The PKD inhibitor CID755673 enhances cardiac function in diabetic db/db mice

Abstract

The development of diabetic cardiomyopathy is a key contributor to heart failure and mortality in obesity and type 2 diabetes (T2D). Current therapeutic interventions for T2D have limited impact on the development of diabetic cardiomyopathy. Clearly, new therapies are urgently needed. A potential therapeutic target is protein kinase D (PKD), which is activated by metabolic insults and implicated in the regulation of cardiac metabolism, contractility and hypertrophy. We therefore hypothesised that PKD inhibition would enhance cardiac function in T2D mice. We first validated the obese and T2D db/db mouse as a model of early stage diabetic cardiomyopathy, which was characterised by both diastolic and systolic dysfunction, without overt alterations in left ventricular morphology. These functional characteristics were also associated with increased PKD2 phosphorylation in the fed state and a gene expression signature characteristic of PKD activation. Acute administration of the PKD inhibitor CID755673 to normal mice reduced both PKD1 and 2 phosphorylation in a time and dose-dependent manner. Chronic CID755673 administration to T2D db/db mice for two weeks reduced expression of the gene expression signature of PKD activation, enhanced indices of both diastolic and systolic left ventricular function and was associated with reduced heart weight. These alterations in cardiac function were independent of changes in glucose homeostasis, insulin action and body composition. These findings suggest that PKD inhibition could be an effective strategy to enhance heart function in obese and diabetic patients and provide an impetus for further mechanistic investigations into the role of PKD in diabetic cardiomyopathy.

Fig 1. Cardiac dysfunction in db/db mice.

(A) Physiological and cardiac morphological measures; (B) Representative M-mode and Doppler images; (C) LV Structural dimensions; (D) E:A ratio; (E) Deceleration time; (F) Ejection time; (G) Ejection fraction, and; (H) Fractional shortening in db/- and db/db mice. Data represented are mean ± SEM, n = 7–8/group. * Denotes significantly different from db/- mice (p<0.05). IVS—intraventricular septum thickness; LVID—left ventricular internal diameter; LVPW—left ventricular posterior wall thickness.

Fig 2. Assessment of PKD activation in db/db mice.

(A) Representative western blots; (B) S916 PKD1 phosphorylation; (C) S916 PKD2 phosphorylation; (D) Representative western blots; (E) S23/24 cTn1 phosphorylation; (F) S498 HDAC5 phosphorylation in db/- and db/db mice in the fed or 4 hr fasted state, and; (G) Heat map of a PKD- dependent gene expression signature in 4 hr fasted db/db mice. Expression values are relative to db/- mice. Data represented are mean ± SEM, n = 7–8/group.

Fig 3. Acute CID755673 administration reduces PKD activity in a time and dose-dependent manner.

(A) Representative western blots; (B) S916 PKD1 phosphorylation; (C) S916 PKD2 phosphorylation in the ventricles of wild type C57BL6 mice 1 hr after drug administration. (D) Representative western blots; (E) S916 PKD1 phosphorylation; (F) S916 PKD2 phosphorylation in the ventricles of wild type C57BL6 mice 4 hr after drug administration. Data represented are mean ± SEM, n = 3/group. * Denotes significantly different from vehicle-treated mice (p<0.05).

Fig 4. The PKD inhibitor CID755673 enhances cardiac function in db/db mice.

(A) Physiological and cardiac morphological measures; (B) Representative M-mode and Doppler images; (C) LV Structural dimensions; (D) E:A ratio; (E) Deceleration time; (F) Ejection time; (G) Ejection fraction, and; (H) Fractional shortening in db/db mice treated with vehicle, 1mg/kg or 10mg/kg CID755673. Data represented are mean ± SEM, n = 7–8. † Denotes significantly different from vehicle mice (p<0.05). # Denotes significantly different from 1mg/kg CID755673-treated mice. Veh—vehicle; IVS—intraventricular septum thickness; LVID—left ventricular internal diameter; LVPW—left ventricular posterior wall thickness.

Fig 5. Assessment of PKD activation in CID755673-treated db/db mice.

(A) Heat map of a PKD- dependent gene expression signature; (B) Representative western blots; (C) S23/24 cTn1 phosphorylation; (D) S498 HDAC5 phosphorylation; (E) KCNH2 gene expression in vehicle, 1mg/kg and 10mg/kg CID755673-treated db/db mice. Expression values are relative to db/- mice. Data represented are mean ± SEM, n = 7–8/group. * Denotes significantly different from vehicle-treated mice (p<0.05).

Fig 6. The CID755673-mediated reduction in heart size is not specific for the LV.

(A) Representative cell size images and quantification; (B) Estimated gross LV mass, and; (C) LV mass normalised to heart weight in db/db mice treated with vehicle, 1mg/kg or 10mg/kg CID755673. Data represented are mean ± SEM, n = 3–8/group. † Denotes significantly different from vehicle mice (p<0.05).