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Ann Thorac Surg 1998;66:1600-1603
© 1998 The Society of Thoracic Surgeons

Carnitine supplementation improves myocardial function in hearts from ischemic diabetic and euglycemic rats

^a Department of Surgery, Tulane University Medical Center, New Orleans, Louisiana, USA

Address reprint requests to Dr Toporoff, The University of Arizona Health Sciences Center, PO Box 245071, Tucson, AZ 85724-5071
e-mail: (toporoff{at}u.arizona.edu)

Presented at the Forty-fourth Annual Meeting of the Southern Thoracic Surgical Association, Naples, FL, Nov 6–8, 1997.

	Abstract

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Background. Nonischemic myocardial dysfunction in patients with diabetes mellitus appears to be attenuated with long-term L-carnitine therapy. The effect of acute L-carnitine supplementation on rat hearts from euglycemic and diabetic animals subjected to ischemia and reperfusion is investigated in this study.

Methods. Study rats had diabetes mellitus induced by streptozocin (65 mg/kg intraperitoneally), and control rats had injection of saline solution (n = 12 per group). About 1 month later, the hearts were suspended on a Langendorff apparatus and perfused with either standard buffered Krebs-Henseleit solution or this standard solution supplemented with L-carnitine (5 mmol/L). After stabilization, normothermic, zero-flow ischemia was instituted for 20 minutes followed by 60 minutes of reperfusion. There were four study groups (n = 6 per group): hearts that were from euglycemic rats and that were perfused with standard buffered Krebs-Henseleit solution (E-STD); hearts that were from diabetic animals and that were perfused with the same standard buffered solution (DM-STD); hearts taken from diabetic animals and perfused with L-carnitine–enriched solution (DM-CAR); and hearts taken from euglycemic rats and perfused with the enriched solution (E-CAR).

Results. At 60 minutes of reperfusion, left ventricular developed pressure was significantly better in hearts from both groups (diabetic and euglycemic) with carnitine supplementation (DM-CAR versus DM-STD and E-CAR versus E-STD, p < 0.01 for both, by analysis of variance). Left ventricular end-diastolic pressure was significantly lower in the DM-CAR group compared with all other groups (p < 0.01 by analysis of variance).

Conclusions. These findings suggest that acute L-carnitine supplementation significantly improves the recovery of the ischemic myocardium in diabetic and euglycemic rats.

	Introduction

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Cardiac dysfunction that occurs in patients with diabetes mellitus (DM) is generally considered to be secondary to the increased incidence of coronary artery disease and silent myocardial infarction noted in these patients; however, many diabetics have myocardial dysfunction despite normal coronary arteriograms. Explanations for myocardial dysfunction in the absence of coronary artery disease in patients with DM include microangiopathy and metabolic derangement [1]. Among the most studied metabolic abnormalities in diabetics is L-carnitine deficiency.

L-Carnitine is a naturally occurring amino acid that is the requisite carrier of long-chain fatty acids across the mitochondrial membrane where they undergo beta-oxidation [2]. Oxidation of long-chain fatty acids is by far the most important aerobic source of adenosine triphosphate in the mammalian heart, and adequate myocardial levels of L-carnitine are essential for normal energy production [3]. Multiple clinical and experimental studies have documented that a carnitine deficiency characterized by decreased myocardial carnitine levels is associated with DM [4, 5], infarcted myocardium [6, 7], and dilated cardiomyopathy [8, 9]. Increasing myocardial carnitine concentration by treatment with L-carnitine has been shown to improve symptoms in these patients [10–12].

A large percentage of patients who undergo a cardiac operation have DM, and increasingly, surgeons are being asked to operate on patients with DM and cardiac dysfunction that is out of proportion to the extent of the coronary artery disease. On the basis of the data suggesting that diabetics have a carnitine deficiency [4, 13], one of us (N.M.) routinely checks carnitine levels in all patients with DM and diminished ventricular function when they are referred for open heart operation. Many such patients have had marked improvement in ventricular function after 6 weeks of oral L-carnitine therapy and then have undergone a successful operation.

This clinical experience combined with experimental evidence that long-term L-carnitine supplementation improves myocardial function [14] and tolerance to ischemia in the presence of DM [13, 15] prompted us to use an established animal model to see if acute carnitine supplementation would attenuate reperfusion injury in hearts from diabetic rats.

	Material and methods

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Experimental protocol
Twelve-week-old Sprague-Dawley rats were anesthetized with sodium pentobarbital (35 mg/kg, intraperitoneally). They were then injected with streptozocin (65 mg/kg in normal saline solution intraperitoneally) (n = 12) to induce DM. Control rats (euglycemic) were injected with normal saline solution (n = 12). The rats were fed a diet of "rat chow" for 4 to 6 weeks. Then they were anesthetized again. After serum glucose levels were determined, the hearts were rapidly excised through a median sternotomy and placed in iced buffered Krebs solution (glucose, 17 mmol/L; EDTA [ethylenediaminetetraacetic acid], 0.5 mmol/L; potassium chloride, 5.9 mmol/L; and magnesium chloride, 1.7 mmol/L).

The hearts were suspended on a Langendorff isolated perfusion apparatus by the aorta and perfused with either standard buffered Krebs solution (290 mOsm) or L-carnitine–enriched buffered Krebs solution (Krebs solution with 5 mmol/L L-carnitine, 293 mOsm) at normothermia (37°C) and a constant pressure of 80 mm Hg. The hearts were allowed to beat spontaneously for 20 minutes as hemodynamic data were collected. After stabilization, the hearts were subjected to 20 minutes of global, normothermic, zero-flow ischemia, followed by 60 minutes of reperfusion with standard buffered Krebs solution or L-carnitine–enriched buffered solution. Hemodynamic data were collected every 5 minutes for 1 hour during reperfusion.

Hemodynamic data collection
Hemodynamic data including heart rate (beats per minute), left ventricular developed pressure (LVDP) (millimeters of mercury), coronary flow (milliliters per minute), and left ventricular end-diastolic pressure (millimeters of mercury) were collected after stabilization and every 5 minutes during reperfusion. Heart rate, LVDP, and left ventricular end-diastolic pressure were assessed using a Millar Mikrotip pressure transducer (Millar Corporation, Houston, TX), and coronary flow was assessed using a Transonic in-line flow probe (No. 2N; Transonic Systems, Ithaca, NY). All data were acquired using a MacLab data acquisition system and Macintosh 7200/75 PowerPC.

Experimental groups
Six hearts from streptozocin-injected rats (diabetic) were perfused with standard buffered Krebs solution (DM-STD), and the other 6 hearts from this group were perfused with L-carnitine–enriched buffered solution (5 mmol/L) (DM-CAR). Six hearts from saline solution-injected rats (euglycemic) were perfused with standard buffered Krebs solution (E-STD), and the other 6 hearts were perfused with L-carnitine–enriched buffered solution (5 mmol/L) (E-CAR).

All animals received humane care in compliance with the "Principles of Laboratory Animal Care" formulated by the National Society for Medical Research and the "Guide for the Care and Use of Laboratory Animals" published by the National Institutes of Health (NIH publication 86-23, revised 1985).

	Results

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Serum glucose levels
Rats that had been injected 4 to 6 weeks previously with streptozocin (diabetic group) had an average serum glucose level of 599 ± 14 mg/dL versus 126 ± 17 mg/dL for euglycemic controls injected with saline solution alone (p < 0.01).

Hemodynamic data
As seen in Table 1, which shows data at 60 minutes of reperfusion, heart rate, coronary flow, and LVDP were expressed as percent recovery from baseline. There were no significant differences between the four groups regarding heart rate and coronary flow. Left ventricular developed pressure was significantly preserved in both the euglycemic group and the diabetic group when the buffered solution was supplemented with carnitine. Hearts from diabetic rats in the L-carnitine–supplemented group (DM-CAR) had an LVDP of 116% ± 13% of baseline compared with an LVDP of 54% ± 12% in the Krebs solution–perfused group (DM-STD) (p < 0.01 by analysis of variance). In hearts from euglycemic rats, the LVDP was 75% ± 8% of baseline after acute L-carnitine supplementation (E-CAR) compared with 39% ± 9% of baseline in the group perfused with standard Krebs solution (E-STD) (p < 0.01 by analysis of variance). There was no significant difference in LVDP between the DM-CAR group and the E-CAR group (p > 0.05 by analysis of variance) at 60 minutes of reperfusion. Likewise, there was no significant difference in LVDP between the DM-STD and E-STD groups at 60 minutes.

	Comment

Top Abstract Introduction Material and methods Results Comment References

 
These experiments suggest that acute supplementation with L-carnitine significantly improves the tolerance of hearts from both diabetic and euglycemic rats for ischemia and reperfusion. The preservation of systolic function indicated by significantly higher LVDP after ischemia and reperfusion in carnitine-supplemented hearts from diabetic rats is not surprising based on the findings of several investigators [13, 15] who have shown that chronic L-carnitine treatment diminishes ischemic injury in diabetic rats. However, the preservation of LVDP after ischemia and reperfusion in hearts excised from euglycemic animals that were acutely supplemented with L-carnitine implies that a much larger patient population may benefit from carnitine treatment than we originally suspected.

Oral carnitine therapy with 2 to 3 g/day for 3 months has been shown to be of clinical benefit for nondiabetic patients with cardiomyopathy [12], arrhythmias [16], and acute myocardial infarction [11, 17]. However, L-carnitine treatment has not been extensively studied by investigators searching for increasingly effective ways to prolong the tolerance of the myocardium toward ischemia and reperfusion. Silverman and associates [18] treated nondiabetic dogs with a dose of carnitine before ischemia and showed a significant improvement in both systolic and diastolic ventricular function when a dose of 100 mg/kg was used. Recently, Azari and coworkers [19] documented smaller infarct size and maintenance of normal excitation-contraction coupling when carnitine-supplemented crystalloid cardioplegia was used to protect isolated hearts excised from nondiabetic rats and subjected to ischemia and reperfusion.

The results of our experiment show that LVDP is preserved after ischemia plus reperfusion in hearts isolated from diabetic (DM-CAR) and euglycemic rats (E-CAR) and perfused with L-carnitine–enriched buffered solution (see Table 1). The LVDP 60 minutes after ischemia did not differ significantly from preischemic (baseline) values in the carnitine-supplemented hearts. However, hearts in the groups without carnitine (DM-STD and E-STD) were significantly injured by ischemia and reperfusion, as at 60 minutes, they approached only half of the LVDP seen before ischemia. Clearly, acute L-carnitine supplementation attenuated ischemia-reperfusion injury in our model, both in diabetic animals and in euglycemic animals.

It is important to note that our results compare the percent recovery of LVDP with baseline (preischemic) values. Although it appears that the carnitine-supplemented hearts from diabetic animals (DM-CAR) recovered more left ventricular function than the carnitine-supplemented hearts from the euglycemic animals (E-CAR) (116% versus 75% recovery), there was no significant difference between the groups by analysis of variance.

The significantly decreased left ventricular end-diastolic pressure observed in hearts from the diabetic L-carnitine–supplemented rats suggests that L-carnitine may be helpful in the preservation of diastolic function of diabetic animals exposed to ischemia plus reperfusion. We are unaware of any previous studies looking at the effects of L-carnitine on the diastolic function of such a group.

The results of this small preliminary study suggest that L-carnitine may be efficacious as an additive to cardioplegia, especially for diabetic patients. We chose a crystalloid-perfused model for our initial work, as this closely approximates the cardioplegia we use clinically. Future studies will determine if acute L-carnitine supplementation is beneficial in a blood-perfused model and if the improved tolerance toward ischemia occurs with hypothermic delivery of L-carnitine–enriched cardioplegia.

Experimental evidence of carnitine deficiency in patients with cardiomyopathy and DM [4, 15] suggests that there is tremendous potential to improve ventricular function with carnitine supplementation. On the basis of the improvement in ventricular function that was observed with acute L-carnitine supplementation in this ischemia-reperfusion model, we believe that there is a large number of patients who potentially may benefit from L-carnitine treatment during an open heart procedure. Further studies in a model that more closely approximates clinical myocardial protection are necessary to determine if L-carnitine supplementation is beneficial and safe.

	References

Top Abstract Introduction Material and methods Results Comment References

 

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