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Genetic deletion of CD38 confers post-ischemic myocardial protection through preserved pyridine nucleotides
Isolated heart perfusion
Isolated heart experiments were performed as described previously . Male, C57Bl/6 mice (control) or CD38-/- mice weighing 25-30 g were heparinized and injected with 100 mg/kg ketamine and 15 mg/kg xylazine intraperitoneally. Hearts were excised, cleaned of non- myocardial tissue, cannulated via the aorta and perfused retrogradely in Langendorff mode with Krebs-Henseleit buffer (KHB) (119 mM NaCl, 17 mM glucose, 2 mM sodium pyruvate, 25 mM NaHCO3, 5.9 mM KCl, 1.2 mM MgCl2, 2.5 mM CaCl2, 0.5 mM NaEDTA). A polyvinylchloride balloon connected to a pressure transducer (ADInstruments, Colorado Springs, CO) was placed in the left ventricle to measure left ventricular developed pressure (LVDP), systolic pressure (ESP), left ventricle end diastolic pressure (LVEDP), and heart rate (HR). An inline flow probe (Transonic, Ithaca, NY) measured CF.
CD38 activity assay
CD38 has been reported to function primarily as an NAD(P)+ase through its hydrolysis of NAD(P)+ to 2’-P-ADPR. To measure this enzyme activity specifically, a substrate analog of NAD+, nicotinamide 1,N6-ethenoadenine dinucleotide (-NAD), was used [13, 17]. WT and CD38-/- hearts subjected to control perfusion, 30 min ischemia, or 30 min ischemia/30 min reperfusion were ground in liquid nitrogen and homogenized in buffer containing 150 mM NaCl, 50 mM Tris, 1 mM EDTA, 1% Triton X-100, and freshly added protease inhibitors. Homogenate totaling 100 μg of protein was added to a 200 μl reaction mixture containing 200 μM -NAD. Reactions were monitored for the conversion of -NAD to strongly fluorescent product etheno- ADP-ribose (-ADPR). Fluorescence was measured at an excitation wavelength of 300 nm and an emission wavelength of 410 nm on a Molecular Devices SpectraMax plate reader. NMN
In experiments utilizing heart tissue, hearts were homogenized in 5 volumes of RIPA buffer (150 mM NaCl, 10 mM Tris, 1 mM EDTA, 0.1% SDS, 0.1% sodium deoxycholate, and 1% Triton X- 100) with freshly added protease inhibitors. Homogenates were allowed to incubate on ice for 30 minutes before centrifugation to pellet cell debris. Supernatants were assayed for protein concentration by the detergent-compatible protein assay.
For western blotting of myoglobin and PTBP1 from coronary effluents, effluent was initially collected from hearts in 1 mL fractions and was immediately frozen in liquid nitrogen. We also performed experiments to test the presence of PTBP1 in coronary effluent after permeabilization of the coronary endothelium. In these experiments, coronary effluent was collected after a 25 L
bolus injection of 0.25% Triton X-100 delivered through a septum-sealed sidearm located directly above the heart with a Hamilton syringe. In some cases, frozen coronary effluent samples were completely lyophilized and reconstituted in 200 L 1X sample loading buffer with 10 mM DTT. In other cases, protein in coronary effluent samples was concentrated using Corning Spin-X 5000 molecular weight cut off filters. For experiments assessing the monomer:dimer ratio of eNOS in hearts from WT and CD38-/- hearts, low temperature PAGE (LT-PAGE) with non-reducing conditions was used. Specifically, reducing agents were omitted from the protein samples prior to electrophoresis, and gels were run at 125V at 4°C with a sample SDS concentration of 2%. Protein samples were separated either on 4-20% gradient or 12% Tris-glycine polyacrylamide gels. Protein from gels was transferred to PVDF membranes and blocked for 1 hour at room temperature (RT) with 5% milk in Tris-buffered saline with 0.1%
Tween-20 (TBST). Membranes were incubated overnight at 4°C with anti-CD38 antibody diluted at 1:2000, anti-eNOS antibody diluted at 1:1000, anti-GAPDH antibody diluted at 1:10,000, anti- myoglobin antibody diluted at 1:2000, or anti-PTBP1 antibody diluted at 1:2000. Membranes were then washed in TBST and incubated for 1 hour with HRP-conjugated secondary antibodies in TBST with 5% milk at RT. Imaging was performed with ECL immunoblotting detection reagents. The intensity of blotting was quantified using ImageJ from the NIH.
Immunohistochemistry of CD38
WT and CD38-/- hearts were perfused with Krebs buffer for 20 minutes and then embedded in OCT compound. Heart sections were then taken and used for immunohistochemistry (IHC) of CD38. Sections were first washed with PBS with 0.1% Tween-20 (PBST) to dissolve residual OCT. Then, sections were fixed with 4% paraformaldehyde, washed briefly with PBS, and blocked with 5% BSA in PBST containing 0.3 M glycine. After 1 hour of blocking at RT, CD38 antibody (ab90) and CD31 antibodies were added at a dilution of 1:100 in PBST overnight at 4°C. After overnight incubation, sections were washed 3×5 minutes with PBST, and secondary antibodies (Alexa Fluor 594 for CD38, Alexa Fluor 488 for CD31) were added at a final dilution of 1:200. Images were taken on an Olympus FV 1000 spectral confocal microscope with a 60X objective. For nuclei staining, DAPI was used at a concentration of ~1 M.