Cardiac-Specific Deletion of Pyruvate Dehydrogenase Impairs Glucose Oxidation Rates and Induces Diastolic Dysfunction

Figures

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  Figure 1. Generation of cardiac-specific Pdha1-deficient (Pdha1Cardiac−/−) mice. (A) Depiction of the Pdha1 gene (11 exons) in Pdha1WT mice and Pdha1Flox mice. Black triangles depict the loxP sites flanking exon 8 of the Pdha1 gene in the Pdha1Flox mice. (B/C) Cardiac-specific Pdha1-deficient mouse was generated by breeding αMHC-MerCreMer transgenic mice expressing a tamoxifen-inducible Cre in cardiac myocytes with Pdha1flox mice. PCR genotyping of mouse offspring showing presence of Cre(B) as amplification of a 100 base pair fragment, or presence of a floxed Pdha1 or wild-type Pdha1(C) gene as amplification of a 380 base pair or 303 base pair fragment, respectively. Amplification of the Il2r was utilized as a positive control for PCR amplification. (D) Schematic model for induction of cardiac-specific Pdha1 knockout by 6-injections of tamoxifen indicating whether mice were injected in the morning (am) or late afternoon (pm) of the day. Mice were allowed a 5 week washout period following the last tamoxifen injection prior to experimentation. (E-H) Western blot analysis of protein lysates of heart (E), liver (F), gastrocnemius muscle (G), and soleus muscle (H) comparing PDH expression in…

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  Figure 2. Baseline parameters in Pdha1Cardiac−/− mice. (A) Body weights, (B) plasma glucose levels, (C) plasma insulin levels, (D) plasma TAG levels, and (E) plasma FFA levels in Pdha1Cardiac−/− mice and their αMHC-MerCreMer control littermates at 5 weeks post-tamoxifen administration (n = 4). Values represent mean ± SEM.

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  Figure 3. Pdha1Cardiac−/− mice exhibit normal systolic function with evidence of diastolic dysfunction. Ultrasound echocardiography was performed in Pdha1Cardiac−/− mice and their αMHC-MerCreMer control littermates to assess indices of systolic function; (A) left ventricular ejection fraction (LVEF), (B) left ventricular fractional shortening (LVFS), and (C) cardiac output (CO). (D) Diastolic function was assessed via measurement of the mitral E/A ratio. Left ventricular wall dimensions were assessed via measurement of posterior wall thickness during systole (LVPWs), (F), posterior wall thickness during diastole (LVPWd), (G), anterior wall thickness during systole (LVAWs), and (H) anterior wall thickness during diastole (LVAWd). Values represent means ± SEM (n = 6–7). Differences were determined using an unpaired, two-tailed Student’s t-test. *P < 0.05.

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  Figure 4. Altered myocardial energy metabolism and normal ex vivo cardiac function in Pdha1Cardiac−/− mice. (A) Glucose oxidation rates, (B) palmitate oxidation rates, (C) cardiac output, (D) left ventricular (LV) work, (E) aortic flow, (F) coronary flow, (G) aortic systolic pressure, and (H) aortic diastolic pressure during isolated aerobic working heart perfusions from Pdha1Cardiac−/− and their αMHC-MerCreMer control littermates (n = 4, 5). Values represent means ± SEM. Differences were determined using an unpaired, two-tailed Student’s t-test. *P < 0.05.

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  Figure 5. Pdha1Cardiac−/− mice exhibit cardiac hypertrophy. Heart weight was normalized to body weight (HW/BW) in Pdha1Cardiac−/− mice and their αMHC-MerCreMer control littermates following completion of 40 min aerobic perfusion. Values represent means ± SEM (n = 5). Differences were determined using an unpaired, two-tailed Student’s t-test. *P < 0.05.

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  Figure 6. Pdha1Cardiac−/− mice exhibit increased sensitivity to Cre recombinase-related mortality due to fatal cardiomyopathy. Survival rates in Pdha1Cardiac−/− mice and their αMHC-MerCreMer control littermates starting from the day of first tamoxifen injection (n = 39). Differences were determined using a Kaplan-Meier survival curve analysis. *P < 0.05.