A SGLT2 inhibitor dapagliflozin suppresses prolonged ventricular-repolarization through augmentation of mitochondrial function in insulin-resistant metabolic syndrome rats

Abstract

Background: Metabolic syndrome (MetS) is a prevalent risk factor for cardiac dysfunction. Although SGLT2-inhibitors have important cardioprotective effects in hyperglycemia, their underlying mechanisms are complex and not completely understood. Therefore, we examined mechanisms of a SGLT2-inhibitor dapagliflozin (DAPA)-related cardioprotection in overweight insulin-resistant MetS-rats comparison with insulin (INSU), behind its glucose-lowering effect.

Methods: A 28-week high-carbohydrate diet-induced MetS-rats received DAPA (5 mg/kg), INSU (0.15 mg/kg) or vehicle for 2 weeks. To validate MetS-induction, we monitored all animals weekly by measuring body weight, blood glucose and HOMO-IR index, electrocardiograms, heart rate, systolic and diastolic pressures.

Results: DAPA-treatment of MetS-rats significantly augmented the increased blood pressure, prolonged Q-R interval, and low heart rate with depressed left ventricular function and relaxation of the aorta. Prolonged-action potentials were preserved with DAPA-treatment, more prominently than INSU-treatment, at most, through the augmentation in depressed voltage-gated K⁺-channel currents. DAPA, more prominently than INSU-treatment, preserved the depolarized mitochondrial membrane potential, and altered mitochondrial protein levels such as Mfn-1, Mfn-2, and Fis-1 as well as provided significant augmentation in cytosolic Ca²⁺-homeostasis. Furthermore, DAPA also induced significant augmentation in voltage-gated Na⁺-currents and intracellular pH, and the cellular levels of increased oxidative stress, protein-thiol oxidation and ADP/ATP ratio in cardiomyocytes from MetS rats. Moreover, DAPA-treatment normalized the increases in the mRNA level of SGLT2 in MetS-rat heart.

Conclusions: Overall, our data provided a new insight into DAPA-associated cardioprotection in MetS rats, including suppression of prolonged ventricular-repolarization through augmentation of mitochondrial function and oxidative stress followed by improvement of fusion-fission proteins, out of its glucose-lowering effect.

Keywords: Diabetes; Electrophysiology; Heart function; Insulin resistance; Oxidative stress; SGLT2 inhibitors.

Fig. 1

General effects of MetS rats treated with either DAPA or INSU. a Body weight (left) and blood glucose level of all groups. b Oral glucose tolerance tests (OGTT) by measuring blood glucose levels before and after orogastric gavage of 1 g/kg glucose administration at 15th min, 30th min, 60th min, 120th min to the rats. c The HOMO-IR index determined as described previously [16]. d The systolic (left) and diastolic (right) pressure changes measured from the tail. e Confirmation of DAPA beneficial effect in MetS rats directly targeting the heart by measuring mRNA levels of SGLT2 in tissue homogenates. Data presenting as mean (± SEM) values. The total number of rats/group; n = 15–17. Significance level at *p < 0.05 vs. CON group and ^#p < 0.05 vs. MetS group

Fig. 2

Assessment of in vivo electrical activity of the hearts from DAPA or INSU administrated Mets rats. a Representative in situ ECG traces and b calculated ECG parameters including the R–R and Q–T intervals from DAPA or INSU treated MetS group compared to either MetS or age-matched control rats. c Heart rate was estimated from ECG traces. The representative pressure changes (inset) of the LVDP changes (d) and the rates of pressure development and decay (± dP/dt) of LVDP (e). Data presenting as mean (± SEM) values. The total number of rats/group/protocol; n = 6–7. Significance level at *p < 0.05 vs. CON group and ^#p < 0.05 vs. MetS group

Fig. 3

Effects of DAPA or INSU treatment on action potential parameters in freshly isolated cardiomyocytes. a Representative single cell action potential traces for all groups. b Action potential duration (APD) at 25, 50, 75, 90% (APD₂₅, APD₅₀, APD₇₅, APD₉₀) of repolarization are examined from DAPA or INSU treated MetS group compared to MetS and age-matched controls. The current–voltage relations (current–voltage, I–V, characteristics) of voltage-dependent Na⁺-channels (c) and L-type Ca²⁺-channels (d) for all groups. The calculated maximum current of Na⁺-channels (− 40 mV) and Ca²⁺-channels (0 mV) are given in the lower parts of the graphs as an inset. The total number of cells/group/protocol; n = 6–7. Significance level at *p < 0.05 vs. CON group and ^#p < 0.05 vs. MetS group

Fig. 4

Effects of DAPA or INSU treatment on the voltage-dependent repolarizing K⁺-channel currents in freshly isolated cardiomyocytes. a Demonstration of representative original repolarizing K⁺-channel traces for all groups. The current–voltage relations (I–V characteristics) of K⁺ channels (b) and maximum outward (c, + 70 mV) and inward currents (d, − 120 mV) in DAPA or INSU treated MetS rats compared to MetS rats and age-matched control rats. The total number of cells/group/protocol; n = 12–15. Significance level at *p < 0.05 vs. CON group and ^#p < 0.05 vs. MetS group

Fig. 5

Changes in Ca²⁺ transients in DAPA or INSU treated MetS rats compared to MetS rats and age-matched controls rats in Fura-2AM loaded cardiomyocytes. The basal level of Ca²⁺ in resting cells (a), the representative original Ca²⁺ transients under electrical stimulation (b, left), the amplitude of transients Ca²⁺ changes and their corresponding kinetics under electrical-field stimulation (b, right), the time course of Ca²⁺-transients (c) in DAPA or INSU treated MetS rats compared to MetS rats and age-matched control rats. d Assessment of sarco(endo)plasmic reticulum (SR) Ca²⁺ content obtained following 10 mM caffeine applications. The total number of cells/group/protocol; n = 19–30. Significance level at *p < 0.05 vs. CON group and ^#p < 0.05 vs. MetS group. TP time to peak, DT50 decreasing time to 50% relaxation

Fig. 6

Confocal imaging of intracellular levels of free Na⁺ and H⁺ and analysis of mitochondrial function in DAPA or INSU treated MetS rats compared to MetS rats and age-matched controls. The level of free Na⁺ ([Na]_i) estimated in cells loaded with Na⁺-sensitive SBFI florescent dye (a) and free H⁺ ([H⁺]_i or [pH]_i) with SNARF-1AM (b). The mitochondrial membrane potential (MMP, as FCCP responses) in isolated JC-1 loaded cardiomyocytes (c). The expression levels of mitofusion proteins Mfn-1 and Mfn-2 and their ratio (d). The expression level of mitofission protein Fis-1 (e). The total number of cells/group/protocol; n = 10–19. Significance level at *p < 0.05 vs. CON group and ^#p < 0.05 vs. MetS group

Fig. 7

Assessment of oxidative stress status in cardiomyocytes in DAPA or INSU treated MetS rats compared to MetS rats and age-matched controls. Intracellular ROS ([ROS]_i) (a) and RNS levels ([RNS]_i) (b) imagined with confocal microscopy in cells loaded with either a specific dyes DCFDA or DAF, respectively. Maximal fluorescence intensity was achieved by a HEPES-buffered solution supplemented with H₂O₂ (100 μM) and NO donor ZipNONO, (100 μM). The free and total protein thiol levels (c) and the ADP/ATP ratio (d) in isolated cardiomyocytes. The total number of cells/group/protocol; n = 15–17. Significance level at *p < 0.05 vs. CON group and ^#p < 0.05 vs. MetS group