Impaired cytosolic NADH shuttling and elevated UCP3 contribute to inefficient citric acid cycle flux support of postischemic cardiac work in diabetic hearts

Abstract

Diabetic hearts are subject to more extensive ischemia/reperfusion (ISC/REP) damage. This study examined the efficiency of citric acid cycle (CAC) flux and the transfer of cytosolic reducing equivalents into the mitochondria for oxidative support of cardiac work following ISC/REP in hearts of c57bl/6 (NORM) and type 2 diabetic, db/db mouse hearts. Flux through the CAC and malate-aspartate shuttle (MA) were monitored via dynamic (13)C NMR of isolated hearts perfused with (13)C palmitate+glucose. MA flux was lower in db/db than NORM. Oxoglutarate malate carrier (OMC) was elevated in the db/db heart, suggesting a compensatory response to low NADHc. Baseline CAC flux per unit work (rate-pressure-product, RPP) was similar between NORM and db/db, but ISC/REP reduced the efficiency of CAC flux/RPP by 20% in db/db. ISC/REP also increased UCP3 transcription, indicating potential for greater uncoupling. Therefore, ISC/REP induces inefficient carbon utilization through the CAC in hearts of diabetic mice due to the combined inefficiencies in NADHc transfer per OMC content and increased uncoupling via UCP3. Ischemia and reperfusion exacerbated pre-existing mitochondrial defects and metabolic limitations in the cytosol of diabetic hearts. These limitations and defects render diabetic hearts more susceptible to inefficient carbon fuel utilization for oxidative energy metabolism.

Keywords: Mitochondria; Oxoglutarate–malate carrier; Reducing equivalents; Uncoupling protein; db/db mouse malate–aspartate shuttle.

Figure 1

Representative spectra from a perfused db/db heart after 8 min ischemia followed by 10 minutes reperfusion. A. Dynamic-mode ¹³C spectra from isolated db/db heart perfused with 0.4 mM [4,6,8,10,12,14,16 – ¹³C₇] palmitate + 10 mM unlabeled glucose. Spectra display progressive ¹³C enrichment of glutamate over 30 minutes. The signals from the 4-, 3-, and 2-carbons of glutamate produced by oxidation of ¹³C palmitate, at 34, 28, and 56 ppm respectively (C4, C3, and C2). C. ¹³C NMR spectrum of extract from db/db heart perfused with ¹³C palmitate + 10 mM unlabeled glucose. The resonance signals from the 4- and 3-carbons of glutamate at 34 and 28 ppm respectively (C4 and C3) are produced by oxidation of ¹³C palmitate and display multiplets from J_13C-_13C coupling due to glutamate isotopomer formation. Note the absence of ¹³C enriched glutamine at 32 ppm the spectrum, which displays enrichment at only the 1.1% natural abundance level that indicates no significant conversion of ¹³C glutamate to glutamine in the heart.

Figure 2

Malate/Aspartate Shuttle Activity. C57bl/6: N = 6; c57bl/6 + ISC/REP: N = 6; db/db: N = 6; db/db + ISC/REP: N = 7. *P < 0.05 vs. c57bl/6; † P < 0.05 vs. c57bl6 ISC/REP.

Figure 3

Myocardial Oxoglutarate Malate Carrier (OMC). A. Myocardial OMC protein content. B. Myocardial OMC mRNA content. C. Western blot from perfused c57bl/6 and db/db tissue with and without ISC/REP. Calsequestrin was used for control protein. *P < 0.05 vs. c57bl/6; † P < 0.05 vs. c57bl6 ISC/REP.

Figure 4

Metabolic Efficiency of Cardiac Function: Citric acid cycle (CAC) flux normalized to rate-pressure product (Vcac/RPP) *P < 0.05, vs. db/db; † P < 0.05 vs. c57bl6 ISC/REP.

Figure 5

UCP2 and UCP3 Content and Expression. A. UCP2 protein content. B. UCP2 mRNA content. C. UCP3 protein content. D. UCP3 mRNA content. E. Western blot for UCP2 from perfused c57bl/6 and db/db tissue with and without ISC/REP. F. Western blot for UCP3 from perfused c57bl/6 and db/db tissue with and without ISC/REP. Calsequestrin was used for control protein. *P < 0.05, vs. c57bl/6; † P < 0.05 vs. c57bl6 ISC/REP; ‡ P < 0.05 vs. db/db.

Figure 6

PPARα Content and Expression. A. PPARα protein content. B. PPARα mRNA content. C. Western blot for PPARα from perfused c57bl/6 and db/db tissue with and without ISC/REP. Calsequestrin was used for control protein. *P < 0.05, vs. c57bl/6; † P < 0.05, vs. c57bl6 ISC/REP.