In vivo [U-13C]glucose labeling to assess heart metabolism in murine models of pressure and volume overload

Fig. 1.. Time course of 13C labeling in mouse hearts following [U-13C]glucose infusion. Mice were infused with [U-13C]glucose for different durations as indicated followed by NMR analysis of heart tissue. Time courses show 13C enrichment of glucose at the C-1 position (A), lactate at the C-3 position (B), and alanine at the C-3 position (C). Enrichment at time 0 is assumed to be 0%. The blue bar estimates the time of maximal 13C enrichment. Data are shown as means ± SD with n = 3 mice/analyzed time point or n = 2 where no SD bars are shown.

Fig. 2.. Shunt- and transverse aortic constriction (TAC)-induced cardiac remodeling. A: left ventricular weight-to-tibia length ratio as a marker of hypertrophy (n = 5–6 mice/group). B: relative wall thickness. C: left ventricular ejection fraction. ShamS, control group for Shunt; ShamT, control group for TAC. n = 8–14 mice/group. Data are presented as means ± SE. **P < 0.01 between Shunt/TAC and the respective Sham control. ns, Not significant by unpaired Student’s t test. Gaps in the x-axis indicate that Shunt and TAC experiments were performed and analyzed separately.

Fig. 3.. Percent 13C enrichment of metabolites in the heart following in vivo infusion of [U-13C]glucose in Shunt and transverse aortic constriction (TAC). A: lactate; B: succinate; C: glutamate; D: glutamine; E: alanine; F: glycine; G: aspartate; and H: glucose. ShamS, control group for Shunt; ShamT, control group for TAC. n = 4–5 mice/group. Data are presented as means ± SE. *P < 0.05 and **P < 0.01 between Shunt/TAC and the respective Sham control. ns, Not significant by unpaired Student’s t test. Gaps in the x-axis indicate that Shunt and TAC experiments were performed and analyzed separately.

Fig. 4.. Percent 13C enrichment of metabolites in blood plasma following in vivo infusion of [U-13C]glucose in Shunt and transverse aortic constriction (TAC). A: lactate; B: succinate; C: glutamate; D: glutamine; E: alanine; F: glycine; G: glucose. ShamS, control group for Shunt; ShamT, control group for TAC. n = 4–5 Mice/group. Data are presented as means ± SE. *P < 0.05 between Shunt/TAC and the respective Sham control. ns, Not significant by unpaired Student’s t test. Gaps in the x-axis indicate that Shunt and TAC experiments were performed and analyzed separately.

Fig. 5.. Effects of Shunt and transverse aortic constriction (TAC) on cardiac glycolysis. A, left: following [U-13C]glucose administration, [1,2,3-13C]lactate is produced through glycolysis. Red circles, 13C-labeled carbons. A, right: 13C enrichment of [1,2,3-13C]lactate in hearts subjected to Shunt, TAC, or respective control surgeries. ShamS, control group for Shunt; ShamT, control group for TAC. B and C: cardiac mRNA expression and protein levels, respectively, of glucose transporters 1 and 4 (Glut1 and Glut4), hexokinase 1 (Hk1), and lactate dehydrogenase A (LDHA). GAPDH served as internal control. n = 4–5 Mice/group. Data are presented as means ± SE. *P < 0.05 and **P < 0.01 or as indicated between Shunt/TAC and the respective Sham controls by unpaired Student’s t test. Gaps in the x-axis indicate that Shunt and TAC experiments were performed and analyzed separately.

Fig. 6.. Effects of Shunt and transverse aortic constriction (TAC) on cardiac tricarboxylic acid (TCA) cycle activity. A, left: following [U-13C]glucose administration, [1,2,3-13C]pyruvate arises from glycolysis, which can enter the TCA cycle via pyruvate dehydrogenase (PDH) or through anaplerotic reactions via pyruvate carboxylase (PC) or malic enzyme (ME). [4,5-13C]glutamate was used as a readout for PDH activity and [2,3-13C]glutamate for anaplerosis. Red circles, 13C-labeled carbons arising from PDH; blue circles, from anaplerotic reactions. A, right: 13C enrichment of [4,5-13C]glutamate and [2,3-13C]glutamate in Shunt, TAC, or respective control hearts. ShamS, control group for Shunt; ShamT, control group for TAC. B: cardiac mRNA expression of pyruvate dehydrogenase-E1α (Pdh-E1α) and pyruvate dehydrogenase kinase-4 (Pdk-4). C: cardiac protein expression of phospho-PDH-E1α (S293, p-PDH-E1α), total PDH-E1α, and PDK-4. D: protein levels of PC. GAPDH served as internal control. n = 4–5 Mice/group. Data are presented as means ± SE. *P < 0.05 and **P < 0.01 between Shunt/TAC and the respective Sham controls. ns, Not significant by unpaired Student’s t test. Gaps in the x-axis indicate that…

Fig. 7.. Effects of Shunt and transverse aortic constriction (TAC) on cardiac glutamine metabolism. A, left: following [U-13C]glucose administration, labeled glutamate arising from pyruvate dehydrogenase (PDH) or anaplerotic reactions can be further processed to glutamine via glutamine synthetase (GS). [4,5-13C]glutamine was used as a readout for glutamine biosynthesis. Red circles, 13C-labeled carbons arising from PDH; blue circles, from anaplerotic reactions. A, right: 13C enrichment of [4,5-13C]glutamine in Shunt, TAC, or respective control hearts. ShamS, control group for Shunt; ShamT, control group for TAC. B: total glutamine concentration in heart extracts. C: protein levels of GS and glutaminase (Gls). D: protein levels of O-GlcNacylation (GlcNAc) and glutamine fructose amidotransferase-2 (GFAT2). n = 4–5 Mice/group. Data are presented as means ± SE. *P < 0.05 and **P < 0.01 between Shunt/TAC and the respective Sham controls. ns, Not significant by unpaired Student’s t test. Gaps in the x-axis indicate that Shunt and TAC experiments were performed and analyzed separately.