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

Abstract

Obesity and type 2 diabetes (T2D) increase the risk for cardiomyopathy, which is the presence of ventricular dysfunction in the absence of underlying coronary artery disease and/or hypertension. As myocardial energy metabolism is altered during obesity/T2D (increased fatty acid oxidation and decreased glucose oxidation), we hypothesized that restricting myocardial glucose oxidation in lean mice devoid of the perturbed metabolic milieu observed in obesity/T2D would produce a cardiomyopathy phenotype, characterized via diastolic dysfunction. We tested our hypothesis via producing mice with a cardiac-specific gene knockout for pyruvate dehydrogenase (PDH, gene name Pdha1), the rate-limiting enzyme for glucose oxidation. Cardiac-specific Pdha1 deficient (Pdha1^Cardiac-/-) mice were generated via crossing a tamoxifen-inducible Cre expressing mouse under the control of the alpha-myosin heavy chain (αMHC-MerCreMer) promoter with a floxed Pdha1 mouse. Energy metabolism and cardiac function were assessed via isolated working heart perfusions and ultrasound echocardiography, respectively. Tamoxifen administration produced an ~85% reduction in PDH protein expression in Pdha1^Cardiac-/- mice versus their control littermates, which resulted in a marked reduction in myocardial glucose oxidation and a corresponding increase in palmitate oxidation. This myocardial metabolism profile did not impair systolic function in Pdha1^Cardiac-/- mice, which had comparable left ventricular ejection fractions and fractional shortenings as their αMHC-MerCreMer control littermates, but did produce diastolic dysfunction as seen via the reduced mitral E/A ratio. Therefore, it does appear that forced restriction of glucose oxidation in the hearts of Pdha1^Cardiac-/- mice is sufficient to produce a cardiomyopathy-like phenotype, independent of the perturbed metabolic milieu observed in obesity and/or T2D.

Keywords: cardiac function; diabetic cardiomyopathy; diastolic dysfunction; glucose oxidation; pyruvate dehydrogenase.

Figure 1

Generation of cardiac-specific Pdha1-deficient (Pdha1^{Cardiac−/−}) mice. (A) Depiction of the Pdha1 gene (11 exons) in Pdha1^WT mice and Pdha1^Flox mice. Black triangles depict the loxP sites flanking exon 8 of the Pdha1 gene in the Pdha1^Flox 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 Pdha1^flox 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 Pdha1^{Cardiac−/−} mice (4) versus their various control littermates [Wild-type mice (1), Pdha1^Flox mice (2), and αMHC-MerCreMer mice (3)] (n = 4–7). Values represent mean ± SEM. Differences were determined using 1-way ANOVA followed by a Bonferroni post-hoc analysis. *P < 0.05, significantly different versus all other genotypes.

Figure 2

Baseline parameters in Pdha1^{Cardiac−/−} mice. (A) Body weights, (B) plasma glucose levels, (C) plasma insulin levels, (D) plasma TAG levels, and (E) plasma FFA levels in Pdha1^{Cardiac−/−} mice and their αMHC-MerCreMer control littermates at 5 weeks post-tamoxifen administration (n = 4). Values represent mean ± SEM.

Figure 3

Pdha1^{Cardiac−/−} mice exhibit normal systolic function with evidence of diastolic dysfunction. Ultrasound echocardiography was performed in Pdha1^{Cardiac−/−} 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.

Figure 4

Altered myocardial energy metabolism and normal ex vivo cardiac function in Pdha1^{Cardiac−/−} 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 Pdha1^{Cardiac−/−} 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.

Figure 5

Pdha1^{Cardiac−/−} mice exhibit cardiac hypertrophy. Heart weight was normalized to body weight (HW/BW) in Pdha1^{Cardiac−/−} 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.

Figure 6

Pdha1^{Cardiac−/−} mice exhibit increased sensitivity to Cre recombinase-related mortality due to fatal cardiomyopathy. Survival rates in Pdha1^{Cardiac−/−} 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.