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Am J Physiol Heart Circ Physiol 293: H394-H401, 2007. First published March 16, 2007; doi:10.1152/ajpheart.00833.2006
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Abolition of reperfusion-induced arrhythmias in hearts from thiamine-deficient rats

Fernando A. Oliveira,1,2 Silvia Guatimosim,2 Carlos H. Castro,2 Diogo T. Galan,1 Sandra Lauton-Santos,1 Angela M. Ribeiro,1 Alvair P. Almeida,2 and Jader S. Cruz1

Departments of 1Biochemistry and Immunology and 2Physiology and Biophysics, Biological Sciences Institute, Universidade Federal de Minas Gerais, Belo Horizonte, Brazil

Submitted 2 August 2006 ; accepted in final form 8 March 2007


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Extensive work has been done regarding the impact of thiamine deprivation on the nervous system. In cardiac tissue, chronic thiamine deficiency is described to cause changes in the myocardium that can be associated with arrhythmias. However, compared with the brain, very little is known about the effects of thiamine deficiency on the heart. Thus this study was undertaken to explore whether thiamine deprivation has a role in cardiac arrhythmogenesis. We examined hearts isolated from thiamine-deprived and control rats. We measured heart rate, diastolic and systolic tension, and contraction and relaxation rates. Whole cell voltage clamp was performed in rat isolated cardiac myocytes to measure L-type Ca2+ current. In addition, we investigated the global intracellular calcium transients by using confocal microscopy in the line-scan mode. The hearts from thiamine-deficient rats did not degenerate into ventricular fibrillation during 30 min of reperfusion after 15 min of coronary occlusion. The antiarrhythmogenic effects were characterized by the arrhythmia severity index. Our results suggest that hearts from thiamine-deficient rats did not experience irreversible arrhythmias. There was no change in L-type Ca2+ current density. Inactivation kinetics of this current in Ca2+-buffered cells was retarded in thiamine-deficient cardiac myocytes. The global Ca2+ release was significantly reduced in thiamine-deficient cardiac myocytes. The amplitude of caffeine-releasable Ca2+ was lower in thiamine-deficient myocytes. In summary, we have found that thiamine deprivation attenuates the incidence and severity of postischemic arrhythmias, possibly through a mechanism involving a decrease in global Ca2+ release.

arrhythmia; calcium current; confocal microscopy

THIAMINE DEFICIENCY CAUSES cardiovascular and neurological damage that presents clinically as beriberi. Thiamine pyrophosphate is an important cofactor in several vital enzymatic reactions involved in metabolism and energy production (34, 39). Following its uptake by the cell, thiamine rapidly undergoes pyrophosphorylation, yielding thiamine pyrophosphate, which is the active substance. Extensive work has been done regarding the impact of thiamine deprivation on the nervous system. However, very little is known about the effects of thiamine deficiency on the myocardium (5). Cappelli et al. (5) have shown evidence that supports the view that cardiac contractility is deeply affected in the rat by thiamine deficiency. Thiamine is also considered a clinically important factor in heart function, and its deficiency has been reported to cause heart failure (28, 33, 35).

The general view that reperfusion of the ischemic myocardium exerts a beneficial effect by preventing the ischemia-induced changes in cardiac performance has been supported by a number of studies; however, reperfusion after a certain critical time has been shown to be deleterious to cardiac function (16). Intracellular Ca2+ has an important role in heart function, and studies have been conducted to investigate possible changes in sarcoplasmic Ca2+ in hearts subjected to ischemia-reperfusion (I/R) (30). Thiamine administration was found to reduce myocardial ischemic lesions in an infarct model and had a marked cytoprotective effect against ischemic damage to the heart (18, 37). It is well known that intracellular metabolic factors are important in maintaining cardiac myocyte structure, energetics, and intracellular homeostasis (2). Depletion of certain metabolic compounds such as ATP and pyruvate may contribute to the deleterious effects observed in heart failure (22).

Ca2+ signaling in heart failure is characterized by depressed contractility and depletion of Ca2+ from sarcoplasmic reticulum (SR) Ca2+ stores. Additionally, asynchronous Ca2+ release and Ca2+-triggered arrhythmias are both common features of diverse heart-failure models. We thus hypothesized that long-term thiamine deficiency may increase the susceptibility to heart I/R injury.

In the present study, we show that thiamine-deficient rats consistently exhibited a failure to develop irreversible reperfusion arrhythmias. We also report the electrophysiological properties of the L-type Ca2+ current (ICa,L) in cardiomyocytes from thiamine-deficient rat hearts. An investigation of the whole cell intracellular Ca2+ concentration ([Ca2+]i) transient revealed that thiamine-deficient heart cells had a slower rate of rise and a decrease in the magnitude of the systolic Ca2+ transient. Our results bring up the possible "beneficial" effect of thiamine deprivation on the reperfusion arrhythmias.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Our investigation conformed with the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health (NIH Publication No. 85-23, revised 1996), and the institutional ethical committee for animal experimentation approved this study (protocol no. 042-CETEA). Experiments were carried out in Wistar rats. Standard rat chow and thiamine-deficient chow were made in our laboratory routinely. The two diets, control and thiamine-deficient, were isocaloric, and the only difference between them was the presence or absence of thiamine in the vitamin mixture used to prepare the chow. Both diets contained 20% protein, 1% cellulose, 5% salt mixture, 1% vitamin mixture, 5% corn oil, 0.4% choline, and 67.6% cornstarch (27, 29).

Male Wistar rats at 2.5–3 mo of age weighing 187 ± 7.3 g (with thiamine, n = 12 animals) and 193 ± 5.6 g (thiamine deficient, n = 24 animals) were housed in individual plastic cages and were allowed free access to standard or thiamine-deficient chow and water. Animals from control and thiamine-deficient groups were treated for 30 days. Chow consumption was recorded daily, and body weight was recorded every seven days throughout this period. We used animals at the 30th day of treatment (with thiamine, 233 ± 6.6 g and thiamine deficient, 213 ± 6.7 g). The food intake (at day 30) for control rats was 14.2 ± 2.3 g and for thiamine-deficient rats was 5.5 ± 3.1 g.

Measurements of blood thiamine. Thiamine level was assessed by the quantitative spectrophotometric transketolase activity described by Warnock (38). Approximately 1.0 ml of whole blood was collected from the tail artery and was stored in an Eppendorf tube containing EDTA as the anticoagulant. The samples were kept refrigerated at –80°C until the day of the analysis. Each sample was assayed twice by using 50 µl of the whole blood for thiamine determinations. Blood thiamine levels were expressed as micromoles of ribose-5-phosphate consumed per hour per milliliter of blood. To perform these measurements, we used a total of 22 animals (10 controls and 12 thiamine deficient). The thiamine blood level was assessed on the 1st, 15th, and 30th days of treatment.

I/R studies. Male Wistar rats were decapitated 10–15 min after intraperitoneal injection of 400 IU heparin. The thorax was opened, and the heart was carefully dissected and perfused through a 1.0 ± 0.3-cm aortic stump with Krebs-Ringer solution containing (in mmol/l) 118.4 NaCl, 4.7 KCl, 1.2 KH2PO4, 1.2 MgSO4·7H2O, 2.5 CaCl2·2H2O, 11.7 glucose, and 26.5 NaHCO3. The perfusion fluid was maintained at 37 ± 1°C, with a pressure of 65 mmHg and constant oxygenation (5% CO2-95% O2). A force transducer was attached through a heart clip to the apex of the ventricles to record the contractile force (tension, in grams). These parameters were recorded on a computer by a data-acquisition system (Biopac Systems, Santa Barbara, CA). A diastolic tension of 0.5–1.0 g was applied to the hearts. Coronary flow was measured by collecting the perfusate over a period of 1 min at regular intervals. After 25–30 min of the equilibration period, the left anterior descending coronary artery was ligated as described previously (21). The ligature was released after 15 min, and reperfusion was performed for an additional 30 min. Cardiac arrhythmias were defined as the presence of ventricular tachycardia and/or ventricular fibrillation after the ligature of the coronary artery was released. We monitored the electrical activity through a bipolar electrocardiogram (data not shown; n = 5). To obtain a quantitative measurement, the arrhythmias were graded arbitrarily by their duration, with duration of 30 min considered irreversible arrhythmia. Therefore, the occurrence of cardiac arrhythmias for up to 3 min was assigned the factor 2; 3–6 min was assigned the factor 4; 6–10 min was assigned the factor 6; 10–15 min was assigned the factor 8; 15–20 min was assigned the factor 10; 20–25 min was assigned the factor 11; and 25–30 min was assigned the factor 12. Values ranging from 2 to 12 were obtained and were denoted as the arrhythmia severity index (ASI) (1, 9).

Electrophysiological experiments. Control and thiamine-deficient ventricular cardiomyocytes from age-matched rats were enzymatically isolated as previously described (10). An EPC-10 (HEKA Electronics) was used to patch clamp single myocytes (whole cell voltage-clamp configuration), and membrane currents were measured (7). Whole cell currents were recorded under conditions that eliminated Na+ and K+ currents. Patch pipettes (1.5–2 M{Omega}) were filled with a solution containing (in mmol/l) 10 NaCl, 120 CsCl, 20 TEA-Cl, 4 Mg-ATP, 5 EGTA, and 10 HEPES (pH 7.2). Myocytes were bathed with an external solution containing (in mmol/l) 140 NaCl, 5 CsCl, 1 MgCl2, 1.8 CaCl2, 5 glucose, 5 HEPES, and 4 4-aminopyridine (pH 7.4). ICa,L was elicited by steps of depolarization ranging from –40 to 60 mV for 300 ms from a holding potential of –40 mV at a frequency of 0.1 Hz. Cell capacitances and ICa,L densities were calculated with Pulse-Fit (HEKA Electronics). Double exponential functions were used to fit the time-dependent current decay and had the following form

Formula 1(1)
where A0 is the time-independent component; Afast and Aslow are the amplitudes of the fast and slow inactivation components, respectively; t is time (in ms); and tau ({tau}) represents the inactivation time constant.

Confocal microscopy. Isolated myocytes were incubated with fluo 4-AM (10 µM; Molecular Probes, Eugene, OR) for 20 min and then were washed with an extracellular solution that contained 1.8 mmol/l Ca2+ to remove the excess dye. Cells were electrically stimulated at 1 Hz to produce steady-state conditions. The confocal line-scan imaging was performed by a Zeiss LSM 510META confocal microscope equipped with an argon laser (488 nm) and a 63x oil-immersion objective. Line-scan images were acquired at sampling rate of 1.54 ms/line. The amplitude of the [Ca2+]i transient evoked by the application of a Ca2+- and Na+-free solution containing 10 mM caffeine (~10 s) was used as an indicator of SR Ca2+ load (31). Cells were subjected to a series of preconditioning pulses (1 Hz) before caffeine was applied. For this series of experiments we used a group of six rats (3 for control and 3 for thiamine deficiency). Digital image processing was performed by using custom-devised routines created with the IDL programming language (Research Systems, Boulder, CO). The Ca2+ level was reported as F/F0 (or as {Delta}F/F0), where F0 is the resting Ca2+ fluorescence.

Statistical analysis. All data are expressed as means ± SE, and the number of cells or experiments is shown as n. Significant differences between groups were determined with a Student's t-test or ANOVA followed by the Bonferroni post hoc test. ASI was analyzed by nonparametric Mann-Whitney test. Probability values of P < 0.05 were considered to be statistically significant.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
We used a rat model to determine whether long-term thiamine deprivation exacerbates myocardial damage (5) that would cause significant changes in cardiac function. Chow intake was significantly reduced in the thiamine-deprived group compared with the control group (see MATERIALS AND METHODS; data not shown). Average blood thiamine levels (30 days of treatment) in micromoles of ribose-5-phosphate consumed per hour per milliliter of blood were 27.4 ± 3.8 (control) and 14.0 ± 8.1 (thiamine deficient) and sedoheptulose production per hour per milliliter was 1.9 ± 0.3 (control) and 0.6 ± 0.1 (thiamine deficient). These values are significantly different (P < 0.05).

Isolated Langendorff-perfused heart preparations were used to examine effects of thiamine deprivation on cardiac function. After the heart had stabilized under ex vivo conditions, we monitored for heart rate, diastolic and systolic tension, ±dT/dt, and coronary flow. The protocol for I/R in isolated perfused hearts is illustrated in Fig. 1. The typical time course of reperfusion injury is shown for the control group (Fig. 1A). As indicated in Fig. 1B, however, hearts from thiamine-deficient rats did not develop irreversible arrhythmias during at least 30 min of reperfusion. The putative antiarrhythmogenic effects of thiamine deficiency were further characterized by determining the ASI. Figure 1C shows the results for the two different experimental groups. The median ASI value for control hearts was 12 (n = 6) and for thiamine-deficient hearts was 2 (n = 5). These results show that hearts from thiamine-deprived rats did not experience irreversible arrhythmias (0/5 of tested animals; P < 0.05; Fig. 1C).


Figure 1
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  		Fig. 1. Thiamine deficiency protects the heart against reperfusion injury. A and B: original records of contraction force (tension, in grams) in 2 representative experiments are shown. Lowercase letters indicate stage of experiment: basal (a), during left anterior descending coronary artery (LAD) occlusion (b), and after reperfusion (c). A: typical time course of reperfusion-induced irreversible contracture in isolated perfused rat heart from control group. B: representative time course of contraction force in isolated perfused rat heart from thiamine-deficient group. C: averaged arrhythmia severity index (ASI) on reperfusion after 15 min of occlusion of the LAD in isolated perfused rat hearts. Numbers above the bars indicate the incidence of irreversible arrhythmias during the reperfusion period. *P < 0.05 for control (hatched bar) vs. thiamine deficient (cross-hatched bar). D: time course of heart rate in isolated perfused rat heart before and during ischemia and after reperfusion. The hearts were perfused according to the Langendorff technique. *P < 0.05 for thiamine-deficient vs. control hearts (ANOVA).

 
Cardiac I/R is associated with alterations in metabolism that, depending on the severity of the ischemic insult, can progress to irreparable tissue damage. Therefore, the observation that hearts from thiamine-deficient rats did not develop irreversible reperfusion arrhythmias was totally unexpected. This apparent paradox was investigated further by analyzing different parameters related to cardiac performance. Hearts from thiamine-deficient animals showed lower heart rates during basal and occlusion intervals compared with control hearts (Fig. 1D). It is interesting to note that reperfusion caused a transient increase in heart rate from thiamine-deficient rats (P < 0.05; n = 5). The control group did not recover during reperfusion. One important point that should be addressed is the variation observed in the spontaneous frequency in the thiamine-deficient hearts. Before the ischemia, the average spontaneous frequency in hearts isolated from thiamine-deficient rats was lower than that measured in the controls (Fig. 1D; basal period). In this condition, ischemia's deleterious effects could have been attenuated because of milder ATP depletion due to lower ATP requirement of the heart preparation contractility at a lower rate. Therefore, contracture or arrhythmias would be less likely to follow during the reperfusion period.

Figure 2 shows the changes in diastolic (Fig. 2A) and systolic (Fig. 2B) tension before (basal), during (occlusion), and after coronary reperfusion. During occlusion (15 min), the diastolic tension did not change for heart from thiamine-deficient rats (P > 0.05; n = 5). On reperfusion, diastolic tension increased dramatically in control hearts with no observed change for the thiamine-deficient hearts (Fig. 2A). The systolic tension during the basal period was significantly lower for thiamine-deficient hearts (P < 0.05; n = 5) compared with control hearts (Fig. 2B). During occlusion, systolic tension decreased. and only for thiamine-deficient hearts could we observe a full recovery to basal levels under reperfusion (Fig. 2B; n = 5). To determine whether or not thiamine deficiency is associated with changes in contraction and/or relaxation rates, we examined hearts isolated from thiamine-deficient and control rats. There were significant changes of the contraction rate (as viewed by +dT/dt values) between the two groups during basal and occlusion intervals. The composite data are depicted in Fig. 2C. However, during reperfusion only thiamine-deficient hearts recovered to basal levels. Figure 2D shows relaxation rates during basal, occlusion, and reperfusion intervals. Under basal conditions, –dT/dt in the thiamine-deprived group remained significantly diminished relative to the control values (P < 0.05; n = 5). During occlusion, both groups show a significant decrease compared with their respective basal condition. However, the recovery of –dT/dt at 30 min reperfusion was complete only for the thiamine-deficient rat hearts (n = 5).


Figure 2
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  		Fig. 2. Cardiac performance of thiamine-supplemented (bullet) and thiamine-deprived ({circ}) hearts. Time course of diastolic (A) and systolic tension (B) in isolated perfused rat heart before and during ischemia and after reperfusion are shown. Hearts were perfused according to the Langendorff technique. *P < 0.05 for thiamine deficient vs. control by ANOVA. Time course of +dT/dt (C) and –dT/dt (D) in isolated perfused rat heart before and during ischemia and after reperfusion are shown. *P < 0.05 for thiamine deficient vs. control by ANOVA.

 
To gain more insight into the mechanism by which thiamine-deprived hearts were less prone to develop reperfusion arrhythmias, we evaluated the ICa,L in isolated cardiac myocytes under whole cell voltage clamp. Figure 3, A and B illustrates typical tracings of ICa,L under our recording conditions in both a control and a thiamine-deficient cell. The ICa,L density (pA/pF) in thiamine-deficient cardiac myocytes was not found to be statistically different from the control cells (Fig. 3C; P > 0.05; n = 10). Cell capacitance was not statistically different (Fig. 3D; P > 0.05). Interestingly, we found that thiamine-deficient and control cells had ICa,L with different decay kinetics. Our data show (Fig. 4, A and B) that in thiamine-deficient cells, ICa,L decays more slowly ({tau}fast = 29.6 ± 0.5 ms and {tau}slow = 89.5 ± 0.3 ms) than it does in control cells ({tau}fast = 13.5 ± 0.2 ms and {tau}slow = 56.8 ± 2.3 ms). It is well established that ICa,L declines with two time constants, inactivating as a result of both Ca2+-dependent and voltage-dependent processes. The results indicate that L-type Ca2+ channel inactivation (fast and slow components) was affected by thiamine deprivation in rat heart myocytes (see DISCUSSION). Figure 4C shows the voltage dependence of ICa,L conductance. The slope of the Boltzmann function used to fit the data and the voltage at which 50% of ICa,L conductance is attained are summarized in Fig. 4D. The parameters were different in thiamine-deficient and control cells. These data indicate that the ICa,L of thiamine-deficient and control myocytes are different.


Figure 3
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  		Fig. 3. L-type Ca2+ current (ICa,L) density is unchanged in thiamine-deprived heart cells. A: representative whole cell Ca2+-current record from control cells. Holding potential was set at –40 mV. Current was evoked by step depolarization to 0 mV for 300-ms duration. B: representative whole cell Ca2+-current record from thiamine-deficient cells. Stimulation protocol was the same as in A. C: voltage dependence of ICa,L, plotted as current density (pA/pF) obtained for test potentials from –40 to +50 mV (control, black symbols; thiamine deficient, gray symbols). Continuous lines were obtained by nonlinear regression analysis. D: cell capacitance. Black bar represents the control condition (+) and open bar represents thiamine deficiency (–).

 

Figure 4
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  		Fig. 4. Inactivation time course and voltage dependence for activation are changed in thiamine-deficient cardiomyocytes. A: inactivation time course in control cells (i), thiamine-deficient cells (ii), and superimposed traces (iii). Smooth lines [white (i) and black (ii) curves] represent best-fit curves to the data determined by a least-squares method using equation 1. B: means ± SE of the fast and slow time constants for the ICa,L inactivation at 0 mV. Black bars depict controls, and gray bars depict thiamine deficient cardiomyocytes. *P < 0.05. C: voltage dependence of activation. Peak ICa,L at test potentials between –40 mV and +20 mV were converted into conductances, normalized, and plotted as a function of test potential. Smooth lines represent best-fit curves to the data determined by a least-squares method using a Boltzmann equation: g/gmax = 1/{1 + exp[(V – V0.5)/k]}, where V0.5 is the voltage at which 50% of the maximum conductance was observed and k is the slope factor. Black smooth line depicts controls, and gray line depicts thiamine-deficient myocytes. D: summary of parameters used to fit the conductance in control and thiamine-deficient cells.*P < 0.05.

 
Figure 5A displays typical line-scan images recorded from field-stimulated control and thiamine-deficient cardiomyocytes loaded with the Ca2+ indicator fluo 4-AM. Thiamine-deficient cardiomyocytes develop significantly smaller (2.57 ± 0.07; n = 503 cells from 7 different animals) and slower (T50, 335 ± 4.5 ms; n = 503 cells) [Ca2+]i transients compared with control (2.79 ± 0.07, n = 469 cells from 6 different animals; T50, 298 ± 3.8 ms, n = 469; P < 0.05) as shown in Fig. 5, B and C. Thiamine deficiency is also associated to prolonged time-to-peak of Ca2+ release (Fig. 5D; P < 0.05). Time-to-peak was increased by 27% in thiamine-deficient cardiomyocytes (52 ± 2.4 ms; n = 503) compared with control cardiomyocytes (41 ± 1.6 ms; n = 469).


Figure 5
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  		Fig. 5. Global Ca2+ transient is altered in thiamine-deficient cardiomyocytes. A: line-scan images (top) and [Ca2+]i-transient records (bottom) from control [Thiamine (+)] and thiamine-deficient [Thiamine (–)] myocytes stimulated at 1 Hz. Ca2+ signal is shown as fluorescence ratio (F/F0), with fluorescence intensity (F) being normalized to intensity at rest before stimulation (F0). B: averaged peak intracellular Ca2+ concentration ([Ca2+]i) transient in control (n = 469 from 6 different animals) and thiamine-deficient myocytes (n = 503 from 7 different animals). C: time to 50% decline of [Ca2+]i transients in control (n = 469) and thiamine-deficient (n = 503) heart cells. D: time to peak of Ca2+ release in control and thiamine-deficient cardiomyocytes. In B, C, and D, closed bar represents control and open bar represents thiamine-deficient group. *P < 0.05 for thiamine-deficient vs. control.

 
We examined SR Ca2+ load in thiamine-deficient and control cells. The amplitude of the global [Ca2+]i transient induced by caffeine application was used as an indicator of SR Ca2+ content. We investigated SR Ca2+ load in stimulated thiamine-deficient and control myocytes. In these experiments, cardiac myocytes were loaded with fluo 4-AM as previously performed and field stimulated (at 1 Hz) for at least 10 s before caffeine (10 mM) was applied. This protocol ensured steady-state SR Ca2+ loading at the time of caffeine application (31). As shown in Fig. 6, the amplitude of the caffeine-induced [Ca2+]i transients was smaller in stimulated thiamine-deficient cells (10.1 ± 0.5, control, n = 21 cells from 3 different rats vs. 7.8 ± 0.5, thiamine deficient, n = 20 cells from 3 different rats; P < 0.05). These data suggest that, during stimulation, SR Ca2+ load is smaller (by ~23%) in thiamine-deficient than in control cardiomyocytes.


Figure 6
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  		Fig. 6. Lower sarcoplasmic reticulum Ca2+ load in thiamine-deficient myocytes. Bar plots of the amplitude of the caffeine-induced [Ca2+]i transient in control (n = 21 cells from 3 different animals; closed bar) and thiamine-deficient (n = 20 cells from 3 different animals; open bar) cardiac myocytes are shown. *P < 0.05.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
One of the most important presentations of cardiovascular disease is ischemia, which leads to tissue hypoxia, cellular necrosis, apoptosis, and, in severe situations, organ dysfunction (11, 26). During the past several years, there have been major improvements in the strategies used to protect myocardial tissue from I/R injury (26). However, the mechanism of I/R injury remains unknown. This study was undertaken to determine whether rats subjected to a thiamine-deficient diet would exacerbate the deleterious effects on cardiac performance caused by ischemia and reperfusion. However, the results of this study indicate, surprisingly, that hearts from thiamine-deficient rats did not develop severe reperfusion arrhythmias. There are few reports where the effects of thiamine deprivation on heart function were studied. As far as we are aware, only three reports (5, 6, 39) have described the effect of thiamine deficiency on contraction amplitude, either on rat isolated cardiac cells in response to external stimulation or on spontaneous contractions. Those studies showed that thiamine deficiency (during 35 days) decreased the rate of contraction. Zangen and Schainberg (39) also reported a significant reduction of spontaneous contraction amplitude. Those results are in agreement with our data, because in our experimental conditions diastolic and systolic rates are smaller in hearts from thiamine-deficient rats compared with control animals.

More importantly, thiamine deprivation seems to protect the heart against reperfusion injury arrhythmias, and, as far as we know, this is the first time that an "antiarrhythmogenic" effect of thiamine deficiency has been shown. It is interesting to note that after a recovery period in which the thiamine-deficient animals were treated with a standard diet (additional 7 days), the hearts of these animals developed irreversible reperfusion arrhythmias similar to the control rats (unpublished results; n = 5).

On the other hand, thiamine deprivation has been reported to cause congestive heart failure (32). The authors reported that a thiamine-deficient diet causes death usually after sudden fatal arrhythmia occurring after 8 wk of treatment in adult rats. Histological examination of heart muscle from those animals showed focal necrosis, especially in the left ventricle, with significant structural alterations in the mitochondria (8). Cardiac function and contractility are closely related to cardiac metabolism and energy production (36). In cardiomyocytes, energy production is related to the number of mitochondria, with these organelles occupying up to 40% of the sarcoplasm. Hence, the total number of mitochondria is an important condition to maintain normal heart functioning (2).

Thiamine is the precursor for the cofactor of both pyruvate dehydrogenase and {alpha}-ketoglutarate dehydrogenase, enzymes that catalyze the oxidative decarboxylation of pyruvate to acetyl-CoA and the oxidative decarboxylation of {alpha}-ketoglutarate to succinyl-CoA, respectively. Thiamine deficiency causes an increase in blood and cellular pyruvate concentration and could impair mitochondrial function.

Pyruvate, an end product of glycolysis, has been shown to improve mechanical performance of the heart under normoxic conditions (4, 22–25). Important to note is the fact that pyruvate also ameliorates the heart's mechanical performance during I/R injury (24, 40). The cellular mechanisms of pyruvate action have recently been explored by Zima et al. (41). They found that pyruvate has a dual effect on SR calcium release, consisting of a direct inhibition of ryanodine receptor channel activity and elevation of SR Ca2+ content. In our experimental conditions (thiamine deficiency), pyruvate will accumulate intracellularly, and as a consequence one would expect an intracellular acidification. Pyruvate has been reported to alter the kinetics of the [Ca2+]i transient (41). Pyruvate slowed the time to peak, which is in line with our results but had no significant effect on the transient decay kinetics. However, the [Ca2+]i-transient amplitude is increased by pyruvate, probably because of its effect on SR Ca2+ load. In the work of Zima et al. (41), there is experimental evidence that both the slowing of the rising phase of the [Ca2+]i transient and the increase in the amplitude are independent of intracellular acidosis. In our model, the SR Ca2+ content was not augmented, most likely because of a reduction in the mitochondrial ATP synthesis implicating a reduction of the cytosolic ATP/ADP concentration ratio and therefore limiting the sarcoplasmic Ca2+ reuptake to the SR. Acidosis inhibits Ca2+ uptake by the SR of rat skinned trabeculae (17), which would be expected to decrease SR Ca2+ content. However, in isolated myocytes, acidosis increases the SR Ca2+ content (14). This apparent discrepancy is reconciled by the proposal that the increase in SR Ca2+ load observed in the intact cell may be a consequence of a rise in resting [Ca2+]i during acidosis, which will increase the amount of Ca2+ available to the SR Ca2+-ATPase to pump it back to the SR because of phosphorylation of phospholamban by Ca2+-calmodulin kinase II (13). Because our results in isolated myocytes showed a significant diminution of SR Ca2+ load (Fig. 6) and not an increase, we cannot rely on the acidosis that presumably follows thiamine deficiency to explain the full recovery after ischemia (Fig. 1).

On the other hand, recent evidence (41) suggests that high concentrations of pyruvate (~10 mM) may also exert proarrhythmogenic effects by inducing cardiac alternans in atrial and ventricular myocytes from the cat heart (3, 15).

These contradictory data could be explained if we consider the pyruvate level as a preconditioning signal ranging from anti- to proarrhythmogenic effects. A mild pyruvate increase could function as antiarrhythmogenic, whereas a high pyruvate concentration could function as a proarrhythmogenic factor.

During a mild thiamine deficiency, pyruvate accumulates and could elicit opening of the mitochondrial permeability transition pore in its low-conductance state, which does not lead to cell death (42) and in fact may play important functions that may contribute to ischemic preconditioning-induced protection (12). Clearly, further studies are necessary to determine whether mitochondrial permeability transition pore opening is in fact the major mechanism leading to the protection effect observed in the present work.

Deficiency of thiamine may produce changes in the glycolytic flux that accumulates some of the substrates needed to maintain ATP/ADP concentration ratio to provide enough metabolic energy to keep the heart working. When we challenged the hearts from thiamine-deficient rats after ~30 days under diet to ischemia, it seems that the myocardium was well adapted to the new situation (like the ischemic preconditioning), and during reperfusion the expected injury is greatly attenuated. One important mechanism of cardioprotection involves the activation of ATP-sensitive K+ channels from mitochondrial inner membrane, and because thiamine deficiency depresses the enzymatic activity of {alpha}-ketoglutarate dehydrogenase present in the Krebs' cycle, we would expect that opening of this channel causes sufficient K+ cycling to uncouple mitochondria, leading to reduced Ca2+ uptake and cardioprotection (19).

Louch and colleagues (20) showed that with a reduction of the T-tubule (TT) density to 30% of control values, there is a slowing of the rate of rise of the [Ca2+]i transient, which is consistent with the present results (Fig. 5D). If the thiamine-deficiency treatment used in the present study leads to a decrease of TT density, we would expect a decrease of ICa,L density due to the fact that L-type Ca2+ channels are assumed to be located preferentially in TT. However, our data demonstrate that ICa,L density showed no difference between the experimental groups (Fig. 3C). Therefore, the absence of changes in ICa,L density argues against the idea that there was a significant reduction of TT density. Besides, the membrane capacitance is not altered under the conditions studied, which makes the reduction in TT density unlikely. However, experiments using staining with di-8-ANEPPS, a fluorescent probe that allows the visualization of the TT network, need to be performed to further test this hypothesis.

Ca2+ is presumed to play an important role in myocardial I/R injury. Our data indicate that ICa,L density is unchanged in thiamine-deficient cardiac myocytes compared with controls. The trigger ICa,L recorded from thiamine-deficient myocytes showed a significant delay on the inactivation kinetics, thereby suggesting an enhancement in the amount of Ca2+ ions entering into thiamine-deficient myocytes. This enhancement is likely balanced by the shift of the ICa,L activation curve toward more depolarized membrane potentials. We measured ICa,L in the absence of Ca2+ release by heavily buffering cytosolic Ca2+ to avoid ICa,L inactivation by Ca2+ release, which could prevent differences in available Ca2+ influx. The global Ca2+ transient is significantly smaller in thiamine-deficient cardiomyocytes, and this could account for the absence of reperfusion injury. We found, at the cellular level, a lower [Ca2+]i that, along with the lower spontaneous heart rate, might act synergistically to produce the cardioprotective effect reported here. Because the difference seen in F/F0 ratio is rather small, and knowing that we did not properly calibrate the fluorescence measurements (a possible limitation of our study), caution should be exercised when interpreting the global Ca2+-transient results. In fact, a marked increase in tissue Ca2+ content has been reported in I/R hearts under various conditions, and the intracellular Ca2+ overload may explain I/R injury to the myocardium (30). Our data indicate that SR Ca2+ content is smaller in thiamine-deficient cardiac myocytes. This may be important to the mechanism involved in the cardioprotection against the reperfusion injury.

It will be important in future work to delineate the intracellular signaling events responsible for those changes that provide cardiac myocyte protection caused by thiamine deficiency.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This study was funded by Conselho Nacional de Pesquisa e Desenvolvimento Cientifico e Tecnológico (Dr. J. S. Cruz and Dr. S. Guatimosim) and Fundação de Amparo a Pesquisa do Estado de Minas Gerais (Drs. J. S. Cruz, S. Guatimosim, and A. P. Almeida) grants. F. A. Oliveira was a fellow of Coordenação de Aperfeiçoamento de Pessoal de Nível Superior.


   ACKNOWLEDGMENTS
 
The confocal experiments were done at the Centro de Microscopia Eletrônica-Instituto de Ciências Biológicas facility.


   FOOTNOTES
 

Address for reprint requests and other correspondence: J. S. Cruz, Dept. of Biochemistry and Immunology, Universidade Federal de Minas Gerais, Belo Horizonte, MG, CEP 31900-901, Brazil (e-mail: jcruz{at}icb.ufmg.br)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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