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J Appl Physiol 100: 150-156, 2006. First published September 15, 2005; doi:10.1152/japplphysiol.00988.2005
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High-dose benfotiamine rescues cardiomyocyte contractile dysfunction in streptozotocin-induced diabetes mellitus

Asli F. Ceylan-Isik,* Shan Wu,* Qun Li, Shi-Yan Li, and Jun Ren

Division of Pharmaceutical Sciences and Center for Cardiovascular Research and Alternative Medicine, University of Wyoming, Laramie, Wyoming

Submitted 15 August 2005 ; accepted in final form 12 September 2005


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 ACKNOWLEDGMENTS
 REFERENCES
 
Diabetic cardiomyopathy is characterized by cardiac dysfunction. This study was designed to examine the effect of benfotiamine, a lipophilic derivative of thiamine, on streptozotocin (STZ)-induced cardiac contractile dysfunction in mouse cardiomyocytes. Adult male FVB mice were made diabetic with a single injection of STZ (200 mg/kg ip). Fourteen days later, control and diabetic (fasting plasma glucose > 13.9 mM) mice were put on benfotiamine therapy (100 mg·kg–1·day–1 ip) for another 14 days. Mechanical and intracellular Ca2+ properties were evaluated in left ventricular myocytes using an IonOptix MyoCam system. The following indexes were evaluated: peak shortening (PS), time to PS (TPS), time to 90% relengthening (TR90), maximal velocity of shortening/relengthening, resting and rise of intracellular Ca2+ in response to electrical stimulus, sarcoplasmic reticulum (SR) Ca2+ load, and intracellular Ca2+ decay rate ({tau}). Two- or four-week STZ treatment led to hyperglycemia, prolonged TPS and TR90, reduced SR Ca2+ load, elevated resting intracellular Ca2+ level and prolonged {tau} associated with normal PS, maximal velocity of shortening/relengthening, and intracellular Ca2+ rise in response to electrical stimulus. Benfotiamine treatment abolished prolongation in TPS, TR90, and {tau}, as well as reduction in SR Ca2+ load without affecting hyperglycemia and elevated resting intracellular Ca2+. Diabetes triggered oxidative stress, measured by GSH-to-GSSG ratio and formation of advanced glycation end product (AGE) in the hearts. Benfotiamine treatment alleviated oxidative stress without affecting AGE or protein carbonyl formation. Collectively, our results indicated that benfotiamine may rescue STZ-induced cardiomyocyte dysfunction but not AGE formation in short-term diabetes.

diabetes; ventricular myocyte; contraction; advanced glycation end product

DIABETES MELLITUS LEADS TO high cardiovascular morbidity and mortality as a result of functional and morphological damage in diabetic hearts (20, 21). A unique myopathic alteration, namely diabetic cardiomyopathy, has been established independent of macro- or microvascular diseases commonly seen in diabetes (20). The most prominent defects of diabetic cardiomyopathy are prolonged duration of contraction and relaxation, as well as reduced cardiac compliance (19–21). The etiology of diabetic cardiomyopathy is rather complex and involves metabolic derangements, depressed autonomic function, and abnormalities in certain hormones or proteins that regulate intracellular ion homeostasis, particularly Ca2+ (8, 16, 20). The high mortality of diabetic heart complications warrants stringent and aggressive treatment against hyperglycemia, hyperinsulinemia, dyslipidemia, and oxidant damage. Classical therapeutic agents against diabetic heart diseases include angiotensin-converting enzyme inhibitors, angiotensin receptor antagonists, digoxin, diuretics, {beta}-blockers, Ca2+ antagonists, and spironolactone. Both enzymatic and nonenzymatic antioxidants have been proven to be effective against diabetic heart complications (15, 24, 29). Insulin-sensitizing or lipid-reducing agents, such as thiazolidinediones and peroxisome proliferator-activated receptor agonists, have also shown promise for various types of diabetic complications (18). In addition to pharmacological interventions, lifestyle changes, such as smoking cessation, weight control, exercise, and dietary restriction, have all been included in the primary care for diabetes mellitus (14). Nevertheless, none of these therapeutic strategies may be considered the panacea for optimal and ultimate management of high morbidity and mortality of diabetic heart complications.

Recent evidence has indicated that treatment of diabetic rats with high doses of thiamine (vitamin B1) prevents diabetic retinopathy through inhibition of advanced glycation end product (AGE) formation and other signaling or metabolic pathways (10). AGE accumulation occurs under hyperglycemic and diabetic environments where elevated oxidative stress is usually present (10). AGE binds to its cell surface receptor, RAGE, resulting in the activation of postreceptor signaling, generation of intracellular reactive oxygen species, and alteration of gene expression (13, 23). The finding that thiamine interrupts AGE formation (10) may be related to a so-called "AGE-breaker" effect, that is, the ability to cleave dicarbonyl bonds formed during advanced glycation (26). The clinical potential of thiamine in diabetic therapeutics is consistent with the notion that this vitamin may be depleted as a result of diabetes-induced oxidative stress or glucose autoxidation, where thiamine is oxidized into the biologically nonfunctional products thiochrome and oxodihydrothiochrome (17). Deficiencies in B series vitamins and folic acid are among the key causative factors leading to diabetic organ damage, consistent with the high incidence of idiopathic dilated cardiomyopathy in patients with vitamin deficiency (25). Group B vitamins, which may be acquired through dietary intake, are important water-soluble vitamins essential for DNA synthesis and repair. To further examine the impact of thiamine supplementation on cardiomyocyte function in diabetes, Type 1 experimental diabetes was induced with a single injection of streptozotocin (STZ) in FVB albino mice. Both control and diabetic mice received 14-day oral treatment of benfotiamine, a lipophilic derivative of thiamine. State-of-the-art cell physiological techniques were employed to examine the mechanical and intracellular Ca2+ properties of isolated ventricular myocytes.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 ACKNOWLEDGMENTS
 REFERENCES
 
Experimental animals and benfotiamine therapy.   The procedures described here were approved by the Institutional Animal Care and Use Committee of the University of Wyoming (Laramie, WY) and were in accordance with National Institutes of Health animal care standards. Eight-week-old male FVB albino mice (16–20 g) were injected with a single dose of STZ (200 mg/kg ip in 0.01 M citrate buffer with a pH of 4.3) (9). Nondiabetic control mice (weight-matched) received citrate buffer only. Fasting plasma glucose was examined after 3 and 14 days of STZ injection, and diabetes was confirmed by fasting plasma glucose value of 13.9 mM or higher using ACCU-CHEK Advantage Glucometer (Boehringer Mannheim Diagnostics, Indianapolis, IN). On day 15 of STZ or citrate injection, both diabetic and nondiabetic control mice were randomly divided into two experimental groups, with only one group being gavaged with benfotiamine (100 mg·kg–1·day–1) for 2 wk. Selection of the benfotiamine dose (100 mg·kg–1·day–1) and duration of treatment were chosen based on previously published data for the thiamine derivative (2, 3, 10). Mice were maintained on a 12:12-h light-dark cycle and were allowed access to food and water ad libitum. A cohort of confirmed diabetic mice (without benfotiamine treatment) was killed on day 15 to evaluate cardiomyocyte function. All other mice were killed 4 wk after STZ or citrate injection. Plasma glucose levels were measured again at the time of death and were included in Table 1.


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  		Table 1. General features of control or streptozotocin-induced diabetic mice with or without benfotiamine treatment

 
Ventricular myocyte isolation procedures.   Hearts were rapidly removed from anesthetized mice and immediately mounted on a temperature-controlled (37°C) Langendorff perfusion system. After perfusion with modified Tyrode solution (Ca2+ free) for 2 min, the heart was digested for 10 min with 0.9 mg/ml collagenase D (Boehringer Mannheim Biochemicals) in modified Tyrode solution. The modified Tyrode solution (pH 7.4) contained the following (in mM): 135 NaCl, 4.0 KCl, 1.0 MgCl2, 10 HEPES, 0.33 NaH2PO4, 10 glucose, and 10 butanedione monoxime, and the solution was gassed with 5% CO2–95% O2. The digested heart was then removed from the cannula, and the left ventricle was cut into small pieces in the modified Tyrode solution. These pieces were gently agitated, and the pellet of cells was resuspended in modified Tyrode solution and allowed to settle for another 20 min at room temperature, during which time extracellular Ca2+ was added incrementally back to 1.20 mM. Isolated myocytes were used for experiments between 1 and 8 h after isolation. Only rod-shaped myocytes with clear edges were selected for recording of mechanical properties and intracellular Ca2+ transients, as described (8).

Cell shortening/relengthening.   Mechanical properties of ventricular myocytes were assessed by using a SoftEdge MyoCam system (IonOptix, Milton, MA) (8). In brief, cells were placed in a Warner chamber mounted on the stage of an inverted microscope (Olympus IX-70, Olympus Optical, Tokyo, Japan) and superfused (~1 ml/min at 25°C) with a buffer containing the following (in mM): 131 NaCl, 4 KCl, 1 CaCl2, 1 MgCl2, 10 glucose, and 10 HEPES, at pH 7.4. The cells were field stimulated with suprathreshold voltage at a frequency of 0.5 Hz, 3-ms duration, using a pair of platinum wires placed on opposite sides of the chamber connected to a FHC stimulator (FHC, Bowdoinham, ME). The polarity of stimulating electrodes was reversed frequently to avoid buildup of electrolyte by-products. The myocyte being studied was displayed on a computer monitor using an IonOptix MyoCam camera, which rapidly scans the image every 8.3 ms. A SoftEdge software (IonOptix) was used to capture changes in cell length during shortening and relengthening. The subphysiological stimulus frequency (0.5 Hz) was chosen to best exemplify contractile properties, including peak shortening (PS) amplitude, maximal velocity of shortening/relengthening (±dL/dt), time to PS (TPS), and time to 90% relengthening (TR90). Higher stimulus frequencies (>1.0 Hz) cannot provide recording of TPS and TR90 with an excellent fidelity limited by the 8.3-ms time interval between two adjacent data points.

Intracellular fluorescence measurement and sarcoplasmic reticulum Ca2+ load.   A separate cohort of murine myocytes was loaded with fura-2 AM (0.5 µM) for 10 min, and fluorescence measurements were recorded with a dual-excitation fluorescence photomultiplier system (Ionoptix), as described (8). Myocytes were placed on an Olympus IX-70 inverted microscope (Olympus, Tokyo, Japan) and imaged through a Fluor x40 oil objective. Cells were exposed to light emitted by a 75-W lamp and passed through either a 360- or a 380-nm filter (bandwidths were ±15 nm), while being stimulated to contract at 0.5 Hz. Fluorescence emissions were detected between 480 and 520 nm by a photomultiplier tube after first illuminating the cells at 360 nm for 0.5 s then at 380 nm for the duration of the recording protocol (333-Hz sampling rate). The 360-nm excitation scan was repeated at the end of the protocol, and qualitative changes in intracellular Ca2+ concentration were inferred from the ratio of the fluorescence intensity at two wavelengths. Sarcoplasmic reticulum (SR) Ca2+ loading capacity was assessed by rapid puff of caffeine (10 mM)-induced intracellular Ca2+ transient intensity in fura-2-loaded ventricular myocytes. Caffeine triggers release of Ca2+ from SR, the major pool of Ca2+ available to contractile proteins in rodent cardiac muscle. Multiple applications of caffeine were given at 5-min intervals to ensure steady state (11).

Protein carbonyl assay.   To assess cardiac oxidative protein damage, the carbonyl content of protein was extracted from mitochondria and was lysed to prevent proteolytic degradation. Nucleic acids were eliminated by treating the samples with 1% streptomycin sulfate for 15 min, followed by a 10-min centrifugation (11,000 g). Protein was precipitated by adding an equal volume of 20% TCA to protein (0.5 mg) and was centrifuged for 1 min. The TCA solution was removed, and the sample was resuspended in 10 mM 2,4-dinitrophenylhydrazine solution. Samples were incubated at room temperature for 15–30 min. Five hundred microliters of 20% TCA were added, and samples were centrifuged for 3 min. The resultant supernatant was discarded; the pellet was washed in ethanol-ethyl acetate and allowed to incubate at room temperature for 10 min. The samples were centrifuged again for 3 min, and the ethanol-ethyl acetate steps were repeated twice more. The precipitate was resuspended in 6 M guanidine solution and centrifuged for 3 min, and insoluble debris was removed. The maximum absorbance (360–390 nm) was read against blank (2 M HCl), and the carbonyl content was calculated using the molar absorption coefficient of 22,000 M–1·cm–1 (11).

GSH/GSSG assay.   Glutathione levels were determined, and the ratio of GSH to GSSG was used as an indicator for oxidative stress. In brief, samples were homogenized in four volumes (wt/vol) of 1% picric acid. Acid homogenates were centrifuged at 16,000 g (30 min), and supernatant fractions were collected. Supernatant fractions were assayed for total GSH and GSSG by the standard recycling method. The procedure consisted of using one-half of each sample for GSSG determination and the other half for GSH. Samples for GSSG determination were incubated at room temperature with 2 µl of 4-vinyl pyridine per 100-µl sample for 1 h after vigorous vortexing. Incubation with 4-vinyl pyridine conjugates any GSH present in the sample so that only GSSG is recycled to GSH without interference by GSH. The GSSG (as GSH x 2) was then subtracted from the total GSH to determine actual GSH level and GSH-to-GSSG ratio (22).

Western blot analysis of AGE and phospholamban.   Tissue samples from ventricles were removed and homogenized in a lysis buffer containing 20 mM Tris (pH 7.4), 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton, 0.1% SDS, and 1% protease inhibitor cocktail. Samples were then sonicated for 15 s and centrifuged at 12,000 g for 20 min at 4°C. The protein concentration of the supernatant was evaluated using Protein Assay Reagent (Bio-Rad, Hercules, CA). Equal amounts (50 µg protein/lane) of the protein from the tissue extraction or prestained molecular weight markers (Gibco-BRL, Gaithersburg, MD) were separated on 10 or 15% SDS-polyacrylamide gels in a minigel apparatus (Mini-PROTEAN II, Bio-Rad) and then were transferred electrophoretically to nitrocellulose membranes (0.2-µm pore size, Bio-Rad Laboratories). Membranes were incubated for 1 h in a blocking solution containing 5% milk in Tris-buffered saline, and then membranes were washed briefly in Tris-buffered saline and incubated overnight at 4°C with mouse anti-AGE monoclonal (1:1,000) (Trans Genic) and mouse anti-phospholamban monoclonal (1:2,000, provided by Dr. Steven Cala from Wayne State University, Detroit, MI) antibodies. After blots were washed to remove excessive primary antibody binding, blots were incubated for 1 h with horseradish peroxidase-conjugated secondary antibody (1:5,000). The membrane was then exposed to 2 ml of a mixture of luminol plus hydrogen peroxide under alkaline conditions (SuperSignal West Dura Extended Duration Substrate, Pierce, Rockford, IL) for 1 min, and the resulting chemiluminescent reaction was detected by Kodak X-OMAT AR Film (Eastman Kodak, Rochester, NY) (8).

Statistical analyses.   An average of five to seven mice were used per group (control and diabetic with or without benfotiamine) for mechanical and intracellular Ca2+ recordings. For each experimental series, data are presented as means ± SE. Statistical significance (P < 0.05) for each variable was estimated by ANOVA by using Dunnett's test as the post hoc analysis.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 ACKNOWLEDGMENTS
 REFERENCES
 
General features of experimental animals.   Four weeks of STZ treatment significantly increased fasting plasma glucose levels and reduced body weight gain. The absolute organ (heart, liver, and kidney) weight or organ-to-body weight ratio was comparable between control and STZ-induced diabetic groups. Two weeks of benfotiamine treatment (100 mg·kg–1·day–1) did not elicit any significant effect on fasting plasma glucose levels, body, organ weight, or organ-to-body weight ratio in either control or STZ diabetic mouse group (Table 1). Two weeks of STZ treatment produced biometric change similar to those found in 4-wk STZ mice compared with their age-matched nondiabetic controls (data not shown).

Cell shortening and relengthening properties of myocytes.   There was no significant difference in resting cell length of ventricular myocytes from control, 2-wk STZ, or 4-wk STZ diabetic mice, with or without benfotiamine treatment. PS amplitude normalized to resting cell length was similar in ventricular myocytes from all five mouse groups examined. Myocytes from diabetic mice (either 2 or 4 wk of STZ treatment) demonstrated significantly prolonged TPS and TR90 compared with those from control groups, consistent with our laboratory's previous findings (8, 29). Interestingly, 2 wk of benfotiamine treatment completely blunted diabetes-induced prolongation of TPS and TR90. Neither the +dL/dt nor –dL/dt was significantly affected by STZ (2- or 4-wk treatment) or benfotiamine treatment (Fig. 1).



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  		Fig. 1. Contractile properties of ventricular myocytes from control, 2-wk streptozotocin (STZ) diabetic mice, and 4-wk STZ diabetic mice, with or without benfotiamine (BT) treatment at a dose of 100 mg·kg–1·day–1 for 2 wk. A: resting cell length. B: peak shortening (PS; as percentage of resting cell length). C: time to peak shortening (TPS). D: time to 90% relengthening (TR90). E: maximal velocity of shortening (+dL/dt). F: maximal velocity of relengthening (–dL/dt). Values are means ± SE; n = 85–86 myocytes from 5–7 mice per group. *P < 0.05 vs. corresponding control group; #P < 0.05 vs. 4-wk STZ diabetic group.

 
Intracellular Ca2+ transient properties and SR Ca2+ load.   We used the membrane-permeant form of fura-2 AM to evaluate the properties of intracellular Ca2+ transients in cardiomyocytes from control, 2-wk STZ, or 4-wk STZ diabetic mice. The time course of fluorescence signal decay was fitted to a single-exponential equation, and the time constant was calculated as a measure of intracellular Ca2+ clearing rate. The fluorescence measurements revealed that both 2- and 4-wk STZ-induced diabetes significantly elevated resting intracellular Ca2+ level, reduced SR Ca2+ release, and prolonged intracellular Ca2+ decay rate without affecting rise of intracellular Ca2+ in response to electrical stimulus. Benfotiamine treatment restored diabetes-induced reduction in SR Ca2+ release and intracellular Ca2+ clearing rate without affecting resting intracellular Ca2+ level. Last but not least, benfotiamine itself suppressed the rise of intracellular Ca2+ in response to electrical stimulus in control myocytes (Fig. 2).



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  		Fig. 2. Intracellular Ca2+ properties of ventricular myocytes from control, 2-wk STZ diabetic mice, and STZ diabetic mice with or without BT (100 mg·kg–1·day–1 for 2 wk) treatment. A: resting intracellular Ca2+ levels. B: increase in intracellular Ca2+ in response to electrical stimuli. C: intracellular Ca2+ transient decay rate. D: sarcoplasmic reticulum (SR) Ca2+ release. Qualitative change in intracellular Ca2+ concentration was inferred from the ratio of the fluorescence intensity at 2 wavelengths (360/380). Values are means ± SE; n = 57–58 myocytes from 5–7 mice per group (n = 17–18 cells per group for D). *P < 0.05 vs. corresponding control group; #P < 0.05 vs. 4-wk STZ diabetic group.

 
Effect of benfotiamine on oxidative stress, protein carbonyl formation, and age accumulation in control and diabetic mouse hearts.   Diabetes is often associated with enhanced oxidative stress, leading to irreversible damage of membrane proteins or lipids (27). The GSH and GSSG levels are commonly used markers for oxidative stress. A low GSH-to-GSSG ratio suggests increased oxidative stress. Results in Fig. 3 indicate that 4-wk STZ treatment-induced diabetes displayed significantly lowered GSH-to-GSSG ratio, suggesting enhanced cardiac oxidative stress. Protein carbonyl formation, an indicator of protein damage, was not altered by 4-wk STZ-induced diabetes or benfotiamine. Two-week STZ treatment also lowered GSH-to-GSSG ratio without affecting protein carbonyl formation, similar to those elicited by 4-wk STZ treatment (data not shown). Because AGE accumulation may serve as a main source for oxidative stress (13, 23), the levels of AGE were evaluated by immunoblot. In agreement with elevated oxidative stress in the STZ-induced diabetic group, the level of AGE formation was increased significantly in 4-wk STZ treatment-induced diabetic hearts. However, benfotiamine treatment failed to alter AGE accumulation in either the control or 4-wk STZ treatment-induced diabetic group (Fig. 4). We also evaluated the effect of benfotiamine on expression of phospholamban, a key intracellular Ca2+ regulatory protein. Our data shown in Fig. 4 indicated that expression of phospholamban was not affected by 4-wk STZ treatment. Benfotiamine treatment itself did not affect expression of phospholamban in control mice, although it significantly attenuated expression of the cardiac regulatory protein under diabetes (Fig. 4). Two weeks of STZ elicited similar influence on AGE accumulation or phospholamban expression, comparable to those found after 4-wk STZ treatment (data not shown).



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  		Fig. 3. Protein carbonyl (A) and GSH-to-GSSG ratio (B) in ventricular tissues from control or STZ-induced diabetic mice with or without BT (100 mg·kg–1·day–1 for 2 wk) treatment. Values are means ± SE; n = 7–12 samples per group. *P < 0.05 vs. corresponding control group.

 


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  		Fig. 4. Western blot analysis of advanced glycation end product (AGE; A) and phospholamban (PLB; B) in ventricular tissues from control or STZ-induced diabetic mice, with or without BT (100 mg·kg–1·day–1 for 2 wk) treatment. Blots show actual gel blotting using anti-AGE and anti-PLB antibodies. C, control; C+B, control+BT; D, diabetic; D+B, diabetic+BT. Values are means ± SE; n = 5–7 samples per group. *P < 0.05 vs. control group.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 ACKNOWLEDGMENTS
 REFERENCES
 
Our study reported for the first time beneficial effects of the lipophilic derivative of thiamine, benfotiamine, on diabetic cardiac dysfunction. In addition, we found that benfotiamine-elicited beneficial effects for diabetic heart dysfunction may be related, at least in part, to reduced oxidative stress but less likely AGE accumulation in diabetic hearts. Our results did not favor any major role of cardiac protein damage or Ca2+ regulatory protein phospholamban in benfotiamine-exerted beneficial effects in diabetes-associated cardiac dysfunction.

Prolonged duration of both contraction and relaxation duration is considered a hallmark of diabetic cardiomyopathy (12, 20, 27). Somewhat similar to our earlier observations using chemically induced or genetic models of diabetes (8, 19, 21), our present study demonstrated prolonged duration of shortening (TPS) and relengthening (TR90) associated with normal PS amplitude and ±dL/dt after only 2 wk of diabetes induction by STZ. These mechanical dysfunctions, which persist through 4 wk of STZ treatment seen in our study, are consistent with our findings of impaired intracellular Ca2+ homeostasis shown as reduced intracellular Ca2+ clearing rate and SR Ca2+ load in diabetic cardiomyocytes. These mechanical defects may be reversed with a 2-wk treatment of benfotiamine, while the thiamine derivative itself exerted little effect on cardiac mechanics in control cardiomyocytes. Several mechanisms have been postulated for the mechanical and intracellular Ca2+ defects in diabetic hearts. Diabetes has been demonstrated to compromise myofilament Ca2+ sensitivity (12, 19) and function of SR Ca2+-ATPase, phospholamban, or Na+/Ca2+ exchange (6, 8). Although the mechanism(s) behind the reduced rise in intracellular Ca2+ in response to electrical stimuli in the control mouse group (Fig. 2B) is unknown, it may be speculated that benfotiamine is capable of promoting myofilament Ca2+ responsiveness, since it maintains peak myocyte shortening amplitude, despite reduced intracellular Ca2+ rise in control myocytes (Fig. 2B). Although short-term STZ treatment has been shown to transiently up- or downregulate certain intracellular Ca2+ regulatory proteins to either diminish or reconcile cardiac contractile function in diabetes (7), our data failed to observe any effect of either diabetes or benfotiamine treatment on phospholamban expression (although the reason behind benfotiamine-induced reduction of phospholamban expression in diabetic hearts is unclear at the present time). Nevertheless, involvement of other Ca2+ regulatory proteins in the beneficial effects of benfotiamine should not be excluded at this time.

Our present study revealed that benfotiamine treatment counters diabetes-induced cardiac mechanical dysfunction at the cellular level, associated with reduction in oxidative stress but not AGE formation or cardiac protein carbonyl formation. This apparent discrepancy in benfotiamine-elicited action on AGE formation and oxidative stress (GSH-to-GSSG ratio) seems to indicate that other mechanism(s) may predominantly contribute to diabetes-induced oxidative stress and cardiac contractile dysfunction in current experimental setting. Possible candidates may include alteration in glucose metabolism and protein kinase C activation (5, 20), although further study is warranted to verify involvement of these signaling pathways and beneficial effects of benfotiamine against diabetic cardiomyopathy. In addition, benfotiamine may directly participate in glucose metabolic regulation. Thiamine and benfotiamine were demonstrated to activate the pentose phosphate pathway enzyme transketolase, facilitating conversion of glyceraldehyde-3-phosphate and fructose-6-phosphate into pentose-5-phosphates (10, 17). Finally, the observation that benfotiamine treatment failed to produce any overt cardiac hypertrophic response should indicate that ventricular remodeling is unlikely to be a concern for the clinical application of benfotiamine. It is noteworthy that we failed to observe cardiac hypertrophy following STZ treatment in our present study. Although STZ has been reported to induce cardiac hypertrophy and does so largely through genomic adaptive or maladaptive process (7), the period of diabetes employed in our present study (4 wk) may not be long enough to induce such cardiac remodeling process.

Experimental limitations.   Our study employed a short-term (2–4 wk) STZ-induced Type 1 diabetic model. This diabetic model may not represent the most prevalent chronic Type 2 diabetes. Further work using ob/ob and db/db Type 2 diabetic models is warranted to provide convincing evidence regarding the therapeutic effectiveness of benfotiamine against diabetic cardiomyopathy. In addition, the potential direct cardiac toxicity of STZ (28) may jeopardize the understanding of therapeutic value of the thiamine derivative in diabetic heart complications. Our present short-term diabetic setting may obscure the long-term or delayed effect of benfotiamine on cardiac AGE accumulation. Longitudinal effect of benfotiamine on diabetic complications should markedly increase our understanding of the therapeutic potential of this thiamine derivative. Therefore, no precise and conclusive statement should be drawn at this point regarding the role of AGE accumulation in benfotiamine treatment regimen. Finally, mechanical and intracellular Ca2+ properties were measured in isolated ventricular myocytes, whereas others (protein carbonyl formation, GSH/GSSG, and immunoblots) were assessed in ventricular tissue. Inclusion of endothelial cells and fibroblasts in ventricular tissues may make it rather difficult to compare data obtained from tissues with those from isolated cardiomyocytes.

In summary, our findings revealed that benfotiamine treatment antagonizes impaired cardiomyocyte contractile function in STZ-induced diabetic mouse hearts, associated with reduced oxidative stress, but is unlikely to be dependent on AGE formation or cardiac protein damage. Given what we know about the ability of benfotiamine, thiamine, and other B series vitamins to promote cell survival and cardiac performance (1–4, 17), the in-depth mechanism of action and clinical value of employing benfotiamine in the treatment of diabetic heart diseases warrants further investigation.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
ACKNOWLEDGMENTS
 REFERENCES
 
This work was supported in part by American Diabetes Association Grant 7–00-RA-21, National Institute on Alcohol Abuse and Alcoholism Grant R15 AA-13575–01, and American Heart Association Pacific Mountain Affiliate Grant 0355521Z to J. Ren. S. Wu was a visiting scholar supported by the China Scholarship Council (Guiyang, Guizhou Province, PR China).


   ACKNOWLEDGMENTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 ACKNOWLEDGMENTS
REFERENCES
 
The authors are grateful to Feng Dong, Karissa H. LaCour, Bonnie H. Ren, and Jesse Zhang for technical assistance.


   FOOTNOTES
 

Address for reprint requests and other correspondence: J. Ren, Division of Pharmaceutical Sciences and Center for Cardiovascular Research and Alternative Medicine, Univ. of Wyoming, Laramie, WY 82071 (e-mail: jren{at}uwyo.edu)

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.

* A. F. Ceylan-Isik and S. Wu contributed equally to this work. Back


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 ACKNOWLEDGMENTS
 REFERENCES
 

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