P53 Acetylation Exerts Critical Roles in Pressure Overload-Induced Coronary Microvascular Dysfunction and Heart Failure in Mice

Abstract

BACKGROUND:

Coronary microvascular dysfunction (CMD) has been shown to contribute to cardiac hypertrophy and heart failure (HF) with preserved ejection fraction. At this point, there are no proven treatments for CMD.

METHODS:

We have shown that histone acetylation may play a critical role in the regulation of CMD. By using a mouse model that replaces lysine with arginine at residues K98/117/161/162R of p53 (p53^4KR), preventing acetylation at these sites, we test the hypothesis that acetylation-deficient p53^4KR could improve coronary microvascular dysfunction and prevent the progression of hypertensive cardiac hypertrophy and HF. Wild-type and p53^4KR mice were subjected to pressure overload (PO) by transverse aortic constriction (TAC) to induce cardiac hypertrophy and HF.

RESULTS:

Echocardiography measurements revealed improved cardiac function together with a reduction of apoptosis and fibrosis in p53^4KR mice. Importantly, myocardial capillary density and coronary flow reserve (CFR) were significantly improved in p53^4KR mice. Moreover, p53^4KR upregulated the expression of cardiac glycolytic enzymes and glucose transporters, as well as the level of fructose-2,6-biphosphate; increased PFK-1 activity; and attenuated cardiac hypertrophy. These changes were accompanied by increased expression of HIF-1α and proangiogenic growth factors. Additionally, the levels of SERCA-2 were significantly upregulated in sham p53^4KR mice as well as in p53^4KR mice after TAC. In vitro, p53^4KR significantly improved endothelial cell (EC) glycolytic function and mitochondrial respiration, and enhanced EC proliferation and angiogenesis. Similarly, acetylation-deficient p53^4KR significantly improved CFR and rescued cardiac dysfunction in SIRT3 KO mice.

CONCLUSIONS:

Our data reveal the importance of p53 acetylation in coronary microvascular function, cardiac function, and remodeling, and may provide a promising approach to improve hypertension-induced CMD and to prevent the transition of cardiac hypertrophy to HF.

Graphical Abstract

Introduction

Hypertension, diabetes, and aging are the major risk factors for the development of heart failure (HF) ^1–4, which is one of the leading causes of mortality and morbidity worldwide ⁵. Clinical studies demonstrate that coronary microvascular dysfunction (CMD), coronary capillary rarefaction, cardiac hypertrophy, and fibrosis play a detrimental role in the progression of HF ^{6, 7}. Recent studies demonstrate that coronary artery remodeling and microvascular rarefaction (impairment of angiogenesis) are two important contributors to reduced coronary flow reserve (CFR) i.e. coronary microvascular dysfunction ^8–14. CFR is an essential predictor of HF diseases such as diastolic dysfunction and HF with preserved ejection fraction (HFpEF) ^{8, 9, 15, 16}. Many pathological conditions such as diabetic and hypertensive cardiac hypertrophy are associated with reduction of CFR^{8, 9, 15}. Moreover, preserved CFR in diabetic patients reduces the number of cardiac events to levels seen in non-diabetic controls¹⁷. Accumulating evidence reveals that restoration of CFR or improvement of CMD have great promise in reducing high mortality in HF. At this point, there are no proven treatments for CMD due to a lack of mechanistic studies. The mechanisms related to CMD are unclear, but endothelial dysfunction and microvascular rarefaction are suggested as two important contributors to reduced CFR ^8–14. Previous studies suggest that coronary microvascular rarefaction contributes to the progression from compensated hypertrophy to HF ^18–20, whereas promotion of angiogenesis improves cardiac function and delays the progression of HF ^21–23. So far, our understanding of the molecular mechanisms of microvascular rarefaction and CMD in HF is still incomplete.

Ablation of p53 has been shown to protect against coronary vascular rarefaction and cardiac injury ^{24, 25}. In contrast, accumulation of p53 impaired cardiac angiogenesis and systolic function ²⁶. P53-mediated impairment of cardiac angiogenesis was observed in animal models of cardiac hypertrophy²⁷. Furthermore, endothelial senescence and dysfunction were associated with acetyl-p53²⁸. These studies suggest that p53 plays a critical role in the development of cardiac and endothelial dysfunction. Our previous study also showed that down-regulation of Sirtuin 3 (SIRT3) was associated with increased acetylation of p53 in cardiomyocytes, whereas overexpression of SIRT3 significantly reduced p53 acetylation and blunted microvascular rarefaction, cardiac fibrosis, and hypertrophy, and improved cardiac function in diabetic mice ²⁹. This study suggests that regulation of p53 activation by acetylation plays a critical role in microvascular and cardiac function. Indeed, p53 activity is finely tuned by the regulation of p53 protein stability via co-activators and inhibitors, as well as post-translational modifications, including acetylation ³⁰. Acetylation of p53 plays a major role in regulating promoter-specific activation of downstream targets during stress responses ³⁰. Previous study demonstrated that the acetylation-deficient p53^4KR (contains lysine to arginine mutations at lysine residues 98, 117, 161, and 162) lost its ability to induce cellular senescence, cell cycle arrest, and apoptosis, and to regulate certain p53-dependent metabolic pathways in cancer cell lines³⁰. However, the role of p53 acetylation in hypertensive pressure overload (PO)-induced CMD and cardiac metabolism and HF is largely unknown.

In this study, we examined whether acetylation-deficient p53^4KR preserves coronary microvascular function, prevents pathological cardiac hypertrophy and fibrosis, and the development of HF in response to chronic PO.

Methods

The data that support the findings of this study are available from the corresponding author upon reasonable request.

Mice

Mice expressing p53 with mutations at lysine residues K98, K117, K161, and K162 (replacing lysine with arginine, p53^4KR) mice on the C57BL/6J background were provided by Dr. Wei Gu at the Columbia University and maintained in the Laboratory Animal Facilities at the University of Mississippi Medical Center (UMMC). This is a global p53^4KR knockin mouse model, which p53^4KR cannot be acetylated only at these four sites. Male C57BL/6J mice (4 months old) were purchased from The Jackson Laboratory (Bar Harbor, ME) and were used as wild type (WT) controls. The SIRT3/p53^4KR mouse was generated by crossing SIRT3KO mouse with p53^4KR mouse. All animals were fed standard laboratory diet (#8640, Teklad, WI) and water and housed in individually ventilated cages. All protocols were approved by the Institutional Animal Care and Use Committee of the UMMC (Protocol ID: 1564 and 1189) and were in compliance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals (NIH Pub. No. 85‐23, Revised 1996).

Transverse Aortic Constriction Procedure

WT and p53^4KR mice were subjected to PO-induced HF via transverse aortic constriction (TAC) for 8 weeks as previously described with minor modifications ^{31, 32}. Mice with overall good health and matching age were included for sham and TAC surgery. WT and age-matched p53^4KR mice were randomly divided into 4 groups: WT sham, WT TAC, p53^4KR sham, and p53^4KR TAC groups. Briefly, the mice were anesthetized with a single intraperitoneal injection of ketamine (50 mg/kg) and xylazine (10 mg/kg) and kept warm. A partial thoracotomy to the second rib was performed to expose the aortic arch. A small piece of 6–0 silk suture was placed between the innominate and left common carotid arteries. Then, a small L-shaped piece of a blunt 27-gauge needle was placed parallel to the transverse aorta. The suture was tied snugly and quickly, and the needle was promptly removed to yield a constriction of 0.4 mm in diameter. The thorax was then closed, and mice were allowed to recover in a warming chamber until they were fully awake. The sham procedure is identical except without ligation of the aorta. Mice were inspected daily, and postoperative analgesia was administrated by injection of 1 ml/kg body weight Carprofen (5 mg/ml) i.p. every 24 h over 3 days. The survival rate was not significantly different between WT and p53^4KR mice after TAC (Supplemental Figure S2D). Mice that died before the expected endpoint (8 weeks after TAC surgery) were excluded from the results. All surviving animals were used for further study. Echocardiogram was conducted on mice at 8 weeks post-surgery. The animals were then euthanized, and the tissue was harvested for further experiments.

Echocardiography

Transthoracic echocardiograms were performed on mice using a Vevo 3100 Preclinical Imaging Platform equipped with an MX400 transducer (FUJIFILM Visual Sonics Inc., Canada). The mice were anesthetized by inhalation of 1–1.5% isoflurane mixed with 100% medical oxygen, and the heart rate was maintained between 450 and 500 beats per minute. To assess systolic function, M-mode cine loops were recorded and analyzed by Vevo LAB software (FUJIFILM Visual Sonics Inc., Canada) in a blinded-manner by a different researcher to measure ejection fraction (EF%), fractional shortening (FS%), and myocardial parameters, including left ventricle (LV) end-systolic diameter (LVESD), LV end-diastolic diameter (LVEDD), LV end-systolic volume (LVESV), LV end-diastolic volume (LVEDV), thickness of the anterior wall (LVAW) and posterior wall (LVPW) at end-systole and end-diastole, stroke volume (SV), and cardiac output (CO) ^32–34.

Transmitral inflow pulsed-wave (PW) Doppler and Tissue Doppler (TD) imaging were used to measure diastolic function. From an apical 4-chamber view, the peak velocity of early (E) and late (A) filling of mitral inflow, isovolumic relaxation time (IVRT), isovolumic contraction time (IVCT), and aortic ejection time (AET) were assessed. The myocardial performance index (MPI) is calculated using the following formula: MPI = (IVRT + IVCT)/AET. In addition, TD images were obtained from the mitral annulus to measure tissue motion velocity in early and late diastole (e’ and a’, respectively) and to calculate E/e’ ratio ^{32, 35–37}.

CFR was assessed by PW Doppler at the left proximal coronary artery (LCA) in a modified parasternal LV short-axis view. Briefly, baseline (1% isoflurane) and hyperemic (2.5% isoflurane) coronary blood flow velocity were recorded and the CFR calculated as the ratio of hyperemic peak diastolic flow velocity to baseline peak diastolic flow velocity ^{32–34, 36, 37}.

Primary Cell Culture

Mouse aortic endothelial cells (MAECs) were isolated from WT or p53^4KR mice (n=3), as previously described with modification ³⁸. Briefly, WT and p53^4KR mice were anesthetized with 2.5% isoflurane. The abdominal and chest area was disinfected and opened from the midline to expose the abdominal aorta and heart. The aorta was then flushed with 1 mL of cold PBS containing 1000U/mL of heparin via injection through the LV. Then, the thoracic aorta was quickly dissected and placed in an ice-cold DMEM medium (Thermo Fisher Scientific, NY). The surrounding adipose tissue was then removed under a microscope. The aorta was cut into 1 mm rings which were then embedded in reduced-growth factor basement membrane matrix (ECM, Thermo Fisher Scientific, NY) and covered with endothelial growth medium (EGM-2, Lonza, MD) supplemented with growth factors and 10% fetal bovine serum (FBS) in a 48-well cell culture plate. The plate was incubated at 37 °C with 5% CO₂. At Day 4, the aortic segments were gently removed without damaging the growing endothelial cells, and cells were allowed to continue to grow for an additional 2–3 days with fresh medium. Then, the cells were trypsinized and re-plated on a gelatin (0.2%, diluted from 2% stock solution, Millipore Sigma, MO) coated 100-mm cell culture plate. The cells were incubated at 37 °C with 5% CO₂ until ~90% confluent and were passaged 2–3 times. Cells between passages 4 and 10 were used for all studies.

Mouse Aortic Ring Sprouting Assay

Ex vivo angiogenesis was evaluated by using isolated mouse aortas as described previously with modification ^{36, 39–42}. Briefly, freshly dissected aorta was gently cleaned in ice-cold DMEM medium and cut into ~1mm rings. One aortic ring was placed in a well of a 96-well culture plate covered with total of 80 μL of ECM gel. Solidified gel was then covered with 120 μL of EGM-2 medium. The culture plate was then incubated at 37 °C with 5% CO₂. Vessel outgrowth was examined on day 5 using an inverted microscope (AMG, Life Technologies, NY). Quantification of vessel sprouting was performed by measuring the relative area of aortic explant outgrowth using ImageJ software.

Histology and Immunofluorescence

Left ventricles were fixed with 10% neutral buffered formalin, processed, embedded in paraffin, and sectioned at 5-μm thickness. Picrosirius red staining was used to evaluate the degree of cardiac interstitial and perivascular fibrosis. 5–10 randomly selected fields per mouse were used, and the fibrotic fraction was calculated as the ratio of Picrosirius red-stained area to total myocardial area.

Freshly collected left ventricular tissues were embedded with optimal cutting temperature compound (OCT) in cryomolds, and tissue sections (10 μm) were prepared by using a cryostat machine (Leica, IL) Tissue sections were dried for 30 minutes at 25 °C, washed 3× in PBS, permeabilized for 20 minutes in 0.2% Triton X-100, and blocked for 1 hour at 25 °C in 5% goat serum and 1% BSA-PBS. Alexa Fluor^™ 488 conjugated wheat germ agglutinin (WGA, Invitrogen, CA), Griffonia Bandeiraea Simplicifolia Isolectin B4 (1:50; IB4, Invitrogen, OR), or unconjugated primary antibodies (p53, HIF-1α, Troponin, and FSP1) was diluted in PBS containing 0.03% Triton X-100 and 1% BSA and added to the sections overnight at 4 °C, followed by 3 washes in PBS and incubation with fluorescent secondary antibodies for 1 hour 25 °C to evaluate the size of cardiomyocyte and cardiac hypertrophy, co-localization, or capillary density, respectively. Microscopic photos of 5 to 10 fields per section per mouse were taken by using the Nikon Eclipse 80i microscope, coupled with an X-Cite^® 120 Fluorescence Illumination system (Nikon Instruments, NY). The cross-sectional area of cardiomyocytes were measured by ImageJ software. Apoptotic cells were labeled by using a DeadEnd^™ Fluorometric TUNEL System according to the manufacture’s protocol (Promega, WI). The result was presented as the number of TUNEL-positive cells per 100 nuclei.

Metabolic Assays

The oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) were measured using the XF^e24 extracellular flux analyzer from Agilent Technologies (Santa Clara, CA), following the manufacturer’s instructions and our previous studies ^{32, 36, 37}. Briefly, cells were seeded at a density of 20000 cells per well one day prior to the assay. The next day, the cells were washed and incubated in unbuffered assay medium supplemented with the following substrates at 37°C in a non-CO₂ incubator for one hour. Glycolysis stress test used assay medium supplemented with glutamine (2 mM). ECARs were measured with injections of glucose (10 mM), oligomycin (1 μM), and 2-deoxyglucose (100 mM). Cellular mitochondrial stress test used assay medium containing glucose (10 mM), pyruvate (1 mM), and glutamine (2 mM). OCRs were measured with injections of oligomycin (1 μM), cyanide p-trifluoromethoxy-phenylhydrazone (1 μM), and rotenone/antimycin A (0.5 μM). All concentrations indicated above are final concentrations in the assay medium. After each assay, the cell nuclei were stained with 4’,6-diamidino-2-phenylindole (DAPI) to count the total number of cells per well for data normalization. The data was expressed as OCR or ECAR/10000 cells.

Tissue Fructose-2,6-biphosphate Assay

Tissue fructose-2,6-biphosphate (F2,6-BP) concentration was determined as previously described ⁴³. Briefly, samples of tissues were weighed and homogenized in NaOH (0.05 M). The resulting mixture was heated for 20 min at 80°C. After cooling, the samples were neutralized with 1M acetic acid in the presence of 20 mM Hepes, and then centrifuged. Samples were incubated at 37 °C for 5 min in the following assay mixture: 50 mM Tris, 5 mM Mg²⁺, 1 mM fructose-6-phosphate (Sigma #F3627), 0.15 mM NADH (Sigma #N4505), excessive PPi-dependent phosphofructokinase (PFK-1, enriched from potato tubers), 0.2U/mL aldolase (Sigma #A2714), 8U/mL triosephosphate isomerase (Sigma #T2507) and 1U/mL glycerol-3-phosphate dehydrogenase (Sigma #10127752001). After the 5 min pre-incubation time, 0.5 mM pyrophosphate was added to start the reaction, and the rate of change in OD_340 nm every 30 seconds was followed for 5 min in a Bio-Rad xMark microplate spectrophotometer (Bio-Rad). Data are expressed as the fold change compared to the WT controls.

Phosphofructokinase Activity Assay

Tissue PFK-1 activity was determined as previously described ⁴⁴. Briefly, samples of tissues were weighed and homogenized in lysis buffer followed by sonication and centrifugation. The reaction was performed using 4 μg of total protein in a 96-well plate containing 80 μL of the reaction buffer (50 mM Tris–HCl, pH 7.5, 5 mM MgCl₂, 5mM ATP (Sigma #A6419), 0.2 mM NADH, 100 mM KCl, 5 mM Na₂HPO₄, 0.1 mM AMP (Sigma #A2252), 5 mM fructose-6-phosphate, 5U/mL triosephosphate isomerase, 1U/mL aldolase, and 10U/mL glycerol-3-phosphate dehydrogenase. Absorbance at 340 nm was read at 37°C every 30 seconds for a period of 30 min in a Bio-Rad xMark microplate spectrophotometer. Data are expressed as the change in absorbance at 340 nm/min/mg of protein.

Cell Proliferation Assay

WT and p53^4KR MAECs were seeded at 8 × 10³ cells/well in a 96-well plate, and cultured for 24 h. The MTT assay (11465007001, Roche Diagnostics, Mannheim, Germany) was performed according to manufacturer’s instructions. Absorbance was measured at 562 nm (reference 750 nm). Cell viability was expressed as fold change as calculated by Absorbance of p53^4KR MAEC/Average absorbance of WT MAEC.

Immunoblot Analysis

Protein extractions from heart ventricular samples or cultured MAECs were prepared in lysis buffer with protease/phosphatase inhibitor cocktail (A32961, ThermoFisher Scientific, NY). Lysates were mixed with Laemmli sample buffer (2X, Bio-Rad, CA) containing 10% mercaptoethanol and boiled at 95°C for 10 minutes. For detection of Collagen-1, the samples were prepared and run under native conditions. The samples were separated by SDS-PAGE gel for 50 minutes at 180V on ice. Separated proteins were then transferred to a PVDF membrane (activated by soaking in 100% methanol for 1 minute) in transfer buffer containing 20% methanol for 1 hour at 100V on ice. Membranes were blocked in 5% milk for 1 hour and washed for 15 minutes 3 times with 1X PBS containing 0.1% Tween-20 (PBST, Millipore Sigma, MA). The membrane was then cut to separate the desired proteins for probing. Membranes were incubated with primary antibodies as indicated in 5% milk overnight at 4°C. The membranes were then washed and incubated with an anti-rabbit (31460) or anti-mouse (31430) secondary antibody conjugated with horseradish peroxidase (1:10000, ThermoFisher Scientific, NY) for 1 hour at room temperature. Secondary antibodies were detected using the ECL Western Blotting Substrate (32106, ThermoFisher Scientific, NY). Samples were analyzed by densitometry by using Image Lab software 6.0 (Bio-Rad, CA) and normalized to loading control.

Statistical Analysis

All data are presented as mean ± SD. Statistical analysis was done in GraphPad Prism 9 (San Diego, CA), Data were tested for normality by using Shapiro-Wilk test. For comparisons between 2 groups, the F test was used to compare variances of the two groups. For equal variance, a two-tailed t test was used. Otherwise, a two-tailed t test with Welch’s correction was performed. When normality was not observed, nonparametric testing was performed with a Mann-Whitney test. For comparisons of 3 or more groups with one factor, Brown-Forsythe test was used to compare whether all groups have equal variances. Normally distributed groups with equal variances were compared with a one-way ANOVA test, followed by Tukey’s post-hoc test for multiple comparisons. Normally distributed groups with unequal variances were compared with one-way Brown-Forsythe test and Welch ANOVA test with a Tamhane T2 correction. Kruskal–Wallis with Dunn’s multiple comparison test was used for nonparametric multiple comparisons. For comparisons of 3 or more groups with two factors, Spearman’s test was used to compare whether all groups have equal variances. Normally distributed groups with equal variances were compared with a two-way ANOVA test, followed by Tukey’s post-hoc test for multiple comparisons. Normally distributed groups with unequal variances or non-normally distributed groups were log-transformed and compared with a two-way ANOVA test, followed by Tukey’s post-hoc test for multiple comparisons. A p value of <0.05 was considered statistically significant. The specific test used for each comparison and p values are summarized in Table S1.

Results

Acetylation-deficient p53^4KR attenuates systolic dysfunction and hypertrophy after TAC

p53^4KR is a knock-in mouse model that expresses p53 with four lysine-to-arginine mutations K98/117/161/162R ⁴⁵. These mutations abolish acetylation of p53 at these four sites, so that this form of p53 fails to regulate cellular senescence, cell cycle arrest and apoptosis, and metabolic pathways ³⁰. To investigate the role of acetylation of p53 in cardiac function during PO, WT and p53^4KR mice were subjected to TAC for 8 weeks. Cardiac parameters measured by echocardiography are shown in supplemental Table S2. The level of acetyl-p53 (K370) was increased in the WT+TAC mice when compared to the WT+sham or p53^4KR+TAC groups (Figure 1A, Supplemental Figure S1A and S1B). Interestingly, the expression of non-acetylated p53 was decreased in the WT+TAC group when compared to the WT+sham or p53^4KR+TAC groups (Figure 1A, Supplemental Figure S1A and S1B). This led to a significant increase in the acetyl-p53/p53 ratio in the WT+TAC mice when compared to the WT+sham or p53^4KR+TAC groups (Figure 1A and 1B). Immunostaining of p53 in the heart showed that p53 was located in the cytosol and nuclei of the endothelial cells, whereas p53 was mainly located in the nuclei of the cardiomyocytes (Supplemental Figure S1C and S1D).

Figure 1.

Effects of acetylation-deficient p53^4KR on systolic function and cardiac hypertrophy. A and B, Representative immunoblots and quantitative analysis of acetyl-p53, p53, β-myosin heavy chain (β-MHC), brain natriuretic peptide (BNP), α-tubulin, and GAPDH in the indicated mouse hearts subjected to either sham or transverse aortic constriction (TAC) procedure for eight weeks (n=6–8). Acetyl-p53(K370), p53, and α-tubulin were from the same membrane. β-MHC and corresponding GAPDH were from the same membrane. BNP and corresponding GAPDH were from the same membrane. C, Left ventricular (LV) ejection fraction (EF) and fractional shortening (FS) measured by echocardiography in the indicated groups (n=6–10). D, Ratio of heart weight to tibia length in the indicated groups (n=6–10). E, LV mass measured and calculated by echocardiography in the indicated groups (n=6–10). F, Representative images of wheat germ agglutinin (WGA)–stained frozen heart sections in the indicated groups. (n=4–5). A minimum of 100 cardiomyocytes from each LV section of each mouse was measured. Bar=25 μm.

The EF and FS were significantly decreased in WT mice at 8 weeks after TAC when compared to the sham controls. However, the systolic function of p53^4KR mice subjected to TAC had no significant change (Figure 1C and Supplemental Figure S2A). The ratio of heart weight to tibia length or body weight was significantly increased in the WT mice subjected to TAC, but not in the p53^4KR mice (Figure 1D and Supplemental Figure S2B). TAC-induced increase of LV mass determined by echocardiography was also significantly attenuated in a p53^4KR mice (Figure 1E). The WT mice+TAC showed gradually decreased cardiac output over time after TAC, while p53 mutant mice+TAC had sustained cardiac output, though it was not statistically different between the two groups (Supplemental Figure S2C). The survival rate was not significantly different between WT and p53^4KR mice after TAC (Supplemental Figure S2D). Immunoblot further showed that the LV β-myosin heavy chain (β-MHC) protein expression was significantly attenuated in p53^4KR mice as compared to the WT mice subjected to TAC (Figure 1A, 1B, and Supplemental Figure S1A). WGA staining also revealed that TAC caused a significantly higher increase in the cross-sectional area of cardiomyocytes in the WT mice as compared with p53^4KR mice (Figure 1F). Western blot analysis also revealed a significant increase in brain natriuretic peptide (BNP) expression, a hypertrophic marker, in the heart of WT+TAC mice, when compared to the WT+sham and p53^4KR+TAC groups (Figure 1A and 1B, and Supplemental Figure S1A). These data suggest that p53 acetylation deficiency ameliorates the development of cardiac hypertrophy and attenuates the deterioration of cardiac function after TAC.

Acetylation-deficient p53^4KR preserves diastolic function and reduces cardiac apoptosis and fibrosis after TAC

We further investigated the role of p53 acetylation deficiency on the diastolic function during PO-induced HF. Doppler measurements are shown in supplemental Table S2. WT mice subjected to TAC exhibited a dramatic increase in E/A ratio (greater than 2, 2.05±0.10 versus 1.32±0.07 in the sham control) and E/e’ ratio (Figure 2A and Supplemental Figure S3A), suggesting elevation of left atrial pressure and development of diastolic dysfunction when compared to the p53^4KR mice after TAC. Interstitial fibrosis was significantly increased in WT mice (but not in p53^4KR mice) after TAC (Figure 2B). Additionally, western blot and immunostaining analysis showed that the expression of SERCA-2 ATPase was significantly upregulated in p53^4KR mice at basal levels and after TAC (Figure 2C and 2D, and Supplemental Figure S3B). Platelet-derived growth factor β-receptor (PDGFR-β) and fibroblast-specific protein 1 (FSP1) regulate tissue fibroblast activation. Interestingly, cardiac PDGFR-β expression was significantly attenuated in p53^4KR mice as compared with WT mice after TAC (Figure 2C, 2D, and Supplemental Figure S3B). The expression of FSP-1 was significantly increased in the WT mice after TAC, although it was not significantly different from the p53^4KR mice after TAC (Figure 2C, 2D, and Supplemental Figure S3B). Cardiac collagen-1 levels were significantly lower in p53^4KR mice as compared with the WT mice at both sham and TAC conditions (Figure 2C and 2D), suggesting that reduced cardiac fibroblast activation may be involved in the attenuation of TAC-induced fibrosis in p53^4KR mouse hearts. In addition, fewer TUNEL positive cells were detected in hearts of p53^4KR mice compared with WT mice after TAC (Figure 2E), indicating that p53 acetylation deficiency attenuated cardiac apoptosis in mice after TAC. To understand which cells are undergoing apoptosis, cells were co-stained for apoptotic marker TUNEL along with either cardiomyocyte marker Troponin-T, or myofibroblast marker FSP-1. Our data showed that the TUNEL positive cells predominantly cardiomyocytes (Supplemental Figure S3C). In addition, there were a few TUNEL positive myofibroblast cells after TAC (Supplemental Figure S3D).

Figure 2.

Effects of acetylation-deficient p53^4KR on diastolic function, cardiac fibrosis, and apoptosis. A, Representative pulsed-wave Doppler and tissue Doppler images from an apical 4-chamber view of WT and p53^4KR mice subjected to either sham or TAC procedure for eight weeks and ratio of the peak velocity of early (E) to late (A) filling of mitral inflow (E/A) in the indicated groups (n=6–9). The ratio of E to the tissue motion velocity in early diastole (e’) was calculated in the indicated groups (n=6–10). B, Representative images of Picrosirius red-stained paraffin-embedded heart sections and quantification of the percentage of interstitial fibrosis area in the indicated groups (n=3–5). Bar=50 μm. C and D, Representative immunoblots and quantitative analysis of PDGFR-β, FSP-1, collagen-1, SERCA2 ATPase, α-tubulin, and GAPDH in the indicated mouse hearts (n=3–4 or 6–8). PDGFR-β and corresponding GAPDH were from the same membrane. FSP-1 and corresponding GAPDH were from the same membrane. SERCA2 ATPase and α-tubulin were from the same membrane. Collagen-1 and corresponding GAPDH were from different membranes. E, Representative images of TUNEL-stained frozen heart sections and quantification of the number of TUNEL-positive cells (green)/field in the indicated groups (n=4). 5–15 randomly selected fields per LV section of each mouse were measured. Bar=25 μm.

Acetylation-deficient p53^4KR preserves capillary density and coronary flow reserve in HF

Deletion of p53 has been shown to promote angiogenesis ²⁴. To determine whether p53 acetylation deficiency is able to stabilize the cardiac microvasculature during PO, isolectin B4-positive area (Figures 3A and 3B) was quantified. The result shows a significant decrease of cardiac capillary density in WT mice after TAC, while cardiac capillary density was preserved in p53^4KR mice after TAC. p53-mediated hypoxia-independent HIF-1α degradation is involved in angiogenesis inhibition following PO ²⁶. We further explored the molecular mechanism by which p53^4KR regulates angiogenic signaling pathways. Cardiac HIF-1α protein expression was significantly elevated in p53^4KR mice but not in WT mice after TAC (Figure 3C, 3D, and Supplemental Figure S4A). To identify where HIF-1α activation was taking place, the expression of HIF-1α in the cardiomyocytes, endothelial cells and fibroblasts were examined by co-staining HIF-1α with cardiomyocyte marker Troponin-T, endothelial marker IB4, or myofibroblast marker FSP-1. Immunohistochemical study revealed that expression of HIF-1α was primarily localized to the cardiomyocyte, whereas endothelial cells and myofibroblasts also showed a few positive cells (Figure 3E and Supplemental Figure S4B and S4C). Cardiac angiopoietin-1 protein expression was significantly higher in p53^4KR mice as compared with WT mice under control condition or after TAC. TAC did not affect cardiac angiopoietin-1 protein expression in WT mice (Figure 3C, 3D, and Supplemental Figure S4A). In addition, cardiac vascular endothelial growth factor (VEGF) protein expression was markedly reduced in WT mice after TAC, while cardiac VEGF protein expression was unchanged in p53^4KR mice (Figure 3C, 3D, and Supplemental Figure S4A). Moreover, the expression of heme oxygenase-1 (HO-1), a direct p53 target with anti-inflammatory, antioxidant, antiapoptotic, and proangiogenic effects, was significantly elevated in p53^4KR mice after TAC (Figure 3C, 3D, and Supplemental Figure S4A). Cardiac vascular cell adhesion molecule 1 (VCAM-1) expression was significantly increased in WT but not in p53^4KR mice after TAC (Figure 3C, 3D, and Supplemental Figure S4A), suggesting that endothelial inflammatory activation was attenuated in p53^4KR mice after TAC. These data suggest that p53 acetylation deficiency preserved cardiac angiogenesis and attenuated endothelial activation after TAC.

Figure 3.

Acetylation-deficient p53^4KR preserves coronary vasculature and coronary flow reserve (CFR). A and B, Representative images of Isolectin B4 (IB4, green; DAPI stains the nuclei, blue)-stained frozen heart sections and quantification of the number of capillaries/100 nuclei in the indicated groups (n=4–5). Bar=50 μm. C and D, Representative immunoblots and quantitative analysis of HIF-1α, HO-1, Ang-1, VEGF, VCAM-1, and GAPDH in the indicated mouse hearts (n=6–8). HIF-1α, HO-1, and corresponding GAPDH were from the same membrane. Ang-1 and corresponding GAPDH were from the same membrane. VEGF and corresponding GAPDH were from the same membrane. VCAM-1 and corresponding GAPDH were from the same membrane. E, Representative images of co-staining of cardiomyocyte marker Troponin-T with HIF-1α in frozen heart sections. F, Representative pulsed-wave Doppler images of the proximal left coronary arteries and CFR of WT and p53^4KR mice subjected to either sham or TAC procedure for eight weeks. CFR was calculated as the ratio of hyperemic peak diastolic flow velocity (2.5% isoflurane) to baseline peak diastolic flow velocity (1% isoflurane) in the indicated groups (n=6–8).

Loss of capillary or microvascular rarefaction is one of the key contributors to the development of coronary microvascular dysfunction; therefore, we further examined whether loss of acetylation of p53 protects microvascular function in PO-induced HF. We measured the coronary flow velocity (Supplemental Table S2) at 1% (baseline) or 2.5% isoflurane (hyperemia) to calculate the CFR. As shown in Figure 3F, PO significantly reduced the CFR in the WT mice, while CFR was preserved in the p53^4KR mice.

P53 acetylation deficiency elevates glycolysis related enzyme and glucose transporters

To explore the molecular events by which p53^4KR regulates cardiac remodeling, we determined the expression levels of some glycolysis-related proteins. Cardiac PFK-1 expression was significantly higher in p53^4KR mice after TAC when compared to the WT+TAC mice, while its expression was decreased in WT+TAC mice when compared to sham mice (Figure 4A, 4B, and Supplemental Figure S5A). In addition, the expression of glucose transporter-1 (Glut-1) was significantly reduced in WT after TAC, but it is not affected by TAC in p53^4KR mice (Figure 4A, 4B, and Supplemental Figure S5A). Cardiac Glut-4 expression was significantly higher in p53^4KR mice after TAC than that of WT mice after TAC, but its expression was not affected in WT mice after TAC (Figure 4A, 4B, and Supplemental Figure S5A).

Figure 4.

Acetylation-deficient p53^4KR increases cardiac glucose transporters and glycolytic function. A and B, Representative immunoblots and quantitative analysis of PFK-1, GLUT-1, GLUT-4, α-tubulin, and GAPDH in the indicated groups. PFK-1 and corresponding GAPDH were from the same membrane. Glut-1 and α-tubulin were from the same membrane. Glut-4 and corresponding GAPDH were from the same membrane. C, Cardiac F2,6-BP level was determined by the coupled-enzymatic assay and expressed as the fold change to the WT sham group. n=3–4. D, Cardiac PFK-1 activity was determined by the coupled-enzymatic assay and expressed as the OD_340nm/min/mg protein. n=5–9.

By using coupled-enzymes methods, we measured the level of glycolytic intermediate F2,6-BP, as well as PFK-1 activity. The basal level of F2,6-BP in the hearts of p53^4KR mice was significantly higher than the WT mice (Figure 4C). WT mice subjected to TAC exhibited a significant elevation in the level of F2,6-BP, whereas no change was observed in p53^4KR mice after TAC (Figure 4C). Moreover, PFK-1 activity in the WT mice after TAC was significantly reduced when compared to the sham controls, whereas it was significantly higher in p53^4KR mice than WT mice after TAC (Figure 4D). These data suggest that p53 acetylation deficiency enhanced glycolytic function by increasing the level of F2,6-BP and enhancing PFK-1 activity.

P53 acetylation deficiency increases glycolysis in the endothelial cell (EC) and aortic sprouting

Since p53^4KR improved glycolytic function and increased expression of cardiac proangiogenic growth factors, we further investigated whether p53^4KR enhanced glycolysis and angiogenesis in isolated ECs from WT and p53^4KR aortas. Cell proliferation was significantly increased in MAECs from p53^4KR mice as compared with MAECs from WT mice (Figure 5A). Moreover, p53^4KR ECs exhibited a significant upregulation of basal glycolysis, glycolytic capacity, and glycolytic reserve when compared to WT ECs (Figure 5B). This was associated with a significant increase in mitochondrial basal respiration and ATP production (Figure 5C). In addition, the vessel explant sprouting study demonstrated a significant increase in the vessel explant sprouting area in the vessel explants of p53^4KR mice as compared to the WT mice (Figure 5D). These data suggest that p53 acetylation deficiency can also promote endothelial glycolysis and ex vivo angiogenesis.

Figure 5.

Acetylation-deficient p53^4KR increases glycolysis in MAECs and aortic sprouting. A, Cell proliferation of p53^4KR MAECs was significantly higher than that of the WT MAECs, as measured by MTT assay. n=8–10. B, Glycolysis stress test and extracellular flux analysis of glycolysis, glycolytic capacity, and glycolytic reserve in MAECs isolated from p53^4KR and WT mice. n=4–5. C, Mitochondrial stress test and extracellular flux analysis of mitochondrial basal and maximal respiration and ATP production in MAECs isolated from p53^4KR and WT mice. n=5. D, Representative images of the aortic ring sprouting assay at day 5 of incubation and quantification of the sprouting area in the indicated groups (n=9–13). Bar=250 μm.

Acetylation-deficient p53^4KR preserves coronary flow reserve and rescues HF in SIRT3KO mice

Our previous study demonstrated that downregulation of SIRT3 leads to a significant increase in acetylation of p53 in mouse hearts²⁹. Here, we further confirmed the level of acetyl-p53 was significantly increased in the SIRT3 KO mice (Supplemental Figure S5B). To validate the p53^4KR-dependent rescue of hypertension-induced CMD, cardiac hypertrophy and HF, the genetic SIRT3 knockout (SIRT3 KO) mouse was crossed with p53^4KR to generate a SIRT3KO/p53^4KR mouse. As shown in Figure 6, knockout of SIRT3 impaired CFR (Figure 6A and 6B), along with systolic and diastolic dysfunction as evidenced by reductions in EF% and FS% (Figure 6C and Supplemental Figure S5C), and elevations in IVRT, MPI and E/e’ ratio (Figure 6D and 6E). Consistent with our TAC-induced HF model, introducing p53^4KR significantly improved CFR and rescued cardiac systolic and diastolic dysfunction in SIRT3KO mice.

Figure 6.

Effects of acetylation-deficient p53^4KR on CFR and cardiac dysfunction in SIRT3KO mice. A and B, Representative pulsed-wave Doppler images of the proximal left coronary arteries and CFR of WT, SIRT3KO, p53^4KR and p53^4KR/SIRT3KO mice. CFR was calculated as the ratio of hyperemic peak diastolic flow velocity (2.5% isoflurane) to baseline peak diastolic flow velocity (1% isoflurane) in the indicated groups (n=7–11). C, Left ventricular (LV) ejection fraction (EF) and fractional shortening (FS) measured by echocardiography in the indicated groups (n=8–10). D, Representative pulsed-wave Doppler and tissue Doppler images from an apical 4-chamber view in the indicated groups. E, The diastolic function parameters, isovolumic relaxation time (IVRT), myocardial performance index (MPI), and ratio of E to the tissue motion velocity in early diastole (e’) were calculated in the indicated groups (n=5–9).

Discussion

In this study, we examined the effect of acetylation-deficient p53 on coronary microvascular dysfunction, cardiac hypertrophy, and the development of HF in response to chronic PO. The main findings are that p53 acetylation deficiency prevents coronary capillary rarefaction and improves CFR while attenuating cardiac remodeling and dysfunction. These changes were associated with enhanced cardiac glycolytic function and increased levels of proangiogenic growth factors, involving PFK-1, glucose transporters, HIF-1α, Ang-1, VEGF, and HO-1. Thus, our data support the importance of p53 acetylation in coronary microvascular dysfunction and cardiac function, as well as cardiac remodeling during PO-induced HF (Supplemental Figure S6).

The tumor suppressor protein p53 plays a critical role in the pathogenesis of heart disease, as evidenced by the increased level of p53 and the number of apoptotic cells in patients with heart disease ⁴⁶. p53 regulates the cellular stress response to DNA damage, oxidative stress, hypoxia, or cytokines, which induces expression of p53 target genes that modulate cell cycle and growth arrest, as well as apoptosis⁴⁷. Thus, by using experimental animals with global, cardiomyocyte-specific, or endothelial-specific p53 gene deletion, as well as pharmacological inhibition of p53, studies have demonstrated that inhibition of p53 effectively attenuated TAC or myocardial infarction (MI)-induced myocardial apoptosis, ventricular remodeling, vascular rarefaction, and HF ^24–26. In addition to the classical regulation of p53 stability by Mdm2, accumulating studies suggest that p53 activity is also rapidly and effectively regulated by post-translational modification, such as phosphorylation and acetylation. Specifically, acetylation of p53 plays a major role in controlling its binding to the promoter region of specific downstream targets during stress responses. Our recent study demonstrated that reduced p53 acetylation was associated with improved microvascular angiogenesis and cardiac function in diabetic mice, exhibiting decreased cardiac fibrosis and hypertrophy ²⁹. A study by Wang et al demonstrated that cells expressing the acetylation-deficient p53^4KR mutant were protected from apoptosis, growth arrest, and ferroptosis ³⁰. The same effect was observed in p53^4KR mice that were tumor prone but failed to develop early-onset tumors ⁴⁵. Our present study demonstrated a significant reduction of apoptosis, although the p53 level was increased in the heart of p53^4KR mice subjected to PO. This result indicates a crucial role of p53-acetylation in mediating apoptosis independent of p53 level, which is consistent with the previous study ³⁰. Our results show that acetylation of p53 at K370 is upregulated in WT+TAC mice relative to p53^4KR+TAC and WT sham mice. These changes resulted a significant increase in the acetyl-p53/p53 ratio in the WT mice after TAC. Therefore, mitigating the hyperacetylation status of p53 is a viable option for attenuating TAC-induced HF by regulating p53 activity. Wang et al., have demonstrated that loss of acetylation at K117 in mice reduced the ability of p53 to mediate apoptosis, while it was still capable of inducing cell senescence and cell cycle arrest ³⁰. By mutating two additional lysine residues (K161 and K162) they found that p53 could no longer regulate cell senescence or cell cycle arrest. Furthermore, Kon et al. found that adding a mutation at the K98 residue abolishes p53-mediated ferroptosis, which is retained in models without this mutation ⁴⁵. They further identified that the mTOR pathway is intact in p53^4KR mice and generated an additional mutation at K136 to create a p53^5KR model which has impaired mTOR pathway activity. Therefore, we believe that individual acetylation sites may regulate different physiological activities of p53, and that altering which acetylation sites are mutated will impact the functional abilities of the p53 protein. While our p53^4KR model has diminished function, it does not inhibit every pathway by which p53 acts, although these 4 sites are critical mediators of various pathways and as such are important for p53 activity. Previous study demonstrated a critical role of p53 acetylation in ferroptosis in which p53^4KR further lost its function to induce ferroptosis when compared to the WT-p53 or p53^3KR (K117/161/162R) in cancer cell lines ³⁰. We have not found that PO-induced HF was correlated with myocardial ferroptosis at 8 weeks after TAC. Although HO-1 was upregulated, the level of glutathione peroxidase 4 and 4-hydroxynonenal were not altered in mouse hearts after PO at this time point (He et al unpublished data). These data suggest that acetylation-deficient p53^4KR functions in mediating apoptosis and ferroptosis in the heart, and thus protects cardiac function.

One of the key features of pressure overload-induced HF is microvascular rarefaction and impairment of CFR. A recent study also revealed that pressure overload induces major transcriptional and metabolic adaptations in cardiac microvascular endothelial cells (MiVEC) resulting in excess interstitial fibrosis and impaired angiogenesis. Our present study showed a very similar cardiac microvascular rarefaction, with reduced coronary blood flow in pressure overload-induced HF. These studies highlight the critical role of microvascular rarefaction and MiVEC as key pathogenic modulators of pressure overload–induced heart disease in mice, suggesting new therapeutic targets to alter angiogenesis in pressure overload–induced cardiac remodeling. Global p53 deficiency or systemic p53 inhibition by pifithrin-α has been shown to increase cardiac angiogenesis and protect the heart from PO- or MI-induced HF ^{25, 26}. In addition, endothelial p53 deletion improved myocardial angiogenesis and attenuated cardiac fibrosis, remodeling, and dysfunction ²⁴. In line with these previous findings, the immunostaining of p53 in the heart showing that p53 is located in the cytosol and nuclei of the endothelial cells, whereas p53 is mainly located in the nuclei of the cardiomyocytes. Our study revealed that p53^4KR acetylation deficiency preserved myocardial capillary density and cardiac contractile function and attenuated cardiac hypertrophy and fibrosis. Endothelial glycolytic function significantly contributes to angiogenic growth factor, VEGF, induced angiogenesis. ECs generate most ATP through glycolysis to drive angiogenesis ⁴⁸. Extracellular flux analysis demonstrates that glycolysis, glucose capacity, and glycolytic reserve were significantly increased in the p53^4KR MAECs and that aortic spouting was enhanced in the p53^4KR aortas compared to the WT mice. Most importantly, p53^4KR improved coronary vascular function as evidenced by increased CFR and preserved diastolic function. CFR is an independent determinant of long-term cardiovascular events, acute coronary syndrome, HF, and cardiac mortality in patients with coronary artery disease ^{49, 50}. Impaired CFR is associated with increased myocardial infarction size, reduced LV ejection fraction, adverse LV remodeling, and reduced long-term survival in animal studies ³³. Our previous studies have demonstrated the impaired CFR is caused, at least in part, by coronary vascular rarefaction due to reduced endothelial glycolytic function, proangiogenic, and HIF signaling in SIRT3KO mice ^{33, 36, 37, 51, 52}. Furthermore, knockout of SIRT3 leads to a significant increase in acetylation of p53 in mouse hearts. Most importantly, cardiomyocyte-specific knockout of SIRT3 causes mitochondrial-specific lysine acetylation and p53 acetylation, which does not occur in the cytosol (Cantrell AC, unpublished study). Our present study showed that acetylation-deficient p53 significantly improves CFR and rescues the HF phenotype of SIRT3 KO mice. Our recent study demonstrates that p53^4KR significantly reduced interstitial fibrosis and cardiomyocyte size in the SIRTKO/p53^4KR mice, via reducing reactive oxygen species (ROS) level and ferroptosis ⁵³. These data suggest a critical role of p53 acetylation in the mitochondria, where SIRT3 is present. Impaired CFR will lead to insufficient perfusion to meet the metabolic demand, causing hypoxia in the heart and promoting the transition from adaptive to maladaptive cardiac remodeling ⁵⁴. Cardiac p53 has been shown to mediate HIF-1α degradation independent of hypoxia following PO ²⁶. Our data showed that HIF-1α and Ang-1 levels were significantly elevated in p53^4KR mice, whereas no changes were observed in WT animals. Also, the level of VEGF was significantly reduced in WT mice but was preserved in p53^4KR mice. Interestingly, HO-1 was identified as a direct p53 target gene that possesses potent proangiogenic, anti-inflammatory, antioxidant, and antiapoptotic effects ^{55, 56}. p53^4KR mice exhibited significantly increased expression of HO-1, along with increased p53 levels, suggesting p53 acetylation deficiency can still regulate the expression of HO-1. Moreover, the level of VCAM-1 was upregulated in WT mice after TAC, whereas no change was observed in p53^4KR mice, suggesting that PO caused an increased inflammatory response in the vascular endothelium which can be suppressed by p53^4KR.

Glucose metabolism plays an important role in diastolic function in heart disease ⁵⁷. A metabolic shift from fatty acid oxidation to glycolysis in cardiomyocytes has been reported during PO-induced cardiac remodeling and diastolic dysfunction ^{58, 59}. In addition, inhibition of glycolysis caused greater impairment of diastolic function ^{60, 61}, whereas increased glycolytic substrates protect against diastolic dysfunction ⁶². Previous studies demonstrated that decreased F2,6-BP level and PFK-1 activity resulted in more profound cardiac hypertrophy, fibrosis, and dysfunction in response to PO ^{63, 64}. Glucose uptake via glucose transporters also plays a critical role in regulating glycolysis, cardiac hypertrophy, and diastolic function ^{65, 66}. Wild type p53 has been shown to possess a repressive effect on transcriptional activity of the GLUT1 and GLUT4 gene promoters ⁶⁷. In the present study, p53^4KR seemed to lose its function to suppress GLUT-1, GLUT-4, and possibly PFK-1, which led to the increased or preserved expression of these proteins. Moreover, coupled-enzyme methods showed that the level of F2,6-BP and PFK-1 activity were significantly increased. Cardiac fibrosis is one of the key factors that contribute to the development of diastolic dysfunction ^{3, 68–70}. Histological analysis demonstrated that fibrosis was significantly reduced in p53^4KR mice along with the levels of PDGFR-β and FSP-1, suggesting that p53^4KR mutations may also preserve the ability of p53 to repress PDGFR-β signaling ⁷¹ and fibroblast activation. A recent study by Liu et al demonstrates that fibroblast-specific p53 deletion induces a proliferative phenotype and facilitates the expression and secretion of ECM proteins that results in excessive interstitial and perivascular fibrosis after TAC ⁷². Considering the decreased expression of non-acetylated p53 and the increased interstitial fibrosis in the WT TAC mice, our data are in consistent with their study and suggest that regulating the activity of p53 by manipulating the acetylation status may be more beneficial and more precise in controlling the outcome than gene deletion of p53, even in a specific cell type. Taken together, these data suggest p53^4KR attenuated PO-induced diastolic dysfunction, at least in part by inducing PFK-1 and glucose transporters, enhancing cardiac glucose utilization, and attenuating cardiac fibrosis.

Calcium ion (Ca²⁺) cycle plays a critical role in the contraction and relaxation of cardiomyocytes. Sarcoplasmic/endoplasmic reticulum Ca²⁺ ATPase 2 (SERCA2 ATPase) functions as a calcium transporter responsible for reuptake of Ca²⁺ by the sarcoplasmic reticulum (SR) during the contraction cycle of the heart, resulting in the myocardial relaxation that characterizes diastole. In HF, SERCA2 ATPase has been found to have impaired activity, causing diastolic dysfunction and diminished myocardial relaxation, while increasing levels of SERCA2 ATPase has been shown to improve heart function to some degree ⁷³. During pressure overload, cardiac-specific overexpression of SERCA2a attenuated cardiac hypertrophy and improved contractility in rodents ^{74, 75}, while inhibition of SERCA2 ATPase by sarcolipin impaired cardiac function in mice treated with isoproterenol. Here, we found for the first time that SERCA2 ATPase is significantly increased in p53^4KR mice compared with WT mice, as well as in p53^4KR + TAC mice compared with WT + TAC. This is likely a contributing factor to the improved cardiac function that p53^4KR mice demonstrate under TAC conditions, as the increased levels of SERCA2 ATPase would cause more calcium reuptake by the SR and thus improve cardiac relaxation during diastole. Although wild-type p53 has been shown to bind to and activate SERCA2 ATPase at the ER⁷⁶, for the first time we showed that p53 is involved in regulating the protein level of SERCA2 ATPase in the heart, and that p53^4KR still retained that ability.

The present study has several limitations. First, we used a global p53^4KR knockin mouse model. Our study could not differentiate the effect of p53^4KR in different cell types, such as cardiomyocyte, endothelial cells, and fibroblasts. Although further study is needed by using tissue/cell specific- p53^4KR knockin mice, the findings from the present study still provide important insights for the effects of p53 acetylation on HF development. Second, we only studied male mice in the present study. Sex difference is an important biological factor that affects TAC-induced HF development, it is possible that female and male p53^4KR mice may have different degrees of cardiac hypertrophy, fibrosis, microvascular and cardiac dysfunction after TAC. However, as cardiac/microvascular dysfunction and remodeling are commonly observed in both male and female mice after TAC, and since p53 exert similar roles in both male and female mice in various disease conditions, p53^4KR is anticipated to exert similar impacts on TAC-induced HF in both sexes. Third, we could not examine whether acetyl-p53 at K98/K117/K161/K172 is upregulated in the heart subjected to TAC due to the lack of antibody that is specific to p53^4KR (not commercially available). However, in our cardiac dysfunction mouse model (SIRT3KO mouse) with hyperacetylation, the level of acetyl-p53 was significantly increased. Moreover, acetylation-deficient p53 (in the SIRT3KO/p53^4KR mice) significantly improved CFR and rescued the HF phenotype of SIRT3 KO mice, suggesting a critical role of p53 acetylation in the heart. Although we did not rule out the possibility that p53^4KR restored hyperacetylation of mitochondrial proteins in SIRT3KO/p53^4KR mice, we think the chances are very low. Theoretically, introducing of p53(4KR) should not affect the acetylation status of other proteins since (a) p53 or p53(4KR) does not possess deacetylase activity; and (b) SIRT3 is still absent in these mice, although it is still possible that p53^4KR affects other deacetylases and indirectly restored hyperacetylation of mitochondrial proteins in SIRT3KO/p53^4KR mice. It was unexpected that acetyl-p53 (K370) was detected around 45 kD, while the total p53 was detected at 53 kD. We freshly isolated primary cardiomyocytes from our WT mice and cultured them in DMEM medium containing 20uM Etoposide and 500 nM Trichostatin A immediately for 5 hours. The band at ~45kD position was dramatically induced when compared to the untreated cells (data not shown). This result suggests that in our mouse model, acetyl-p53 (K370) may be differently processed in the heart resulting in different molecular weights from other cell lines, and this isoform cannot be detected by our total p53 antibody. The acetyl-p53 (K370) at 45 kD may be one of the major isoforms in the cardiac tissue of our mouse strain. Indeed, there are several isoforms of p53 have been discovered in cancer cell lines, such as p53β, p53γ, Δ40p53 (α, β, γ), Δ133p53 (α, β, γ), etc, although they have not been reported in heart tissue ^{77, 78}. The molecular weight of these isoforms ranges from 26 to 48 kD. It is possible that our anti-acetyl-p53 (K370) antibody was detecting one of these isoforms, particularly, the Δ40p53, which is about 42–47 kD. Interestingly, Δ40p53 lacks one of the two transactivation domains at the NH₂-terminal ⁷⁸. While our total p53 antibody is specific to a sequence of amino acids at the NH₂-terminal, the K370 is located at the COOH-terminal, according to the manufacturer’s datasheets. Therefore, our total p53 antibody is potentially not able to detect this isoform, but the anti-acetyl-p53 (K370) antibody can. Nonetheless, the expression of acetyl-p53 (K370) does not influence the main findings and conclusion of the present study.

In conclusion, our findings suggest that acetylation-deficient p53 failed to suppress HIF-1α and PDGFR-β signaling, PFK-1 activity, and transcription of glucose transporters during PO-induced HF, but it can still induce SERCA2 ATPase, HO-1, proangiogenic growth factors, and endothelial anti-inflammatory signaling, despite the loss of its pro-apoptotic function. All of these contribute to myocardial angiogenesis, improved CFR, and attenuated cardiac dysfunction and remodeling. Thus, our data support the importance of p53 acetylation in CMD and cardiac function and may provide a promising approach to improve coronary vascular function and PO-induced cardiac remodeling in order to prevent the transition to HF.

Highlights.

• p53 with lysine to arginine mutations at residues K98, K117, K161, and K162 (acetylation-deficient p53^4KR) prevents coronary capillary rarefaction and improves coronary flow reserve while attenuating cardiac remodeling and dysfunction.

• p53^4KR is not able to suppress HIF-1α and PDGFR-β signaling, PFK-1 activity, and expression of glucose transporters during pressure overload-induced heart failure, but it can still induce SERCA2 ATPase, HO-1, proangiogenic growth factors, and endothelial anti-inflammatory signaling, despite the loss of its pro-apoptotic function.

• Our data support the importance of p53 acetylation in coronary microvascular and cardiac dysfunction during pressure overload-induced heart failure and provide a promising approach to improve coronary vascular function and cardiac remodeling in order to prevent the transition to heart failure.

Nonstandard Abbreviations and Acronyms

AET

aortic ejection time

Ang-1

angiopoietin 1

BNP

brain natriuretic peptide

CFR

coronary flow reserve

CMD

coronary microvascular dysfunction

CO

cardiac output

ECAR

extracellular acidification rate

ECM

extracellular basement membrane matrix

EF

ejection fraction

F2,6-BP

fructose-2,6-biphosphate

FBS

fetal bovine serum

FS

fractional shortening

FSP-1

fibroblast-specific protein 1

GAPDH

glyceraldehyde 3-phosphate dehydrogenase

Glut

glucose transporter

HF

heart failure

HFpEF

HF with preserved ejection fraction

HIF-1α

hypoxia-inducible factor-1α

HO-1

heme oxygenase-1

IB4

isolectin B4

IVCT

isovolumic contraction time

IVRT

isovolumic relaxation time

LCA

left proximal coronary artery

LVAW

left ventricle anterior wall

LVPW

left ventricle posterior wall

LVEDD

left ventricle end-diastolic diameter

LVESD

left ventricle end-systolic diameter

LVEDV

left ventricle end-diastolic volume

LVESV

left ventricle end-systolic volume

β-MHC

β-myosin heavy chain

MAECs

mouse aortic endothelial cells

MPI

myocardial performance index

OCR

oxygen consumption rate

OCT

optimal cutting temperature compound

PDGFR-β

platelet derived growth factor receptor-β

PFK-1

phosphofructokinase 1

PO

pressure overload

PW Doppler

pulsed-wave Doppler

p53^4KR

mutant p53 contains lysine to arginine mutations at lysine residues 98, 117, 161, and 162

SERCA2 ATPase

sarco/endoplasmic reticulum calcium ATPase

SIRT3

Sirtuin 3

SV

stroke volume

TAC

transverse aortic constriction

TD

tissue Doppler

VCAM-1

vascular cell adhesion molecule 1

VEGF

vascular endothelial growth factor

WGA

wheat germ agglutinin

WT

wild-type