: 27 August 1998, 1. Revision received: 6 January 1999, Returned for 2. revision: 5 February 1999, 2. Revision received: 25 February 1999, Accepted: 3 March 1999     

Regulation of the isozymes of protein kinase C in the surviving rat myocardium after myocardial infarction: distinct modulation for PKC-alpha and for PKC-delta

Objective The goal of this study was to clarify the regulation of the isozymes of protein kinase C (PKC) in the process of remodeling after myocardial infarction. Methods An in vivo model of regional myocardial infarction induced by ligation of the left anterior coronary artery in rats was used. Hemodynamic parameters and the heart and lung weights were determined 1 week and 1, 2 and 3 months after operation. In transmural biopsies from the non-ischemic left ventricular wall of the infarcted heart, PKC activity (ELISA) and the expression of its major isozymes, PKC-α, PKC-δ and PKC-ε (Westernblot analysis) were determined. Results As early as one week after myocardial infarction, heart weight and left ventricular enddiastolic pressures were significantly increased. Lung weights increased after 2 – 3 months, indicating progressive pulmonary congestion. The activity of PKC was significantly increased about 1.8-fold after 1 week, decreasing progressively in the later time course. Whereas the expression of PKC-ε did not change, PKC-α was increased after 1 month (157 %) and then returned to baseline values. In contrast, PKC-δ expression was significantly augmented after 2 and 3 months of myocardial infarction (187 %). Conclusions These data demonstrate for the first time that in the remodeling heart after myocardial infarction, a subtype-selective regulation of the PKC isozymes occurs: The upregulation of PKC-α coincides with the development of hypertrophy, whereas the extensive upregulation of PKC-δ outlasts the process of developing hypertrophy and persists in the failing heart. The trigger mechanisms for this newly characterized process remains to be elucidated. Received: 25 October 2001, Returned for revision: 3 December 2001, Revision received: 19 December 2001, Accepted: 20 December 2001     

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     

Spatial heterogeneity of myocardial perfusion and metabolism

Left ventricular myocardium is characterized by a substantial spatial heterogeneity of both perfusion and metabolism. Under resting conditions, the transmural gradient of myocardial oxygen consumption (MVO₂) from the subepi- to the subendocardial layer exceeds that of coronary flow, resulting in a lower subendocardial PO₂, altered kinetics of oxidative phosphorylation, and enhanced free cytosolic adenosine. Within each layer, there is a major spatial variability of perfusion: Local flow rates in individual myocardial samples (200 mg) range from 20–250% of the mean myocardial blood flow. Low flow areas (<50% of mean flow) display a rather low uptake of fatty acids and glucose; the uptake of these substrates increases in proportion to local flow. There is also a close relationship between local perfusion and the local turnover of the tricarboxylic acid cycle and, thus, MVO₂ as was recently demonstrated using ¹³C NMR techniques. Consequently, within the well perfused left ventricular myocardium local MVO₂ and, thus, energy turnover varies more than 3-fold between low and high flow areas. Low flow areas are not ischemic, since local lactate, adenosine, and ATP are comparable to mean flow areas. When coronary perfusion pressure is reduced, the transmural perfusion gradient reverses resulting in impaired energy status and enhanced adenosine predominantly in the subendocardium. The rise in local adenosine or lactate requires a decrease of the individual local flow by more than 50% of its pre-ischemic value. It, thus, appears that not the absolute level of local flow predicts the impact of ischemia but its relative change. Received: 30 June 1998, Returned for 1. revision: 18 August 1998, 1. Revision received: 2 September 1998, Accepted: 3 September 1998     

Cell therapy enhances function of remote non-infarcted myocardium

Cell transplantation improves cardiac function after myocardial infarction; however, the underlying mechanisms are not well-understood. Therefore, the goals of this study were to determine if neonatal rat cardiomyocytes transplanted into adult rat hearts 1 week after infarction would, after 8–10 weeks: 1) improve global myocardial function, 2) contract in a Ca²⁺ dependent manner, 3) influence mechanical properties of remote uninjured myocardium and 4) alter passive mechanical properties of infarct regions. The cardiomyocytes formed small grafts of ultrastructurally maturing myocardium that enhanced fractional shortening compared to non-treated infarcted hearts. Chemically demembranated tissue strips of cardiomyocyte grafts produced force when activated by Ca²⁺, whereas scar tissue did not. Furthermore, the Ca²⁺ sensitivity of force was greater in cardiomyocyte grafts compared to control myocardium. Surprisingly, cardiomyocytes grafts isolated in the infarct zone increased Ca²⁺ sensitivity of remote uninjured myocardium to levels greater than either remote myocardium from non-treated infarcted hearts or sham-operated controls. Enhanced calcium sensitivity was associated with decreased phosphorylation of cTnT, tropomyosin and MLC2, but not changes in myosin or troponin isoforms. Passive compliance of grafts resembled normal myocardium, while infarct tissue distant from grafts had compliance typical of scar. Thus, cardiomyocyte grafts are contractile, improve local tissue compliance and enhance calcium sensitivity of remote myocardium. Because the volume of remote myocardium greatly exceeds that of the grafts, this enhanced calcium sensitivity may be a major contributor to global improvements in ventricular function after cell transplantation.     

Chymase inhibition prevents cardiac fibrosis and dysfunction after myocardial infarction in rats.

Human chymase activates not only angiotensin II but also transforming growth factor-beta, a major stimulator of myocardial fibrosis, while rat chymase activates transforming growth factor-beta, but not angiotensin II. To clarify the role of chymase-dependent transforming growth factor-beta activation, we evaluated whether chymase inhibition prevents cardiac fibrosis and cardiac dysfunction after myocardial infarction in rats. Myocardial infarction was induced by ligation of the left anterior descending coronary artery. One day after the ligation, rats were randomized into 2 groups: 1) a chymase-treated group that received 10 mg/kg per day of the chymase inhibitor NK3201 orally for 4 weeks; and 2) a vehicle group of non-treated rats with myocardial infarction. We also included a control group who underwent sham-operation and no treatment. Four weeks after ligation, echocardiography revealed that chymase inhibitor treatment reduced the akinetic area and increased fractional area change but did not significantly change left ventricular end-diastolic area. Chymase inhibition significantly reduced left ventricular end-diastolic pressure, increased the maximal end-systolic pressure-volume relationship and decreased the time constant of left ventricular relaxation. Chymase activity in the non-infarcted myocardium was significantly increased in the vehicle group, but it was significantly reduced by chymase inhibitor treatment. The fibrotic area in the cardiac tissues and the mRNA levels of collagen I and collagen III were also significantly lower in the chymase inhibitor-treated group than in the vehicle group. Therefore, the pathway forming chymase-dependent transforming growth factor-beta may play an important role in myocardial fibrosis and cardiac dysfunction rather than left ventricular dilatation after myocardial infarction.     

Frequency dependent force generation correlates with sarcoplasmic calcium ATPase activity in human myocardium

Objective: In congestive heart failure both a decreased function of the sarcoplasmic Ca²⁺-ATPase and a negative force-frequency relationship have been shown. This study aimed to investigate a possible relationship between frequency potentiation, sarcoplasmic Ca²⁺-ATPase activity, and SERCA2 protein expression in human myocardium. Methods: Frequency potentiation was studied in electrically stimulated, isometric, left ventricular papillary muscle strip preparations (37°C, 0.5–3.0 Hz) from terminally failing (NYHA IV; n=5, dilated cardiomyopathy) and nonfailing (donor hearts, n=5) human myocardium. In the identical samples the Ca²⁺-ATPase activity (NADH coupled assay) and the protein expression of sarcoplasmic Ca²⁺-ATPase (SERCA2), phospholamban, and calsequestrin (western blot) were determined. The frequency dependent change in the force of contraction and V_max of the Ca²⁺-ATPase activity and the protein expression of SERCA2 were correlated with each other. Results: In terminally failing myocardium the force-frequency relationship was negative (2.0 Hz vs. 0.5 Hz: –0.2±0.1 ΔmN) contrasting a positive rate dependent potentiation of force in nonfailing tissue (2.0 Hz vs. 0.5 Hz: +0.8±0.2 ΔmN; p<0.01). In failing myocardium the corresponding maximal sarcoplasmic Ca²⁺-ATPase activity (V_max) was reduced significantly compared to nonfailing myocardium (174±24 vs. 296±31 nmol ATP/mg·min, p<0.01). The protein expression of SERCA2, phospholamban, and calsequestrin remained unchanged in failing myocardium. The maximal Ca²⁺-ATPase activity significantly correlated with the frequency dependent change in force of contraction (2 Hz vs. 0.5 Hz: r=0.88, p=0.001; 3 Hz vs. 0.5 Hz: r=0.84, p=0.004). No correlation between protein expression of SERCA2 and Ca²⁺-ATPase activity or change in force of contraction was observed. Conclusion: Due to a significant correlation between sarcoplasmic Ca²⁺-ATPase activity and frequency potentiation, the negative rate dependent force potentiation in human heart failure could be at least in part be attributed to decreased function of the sarcoplasmic Ca²⁺-ATPase. Received: 8 January 1998, Accepted: 2 June 1998     

PCI术后AMI患者非梗死区冠脉血流储备的变化及其对左室功能的影响

目的 观察和分析急性心肌梗死（AMI）患者PCI术后非梗死区冠脉血流储备（CFR）的变化及其对左室功能的影响。方法 22名AMI患者PCI术后1周行二维超声心动图和多巴酚丁胺负荷实时心肌声学造影（MCE）检查,测量左室功能和梗死区、非梗死区CFR,比较非梗死区CFR与梗死区及正常对照组CFR;根据非梗死区CFR值将患者分为两组,比较两组远期左室功能的变化。结果 非梗死区CFR值与正常对照组相比明显下降,非梗死区CFR与左室舒张末期容积呈负相关。结论 AMI后非梗死区心肌同样存在微循环功能障碍,非梗死区CFR值能预测AMI后远期左室功能。     

Regulation of myocardial Na+/H+ exchanger activity

The Na⁺/H⁺ exchanger is a plasma membrane protein, present in the myocardium, which removes intracellular protons and exchanges them with extracellular Na⁺. The protein comprises an N-terminal, hydrophobic, integral membrane domain that transports the ions and a C-terminal, hydrophilic region that regulates the N-terminal domain. The C-terminal domain has several sub-domains, including one region that binds calmodulin and another that is phosphorylated by protein kinases. The Na⁺/H⁺ exchanger is activated by angiotensin, endothelin and α1-adrenergic stimulation. These effectors increase phosphorylation of the C-terminal domain by protein kinases, and G proteins have been implicated in this, but their role remains to be defined. It has recently been shown that ischemia and other stimuli lead to an increased expression of the Na⁺/H⁺ exchanger in the myocardium. The role of this increased expression in the pathology of ischemia and reperfusion-mediated myocardial damage has yet to be determined. Recent evidence suggests that the Na⁺/H⁺ exchanger may play a key role in hypertrophy of the myocardium, and that its activation through G protein-coupled receptors may be important in mediating its effects. Received: 23 April 2001 / Accepted: 14 May 2001