In our studies we found that stabilized expression of HIF-1α in heart led to better recovery of function and less tissue death after 30 minutes of global ischemia, via mechanisms that preserve the mitochondrial polarization. Our group previously showed that HIF-1α conferred ischemic tolerance by allowing cardiomyocytes to use fumarate as an alternative terminal electron acceptor to sustain anaerobic mitochondrial polarization. The source of fumarate was identified as the purine nucleotide cycle (PNC). Here we discovered that HIF-1α upregulates AMP deaminase 2 (AMPD2), the entry point to the PNC. The combination of glycolysis and the PNC may protect the heart's nucleotide resources. We subsequently examined the effects that HIF-1α exerts on nucleotide metabolism in the ischemic heart. We found that HIF-1α expression reduces adenosine accumulation in the ischemic heart. As ATP is depleted during ischemia, AMP accumulates. Our results suggest that AMP metabolism is shunted towards AMPD2 rather than the adenosine producing 5'-nucleotidase pathway. Subsequently, we treated hearts with the PNC inhibitor hadacidin followed by 30 minutes of global ischemia. Inclusion of hadacidin reduced ATP and adenylate energy charge in the hearts. These findings allow us to propose that activity of the PNC prevents the F0F1 ATP synthase from consuming glycolytic ATP in order to maintain mitochondrial polarization during ischemia. Thus, the PNC provides ATP sparing effects and preserves the energy charge in the ischemic heart. The fact that ATP and adenylate energy charge is better preserved during the initial 20 minutes of ischemia in HIF-1α expressing hearts is supportive of our observation that HIF-1α upregulates the PNC. HIF-1α also upregulates adenosine deaminase, which degrades adenosine. The limitation of adenosine accumulation may help HIF-1α expressing hearts avoid toxicity due to chronic adenosine exposure. Finally, we found that HIF-1α induces the expression of the nucleotide salvage enzyme hypoxanthine phosphoribosyl transferase (HPRT). Upon reperfusion HPRT serves to reincorporate the nucleotide degradation product, hypoxanthine, into the adenylate pool and may prevent the production of reactive oxygen species. Collectively, HIF-1α robustly protects the heart from ischemic stress and it upregulates several pathways whose cardioprotective role may extend beyond the remodeling of nucleotide metabolism.

These studies have examined the effect of hypoxia inducible factor 1α (HIF-1α) on nucleotide metabolism in the ischemic heart using a genetic mouse model with heart-specific and regulated expression of a stable form of HIF-1α. We find that AMP deaminase (AMPD), the entry point of the purine nucleotide cycle (PNC), is induced by HIF-1α at the level of mRNA, protein, and activity. AMP that accumulates during ischemia can be metabolized to adenosine by 5'-nucleotidase or to IMP by AMPD. Consistent with the finding of AMPD induction, adenosine accumulation during ischemia was much attenuated in HIF-1α-expressing hearts. Further investigation of nucleotide salvage enzymes found that hypoxanthine phosphoribosyl transferase (HPRT) is also upregulated in HIF-1α-expressing hearts. Treatment of hearts with an inhibitor of the PNC, hadacidin, hastens the fall of the adenylate energy charge during ischemia and the accumulation of AMP. The results provide new insight into the role of the PNC in the heart, especially as it relates to ischemia, and indicate that HIF-1α regulates nucleotide metabolism as a compensatory response to hypoxia.