Myocardial protection from ischemic preconditioning is not blocked by sub-chronic inhibition of carnitine palmitoyltransferase I

Life Sciences 77 (2005) 2004 – 2017 www.elsevier.com/locate/lifescie

Myocardial protection from ischemic preconditioning is not blocked by sub-chronic inhibition of carnitine palmitoyltransferase I Claudia Penna, Daniele Mancardi, Donatella Gattullo, Pasquale PagliaroT Dipartimento di Scienze Cliniche e Biologiche dell’Universita` degli Studi di Torino, Orbassano (TO), Italy Received 19 November 2004; accepted 21 March 2005

Abstract Ischemic preconditioning (IP) triggers cardioprotection via a signaling pathway that converges on mitochondria. The effects of the inhibition of carnitine palmitoyltransferase I (CPT-I), a key enzyme for transport of long chain fatty acids (LCFA) into the mitochondria, on ischemia/reperfusion (I/R) injury are unknown. Here we investigated, in isolated perfused rat hearts, whether sub-chronic CPT-I inhibition (5 days i.p. injection of 25 mg/kg/day of Etomoxir) affects I/R-induced damages and whether cardioprotection by IP can be induced after this inhibition. Effects of global ischemia (30 min) and reperfusion (120 min) were examined in hearts harvested from Control (untreated), Vehicle- or Etomoxir-treated animals. In subsets of hearts from the three treated groups, IP was induced by three cycles of 3 min ischemia followed by 10 min reperfusion prior to I/R. The extent of I/R injury under each condition was assessed by changes in infarct size as well as in myocardial contractility. Postischemic contractility, as indexed by developed pressure and dP/ dt max, was similarly affected by I/R, and was similarly improved with IP in Control, Vehicle or Etomoxir treated animals. Infarct size was also similar in the three subsets without IP, and was significantly reduced by IP regardless of CPT-I inhibition. We conclude that CPT-I inhibition does not affect I/R damages. Our data also show that IP affords myocardial protection in CPT-I inhibited hearts to a degree similar to untreated

T Corresponding author. Dipartimento di Scienze Cliniche e Biologiche Universita` di Torino, Ospedale S. Luigi, Regione Gonzole, 10043 ORBASSANO (TO), Italy. Tel.: +39 11 6705430/7710; fax: +39 11 9038639. E-mail address: [email protected] (P. Pagliaro). 0024-3205/$ - see front matter D 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.lfs.2005.03.017

C. Penna et al. / Life Sciences 77 (2005) 2004–2017

2005

animals, suggesting that a long-term treatment with the metabolic anti-ischemic agent Etomoxir does not impede the possibility to afford cardioprotection by ischemic preconditioning. D 2005 Elsevier Inc. All rights reserved. Keywords: Ischemia/reperfusion; Preconditioning; Myocardial necrosis; Mitochondria; Carnitine palmitoyltransferase

Introduction Long chain fatty acids (LCFA) are the major oxidation fuel of the healthy heart in vivo and in vitro, while carbohydrates, especially lactate and glucose, provide the remaining energy source (Neely et al., 1969; Morgan et al., 1984; Jeffrey et al., 1995). However, in several pathological conditions cardiac glucose uptake is higher and fatty acid oxidation is likely to be reduced (Olson, 1959; Wikstrom et al., 1997; Recchia et al., 1998; Osorio et al., 2002). Numerous studies have aimed to establish whether the oxidation of a given type of substrate vs. another energy source results in a more or less advantageous environment for cardiac energetics. The prevailing idea is that the preferential oxidation of glucose improves cardiac efficiency of the ischemic heart (Simonsen and Kjekshus, 1978; Burkhoff et al., 1991, 1995; Korvald et al., 2000) and therefore glucose oxidation is considered beneficial during myocardial hypoperfusion (Pepine and Wolff, 1999; Taniguchi et al., 2001). In fact, compared with fat oxidation, carbohydrate utilization increases the ratio between adenosine triphosphate (ATP) synthesis and consumed oxygen (Starnes et al., 1985). We recently demonstrated that inhibition of LCFA oxidation by inhibition of carnitine palmitoyltransferase I (CPT-I), a key enzyme for transport of LCFA into the mitochondria, impedes an adequate contractile response of the isolated heart to increased pre-load or flow, whereas the inotropic response to adrenergic h-receptor stimulation is insensitive to changes in substrate availability (Pagliaro et al., 2002). The above studies suggest that inhibition of LCFA oxidation may improve cardiac efficiency of ischemic heart, but may endanger contractile function of normal perfused hearts. Ischemic preconditioning (IP), which is obtained by brief episodes of coronary occlusion (few minutes), is well known to induce myocardial protection against the injury caused by sustained ischemia followed by reperfusion (Murry et al., 1986). Among other effects, IP induces a sort of bmetabolic hibernationQ characterized by a reduction in the ATP pool combined with a reduction in ATP hydrolytic rate during subsequent ischemic stress (Murry et al., 1990; Reimer, 1996; Jennings et al., 2001; Schulz et al., 2001). Mitochondrial F0F1 ATPsynthase may play an important role on this respect (Das and Harris, 1990; Vander Heide et al., 1996; Vuorinen et al., 1995; Green et al., 1998; Bosetti et al., 2000; Penna et al., 2004). Importantly, mitochondrial K+ATP channel activation plays a pivotal role in the pathway leading to myocardial protection by IP (Yellon and Downey, 2003; O’Rourke, 2004). Several reviews have been recently published on the critical role played by mitochondria in cardioprotection (Yellon and Downey, 2003; Marin-Garcia and Goldenthal, 2004; Murphy, 2004; O’Rourke, 2004). All the above studies strongly suggest that mitochondrial function is crucial to obtain protection by IP. In particular, ischemia is hypothesized to promote preconditioning, via mitochondria uncoupling; thus metabolic anti-ischemic agents may act against the IP-inducing cardioprotection (Opie, 2003).

2006

C. Penna et al. / Life Sciences 77 (2005) 2004–2017

Therefore, it can be argued that using an inhibitor of fatty acid oxidation, the mitochondrial and contractile function may be altered in a way that precludes the possibility to protect the heart by ischemic preconditioning. As a matter of fact, myocardium of patients with poor contractile function, in which fatty acid oxidation is likely to be reduced (Olson, 1959; Wikstrom et al., 1997; Recchia et al., 1998; Osorio et al., 2002), cannot be protected by IP (Ghosh et al., 2001; Galinanes and Fowler, 2004). However, conflicting conclusions exist regarding the possibility to protect the hearts with preconditioning ischemia, after inhibiting long chain 3-ketoacyl CoA thiolase, a key enzyme of fatty acid betaoxidation (Minners et al., 2000; Kara et al., 2004). Nothing is known about mitochondrial CPT-I and preconditioning. To verify whether CPT-I inhibition may preclude the possibility to precondition the heart with ischemia, we treated rats with Etomoxir, an oxirane carboxylic acid derivative that specifically inactivates CPT-I, thus avoiding degradation of endogenous pool triglycerides and LCFA oxidation (Schmidt-Schweda and Holubarsch, 2000; Zarain-Herzberg and Rupp, 2002). Hearts harvested from rats treated with Etomoxir or with the vehicle used to dissolve the drug were exposed to ischemia/reperfusion with and without IP. Contractile function and infarct size observed in these hearts were compared to those obtained in hearts harvested from untreated animals.

Methods Animals Male Wistar rats (n = 42; body weight 450–500 g; 4–5 months old) were housed in identical cages and were allowed access to tap water and a standard rodent diet ad libitum. The animals received humane care in compliance with the Guide for the Care and Use of Laboratory Animals by the U.S. National Research Council and in accordance with Italian law (DL-116, Jan. 27, 1992). The animals were randomly assigned to one of the treatment groups described below (n = 14 in each group). Animals of the Group 1 (Control Group) were housed in the cages for 15 days without any treatment before to be used for the experiment. Group 2 animals (Vehicle Group) were housed in the cages for 10 days without any treatment, then were treated daily, for 5 days, with an intra-peritoneal injection (i.p.) of 2 ml saline (37 8C), before to be used for the experiment. Animals of Group 3 (Etomoxir Group), were housed together to Vehicle Group animals, and received for 5 days 25 mg/kg/day of Etomoxir {(R)-(+)-ethyl 2(6-(4-chlorophenoxy)hexyl)-oxirane-2-carboxylate}, dissolved in 2 ml of warm (37 8C) saline, via i.p. injection (Svedberg et al., 1991). Isolated heart perfusion After 15 days of housing in the animal facilities (i.e. the last treatment day for animals of Groups 2 and 3), each animal was treated with heparin (2500 U, i.m.). Then, 10 min after, animals were anesthetized with urethane (1 g/kg, i.p.), the chest was opened, the heart was rapidly excised, and placed in ice-cold buffer solution and weighed. Isolated rat hearts were attached to the perfusion apparatus and retrogradely perfused with oxygenated Krebs–Henseleit buffer (127 mM NaCl, 17.7

C. Penna et al. / Life Sciences 77 (2005) 2004–2017

2007

mM NaHCO3, 5.1 mM KCl, 1.5 mM CaCl2, 1.26 mM MgCl2, 11 mM d-glucose and gassed with 95% O2 and 5% CO2). The time required between excision and restoration of perfusion was less than 30 s. The hearts were instrumented as previously described and pump-perfused at constant flow (Paolocci et al., 2000; Rastaldo et al., 2001). The constant flow was maintained to obtain a typical coronary perfusion pressure of 85 mm Hg. A small hole in the left ventricular wall allowed drainage of the thebesian flow, and a polyvinyl-chloride balloon was placed into the left ventricle and connected to an electromanometer for recording of left ventricular pressure (LVP). The hearts were electrically paced at 280–300 bpm and kept in a temperature-controlled chamber (37 8C). Coronary perfusion pressure (CPP) and coronary flow (CF) were monitored with a second electromanometer and an electromagnetic flow-probe, respectively, both placed along the perfusion line. Coronary flow, CPP, and LVP were recorded and analyzed using a Lab-View software (National Instruments, USA), which also allowed quantification of the maximum rate of increase of LVP during systole (dP/dt max). Experimental protocols Each heart was allowed to stabilize for 30 min at which time baseline parameters were recorded. Typically, CF (9F 2 ml/min/g wet weight) was adjusted to obtain the desired CPP (85 mm Hg) within the first 10 min and kept constant thereon. During this stabilization period the ventricular volume (VV) was adjusted, and thereon kept constant, to obtain an end diastolic left ventricular pressure of 5 mm Hg (Pagliaro et al., 2003). After this stabilization period, hearts of each Group were randomly assigned to the treatment subset described below. Seven hearts of each Group (subsets 1A, 2A and 3A; total n =21) were perfused with buffer for an additional 29 min after stabilization, while the other seven hearts of each Group (subsets 1B, 2B and 3B; total n = 21) underwent IP protocol, which consisted of three cycles of 3 min global ischemia followed by 5 min of reperfusion and a final 10 min buffer washout period. All hearts were then subjected to 30 min of global, normothermic, no-flow ischemia followed by 120 min of reperfusion (I/R). Pacing was discontinued at the beginning of the ischemic period and restarted after the third minute of reperfusion. Experimental compounds Etomoxir was a generous gift of Medigene (Germany). All other chemicals were purchased from Sigma (St. Louis, MO, USA). Assessment of ventricular function Changes in left ventricular end-diastolic pressure (LVEDP), developed LVP, and dP/dt max values induced by the I/R protocol were continuously monitored. The difference between LVEDP before the end-reperfusion (mean value of the last minute of reperfusion) and during pre-ischemic conditions (mean value of the last minute of washout/buffer only) was used as an index of the extent of contracture development. Maximal recovery of developed LVP and dP/dt max during reperfusion was also compared with respective pre-ischemic values.

2008

C. Penna et al. / Life Sciences 77 (2005) 2004–2017

Assessment of myocardial injury Infarct areas were assessed in a blinded fashion at the end of the experiment. Directly after reperfusion, each heart was rapidly removed from the perfusion apparatus, and the left ventricle (LV) was dissected into 2–3 mm circumferential slices. Following 15 min of incubation at 37 8C in 0.1% solution of nitro blue tetrazolium in phosphate buffer (Ma et al., 1999; Pagliaro et al., 2003), unstained necrotic tissue was carefully separated from stained viable tissue by an independent observer who was not aware of the nature of the intervention. The weights of the necrotic and non-necrotic tissues were then determined, and the necrotic mass was expressed as percentage of the total left ventricular mass. In fact, though in this model the whole heart underwent ischemia, only the LV had a fixed volume and preload; therefore only the LV mass was considered as risk area. Statistical analysis All values are presented as meansFS.E.M. To compare the cardiac responses of different groups we also report data as percent changes from baseline values. Two sets of comparisons were made for each of the considered hemodynamic variables: one between baseline data and the other between maximal effect in response to I/R. Comparisons were performed using two-way analysis of variance (ANOVA) for repeated measures (factors: group and condition). One-way ANOVA was used to compare between subsets the cardiac weight, LV weight and the cardiac to body weight ratio. Post hoc comparison between groups at an individual time point was performed using Student’s t-test for unpaired data. Contrasts between conditions in the same subset were analyzed using Student’s t-test for paired data. Student’s t-test was performed with Bonferroni correction. Significance was accepted at a P level of b 0.05.

Results Cardiac weight (1.33 F0.01 g; n =42), LV weight (0.89 F0.006 g; n = 42) and the cardiac to body weight ratio (0.003F0.00003; n = 42) were equivalent among the three treatment Groups. In particular, in Group 3 hearts (Etomoxir Group; n = 14) cardiac weight, LV weight and the cardiac weight to body weight ratio were slightly, but not significantly, higher than other groups, being 1.40F0.01 g, 0.92F0.009 g, and 0.0034F0.0008, respectively. Hearts of the three Groups (Control, n = 14; Vehicle, n = 14; and Etomoxir, n =14) were randomly assigned to subsets that underwent I/R only (subsets 1A, 2A, and 3A, n = 7 for each subset) or IP prior to I/R (subsets 1B, 2B, and 3B, n = 7 for each subset). Cardiac weight, LV weight and the cardiac to body weight ratio were also equivalent among the six subsets (Table 1). Hearts subjected to 30 min ischemia and 120 min reperfusion only (subsets 1A, 2A and 3A) 1. Preischemic function. Baseline cardiac function after stabilization and prior to ischemia observed in these three subsets of hearts are reported in Table 1. Hearts treated with Etomoxir (subset 3A) showed significantly lower levels of developed LVP and dP/dt max in comparison to 1A and 2A subsets.

C. Penna et al. / Life Sciences 77 (2005) 2004–2017

2009

Table 1 Baseline hemodynamic and morphological parameters Non-preconditioned

Ischemic preconditioned Control (Subset 1 A)

Vehicle (Subset 2 A)

Etomoxir (Subset 3 A)

Control (Subset 1 B)

Vehicle (Subset 2 B)

Etomoxir (Subset 3 B)

End diastolic LVP 5F1 4F2 6F1 6F1 3 F1 4F1 (mm Hg) Developed LVP 111 F13 103F 10 86 F 6T 91 F 6 95 F 5 82 F 2T (mm Hg) dP/dt max (mm Hg/s) 2810F 196 2724 F180 2195F 210T 2649F 108 2703F 126 2021F 265T CPP (mm Hg) 85 F 5 86 F5 88 F 3 87 F 4 85 F 4 83 F 5 Cardiac weight (g) 1.32 F0.02 1.29F 0.05 1.41 F 0.05 1.40F 0.04 1.31 F 0.06 1.29 F0.01 LV weight (g) 0.91 F0.02 0.86F 0.03 0.92 F 0.04 0.92F 0.04 0.89 F 0.02 0.87 F0.02 C/B weight ratio 0.0030 F0.0001 0.0029 F 0.0002 0.0034 F 0.0004 0.0034F 0.0004 0.0029 F 0.0001 0.0029F 0.0001 LVP= left ventricular pressure; dP/dt max = recovery of maximum rate of increase in LVP during systole; CPP= coronary perfusion pressure; C/B =cardiac/body. T P b0.05 vs. Control or Vehicle.

2. Postischemic function. Cardiac function data observed at the end of reperfusion in these three subsets of hearts are reported in Table 2. Contracture development during reperfusion LVEDP increased during ischemia/reperfusion in a similar fashion among these three subsets (Table 2). Postischemic contractile function Maximal percent recovery of developed LVP and dP/dt max during reperfusion compared to preischemic values is reported in Fig. 1A and B. Left ventricle contractile function, indexed by developed LVP and dP/dt max, was found to be similarly impaired in these three subsets at reperfusion. In fact, percent recovery of LVP and dP/dt max from myocardial stunning was similar in Control (1A) Vehicle (2A) and Etomoxir-treated (3A) hearts (Fig. 1).

Table 2 End-reperfusion hemodynamic parameters Non-preconditioned

End diastolic LVP (mm Hg) Developed LVP (mm Hg) dP/dtmax (mm Hg/s) CPP (mm Hg)

Ischemic preconditioned Control (Subset 1 A)

Vehicle (Subset 2 A)

Etomoxir (Subset 3 A)

Control (Subset 1 B)

Vehicle (Subset 2 B)

Etomoxir (Subset 3 B)

50 F 10 40 F 11 1205F 117 146F10

48 F 12 40 F 10 1105F 242 144F 10

51 F 7 35 F 8 748F 152 138F 6

31 F 7T 57 F 8T 1352F 98T 140F 8

33 F 10T 58 F 5T 1325F 106T 138F 8

31 F 6T 55 F 5T 1225F 95T 136F 10

LVP= left ventricular pressure; dP/dt max = recovery of maximum rate of increase in LVP during systole; CPP= coronary perfusion pressure. T P b0.05 vs. Non-Preconditioned.

C. Penna et al. / Life Sciences 77 (2005) 2004–2017

A % recovery during reperfusion

#

#

#

#

Vehicle+IP (2B)

Developed LVP 80

Control+IP (1B)

2010

#

60

40

20

0

dP/dtmax

B

#

60

40

Non-Preconditioned

Etomoxir +IP (3B)

Etomoxir (3A)

0

Vehicle (2A)

20

Control (1A)

% recovery during reperfusion

80

Preconditioned

Fig. 1. Postischemic heart contractile function. (A) Recovery of developed left ventricular pressure (LVP) and (B) recovery of maximum rate of increase in LVP during systole (dP/dt max) following 30 min global, no-flow ischemia and 120 min reperfusion. Results are presented as mean F S.E.M. #P b 0.05 vs. non-preconditioned.

Myocardial injury after ischemia/reperfusion Total infarct area (Fig. 2), expressed as a percentage of left ventricular mass, was 65F6%, 61F6%, and 54F7% in the Control (1A), Vehicle (2A) and Etomoxir-treated (3A) hearts ( P = NS among these three non-preconditioned subsets). Hearts subjected to 30 min ischemia and 120 reperfusion after IP (subsets 1B, 2B and 3B) and comparisons with subsets without IP (subsets 1A, 2A and 3A) 1. Preischemic function. Table 1 also displays baseline cardiac function after stabilization and prior to ischemia observed in the three preconditioned subsets of hearts (1B, 2B and 3B). In particular, hearts treated with Etomoxir (subset 3B) showed slight, but significantly lower levels of developed LVP and

C. Penna et al. / Life Sciences 77 (2005) 2004–2017

2011

Infarct Size

100

# 40

#

#

Etomoxir+IP(3B)

60

Vehicle+IP (2B)

% of LV

80

Non-Preconditioned

Control+IP (1B)

Etomoxir (3A)

Vehicle (2A)

0

Control (1A)

20

Preconditioned

Fig. 2. Infarct size (percentage of left ventricle (LV)) following 30 min of global no-flow ischemia and 120 min reperfusion. Results are presented as mean F S.E.M. #P b0.05 vs. non-preconditioned.

dP/dt max in comparison to 1B and 2B subsets. There were no differences in baseline function between hearts of subsets A and B of each Group. 2. Postischemic function. Table 2 also displays cardiac function data at the end of reperfusion observed in the three preconditioned subsets (1B, 2B and 3B). Contracture development during reperfusion LVEDP increased during ischemia/reperfusion in a similar fashion among the three subsets that underwent IP (subsets 1B, 2B and 3B). However, the levels of LVEDP reached in these three subsets are lower than those reached in non-preconditioned hearts (subsets 1A vs. 1B, 2A vs. 2B and 3A vs. 3B; P b 0.05 for all). Postischemic contractile function Maximal percent recovery of developed LVP and dP/dt max during reperfusion compared to preischemic values is reported in Fig. 1A and B. Left ventricle contractile function at reperfusion, indexed by developed LVP and dP/dt max, was found to be similarly impaired in the three subsets that underwent IP. In fact, percent recovery of LVP and dP/dt max from myocardial stunning was similar in Control (1B), Vehicle (2B) and Etomoxirtreated (3B) hearts (Fig. 1). However, the levels of LVP and dP/dt max reached in these three subsets are higher than those reached in non-preconditioned hearts (subsets 1A vs. 1B, 2A vs. 2B and 3A vs. 3B; P b 0.05 for all). Myocardial injury after ischemia/reperfusion In the subsets of hearts that underwent IP, total infarct area (Fig. 2), expressed as a percentage of left ventricular mass, was 36F 7%, 35 F6%, and 33F6% in the Control (1B), Vehicle (2B) and Etomoxirtreated (3B) hearts, respectively ( P = NS among these three preconditioned subsets). However, the infarct

2012

C. Penna et al. / Life Sciences 77 (2005) 2004–2017

size achieved in each of these three preconditioned subsets was significantly smaller than that reached in non-preconditioned hearts (subsets 1A vs. 1B, 2A vs. 2B and 3A vs. 3B; P b 0.05 for all). During reperfusion CPP increased similarly in all subsets. This meant that, in this model of constant flow perfused heart, the vascular resistance was similarly increased in all hearts after the 30 min ischemia. Finally, it is noteworthy that IP-induced myocardial savage was neither a function of CPP at reperfusion nor a function of pre-ischemic levels of LVP.

Discussion This study shows, for the first time, that early IP affords myocardial protection regardless of subchronic (5-days) CPT-I inhibition. In our experiments, we blocked CPT-I, with 5-day treatment with Etomoxir, to avoid degradation of endogenous pool triglycerides (Schmitz et al., 1995; Zarain-Herzberg and Rupp, 2002; Mengi and Dhalla, 2004). The hearts isolated from rats treated with Etomoxir developed a slightly lower LV pressure during baseline conditions in comparison with control hearts or hearts treated with the vehicle only. Even so, the infarct size in the hearts of Etomoxir treated animals was not significantly smaller than controls. Moreover, when preconditioned by three cycles of brief periods of ischemia the infarct size and percent recovery of heart function of Etomoxir-treated animals were similar to that of vehicle- and control-preconditioned subsets. Our findings suggest that in hearts in which CPT-I is chronically blocked by Etomoxir the cardiac function may be worsened when the heart is supplied with glucose as only substrate. This experimental condition (reduced fatty acid oxidation and poor contractile function) may resemble some, but not all, features of the heart failure condition in which it is known that cardiac glucose uptake is higher and fatty acid oxidation is likely to be reduced (Olson, 1959; Wikstrom et al., 1997; Recchia et al., 1998; Osorio et al., 2002). We previously showed that in the isolated rat heart, the Frank-Starling mechanism and Gregg effect are sensitive to the type of metabolic substrate used after acute CPT-I inhibition. Fatty acid oxidation was necessary for an adequate contractile response of the isolated heart to myocardial stretch by increasing VV (Frank-Starling mechanism) or CF (Gregg effect), but not to h-receptor stimulation (Pagliaro et al., 2002). In these experiments of acute CPT-inhibition, however, the basal function of the heart was not altered. We do not know why after 5 days of CPT-I inhibition by Etomoxir the isolated hearts showed a lower basal contractility. This was for us a surprising new finding. On the basis of our previous observation on CPT-I inhibition and Gregg effect (Pagliaro et al., 2002), we can speculate that this impaired function is due to the higher CF achieved in vitro preparation when compared to the in vivo condition. To clarify this point was beyond the aims of the present study. Nevertheless, the fact that infarct size and the extension of cardiodepression in non-preconditioned hearts, as well as the extension of myocardial protection in preconditioned hearts, were not a function of pre-ischemic levels of contractile parameters (LVP and dP/dt max), suggests that the protection of the hearts by IP is not dependent on reduced oxygen demand by myocardial depression. Our data indicate that, though LCFA cannot enter mitochondria, IP is still able to protect the heart. Whether or not protection by IP in Etomoxir treated heart requires mitochondrial K+ATP channel opening will be ascertained in future studies. The opening of these channels seems a compulsory step of cardioprotection by preconditioning ischemia (Ghosh et al., 2001; Yellon and Downey, 2003; Galinanes

C. Penna et al. / Life Sciences 77 (2005) 2004–2017

2013

and Fowler, 2004; O’Rourke, 2004); therefore there are no reasons to postulate that, after Etomoxir, IP does not require mitochondrial K+ATP channel opening. Trimetazidine another metabolic anti-ischemic agent, which is widely used clinically, acts by inhibiting long chain 3-ketoacyl CoA thiolase, a key enzyme of fatty acid beta-oxidation. Very recently, it has been reported that Trimetazidine preserves the effects of ischemic preconditioning and pharmacological preconditioning, and is able to mimic IP in anesthetized rats (Kara et al., 2004). Moreover, in isolated rat hearts, it has been reported that Trimetazidine significantly reduced protection afforded by both adenosine- and ischemia-induced preconditioning (Minners et al., 2000). It is known that protection by adenosine can bypass mitochondrial K+ATP channel opening (Yellon and Downey, 2003). It is not clear, however, why, differently from Etomoxir and differently from what seen in the anaesthetized rat, Trimetazidine abolished IP-induced protection in isolated hearts in the study of Minners et al. (2000). Besides the different mechanisms of action of the drugs, difference may be also due to the fact that Trimetazidine was used acutely (Minners et al., 2000; Kara et al., 2004), while in our study Etomoxir was used in a long-term fashion. Nevertheless, the precise mechanism for the interaction between this drug and various preconditioning stimuli is unknown. Trimetazidine is also a weak CPT-I inhibitor, thus a role of CPT-I may be hypothesized. However, the current findings, obtained with a more specific CPT-I inhibitor, provide evidence against a role of CPT-I to modulate cardioprotection. Lopaschuk et al. previously demonstrated that the effects of Etomoxir were dose-dependent. Low doses of Etomoxir decreased long chain acylcarnitine and long chain acyl-coenzyme A levels but did not prevent depressed function in reperfused ischemic hearts, whereas high doses prevented ischemiainduced dysfunction. These investigators further demonstrated that Etomoxir improves ventricular function in reperfused ischemic hearts in the presence of 1.2 mM palmitate by increasing glucose oxidation (Lopaschuk et al., 1988, 1989). Thus Etomoxir may improve recovery of function by overcoming fatty acid inhibition of glucose oxidation. A protective role of increased glucose uptake in the preconditioning paradigm has been previously suggested (Opie and Sack, 2002). Critique of the model and methodological considerations We chose to treat the animals for 5 days (including the day of heart isolation) with a relatively high dose of Etomoxir to be sure that the drug achieved the blockage of CPT-I in a similar fashion of what can happen in clinical situations (Hayashi et al., 2001). The fact that the hearts of Etomoxir-treated animals showed reduced basal contractility supports the effectiveness of the used dose. By reducing the number of independent variables to a reasonable minimum, the isolated crystalloidperfused heart model appears suitable for studying the effects of ischemia/reperfusion on cardiac performance and infarct size (Morgan et al., 1984; Starnes et al., 1985; Lopaschuk et al., 1988; Jeffrey et al., 1995; Minners et al., 2000; Pagliaro et al., 2003). The non-use of perfusate containing LCFA, lactate and/or pyruvate as additional substrates may be a limitation of the protocol. However, historically, many studies on ischemia/reperfusion in isolated hearts have been performed by supplying glucose as the sole exogenous substrate (e.g., Morgan et al., 1984; Starnes et al., 1985; Jeffrey et al., 1995; Minners et al., 2000; Pagliaro et al., 2003). Moreover, during initial transition from normal perfusion to no-flow condition, isolated hearts can still derive part of their energy requirements from endogenous reserves of LCFA (Starnes et al., 1985). For these reasons and to keep the number of variables to a reasonable minimum we did not add other substrates

2014

C. Penna et al. / Life Sciences 77 (2005) 2004–2017

and/or insulin to the perfusate. Notably, insulin per se has multiple anti-ischemic mechanisms (Opie, 2003). LCFA are known to increase the severity of injury during acute myocardial ischemia (Lopaschuk et al., 1988). Finally, our study is limited by the absence of measurements of cardiac metabolites and high-energy phosphates. The classical method to determine the rate of substrate utilization is based on isotopic tracer infusion. Although we did not use isotopic tracking of metabolic fate of substrates, cardiac metabolism of endogenous and exogenous carbohydrates and fatty acids in isolated hearts, as well as Etomoxir effects on CPT-I, have been widely investigated and are already well defined (Schmitz et al., 1995; Schmidt-Schweda and Holubarsch, 2000; Zarain-Herzberg and Rupp, 2002; Mengi and Dhalla, 2004). Moreover, in the present study, in the absence of any exogenous LCFA supply during heart isolation, the effects of Etomoxir on substrate metabolism were likely caused by inhibition of endogenous LCFA oxidation. As isotopic tracers cannot allow measurements of the rate of oxidation of endogenous substrate, they would have not been useful in our study unless we had adopted challenging protocols of endogenous pool priming with labeled substrate. This was beyond our aims. Several in vivo studies (Schmitz et al., 1995; Schmidt-Schweda and Holubarsch, 2000; ZarainHerzberg and Rupp, 2002; Opie, 2003) reported an improvement of the function of bsickQ hearts during Etomoxir treatment. This improvement has been attributed to bselective changes in dysregulated gene expression of hypertrophied cardiomyocytesQ (Zarain-Herzberg and Rupp, 2002). The potential effects of Etomoxir on gene transcription are possible also in our study considering the 5-day treatment regimen. It is known that Etomoxir activates peroxisome prolifator-activated receptor (PPAR) (Muoio et al., 2002). It is also reported that treatment with Etomoxir initially increases the expression of acyl-CoA oxidase and cytosolic acyl-CoA thioesterase (Cabrero et al., 1999; Djouadi et al., 1999), while prolonged (5–10 days) treatment increases the expression of medium-chain acyl-CoA dehydrogenase, glutathione peroxidase, and NF-nB (Cabrero et al., 1999; Djouadi et al., 1999). Although we hypothesize that basal cardiodepression by Etomoxir may be a feature of in vitro conditions (see above), we cannot exclude that PPAR, NF-nB and overexpressed enzymes participate to the worsening of basal function and modulation of ischemic injury. Nevertheless, the increased expression of genes occurs in response to the metabolic stress imposed by the inhibition of CPT-I (Cabrero et al., 1999), which, however, does not block myocardial protection by IP. Of course, specifically designed studies are required to ascertain 1) whether improvement of function of bsickQ heart can also be obtained in vitro, and 2) whether CPT-I inhibition also in bsickQ heart does not affect the possibility to obtain cardioprotection by preconditioning. In conclusion, we show that the administration of the CPT-I blocker Etomoxir for 5 days does not affect I/R damages in isolated rat hearts perfused with glucose only. Our data also show that IP affords myocardial protection in CPT-I inhibited hearts to a degree similar to untreated animals, suggesting that inhibition of CPT-I does not impede the possibility to afford cardioprotection by ischemic preconditioning.

Acknowledgments This work was supported by Compagnia di S. Paolo, MURST (ex 40%), University of Turin (ex 60%) (Dr Gattullo, Dr Pagliaro) and dIstituto di Ricerche CardiovascolariT (Dr Penna). The authors thank Medigene (Germany) for the supply of Etomoxir.

C. Penna et al. / Life Sciences 77 (2005) 2004–2017

2015

References Bosetti, F., Yu, G., Zucchi, R., Ronca-Testoni, S., Solaini, G., 2000. Myocardial ischemic preconditioning and mitochondrial F1F0-ATPase activity. Molecular and Cellular Biochemistry 215 (1–2), 31 – 37. Burkhoff, D., Weiss, R.G., Schulman, S.P., Kalil-Filho, R., Wannenburg, T., Gerstenblith, G., 1991. Influence of metabolic substrate on rat heart function and metabolism at different coronary flows. American Journal of Physiology 261 (3 Pt 2), H741 – H750. Burkhoff, D., Stennett, R.A., Ogino, K., 1995. Mechanisms of the Frank-Starling phenomena studied in intact hearts. Advances in Experimental Medicine and Biology 382, 155 – 161. Cabrero, A., Alegret, M., Sanchez, R., Adzet, T., Laguna, J.C., Vazquez, M., 1999. Etomoxir, sodium 2-[6-(4chlorophenoxy)hexyl]oxirane-2-carboxylate,up-regulates uncoupling protein-3 mRNA levels in primary culture of rat preadipocytes. Biochemical and Biophysical Research Communications 263 (1), 87 – 93. Das, A.M., Harris, D.A., 1990. Regulation of the mitochondrial ATP synthase in intact rat cardiomyocytes. Biochemical Journal 266 (2), 355 – 361. Djouadi, F., Brandt, J.M., Weinheimer, C.J., Leone, T.C., Gonzalez, F.J., Kelly, D.P., 1999. The role of the peroxisome proliferator-activated receptor alpha (PPAR alpha) in the control of cardiac lipid metabolism. Prostaglandins, Leukotrienes and Essential Fatty Acids 60 (5–6), 339 – 343. Galinanes, M., Fowler, A.G., 2004. Role of clinical pathologies in myocardial injury following ischaemia and reperfusion. Cardiovascular Research 61 (3), 512 – 521. Ghosh, S., Standen, N.B., Galinianes, M., 2001. Failure to precondition pathological human myocardium. Journal of the American College of Cardiology 37 (3), 711 – 718. Green, D.W., Murray, H.N., Sleph, P.G., Wang, F.L., Baird, A.J., Rogers, W.L., Grover, G.J., 1998. Preconditioning in rat hearts is independent of mitochondrial F1F0 ATPase inhibition. American Journal of Physiology 274 (1 Pt 2), H90 – H97. Hayashi, K., Okumura, K., Matsui, H., Murase, K., Kamiya, H., Saburi, Y., Numaguchi, Y., Toki, Y., Hayakawa, T., 2001. Involvement of 1,2-diacylglycerol in improvement of heart function by Etomoxir in diabetic rats. Life Science 68 (13), 1515 – 1526. Jeffrey, F.M., Diczku, V., Sherry, A.D., Malloy, C.R., 1995. Substrate selection in the isolated working rat heart: effects of reperfusion, afterload, and concentration. Basic Research in Cardiology 90 (5), 388 – 396. Jennings, R.B., Sebbag, L., Schwartz, L.M., Crago, M.S., Reimer, K.A., 2001. Metabolism of preconditioned myocardium: effect of loss and reinstatement of cardioprotection. Journal of Molecular and Cellular Cardiology 33 (9), 1571 – 1588. Kara, A.F., Demiryurek, S., Celik, A., Tarakcioglu, M., Demiryurek, A.T., 2004. Effects of trimetazidine on myocardial preconditioning in anesthetized rats. European Journal of Pharmacology 503 (1–3), 135 – 145. Korvald, C., Elvenes, O.P., Myrmel, T., 2000. Myocardial substrate metabolism influences left ventricular energetics in vivo. American Journal of Physiology. Heart and Circulatory Physiology 278 (4), H1345 – H1351. Lopaschuk, G.D., Wall, S.R., Olley, P.M., Davies, N.J., 1988. Etomoxir, a carnitine palmitoyltransferase I inhibitor, protects hearts from fatty acid-induced ischemic injury independent of changes in long chain acylcarnitine. Circulation Research 63 (6), 1036 – 1043. Lopaschuk, G.D., McNeil, G.F., McVeigh, J.J., 1989. Glucose oxidation is stimulated in reperfused ischemic hearts with the carnitine palmitoyltransferase 1 inhibitor, Etomoxir. Molecular and Cellular Biochemistry 88 (1–2), 175 – 179. Ma, X.L., Gao, F., Liu, G.L., Lopez, B.L., Christopher, T.A., Fukuto, J.M., Wink, D.A., Feelisch, M., 1999. Opposite effects of nitric oxide and nitroxyl on postischemic myocardial injury. Proceedings of the National Academy of Sciences of the United States of America 96 (25), 14617 – 14622. Marin-Garcia, J., Goldenthal, M.J., 2004. Mitochondria play a critical role in cardioprotection. Journal of Cardiac Failure 10 (1), 55 – 66. Mengi, S.A., Dhalla, N.S., 2004. Carnitine palmitoyltransferase-I, a new target for the treatment of heart failure: perspectives on a shift in myocardial metabolism as a therapeutic intervention. American Journal of Cardiovascular Drugs 4 (4), 201 – 209. Minners, J., van den Bos, E.J., Yellon, D.M., Schwalb, H., Opie, L.H., Sack, M.N., 2000. Dinitrophenol, cyclosporin A, and trimetazidine modulate preconditioning in the isolated rat heart: support for a mitochondrial role in cardioprotection. Cardiovascular Research 47 (1), 68 – 73. Morgan, H.E., Neely, J.R., Kira, Y., 1984. Factors determining the utilization of glucose in isolated rat hearts. Basic Research in Cardiology 79 (3), 292 – 299.

2016

C. Penna et al. / Life Sciences 77 (2005) 2004–2017

Muoio, D.M., Way, J.M., Tanner, C.J., Winegar, D.A., Kliewer, S.A., Houmard, J.A., Kraus, W.E., Dohm, G.L., 2002. Peroxisome proliferator-activated receptor-alpha regulates fatty acid utilization in primary human skeletal muscle cells. Diabetes 51 (4), 901 – 909. Murphy, E., 2004. Primary and secondary signaling pathways in early preconditioning that converge on the mitochondria to produce cardioprotection. Circulation Research 94 (1), 7 – 16. Murry, C.E., Jennings, R.B., Reimer, K.A., 1986. Preconditioning with ischemia: a delay of lethal cell injury in ischemic myocardium. Circulation 74 (5), 1124 – 1136. Murry, C.E., Richard, V.J., Reimer, K.A., Jennings, R.B., 1990. Ischemic preconditioning slows energy metabolism and delays ultrastructural damage during a sustained ischemic episode. Circulation Research 66 (4), 913 – 931. Neely, J.R., Bowman, R.H., Morgan, H.E., 1969. Effects of ventricular pressure development and palmitate on glucose transport. American Journal of Physiology 216 (4), 804 – 811. Olson, R.E., 1959. Myocardial metabolism in congestive heart failure. Journal of Chronic Diseases 9 (5), 442 – 464. Opie, L.H., 2003. Preconditioning and metabolic anti-ischaemic agents. European Heart Journal 24 (20), 1854 – 1856. Opie, L.H., Sack, M.N., 2002. Metabolic plasticity and the promotion of cardiac protection in ischemia and ischemic preconditioning. Journal of Molecular and Cellular Cardiology 34 (9), 1077 – 1089. O’Rourke, B., 2004. Evidence for mitochondrial K+ channels and their role in cardioprotection. Circulation Research 94 (4), 420 – 432. Osorio, J.C., Stanley, W.C., Linke, A., Castellari, M., Diep, Q.N., Panchal, A.R., Hintze, T.H., Lopaschuk, G.D., Recchia, F.A., 2002. Impaired myocardial fatty acid oxidation and reduced protein expression of retinoid X receptor-alpha in pacinginduced heart failure. Circulation 106 (5), 606 – 612. Pagliaro, P., Chiribiri, A., Gattullo, D., Penna, C., Rastaldo, R., Recchia, F.A., 2002. Fatty acids are important for the FrankStarling mechanism and Gregg effect but not for catecholamine response in isolated rat hearts. Acta Physiologica Scandinavica 176 (3), 167 – 176. Pagliaro, P., Mancardi, D., Rastaldo, R., Penna, C., Gattullo, D., Miranda, K.M., Feelisch, M., Wink, D.A., Kass, D.A., Paolocci, N., 2003. Nitroxyl affords thiol-sensitive myocardial protective effects akin to early preconditioning. Free Radical Biology & Medicine 34 (1), 33 – 43. Paolocci, N., Ekelund, U.E., Isoda, T., Ozaki, M., Vandegaer, K., Georgakopoulos, D., Harrison, R.W., Kass, D.A., Hare, J.M., 2000. cGMP-independent inotropic effects of nitric oxide and peroxynitrite donors: potential role for nitrosylation. American Journal of Physiology. Heart and Circulatory Physiology 279 (4), H1982 – H1988. Penna, C., Pagliaro, P., Rastaldo, R., Di Pancrazio, F., Lippe, G., Gattullo, D., Mancardi, D., Samaja, M., Losano, G., Mavelli, I., 2004. F0F1 ATP synthase activity is differently modulated by coronary reactive hyperemia before and after ischemic preconditioning in the goat. American Journal of Physiology. Heart and Circulatory Physiology 287 (5), H2192 – H2200. Pepine, C.J., Wolff, A.A., 1999. A controlled trial with a novel anti-ischemic agent, ranolazine, in chronic stable angina pectoris that is responsive to conventional antianginal agents. Ranolazine Study Group. American Journal of Cardiology 84 (1), 46 – 50. Rastaldo, R., Paolocci, N., Chiribiri, A., Penna, C., Gattullo, D., Pagliaro, P., 2001. Cytochrome P-450 metabolite of arachidonic acid mediates bradykinin-induced negative inotropic effect. American Journal of Physiology. Heart and Circulatory Physiology 280 (6), H2823 – H2832. Recchia, F.A., McConnell, P.I., Bernstein, R.D., Vogel, T.R., Xu, X., Hintze, T.H., 1998. Reduced nitric oxide production and altered myocardial metabolism during the decompensation of pacing-induced heart failure in the conscious dog. Circulation Research 83 (10), 969 – 979. Reimer, K.A., 1996. The slowing of ischemic energy demand in preconditioned myocardium. Annals of the New York Academy of Sciences 793, 13 – 26. Schmidt-Schweda, S., Holubarsch, C., 2000. First clinical trial with Etomoxir in patients with chronic congestive heart failure. Clinical Science (London) 99 (1), 27 – 35. Schmitz, F.J., Rosen, P., Reinauer, H., 1995. Improvement of myocardial function and metabolism in diabetic rats by the carnitine palmitoyl transferase inhibitor Etomoxir. Hormone and Metabolic Research 27 (12), 515 – 522. Schulz, R., Cohen, M.V., Behrends, M., Downey, J.M., Heusch, G., 2001. Signal transduction of ischemic preconditioning. Cardiovascular Research 52 (2), 181 – 198. Simonsen, S., Kjekshus, J.K., 1978. The effect of free fatty acids on myocardial oxygen consumption during atrial pacing and catecholamine infusion in man. Circulation 58 (3 Pt 1), 484 – 491.

C. Penna et al. / Life Sciences 77 (2005) 2004–2017

2017

Starnes, J.W., Wilson, D.F, Erecinska, M., 1985. Substrate dependence of metabolic state and coronary flow in perfused rat heart. American Journal of Physiology 249 (4 Pt 2), H799 – H806. Svedberg, J., Bjorntorp, P., Lonnroth, P., Smith, U., 1991. Prevention of inhibitory effect of free fatty acids on insulin binding and action in isolated rat hepatocytes by Etomoxir. Diabetes 40 (6), 783 – 786. Taniguchi, M., Wilson, C., Hunter, C.A., Pehowich, D.J., Clanachan, A.S., Lopaschuk, G.D., 2001. Dichloroacetate improves cardiac efficiency after ischemia independent of changes in mitochondrial proton leak. American Journal of Physiology. Heart and Circulatory Physiology 280 (4), H1762 – H1769. Vander Heide, R.S., Hill, M.L., Reimer, K.A., Jennings, R.B., 1996. Effect of reversible ischemia on the activity of the mitochondrial ATPase: relationship to ischemic preconditioning. Journal of Molecular and Cellular Cardiology 28 (1), 103 – 112. Vuorinen, K., Ylitalo, K., Peuhkurinen, K., Raatikainen, P., Ala-Rami, A., Hassinen, I.E., 1995. Mechanisms of ischemic preconditioning in rat myocardium. Roles of adenosine, cellular energy state, and mitochondrial F1F0-ATPase. Circulation 91 (11), 2810 – 2818. Wikstrom, B.G., Ronquist, G., Waldenstrom, A., 1997. No further improvement of ischaemic myocardial metabolism by combining preconditioning with beta-blockade: an in vivo experimental study in the pig heart using a microdialysis technique. Acta Physiologica Scandinavica 159 (1), 23 – 32. Yellon, D.M., Downey, J.M., 2003. Preconditioning the myocardium: from cellular physiology to clinical cardiology. Physiological Reviews 83 (4), 1113 – 1151. Zarain-Herzberg, A., Rupp, H., 2002. Therapeutic potential of CPT I inhibitors: cardiac gene transcription as a target. Expert Opinion on Investigational Drugs 11 (3), 345 – 356.