Genetic deletion of CD38 extend to postischemic myocardial protection through preserved pyridine nucleotides

After establishing similar expression of eNOS in each group, we explored the levels of its substrates/cofactors with I/R injury. Using endothelial permeabilization, we measured the endothelial NADP(H) pool [7, 18]. While baseline levels were similar in both groups, with I/R greater than 80% depletion of NADP(H) occurred in WT coronary endothelium, with only ~ 20% depletion in CD38-/- hearts (Fig. 5A). Thus, NADP(H) depletion in WT hearts is more severe in the endothelium relative to the whole heart, likely due to the much higher expression of CD38 in endothelial cells as noted above [7]. We also showed that while BH4 levels were similar at baseline, BH4 levels recovered over 2-fold higher in CD38-/- hearts compared to WT after 30 minutes of reperfusion (Fig. 5B). With depletion of whole heart and endothelial NADP(H) levels, we reasoned that the activity of NADPH-dependent enzymes in the BH4 recycling and de novo synthesis pathways may also be affected. These are DHFR, which catalyzes the conversion of BH2 to BH4 in the recycling pathway, and SPR which catalyzes the final step in BH4 synthesis by converting 6-pyruvoyl tetrahydrobiopterin to BH4 in the de novo synthesis pathway [43, 44]. Thus, post-ischemic BH4 recycling from BH2, or BH4 synthesis from GTP, could be impaired in WT hearts but protected in CD38-/- hearts due to preserved myocardial and endothelial NADPH levels.

With increased recovery of NADPH and BH4 in CD38-/- hearts, both of which are essential for NO production by eNOS, we hypothesized that increased production of cGMP would occur due to NO binding to and activation of soluble guanylate cyclase (sGC). cGMP levels were similar between groups at baseline, demonstrating normal NOS-dependent function pre-ischemia. Following I/R, however, WT hearts exhibited very low levels ~10-20% of baseline pre-ischemic levels of myocardial cGMP, while levels in CD38-/- hearts were largely preserved (~80% of baseline) (Fig. 5C). Thus, NO signaling through sGC is largely lost in WT hearts but preserved with CD38-/-.

The physiological significance of higher post-ischemic cGMP levels in CD38-/- hearts was evaluated by measuring the change in CF with L-NAME (1 mM) with and without prior liposomal NADPH infusions. We found that there was significantly higher NOS-dependent CF in CD38-/- compared to WT hearts after I/R. Recovery of NOS-dependent CF was approximately 95% in CD38-/- hearts, but only 25% in WT hearts (Fig 5D). However, NADPH supplementation fully restored NOS-dependent CF in WT hearts, with little additional effect in CD38-/- hearts which had intrinsically preserved NADP(H) levels. This is consistent with our previous studies where replenishment of NADPH after I/R led to near complete recovery of NOS-dependent CF, with tandem replenishment of NADPH and BH4 restoring it completely [7]. Buy NMN

Consistent with the above, we found that NO production was severely decreased following I/R in WT hearts but much less so in CD38-/- hearts. This was found along with increased NOS-dependent superoxide production in WT but not in CD38-/- hearts, suggesting that CD38 knockout prevents post-ischemic NOS uncoupling (Fig 7). Aside from measuring BH4 as a potential mechanism of NOS uncoupling, we also assessed the eNOS monomer:dimer ratio which has been considered as a mechanism of NOS dysfunction and uncoupling, possibly regulated by BH4 availability [27, 29, 45, 46]. Consistently, we found increased monomer:dimer ratio following I/R in WT hearts, perhaps due to the lower BH4 levels found in these hearts after I/R. Further supporting this, we found that the increased monomer:dimer occurring with I/R was largely prevented in the CD38-/- hearts which have higher post-ischemic BH4 levels (Fig 6).

Another mechanism of NOS uncoupling is due to the S-glutathionylation of eNOS, a post-translational modification (PTM) of cysteine residues determined in large part by the glutathione redox state [47, 48]. Interestingly, we found higher myocardial levels of GSH at baseline in CD38-/- compared to WT hearts. While I/R had a similar effect in each with around 30% depletion, the resulting GSH levels were higher in CD38-/- hearts. In the endothelium, where NADP(H) is more severely depleted, GSH recovery was higher in CD38-/- compared to WT hearts (Fig 8B). As the reduction of oxidized glutathione (GSSG) to GSH by glutathione reductase is NADPH-dependent [30], the higher concentration of NADPH throughout I/R could help maintain GSH levels minimizing the formation and loss of GSSG that is accompanied by wash out from the heart. This could further lead to the preservation of the cellular glutathione pool that was observed in the CD38-/- hearts. Further studies on post-ischemic PTMs of eNOS will be needed to determine how they may be modulated by CD38 activation and how this further contributes to NOS dysfunction and/or uncoupling.

With regard to the effect of CD38 knockout on physiological function, we observed that the recovery of total CF, similar to NOS-dependent CF, was higher in CD38-/- hearts (Fig. 9D). This accompanied the increased recovery of LV contractile function, with higher LVDP and RPP and significantly reduced post-ischemic LVEDP in CD38-/- hearts (Fig. 9A, B, C). Strong protection against I/R injury was also evidenced by the dramatic reduction in enzyme release (both myoglobin and CK), and LV infarct size seen in CD38-/- hearts, where only small infarction occurred compared to much larger infarction in WT hearts (Figs. 10 and 12). Measurement of the release of PTBP1, a cytosolic endothelial protein, demonstrated negligible endothelial cell death following I/R (Fig. 11). Therefore, while some depletion of NAD(P)(H) in the whole heart could be attributed to cell washout, as observed with myoglobin and CK, cellular washout of NAD(P)(H) from the endothelium would be negligible. Thus, endothelial cell depletion of NAD(P)(H) is due primarily to CD38-mediated depletion.