Dapagliflozin Improves Cardiac Hemodynamics and Mitigates Arrhythmogenesis in Mitral Regurgitation-Induced Myocardial Dysfunction

Abstract

Background Mitral regurgitation (MR) is a major contributor for heart failure (HF) and atrial fibrillation. Despite the advancement of MR surgeries, an effective medical therapy to mitigate MR progression is lacking. Sodium glucose cotransporter 2 inhibitors, a new class of antidiabetic drugs, has shown measurable benefits in reduction of HF hospitalization and cardiovascular mortality but the mechanism is unclear. We hypothesized that dapagliflozin (DAPA), a sodium glucose cotransporter 2 inhibitor, can improve cardiac hemodynamics in MR-induced HF. Methods and Results Using a novel, mini-invasive technique, we established a MR model in rats, in which MR induced left heart dilatation and functional decline. Half of the rats were randomized to be administered with DAPA at 10 mg/kg per day for 6 weeks. After evaluation of electrocardiography and echocardiography, hemodynamic studies were performed, followed by postmortem tissue analyses. Results showed that DAPA partially rescued MR-induced impairment including partial restoration of left ventricular ejection fraction and end-systolic pressure volume relationship. Despite no significant changes in electrocardiography at rest, rats treated with DAPA exhibited lower inducibility and decreased duration of pacing-induced atrial fibrillation. DAPA also significantly attenuated cardiac fibrosis, cardiac expression of apoptosis, and endoplasmic reticulum stress-associated proteins. Conclusions DAPA was able to suppress cardiac fibrosis and endoplasmic reticulum stress and improve hemodynamics in an MR-induced HF rat model. The demonstrated DAPA effect on the heart and its association with key molecular contributors in eliciting its cardio-protective function, provides a plausible point of DAPA as a potential strategy for MR-induced HF.

Keywords: ER stress; SGLT2 inhibitor; apoptosis; cardiac fibrosis; dapagliflozin; heart failure; mitral regurgitation.

Conflict of interest statement

None.

Figures

Figure 1. The establishment of mitral regurgitation (MR) in rats using echo‐guided mini‐invasive puncture (22G needle) technique.

A, An illustration of echocardiography guided approach of MR creation using a needle puncture via left ventricular apex. B, At parasternal long axis view, the mitral regurgitant jet backward into left atrium was observed immediately after the puncture using color Doppler echocardiography. C, In the postmortem study, 1 significant hole was noticed at the anterior leaflet of the mitral valve (arrow).

Figure 2. The study design of dapagliflozin on cardiac remodeling in rats with mitral regurgitation (MR).

After the surgery of MR, the rats received dapagliflozin at 10 mg/kg per day for 6 weeks orally. The heart rate, blood pressure, and cardiac function were observed by echocardiography every 2 weeks. At the end of the experiments, the section of heart were stained using Masson trichrome for the quantification of myocardial fibrosis. The apoptotic effect in heart tissue was measured by TUNEL (terminal transferase‐mediated dUTP nick‐end labeling) staining. The apoptosis‐ and endoplasmic reticulum stress‐associated protein were measured by Western blot. DAPA indicates dapagliflozin; and PV, pressure‐volume.

Figure 3. Dapagliflozin (DAPA) partially rescued mitral regurgitation (MR)‐induced myocardial dysfunction in echocardiographic assessments.

A, Sequential measurements of echocardiography are shown in the Sham, Sham+DAPA, MR, and MR+DAPA groups; (B) Echocardiographic measurements of left atrium (LA) diameter, (C) aorta diameter, (D) left ventricular internal dimension at end‐diastole (LVIDd), (E) interventricular septal thickness at end‐diastole (IVSd), (F) ejection fraction (EF), (G) fractional shortening (FS), and (H) vena contracta (VC) are shown for each group. Data are expressed using mean±SD. Kruskal‐Wallis test and Dunn's post hoc analysis for analysis of group differences. *P<0.05 for difference from each group. (N=6–12).

Figure 4. Dapagliflozin (DAPA) attenuated mitral regurgitation (MR)‐induced hemodynamic decline in rats.

A, Representative pressure‐volume images in the Sham, MR, and MR+DAPA groups were obtained from Millar catheterization. Hemodynamic measurements obtained in the 3 groups are shown in (B) the mean end‑systolic volume (Ves), end‑diastolic volume (Ved), end‐systolic pressure (Pes), and end‐diastolic pressure (Ped), (C) maximal velocity of pressure rise (+dP/dt) and fall (−dP/dt), (D) mean arterial elastance (Ea), the time constant of isovolumic pressure decay (tau), (E) mean slopes of the ESPVR and the EDPVR are shown for each group. Data are presented as the means± SD. Kruskal‐Wallis test and Dunn's post hoc analysis for analysis of group differences. *P<0.05 for difference from each group. (N=6). EDPVR, end‐diastolic pressure‐volume relationship; ESPVR, end‐systolic pressure‐volume relationship.

Figure 5. Dapagliflozin (DAPA) suppressed the MR‐triggered atrial fibrillation (AF) and the upregulation of arrhythmogenic protein expressions in rats.

A, The illustration of retrograde perfusion, ECG recordings, and programmed atrial burst pacing for AF provocation. B, Representative AF episodes after atrial burst pacing are shown in the Sham, MR, and MR+DAPA groups. C, The inducibility and (D) duration of AF, in each group, was measured. E, The expression of pRyR2/RyR2 ratio and Connexin 43 was evaluated by western blot. The relative expression level of each protein was quantified by densitometry and normalized to the GAPDH (glyceraldehyde 3‐phosphate dehydrogenase) level. Data are expressed using mean± SD. Kruskal‐Wallis test and Dunn's post hoc analysis for analysis of group differences. *P<0.05, and **P<0.01 for difference from each group. (N=3–6).

Figure 6. Dapagliflozin (DAPA) attenuated mitral regurgitation (MR)‐induced cardiac injury and fibrosis.

Effects of dapagliflozin on (A) survival rate in the Sham, MR, and MR+DAPA groups. B, Representative images of harvested hearts. C, The quantitative analysis of heart weight/body weight, (D) heart weight/tibial length, (E) the wet to dry lung weight ratio, (F) hematoxylin‐eosin staining of heart sections, and (G) Masson trichrome staining of left ventricular tissue in indicated groups; scale bars, 100 µm. H, Quantification of cardiac fibrosis in indicated groups of rat. Data are expressed using mean± SD. Kruskal‐Wallis test and Dunn's post hoc analysis for analysis of group differences. *P<0.05, and* ***P<0.0001 for difference from each group. (N=6–12).

Figure 7. Dapagliflozin (DAPA) suppressed cardiomyocyte apoptosis with mitral regurgitation (MR).

A, Representative apoptotic cells by terminal deoxynucleotidyl transferase–mediated UTP nick‐end labeling (TUNEL) analysis (upper panel). Quantification of cardiac apoptosis in indicated groups of rats (lower panel). (Green: TUNEL, Red: F‐actin, Blue: DAPI). B, Expression of apoptosis associated protein were measured by Western blot. Representative BAX, cleaved caspase 3/caspase 3 ratio, and BCL2 expression in the left ventricle in each group of rats (upper panel). The relative expression level of each protein was quantified by densitometry and normalized to the GAPDH (glyceraldehyde 3‐phosphate dehydrogenase) level (lower panel). Data are expressed using mean± SD. Kruskal‐Wallis test and Dunn's post hoc analysis for analysis of group differences. *P<0.05, **P<0.01, and ***P<0.001 for difference from each group. (N=3–6). DAPI indicates 4', 6‐diamidino‐2‐phenylindole; TUNEL, terminal deoxynucleotidal transferase–mediated biotin–deoxyuridine triphosphate nick‐end labeling; and UTP, uridine triphosphate.

Figure 8. Dapagliflozin (DAPA) attenuates mitral regurgitation (MR) induced endoplasmic reticulum (ER) stress in hearts of rats with MR.

The expression of ER stress‐associated proteins were measured by Western blot. Representative GRP78 (glucose‐regulated protein 78), CHOP (C/EBP‐homologous protein), p‐PERK (phospho‐protein kinase RNA‐like endoplasmic reticulum kinase)/PERK ratio, eIF2α (eukaryotic translation initiation factor 2α), and ATF4 (activating transcription factor 6) in the left ventricles in each group of rats (left panel). The relative expression level of each protein was quantified by densitometry and normalized to the control level (right panel). Data are expressed using mean± SD. Kruskal‐Wallis test and Dunn's post hoc analysis for analysis of group differences. *P<0.05, **P<0.01 for difference from each group. (N=3–6).

Figure 9. The summary of a postulated regulatory mechanism of action of dapagliflozin is shown to highlight its effect on improvement of mitral regurgitation (MR)‐induced cardiomyopathy.

In left atrium, through differentially regulating critical proteins‐ by reducing RyR2 levels and increasing Connexin 43 expression, Dapagliflozin partially suppresses the inducibility of atrial fibrillation. In left ventricle, Dapagliflozin protects against MR induced apoptosis through suppressing ER stress pathway. AF indicates atrial fibrillation; ER, endoplasmic reticulum; LA, left atrium; and LV, left ventricle.