Original article
Benfotiamine improves functional recovery of the infarcted heart via activation of pro-survival G6PD/Akt signaling pathway and modulation of neurohormonal response

https://doi.org/10.1016/j.yjmcc.2010.05.014Get rights and content

Abstract

Benfotiamine (BFT) is a transketolase activator that directs glucose to the pentose phosphate pathway. The present study investigated whether BFT improves the recovery after myocardial infarction (MI) and explored underlying mechanisms of protection. Non-diabetic and streptozotocin-induced type 1 diabetic mice were supplemented with BFT (70 mg/kg/day in drinking water) for 4 weeks and then subjected to MI or sham operation. Cardiac function was monitored by echocardiography. At two weeks post-MI, intra-ventricular pressure was measured by Millar tip-catheter and hearts were collected for biochemical, immunohistochemical and expressional analyses. No treatment effect was observed in sham-operated mice. Post-MI mortality was higher in diabetic mice and hemodynamic studies confirmed the worsening effect of diabetes on functional recovery. Furthermore, diabetic mice demonstrated increased cardiomyocyte apoptosis, reduced reparative angiogenesis, larger scars, enhanced oxidative stress, and blunted activation of the pro-survival VEGF receptor-2/Akt/Pim-1 signaling pathway. BFT improved post-MI survival, functional recovery and neovascularization and reduced cardiomyocyte apoptosis and neurohormonal activation in diabetic as well as in non-diabetic mice. In addition, BFT stimulated the activity of pentose phosphate pathway enzymes, leading to reduction of oxidative stress, phosphorylation/activation of VEGF receptor-2 and Akt and increased Pim-1, pBad and Bcl-2 levels. These effects were contrasted on silencing glucose-6-phosphate dehydrogenase, the key enzyme in pentose phosphate pathway, or inhibiting Akt. BFT benefits post-MI recovery through stimulation of pro-survival mechanisms and containment of neurohormonal response. These results may have implications for the treatment of myocardial ischemia.

Research Highlights

► Role of pentose shunt pathway in modulation of the post-MI recovery is not known. ► Results of the present study demonstrates a novel treatment modality for chronic MI through activation of glucose-6-phosphate dehydrogenase and transketolase, the major enzymes involved in the pentose shunt pathway. ► Importantly, this study enlighten the mechanism behind poor recovery after MI in diabetic patients.

Introduction

Myocardial infarction (MI) is the leading cause of death worldwide. Patients with diabetes mellitus (DM) have less favorable outcome after MI [1], [2] or coronary artery revascularization [3], [4], and are at high risk for developing heart failure compared to non-diabetic subjects [5], [6]. Furthermore, DM is known to directly affect myocardial structure and function independently of coronary artery disease [7], [8]. These considerations underscore the urgent need of mechanistic treatments for the cure of MI, especially in the high-risk population of diabetic patients.

Recent studies have highlighted the importance of the pentose phosphate pathway for preservation of cardiomyocyte contractility in ischemia. Under conditions of increased oxidative stress, the activity of glucose-6-phosphate dehydrogenase (G6PD), the rate-limiting enzyme of pentose phosphate pathway, is rapidly increased in cardiomyocytes with consequent neutralization of free radical injury [9]. Furthermore, translocation of G6PD to the plasma membrane of endothelial cells reportedly induces the activation of vascular endothelial growth factor (VEGF) receptor 2 (VEGFR2), protein kinase B (PKB/Akt) and endothelial nitric oxide synthase (eNOS), thereby leading to promotion of angiogenesis [10], [11]. In DM, an impaired activity of G6PD and transketolase, the other pivotal enzyme that shunts glucose metabolites to the pentose pathway, results in depletion of reducing agents and accumulation of glycolysis end-products, which have deleterious effects for cardiovascular cells, such as endothelial cells, mural cells (pericytes and vascular smooth muscle cells) and cardiomyocytes [12], [13]. Of note, similar to diabetic subjects, mice with partial deficiency of G6PD demonstrate impaired angiogenesis and increased myocardial dysfunction following ischemia–reperfusion [10], [14]. However, no information exists on whether induction of G6PD-related anti-oxidative mechanism by ischemia translates in the activation of the VEGFR2/Akt signaling pathway in cardiomyocytes and whether this homeostatic response is maintained or disrupted in DM.

Benfotiamine (BFT), a vitamin B1 analogue and an activator of transketolase, reportedly ameliorates DM-induced vascular complications and healing of ischemic limbs [15], [16], [17], [18]. Furthermore, we showed that BFT prevents DM-induced diastolic dysfunction and heart failure through activation of Akt and its downstream target, proviral integration site for Moloney murine leukemia virus-1 (Pim-1) [19].

The present study investigates whether treatment with BFT protects the heart from ischemic injury and explores cellular and molecular mechanisms of cardioprotection. Results indicate that BFT significantly aids post-MI functional recovery by protecting cardiomyocytes from apoptosis through phosphorylation of VEGFR2 and Akt and activation of Pim-1. Furthermore, BFT contained the excessive activation of neurohormonal systems in the infarcted heart.

Section snippets

Ethics

Experiments were performed in accordance with the Guide for the Care and Use of Laboratory Animals (the Institute of Laboratory Animal Resources, 1996) and with approval of the British Home Office and the University of Bristol.

Experimental protocol

As summarized in Supplemental Fig. 1, 8-week old male CD1 mice (Charles River, UK) were made diabetic using streptozotocin (STZ; 40 mg/kg body weight i.p. daily for 5 days), while age- and gender-matched controls were injected with STZ-vehicle [18]. DM was confirmed by

BFT improves post-MI survival

At 14 days post-MI, only 25% of the diabetic mice in the vehicle group survived as compared to 50% of non-diabetic controls (P < 0.0001, Fig. 1). Treatment with BFT markedly improved the survival of both non-diabetic (80%) and diabetic mice (50%) as compared with the respective vehicle-treated group (P < 0.001).

BFT accelerates post-MI functional recovery

As shown in Table 1, echocardiography measurements before MI induction showed the presence of diastolic dysfunction in diabetic mice, as denoted by significantly decreased E/A ratio (P < 0.01

Discussion

Post-MI mortality and morbidity are remarkable in patients with DM [30], [31]. Different mechanisms have been proposed for the worsened recovery of diabetic infarcted hearts. Data from both diabetic patients [32] and streptozotocin-induced diabetic mice [33] suggest that a consistent loss of myocytes occurs as a consequence of oxidative stress-triggered apoptosis in the area at risk, leading to larger scars and maladaptive remodelling [34]. The present study newly shows that vitamin B1 analogue

Acknowledgments

We thank Professor Takayuki Sato, Kochi Medical School, Japan for his kind scientific advice on markers of neurohormonal activation and Mr. Paul Salvage for his kind technical assistance in immunohistochemical analyses.

Funding sources: This study was supported by a project grant from UK Diabetes Research Foundation (RD06/0003413) and Resolve (Resolve chronic inflammation and achieve healthy ageing, 202047). CE holds a British Heart Foundation (BHF) Basic Science fellowship (BS/05/01).

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