Post‐ischemic Cardiac Protection by NADPH Supplementation or CD38 Inhibition is Dependent on Endothelial Nitric Oxide Synthase (eNOS)
Authors: Boslett James , Zweier Jay L. 
Published: November 2023

Introduction

Recently, we showed that CD38 activation in myocardial ischemia/reperfusion (I/R) injury causes depletion of the NADP(H) pool of the heart with near total depletion from the cardiac endothelium (1). This in turn causes decreased NO production from eNOS, limiting endothelial‐dependent vasodilation. Inhibiting CD38 protected the heart with higher recovery of endothelial vasodilatory function, left ventricular contractile function and decreased infarction. To test the importance of eNOS in this myocardial protection, hearts from WT and eNOS‐deficient (eNOS−/−) mice were studied with and without pre‐ischemic treatment with NADPH or CD38 inhibitor a‐NAD.

Methods

Isolated hearts of WT and eNOS−/− mice were subjected to one of three treatments prior to 30 min ischemia/120 min reperfusion: vehicle control (Krebs' buffer), liposomal NADPH (175 mM), or CD38 inhibitor a‐NAD (5 mM). Contractile function and coronary flow were monitored during experiments with an intraventricular balloon and Doppler flow probe, respectively. After 120 min reperfusion, hearts were stained with 2,3,5‐triphenyltetrazolium chloride (TTC) for measurement of infarct size. In a parallel set of WT and eNOS−/− hearts after 30 min ischemia/30 min reperfusion, hearts were infused with 175 mM Liposomal NADPH to determine the acute effects of NADPH after I/R on coronary flow.

Results

In WT hearts, either NADPH or α‐NAD treatment enhanced the recovery of contractile function, with higher left ventricular developed pressure (LVDP) and lower LV end diastolic pressure (LVEDP) compared to control hearts (Table). Recovery of coronary flow (CF) was also improved by the treatments. In eNOS−/− hearts, no significant changes were seen with either treatment, though recovery of LVDP and CF were slightly higher in a‐NAD‐treated and NADPH‐treated compared to control hearts. Liposomal NADPH decreased infarct size only in WT but not in eNOS−/− hearts (Table). α‐NAD decreased infarct size significantly in WT hearts with only a small statistically insignificant effect in eNOS−/− hearts (P<0.23). With post‐ischemic NADPH infusions after 30 min reperfusion, only WT hearts responded with acute increases in coronary flow (~30% of baseline), while there was no effect of NADPH on CF in eNOS−/− hearts.

Conclusion

Utilizing eNOS−/− hearts, we show that CD38 inhibition or pre‐ischemic administration of NADPH protects the heart in an eNOS‐dependent manner. In WT hearts, either a‐NAD or NADPH treatment increase the recovery of left ventricular function and decrease infarct size after 30 min ischemia and reperfusion. Thus, CD38‐mediated endothelial dysfunction contributes to myocardial injury and infarction in the postischemic heart.

Cardiac function and infarct size values after 30 min ischemia

Background and Treatment
LVDP (% of baseline)
LVEDP (mmHg)
CF (% of baseline)
Infarct Size (% of LV)

WT
12.7 ± 2.4
73.0 ± 3.4
58.3 ± 2.9
46.1 ± 5.8

WT+α‐NAD
28.9 ± 5.1**
55.8 ± 6.3*
87.8 ± 5.0**
32.6 ± 3.9*

WT + NADPH
22.3 ± 3.4*
57.5 ± 2.4**
73.5 ± 4.1**
32.5 ± 2.2*

eNOS−/−
16.8 ± 4.1
72.9 ± 4.1
60.5 ± 5.6
32.8 ± 3.2

eNOS−/−+α‐NAD
24.0 ± 2.6
72.3 ± 3.7
72.9 ± 4.9
26.0 ± 3.4

eNOS−/− + NADPH
17.3 ± 3.4
68.6 ± 4.6
76.0 ± 8.0
32.6 ± 1.7