Canagliflozin Suppresses Atrial Remodeling in a Canine Atrial Fibrillation Model

Figures

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  Figure 1. Schematic of the study protocol. Each dog underwent initial surgery and was allowed to recover for 1 week without pacing before the start of atrial rapid pacing (Day 0). Atrial rapid pacing (400 bpm) was performed for 3 weeks in the pacing control and pacing+CAN groups. In the pacing+CAN group, CAN (3 mg/kg per day) was orally administered from Day −3 until Day 21. Atrial tissue was sampled from each dog at the end of the study period. CAN indicates canagliflozin; EPS, electrophysiological study; and LA, left atrium.

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  Figure 2. Changes in the AERP over the time course in the groups with and without the CAN administration. During rapid atrial pacing, gradual AERP shortening were observed in the pacing control group. On the other hand, the AERP shortening was smaller in the pacing+CAN group than in the pacing control group and this difference tended to become significant at Day 21 ([A], ΔAERP for 3 weeks, pacing control vs pacing+CAN; P=0.06 [generalized linear mixed model]). The points‐sets of closed circles and triangles connected by dashed lines indicate the individuals of the control and treatment groups, respectively. Furthermore, (B) indicated absolute values of AERP. In hierarchical statistical analyses, the ΔAERP was modeled with Gaussian distribution and an identity link function. Each model included the date and condition (treated or not) as fixed effects and their interaction and the subject ID as random effects (ie, random intercept mixed model). See text for the details. AERP indicates atrial effective refractory period; AF, atrial fibrillation; and CAN, canagliflozin.

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  Figure 3. Changes in CV over the time course in the groups with and without the CAN administration. The ΔCV was evaluated as the reciprocal indirectly by measuring the delta conduction time as above although the delta conduction time was expressed as conduction data. The pacing control group exhibited a gradual CV decrease during the 3‐week rapid atrial pacing protocol. In contrast, the degree of CV decrease was smaller in the pacing+CAN group than in the pacing control group and the difference became significant at Day 21 ([A], ΔCV for 3 weeks, pacing control vs pacing+CAN; P=0.009 [generalized linear mixed model]). The points‐sets of closed circles and triangles connected by dashed lines indicate the individuals of the control and treatment groups, respectively. Furthermore, (B) indicated absolute values of CV. In hierarchical statistical analyses, ΔCV was modeled with Gaussian distribution and an identity link function. Each model included the date and condition (treated or not) as fixed effects and their interaction and the subject ID as random effects (ie, random intercept mixed model). See text for the details. AF indicates atrial fibrillation; CAN, canagliflozin; and CV, conduction…

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  Figure 4. Changes in the inducibility of AF over the time course in the left atrium with and without the CAN administration. To evaluate AF inducibility, the incidence of AF induction was evaluated with atrial burst pacing for 3 seconds at the minimal pacing cycle length necessary to achieve 1:1 atrial capture at the LA pacing site. When AF was induced, its duration was measured and AF inducibility was calculated as the ratio (%) of successful AF inductions to the total number of AF induction trials with atrial burst pacing. The AF inducibility gradually increased in the pacing control group, while such an increase was suppressed in the pacing+CAN group, and the difference became significant at Day 21 (% AF inducibility for 3 weeks, pacing control vs pacing+CAN; P=0.011 [generalized linear mixed model]). The AF inducibility was modeled with binomial distribution and a logit link function. Each model included the date and condition (treated or not) as fixed effects and their interaction and the subject ID as random effects (ie, random intercept mixed model). See text for the details. AF indicates atrial fibrillation; and CAN, canagliflozin.

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  Figure 5. Carbohydrate metabolism evaluation. There was no significant difference in the FBS level between the pacing control and pacing+CAN groups (A). In contrast, the levels of plasma ketone bodies, including TKBs, AA, and β‐HA, were higher in the pacing+CAN group than in the pacing control group (B through D). The points‐sets of closed circles and triangles connected by dashed lines indicate the individuals of the control and treatment groups, respectively. In hierarchical statistical analyses, response variables—specifically, log‐transformed FBS, TKBs, AA, and β‐HA—were modeled with Gaussian distribution and an identity link function. Each model included the date and the condition (treated or not) as fixed effects and the subject ID as the random effect. AA indicates acetoacetic acid; CAN, canagliflozin; FBS, fasting blood glucose; TKBs, total ketone bodies; and β‐HA, β‐hydroxybutyric acid.

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  Figure 6. Histological findings in the left atrial tissue. Histological changes, such as the irregularity of cardiomyocytes and interstitial fibrosis, were advanced in the pacing control group when compared with in the nonpacing group. In contrast, the degree of tissue fibrosis was suppressed in the pacing+CAN group relative to in the pacing control group (A). The degree of fibrosis (% area of fibrotic tissue on Azan staining) at the end of the study protocol was higher in the pacing control group than in the nonpacing group (P<0.05) and lower in the pacing+CAN group than in the pacing control group (P<0.05) (B). In hierarchical statistical analysis, the response variable, log‐transformed % area of fibrotic tissue on Azan staining, was modeled with Gaussian distribution and an identity link function. The fixed effect and the random effect were the experimental conditions and subject ID, respectively. CAN indicates canagliflozin; and HE, hematoxylin and eosin.

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  Figure 7. Immunofluorescent staining of DHE in the left atrial tissue. DHE is clearly observable in the myocardial nucleus (indicated by solid arrows) in the pacing control group when compared with the non‐pacing group and could be localized by a merged image of DHE and DAPI staining. As this expression is negated by PEG‐SOD, it is considered to reflect enhanced ROS expression. In contrast, this expression of DHE was suppressed in the pacing+CAN group relative to in the pacing control group (A). The NII (an indicator of ROS expression) was higher in the pacing control group than in the nonpacing group (P<0.05) and lower in the pacing+CAN group than in the pacing control group (P<0.05) (B). The ΔNII was calculated by subtracting the mean NII of the non‐pacing group. In hierarchical statistical analysis, the response variable, ΔNII, was modeled with Gaussian distribution and an identity link function. The fixed effect and the random effect were the experimental conditions and subject ID, respectively. CAN indicates canagliflozin; DAPI, 4,6‐diamidino‐2‐phenylindole; DHE, dihydroethydium; NII, nuclear immunofluorescent intensity; PEG‐SOD, polyethylene glycol–superoxide dismutase; and ROS,…