Knockout of p53 Decreases Cardiac Injury by Reducing ROS Generation during Ischemia‐Reperfusion
Authors: Lesnefsky Edward , Thompson Jeremy , Hu Ying , Chen Qun 
Published: November 2023

BACKGROUND

p53 is a tumor suppressor protein. Its content is usually maintained at a very low level through rapid ubiquitination and degradation. During stress conditions including ischemia (ISC) ‐ reperfusion (REP), cytosolic p53 content is dramatically increased due to decreased degradation. p53 is also translocated to mitochondria and causes mitochondrial damage during ISC‐REP through permeation of the outer mitochondria membrane. We hypothesized that knockout(KO) of p53 in the heart can decrease cardiac injury during ISC‐REP by protecting cardiac mitochondria.

METHODS

Floxed p53 mice were crossed with mice carrying the α‐myosin heavy chain cre to generate cardiac specific p53 KO mice. Isolated, buffer perfused hearts from p53 KO and wild type (WT) underwent 25 min stop flow 37° CISC and 30 min REP. Time control hearts were perfused without ISC‐REP. Mitochondria were isolated at end REP and OXPHOS, calcium retention capacity (CRC, nmol calcium/mg protein to assess sensitivity to MPTP‐mitochondria permeability transition pore opening) and net H2O2 generation (amplex red assay) was measured.

RESULTS

KO of p53 decreased cardiac injury following ISC‐REP shown by decreased infarct size and improved functional recovery [increased left ventricular developed pressure (LVDP) and decreased end diastolic pressure(LVEDP)] compared to WT (Table). The rate of OXPHOS was decreased in both KO and WT hearts compared to corresponding time control using glutamate+malate as complex I substrates. Compared to WT, KO of p53 slightly improved the OXPHOS in hearts following ISC‐REP. Deletion ofp53 decreased release of cytochrome c from into cytosol [Ratio of cytochrome c/GAPDH in mitochondria following ISC‐REP. Mean ± SEM: 2.09±0.51 (WT) vs. 0.76±0.08(KO), p<0.05, n=4 in each group], consistent with the notion that relocation of p53 to mitochondria permeated the outer mitochondrial membrane. ISC‐REP increased H2O2 generation in both WT and KO mice, but was attenuated in p53 KO with succinate+rotenoneas substrate). ISC‐REP decreased the CRCin both WT and KO versus time control. KO of p53 did not improve the CRC following ISC‐REP.

CONCLUSION

KO of p53 decreases release of cytochrome c from mitochondria with less cardiac injury during ISC‐REP, likely via retention of cytochrome c despite susceptibility to MPTP. Decreased H2O2 generation from sites distal to complex I, likely complex III, also contribute to p53 KO‐mediated cardio protection.

Support or Funding Information

Supported by a Grant‐in‐Aid (15GRNT24480123)from the American Heart Association and Medical Research Service Merit Review Award (2IO1BX001355‐01A2) from the Department of Veterans Affairs and the Pauley Heart Center, Virginia Commonwealth University.

Isolated hearts
Infarct size
LVDP mmHg
LVEDP mmHg

WT‐IR (n=7)
30.3 ± 3.2
15 ± 4
44 ± 3

p53 KO‐IR (n=7)
21.3 ± 2.8*
30 ± 5*
30 ± 5*

Mitochondria
OXPHOS
CRC
H2O2

WT‐control
303±23
893 ± 49
481 ± 22

WT ISC‐REP
101±13†
366 ± 26†
746 ± 39†

KO‐control
297±37
920 ± 54
423 ± 32

KO ISC‐REP
155±4*†
400 ± 21†
537± 43*†

Mean ± SEM;

p<0.05 p53 KO vs. corresponding WT;

p<0.05 ISC‐REP vs. time control, n=3–8 in each group.