Ketogenic Diet Regulates Cardiac Remodeling and Calcium Homeostasis in Diabetic Rat Cardiomyopathy

Figures

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  Figure 1. Effects of a ketogenic diet (KD) on echocardiograms in the control rats (control), diabetes mellitus (DM) rats on a normal diet (DM + ND), and DM rats on the KD (DM + KD). Average data of the left ventricular internal dimension at end-diastole (LVIDd), left ventricular internal dimension at end-systole (LVIDs), end-diastolic volume (EDV), end-systolic volume (ESV), ejection fraction (EF), fractional shortening (FS), and left ventricular (LV) mass. Number of rats (N) = 7 per group. Statistical significance was assessed using a one-way analysis of variance (ANOVA) with Tukey’s honest significant difference (HSD) test. ** p < 0.01; *** p < 0.005.

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  Figure 2. Changes in the electrocardiogram and action potentials (Aps) of ventricular myocytes in the control rats (control), diabetes mellitus (DM) rats on a normal diet (DM + ND), and DM rats on the ketogenic diet (DM + KD). (A) Average data and representative tracings of the electrocardiograms before and after treatment in ventricular myocytes of the control (N = 7), DM + ND (N = 7), and DM + KD (N = 7) rats. (B) Average data and representative AP tracings in the ventricular myocytes of the control (n = 13), DM + ND (n = 18), and DM + KD (n = 16). Abbreviations: QT: QT interval, RR: RR interval, QTc: corrected QT interval, N: number of rats, n: number of cardiomyocytes isolated from the rats, RMP: resting membrane potential, APA: action potential amplitude, APD20, APD50, and APD90: action potential durations of repolarization at 20%, 50%, and 90%, respectively, number of rats (N) = 7 per group, n: number of cardiomyocytes isolated from the rats. Statistical significance was assessed using a one-way analysis of variance (ANOVA) with Tukey’s honest significant difference (HSD) test. * p < 0.05; *** p < 0.005.

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  Figure 3. [Ca2+]i transients, Ca2+ stores measured from caffeine (20 mM)-induced Ca2+ transients, and Ca2+ leak in the ventricular myocytes of the control rats (control), diabetes mellitus (DM) rats on a normal diet (DM + ND), and DM rats on the ketogenic diet (DM + KD). (A) Average data and representative tracings of [Ca2+]i transients and decay time of [Ca2+]i transients in the ventricular myocytes of the control (n = 37), DM + ND (n = 25), and DM + KD (n = 29) rats. (B) Average data and representative tracings of Ca2+ stores in the ventricular myocytes of the control (n = 31), DM + ND (n = 20), and DM + KD (n = 17) rats. (C) Average data and representative tracing of the incidence and frequency of Ca2+ leak in the ventricular myocytes of the control (n = 11), DM + ND (n = 12), and DM + KD (n = 19) rats. Abbreviations: [Ca2+]i: intracellular calcium, Ca2+:calcium, n: number of cardiomyocytes isolated from the rats. Statistical significance was assessed using a one-way analysis of variance (ANOVA) with Tukey’s honest significant difference (HSD) test. * p < 0.05; *** p < 0.005.

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  Figure 4. ICa-L and NCX current in the ventricular myocytes of the control rats (control), diabetes mellitus (DM) rats on a normal diet (DM + ND), and DM rats on the ketogenic diet (DM + KD). (A) Representative tracings of ICa-L current and the current–voltage relationship of ICa-L in the ventricular myocytes of the control (n = 16), DM + ND (n = 14), and DM + KD (n = 13) rats. (B) Representative tracings of NCX current and the current–voltage relationship of NCX current in the ventricular myocytes of the control (n = 12), DM + ND (n = 21), and DM + KD (n = 12) rats. Abbreviations: ICa-L: L-type calcium current, NCX: sodium/calcium exchanger, n: number of cardiomyocytes isolated from the rats. Statistical significance was assessed using a one-way analysis of variance (ANOVA) with Tukey’s honest significant difference (HSD) test. # p < 0.05 vs. the controls; ## p < 0.01 vs. the controls; ### p < 0.005 vs. the controls; ** p < 0.01 vs. DM + KD rats; *** p < 0.005 vs. DM + KD rats.

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  Figure 5. INa-Late and Na+/H+ exchanger current in the ventricular myocytes of the control rats (control), diabetes mellitus (DM) rats on a normal diet (DM + ND), and DM rats on the ketogenic diet (DM + KD). (A) Average data and representative tracing of INa-Late current in the ventricular myocytes of the (a) control (n = 17), (b) DM + ND (n = 16), and (c) DM + KD (n = 13) rats. (B) Average data and representative tracings of Na+/H+ exchanger current in the ventricular myocytes of the control (n = 11), DM + ND (n = 14), and DM + KD (n = 14) rats. Abbreviations: INa-Late: late sodium current, Na+/H+ exchanger: sodium/hydrogen exchanger, n: number of cardiomyocytes isolated from the rats. Statistical significance was assessed using a one-way analysis of variance (ANOVA) with Tukey’s honest significant difference (HSD) test. ** p < 0.01 vs. control; * p < 0.05 vs. DM + KD.

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  Figure 6. Oxidative stress and cytosolic sodium (Na+) levels in the ventricular myocytes of the control rats (control), diabetes mellitus (DM) rats on a normal diet (DM + ND), and DM rats on the ketogenic diet (DM + KD). (A) Average data of cytosolic ROS levels and examples of cytosolic ROS in the ventricular myocytes of the control (n = 31), DM + ND (n = 54), and DM + KD (n = 24) rats. Scale bar, 10 μm. (B) Average data of cytosolic Na+ levels and an example in the ventricular myocytes of the control (n = 22), DM + ND (n = 42), and DM + KD (n = 42) rats. Scale bar, 10 μm Abbreviations: ROS: reactive oxygen species, n: number of cardiomyocytes isolated from the rats. Statistical significance was assessed using a one-way analysis of variance (ANOVA) with Tukey’s honest significant difference (HSD) test. * p < 0.05; *** p < 0.005.

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  Figure 7. Calcium regulating proteins in the ventricular myocytes of the control rats (control), diabetes mellitus (DM) rats on a normal diet (DM + ND), and DM rats on a ketogenic diet (DM + KD). Average data and representative immunoblots of (A) pRyR2 at serine 2808/RyR2 ratio (N = 6 per group), (B) pCaMKIIδ/CaMKII-δ (N = 6 per group), (C) pPLB-S16/PLB (N = 6 per group), (D) NCX (N = 5 per group), (E) SERCA2a (N = 6 per group), and (F) Cav1.2 (N = 6 per group) in the ventricular myocytes of the control, DM + ND, and DM + KD rats. Densitometry was normalized to GAPDH as the internal control. Abbreviations: RyR2: ryanodine receptor 2, pRyR2: phosphorylated RyR2, NCX: sodium/calcium exchanger, SERCA2a: sarcoplasmic reticulum calcium ATPase, Cav1.2: voltage-dependent L-type alpha 1C subunit, CaMKII-δ: calcium/calmodulin-dependent protein kinase II, pCaMKII-δ: phosphorylated CaMKII-δ, PLB: phospholamban, pPLB: phosphorylated PLB. N: number of rats. Statistical significance was tested using a one-way analysis of variance (ANOVA) with Tukey’s honest significant difference (HSD) test. * p < 0.05, ** p < 0.01, *** p < 0.005.

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  Figure 8. Schematic diagram of the proposed mechanism of action underlying the effects of a KD in DM hearts. A KD may reverse DM-induced Ca2+/Na+ dysregulation by reducing ROS and ionic channel modification in the cardiomyocytes, leading to the correction of prolonged QTc intervals and improvement in diabetic cardiomyopathy. Abbreviations: APD: action potential duration, DM: diabetes mellitus, Ca2+: calcium, Cav1.2: Ca2+ channel, voltage-dependent, L-type alpha 1C subunit, H+: hydrogen, [Na+]i: intracellular sodium, INa-Late: late sodium current, Na+: sodium, NCX: Na+/Ca2+ exchanger, QTc: corrected QT interval, ROS: reactive oxygen species, RyR2: ryanodine receptor 2, pRyR2: phosphorylated RyR2, SR: sarcoplasmic reticulum, SERCA: sarcoplasmic reticulum ATPase, STZ: streptozotocin.

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  Figure 9. Schematic of our experimental design. The IACUC approval number is LAC 2020-0478. Abbreviations: KD: ketogenic diet, DM: diabetes mellitus, Ca2+: calcium, SR: sarcoplasmic reticulum, Na+: sodium.