The Drug Candidate BGP-15 Delays the Onset of Diastolic Dysfunction in the Goto-Kakizaki Rat Model of Diabetic Cardiomyopathy

Abstract

Background and Aims: Diabetic cardiomyopathy (DCM) is an emerging problem worldwide due to an increase in the incidence of type 2 diabetes. Animal studies have indicated that metformin and pioglitazone can prevent DCM partly by normalizing insulin resistance, and partly by other, pleiotropic mechanisms. One clinical study has evidenced the insulin-senzitizing effect of the drug candidate BGP-15, along with additional animal studies that have confirmed its beneficial effects in models of diabetes, muscular dystrophy and heart failure, with the drug affecting chaperones, contractile proteins and mitochondria. Our aim was to investigate whether the inzulin-senzitizer BGP-15 exert any additive cardiovascular effects compared to metformin or pioglitazone, using Goto-Kakizaki (GotoK) rats. Methods: Rats were divided into five groups: (I) healthy control (Wistar), (II) diseased (GotoK), and GotoK rats treated with: (III) BGP-15, (IV) metformin, and (V) pioglitazone, respectively, for 12 weeks. Metabolic parameters and insulin levels were determined at the endpoint. Doppler echocardiography was carried out to estimate diabetes-associated cardiac dysfunction. Thoracotomy was performed after the vascular status of rats was evaluated using an isolated aortic ring method. Furthermore, western blot assays were carried out to determine expression or phosphorylation levels of selected proteins that take part in myocyte relaxation. Results: BGP-15 restored diastolic parameters (e'/a', E/e', LAP, E and A wave) and improved Tei-index compared to untreated GotoK rats. Vascular status was unaffected by BGP-15. Expression of sarco/endoplasmic reticulum Ca²⁺-ATPase (SERCA2a) and phosphodiesterase 9A (PDE9A) were unchanged by the treatments, but the phosphorylation level of vasodilator-stimulated phosphoprotein (VASP) and phospholamban (PLB) increased in BGP-15-treated rats, in comparison to GotoK. Conclusions: Even though the BGP-15-treatment did not interfere significantly with glucose homeostasis and vascular status, it considerably enhanced diastolic function, by affecting the SERCA/phospholamban pathway in GotoK rats. Although it requires further investigation, BGP-15 may offer a new therapeutic approach in DCM.

Keywords: BGP-15; Goto-Kakizaki; diastolic dysfunction; echocardiography; endothelial dysfunction; metformin; pioglitazone; type 2 diabetes.

Figure 1

Metabolic parameters in control (Wistar) and diabetic (GotoK) rats treated with vehicle (GotoK), BGP-15 (GotoK + BGP15), metformin (GotoK + MET) and pioglitazone (GotoK + PIO). (a) calculated body weight gain in per cent; (b) calculated fasting glucose/insulin ratio; (c) calculated homeostasis model assessment-estimated insulin resistance (HOMA-IR) index; (d) calculated HOMA-B index. Values are presented as mean ±SEM; asterisks (*) denotes significance (Kruskal-Wallis test with Dunn’s post-test, n = 6/group, * p < 0.05; ** p < 0.01).

Figure 2

Relaxant effect of acetylcholine (Ach) on the abdominal aorta isolated from Wistar and Goto-Kakizaki rats, and GotoK rats treated orally with BGP-15, metformin or pioglitazone. All aortic rings underwent a pre-contraction elicited by norepinephrine before the administration of Ach. The axis x shows the common logarithm of molar concentration of Ach, while the axis y denotes the effect as a percentage decrease of the initial tension of aortic rings. The symbols represent the effect of Ach averaged within the groups (±SEM). Asterisks indicate the significance level of differences between responses to Ach in GotoK and pioglitazone-treated GotoK rats (* p < 0.05). n = 6/group, one-way ANOVA (with Geisser–Greenhouse correction) followed by Tukey post-testing.

Figure 3

Echocardiographic parameters of rat groups. Data and representative images obtained from healthy control (Wistar) and diabetic GotoK rats treated with vehicle (GotoK), 10 mg/kg BGP-15 (GotoK + BGP15), 100 mg/kg metformin (GotoK + MET) and 10 mg/kg pioglitazone (GotoK + PIO). (a) representative images of septal annular tissue velocities (s′, e′ and a′ waves) of rats, recorded by TDI echocardiography; (b) representative images of mitral inflow velocities (E and A waves), obtained by Doppler imaging, and representative parasternal long axis views of the left ventricle, obtained by M-mode (EF: ejection fraction); (c) graph of septal e′/a′ ratio of treatment groups; (d) calculated E/e′ ratios of rats; (e) graph of mitral valve (MV) atrial (A)-wave velocities; (f) myocardial performance, shown as Tei-index of treatment groups. All data is presented as mean SEM, n = 6/group, Kruskal-Wallis test with Dunn’s post-test. Asterisks denote the level of significance (* p < 0.05; ** p < 0.01).

Figure 4

Expression SERCA2a and PDE9A proteins, with pPLB/PLB and pVASP/VASP ratios, obtained by Western blot. (a) phospho(Ser16)-phospholamban (pPLB) to phospholamban (PLB) ratios in groups of rats; (b) phopsho(Ser239)-vasodilator-stimulated phosphoprotein (pVASP) to vasodilator-stimulated phosphoprotein (VASP) ratios; (c) sarcoplasmic/endoplasmic reticulum calcium ATPase 2a (SERCA2a) levels, normalized to GAPDH as a housekeeping protein; (d) expression level of phosphodiesterase 9A (PDE9A) levels, normalized to GAPDH. All data is presented as mean ±SEM, data was averaged from 3 independent experiment (n = 6/group), Kruskal-Wallis test with Dunn’s post-test. Asterisks denote the level of significance (* p < 0.05; ** p < 0.01).

Figure 5

Flow-chart of study design. After acclimatization, healthy control (Wistar) and diabetic GotoK rats were randomly divided into subgroups, as follows: (I) Wistar control, treated with vehicle; (II) diseased GotoK treated with vehicle (GotoK), (III) GotoK treated with 10 mg/kg BGP-15 (GotoK + BGP15), (IV) GotoK trated with 100 mg/kg metformin (GotoK + MET), and (V) GotoK treated with 10 mg/kg pioglitazone (GotoK + PIO), for 12 weeks. At the endpoint, echocardiography was carried out, serum parameters were measured, and after thoracotomy, aortic rings were cut off for ex vivo experiments, and left ventricle myocardium was frozen for further analyses (Western blot).

Figure 6

Chemical structure of O-(3-piperidino-2-hydroxy-1-propyl)-nicotinic amidoxime, known as BGP-15, a hydroxamic-acid derivative small molecule (the figure was made using ChemDraw Ultra software ver.12., CambridgeSoft, Perkin Elmer, Waltham, MA, USA).

Figure 7

Representative images of echocardiographic experiments. (a) Left-ventricle outflow tract (LVOT), visualized by Doppler echocardiography; (b) parasternal long axis, M-mode image of left ventricle; (c) 3-chamber view, mitral annular velocities, tissue Doppler mode; (d) mitral inflow velocities, visualized by Doppler echocardiography.