Cardiac phosphatase-deficient 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase increases glycolysis, hypertrophy, and myocyte resistance to hypoxia

Abstract

During ischemia and heart failure, there is an increase in cardiac glycolysis. To understand if this is beneficial or detrimental to the heart, we chronically elevated glycolysis by cardiac-specific overexpression of phosphatase-deficient 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFK-2) in transgenic mice. PFK-2 controls the level of fructose-2,6-bisphosphate (Fru-2,6-P2), an important regulator of phosphofructokinase and glycolysis. Transgenic mice had over a threefold elevation in levels of Fru-2,6-P2. Cardiac metabolites upstream of phosphofructokinase were significantly reduced, as would be expected by the activation of phosphofructokinase. In perfused hearts, the transgene caused a significant increase in glycolysis that was less sensitive to inhibition by palmitate. Conversely, oxidation of palmitate was reduced by close to 50%. The elevation in glycolysis made isolated cardiomyocytes highly resistant to contractile inhibition by hypoxia, but in vivo the transgene had no effect on ischemia-reperfusion injury. Transgenic hearts exhibited pathology: the heart weight-to-body weight ratio was increased 17%, cardiomyocyte length was greater, and cardiac fibrosis was increased. However, the transgene did not change insulin sensitivity. These results show that the elevation in glycolysis provides acute benefits against hypoxia, but the chronic increase in glycolysis or reduction in fatty acid oxidation interferes with normal cardiac metabolism, which may be detrimental to the heart.

Fig. 1

Transgenic hearts overexpress 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFK-2) protein and have elevated fructose-2,6-bisphosphate (Fru-2,6-P₂). A: for the Western blot, cardiac protein was analyzed using rabbit anti-rat liver PFK-2 antibody. Extracts from 3 Mk1 hearts are shown. Similar results were obtained in all Mk transgenic lines. B: for the measurement of Fru-2,6-P₂, hearts were extracted with 50 mM NaOH and estimated using pyrophosphate-dependent fructose-6-phosphate 1-phosphotransferase. Transgenic Fru-2,6-P₂ values were significantly higher than those in FVB hearts (*P < 0.01 by ANOVA). No significant differences were obtained within Mk groups. The number of animals used were 4 FVB, 4 Mk1, 4 Mk2, and 3 Mk5 mice.

Fig. 2

The kinase-active PFK-2 transgene increases glycolysis in Langendorff-perfused hearts. A and C: glycolysis (A) and lactate release (C) without palmitate. B and D: glycolysis (B) and lactate release (D) in the presence of 0.4 mM palmitate. The shaded box shows the addition of 200 μU/ml insulin. Values for FVB and Mk hearts were compared by two-way ANOVA (*P < 0.05). Values are means ± SE for at least 5 mice/time point.

Fig. 3

Effect of palmitate and insulin (Ins) on glycolysis in FVB mice and Mk mice. Preinsulin glycolysis was reduced by 55% (*P < 0.02) in FVB mice (A) but only by 18% in Mk mice (B; P = 0.24). In the presence of insulin, palmitate did not reduce glycolysis in FVB or Mk hearts. C and D: insulin elevated glycolysis in FVB (C) and Mk (D) hearts with or without palmitate (#P < 0.02, preinsulin vs. insulin by paired t-test). The relative effect of insulin was greater in the presence of palmitate because insulin decreased palmitate inhibition of glycolysis. Data are from Fig. 2 at 30 and 80 min.

Fig. 4

The Mk transgene reduces palmitate oxidation in Langendorff-perfused hearts. A: FVB palmitate oxidation values were greater than Mk values at all time points (P < 0.01 by two-way ANOVA, n = 5 FVB and 7 Mk hearts). B: effect of insulin on palmitate oxidation by replotting the 30- and 80-min results from A. Insulin reduced palmitate oxidation by 32% in FVB hearts and by 42% in Mk hearts (#P < 0.01 by paired t-test).

Fig. 5

Overexpression of kinase-active PFK-2 improves contractility under hypoxia. Cardiomyocytes were isolated and incubated under normoxic or hypoxic conditions. The contractile properties of ventricular myocytes from FVB control and Mk transgenic mouse hearts was measured by video-based edge detection. Graphs show the percentage of shortening under normoxic (A) and hypoxic (B) conditions as well as the maximal velocities of cell shortening and relengthening (±dL/dt) under normoxia (C) and hypoxia (D). Values are means ± SE for 60 – 80 cells from 4 mice/group. *P < 0.02, Mk vs. FVB by Student’s t-test.

Fig. 6

Myocardial infarct size (INF) was determined after 40 min of in vivo coronary occlusion and 24 h of reperfusion. The area at risk (AAR) with respect to the left ventricle (LV) was not significantly different between FVB wild-type and Mk transgenic mice, indicating consistent execution of the surgical protocol between the two groups. The amount of nonviable myocardium, according to triphenyltetrazolium negativity, was also similar between the two groups. This was true whether INF was expressed relative to the AAR (INF/AAR) or to the entire LV (INF/LV). P = not significant (NS) for all comparisons. n = 6 mice/group.

Fig. 7

Real-time PCR showed no elevation of brain natriuretic peptide (A) and β-myosin heavy chain (B) mRNA in MK transgenic mice. RQ, relative quantity of each mRNA normalized to the standard mRNA, ND4. Values are means ± SE for 4 mice/group. Values were not significantly different by Student’s t-test.

Fig. 8

Fibrosis in Mk hearts. Cardiac morphology was visualized by hematoxylin and eosin (H&E) staining, and collagen accumulation was visualized by Sirius red staining at a magnification of ×40. A: representative staining of FVB and Mk hearts. B: average (±SE) scores for Sirius red staining from 60 photographs taken from 3 FVB and 3 Mk mouse hearts. Staining was rated by a blinded observer on a scale of 0–2, where 0 indicates mild interstitial accumulation of collagen, 1 indicates increased interstitial accumulation of collagen, and 2 severe interstitial accumulation of collagen. Values are means ± SE and were analyzed by Student’s t-test (*P < 0.01).