Abbreviations: I/R: ischemia-reperfusion; H/R, hypoxia/reoxygenation;
PFKFB3, 6-phosphofructo-2-kinase/fructose-2, 6-bisphosphatase isoform 3;
AAV; adeno-associated virus
What is already known:
Pinocembrin protects myocardial from ischemic injury in animals.
What this study adds:
Pinocembrin postconditioning significantly reduced the infarct size and
improved cardiac contractile function after acute myocardial I/R.
Acute cardioprotective benefits of pinocembrin are mediated in part via
glycolytic stimulation through PFKFB3.
What is the clinical significance:
Pinocembrin may be an effective agent for improving tissue perfusion in
I/R-related diseases.
Introduction
Cardiovascular disease remains the leading cause of death worldwide
(Bice & Baxter, 2015). Currently, there are few effective drugs to
protect the heart after ischemia/reperfusion (I/R) injury. Therefore, it
has become a hot research field to find the molecular mechanism of
coronary artery disease progression and development that can protect
myocardium from I/R injury. With this in mind, more and more attention
is focused on pharmacological interventions because it can induce
cardioprotection and easy to implement (Zheng et al., 2017). However,
for patients with ischemic heart disease, there are still many
restrictive drugs available clinically.
Traditional Chinese medicine has a history of thousands of years and
provides a large amount of medicinal materials. Pinomline
(5,7-dihydroxyflavone) is an abundant flavonoid isolated from propolis
and some plants. It has various biological functions such as
anti-inflammatory, anti-oxidation, anti-bacterial, etc. (Hanieh et al.,
2017). Previous studies have confirmed that pinocembrin confer
neuroprotective effects during cerebral ischemic I/R (Saad, Abdel Salam,
Kenawy, & Attia, 2015; Shi et al., 2011; Tao, Shen, Sun, Chen, & Yan,
2018). Recent research has also tried to determine whether pinocembrin
is beneficial for cardiac injury. For example, several studies suggest
that pinocembrin can improve the cardiac function of myocardial I/R
rats, reduce ventricular arrhythmia, and reduce the area of myocardial
infarction (Lungkaphin et al., 2015; Zhang, Xu, Hu, Yu, & Bai, 2018).
However, it is still unclear whether pinocembrin given at the onset of
reperfusion has cardioprotective effects, which is a more clinically
effective method. In addition, the underlying mechanism by which
pinocembrin can provide cardioprotection is largely unknown.
Recent studies have confirmed that there are changes in energy
metabolism in many human diseases, and targeted energy metabolism may
have therapeutic potential on these diseases (Gohil et al., 2010). Due
to the mechanical function of the heart, it is an organ with high energy
requirements. In general, most of the energy (about 70%) of a healthy
heart comes from the β-oxidation of fatty acids, and the rest of the
energy comes from glucose oxidation (Lopaschuk, Ussher, Folmes, Jaswal,
& Stanley, 2010). Nevertheless, in a pathological environment,
substrate utilization may change (Lionetti, Stanley, & Recchia, 2011).
In patients with diabetes, circulating blood glucose levels at admission
are related to the clinical outcome after acute myocardial infarction
(AMI), suggesting that it may be related to myocardial metabolism
(Malmberg, Norhammar, Wedel, & Ryden, 1999). Metabolic shift from
β-oxidation to glycolysis metabolism will reduce the cell’s need for
oxygen by 11-13%, and NAD+ precursors have been shown to activate
cellular glycolysis to protect the heart from ischemic injury
(Nadtochiy, Wang, Nehrke, Munger, & Brookes, 2018). It is worth noting
that redirect energy metabolism to glycolysis can reduce oxidative
damage and inhibit apoptosis (Hunter, Hendrikse, & Renan, 2007; Jeong,
Kim, Cho, & Kim, 2004; Vaughn & Deshmukh, 2008). However, few agents
that target energy metabolism are clinically safe and useful for
patients. At the same time, it is unclear whether and how pinocembrin
regulates acute myocardial I/R glycolysis.
To address these issues, this study was designed to (i) the current
study was designed to determine the role of pinocembrin in rat and mouse
cardiac I/R injury ex vivo and in vivo respectively. (ii)
clarify the effects of pinocembrin on the glycolytic metabolism during
I/R; (iii) explore the underlying molecular basis that contributes to
the pinocembrin-induced cardioprotection. Our results provide new
insight into the mechanism of pinocembrin-induced cardioprotection and
suggest a potential application value of pinocembrin in the protection
of hearts against I/R injury.
Methods
2.1 Chemicals and reagents
Pinocembrin were obtained from Sigma-Aldrich (St. Louis, MO, USA).
3-(3-Pyridinyl)-1-(4-pyridinyl)-2- propen-1-one (3PO) was from EMD
Millipore (MA, USA). All other reagents were from Sigma-Aldrich (St.
Louis, MO, USA).
2.2 Animals
250-300 g Adult Male Sprague-Dawley (SD) rats and C57BL/6 mice (age,
8-10 weeks) from Shanghai Slac Laboratory Animal Co. Ltd. were cared in
accordance with the Guidelines for Care and Use of Laboratory Animals
published by the US National Institutes of Health (NIH Publication, 8th
Edition, 2011). Animal procedures were approved by the Institutional
Review Board of Shanghai University of Health and Medicine (Shanghai,
China).
2.3 I/R injury model in Langendorff-perfused rat hearts
As mentioned before, the heart was rapidly excised at 37 °C and perfused
with Krebs-Henseleit buffer (KHB) at a constant pressure of 80 mmHg
using Langendorff technique (Xie et al., 2005). Left ventricular (LV)
pressure was monitored with a water-filled latex balloon connected to a
pressure sensor (AD instrument, Bella Vista, New South Wales, Australia)
and inserted into the left ventricle cavity to achieve left ventricular
end-diastolic pressure (LVEDP) at 0 to 10 mm Hg. PowerLab system (AD
instrument, Australia) was used to monitor left ventricular imaging
pressure (LVDP), left ventricular imaging pressure (LVEDP), maximum rate
of pressure changes over time (+ dP/dtmax) and pressure decay over time
(-dP/dtmax). Pinocembrin’s final concentration of 10, 30 and 100 µM
perfusate was added with 5 minutes of reperfusion. In order to assess
the infarct size, the isolated rat heart was re-perfused for 2 hours
after 30 minutes of ischemia. The slices were incubated in 1% w/v
triphenyltetrazolium chloride (TTC, pH 7.4) for 15 min, and then fixed
in 10% formaldehyde. Image-Pro-Plus software (media cybernetics) was
used to calculate the infarct area. The infarct area was expressed as a
percentage of the LV area at risk.
2.4 Myocardial I/R Model
Surgical ligation of the left coronary artery (LCA) was performed as
described previously (Qin et al., 2017). Briefly, mice were anesthetized
with ketamine (50 mg/kg) and pentobarbital sodium (50 mg/kg) by
intraperitoneal injection, followed by orally intubated and ventilated.
The core body temperature is always maintained at 37 °C. An internal
sternotomy was then performed with electrocautery, and then the proximal
LCA was displayed and ligated. After 30 minutes of coronary artery
occlusion, the suture was cut and the blood vessels were allowed to
reperfusion. After 24 hours of reperfusion, the mice were anesthetized
with isoflurane. Transthoracic echocardiography was used to determine
the left ventricular ejection fraction. After reperfusion, blood samples
were collected and centrifuged at 3000 rpm for 10 minutes for cardiac
troponin-T (cTnT) and lactate dehydrogenase (LDH) measurements.
Measurement of area at risk and infarct size was performed as reported
previously. Each of the myocardial slices were weighed and the areas of
infarction, risk, and nonischemic left ventricle were assessed by a
blinded observer using computer-assisted planimetry (NIH ImageJ 1.37).
2.5 Isolation and culture of primary cardiomyocytes
Continuous enzymatic digestion and isolation were used to obtain
neonatal rat and mouse cardiomyocytes (Irvine et al., 2013; Irvine et
al., 2012). Neonatal rats were decapitated and hearts were immediately
placed in HBSS. The ventricle was taken and digested with trypsin
overnight at 4 °C and collagenase four times at 37 °C for 10 min.
Cardiomyocytes were suspended in sterile DMEM, supplemented with
penicillin 100Uml-1, streptomycin 100
mgml-1 and 10% fetal bovine serum. The cells were
pre-plated twice (37 °C for 45 minutes) to reduce fibroblast
contamination. Hypoxia/reoxygenation (H/R, which simulated MI/R in
vivo ) was employed as previously described (Hou et al., 2019).
2.6 Measurement of LDH release and cTnT release
Necrotic cell death was evaluated by supernatant LDH activity, as in
previous studies (Kishi et al., 2015). A spectrophotometric kit (Nanjing
jiancheng, Jiangsu, China) was used according to the manufacturer’s
instruction. In short, 20μl supernatants were collected in a 96-well,
then 25μl matrix buffer and 5μl coenzyme I was added to the 96-well. The
mixture was incubated at 37°C for 15 min. After 25μl
2,4-dinitrophenylhydrazine was added to each well and incubated at 37°C
for 15 min, 250μl 0.4M NaOH was added to each well and the mixture was
incubated at room temperature for 5 min. The absorbance value was
measured at 450nm with the spectrophotometer (BioTek, VT, USA) and the
LDH activity was calculated. Plasma
cTnT levels as an indicator of cardiomyocyte damage was measured using a
mouse cTnT ELISA kit (Elabscience Biotechnology Co., Ltd, Wuhan, China)
according to the manufacturer’s instruction.
2.7 Quantitative real-time PCR
We isolated total RNA was from the heart tissue with Trizol Reagent
(Invitrogen, Carlsbad, CA, USA). Relative quantitation by real-time PCR
involved SYBR Green detection of PCR products in real time with the ABI
PRISM 7700 Sequence Detection System (Applied Biosystems). The primers
were list in Supplemental Table 1. The reactions were conducted in
triplicate by heating the reactant to 95 °C for 5 min, followed by 40
cycles of 94 °C for 30 s, 58 °C for 30 s and 72 °C for 30 s.
2.8 Western blot analysis
Left ventricles were homogenized and the cells were lysed as described
previously (Matsushima et al., 2013). The samples were analyzed by
SDS-PAGE. Transfer the protein to a polyvinylidene fluoride microporous
membrane (Bio Rad) with primary antibody PFKFB3 (Abcam, USA; 1: 1000)
and anti-GAPDH (internal control; Kangcheng Co., Ltd., Shanghai, China;
1: 8000). The secondary antibody was coupled to horseradish peroxidase
(Cell Signaling Technology, 1: 6000). Enhanced ECL detection kit
(Amersham Pharmacia Biotech) was employed to visualize the
immunoreaction, followed by exposing to film and quantified with a video
documentation system (Gel Doc 2000, Bio-Rad).
2.9 Generation and administration of adeno-associated virus (AAV)
Serotype 9 AAV vectors (AAV9) encoding shNC or shPFKFB3 (AAV9-shNC and
AAV9–shPFKFB3) were prepared as previously described (Xie et al.,
2016). 3 x 1011 vg
of AAV9-shNC or AAV9–shPFKFB3 were
injected
intravenously into tail veins as previously described (Somanathan et
al., 2014) of 4-5 weeks old male C57 mice. Sham or myocardial ischemia
surgeries were conducted 4 weeks after AAV9 injection.
2.10 Seahorse extracellular flux analyzer assays
Cellular bioenergetics was measured using a Seahorse XFe24 extracellular
flux analyzer in intact cardiomyocytes. We conducted glycolysis stress
testing according to the manufacturer’s instructions previously reported
(He et al., 2017). Glycolysis stress test: Cells were incubated in
glucose-free Seahorse assay media supplemented with 1mM pyruvate at
37oC in incubator without CO2 for 1-h prior to the
assay. Injectors were loaded to add 20mM Glucose, 1μM Oligomycin and
100mM 2 Deoxy-Glucose (2-DG) and glycolysis, glycolytic capacity and
glycolytic reserves were calculated as extracellular acidification rate
(ECAR).
2.11 Statistical analysis
Data were presented as means ± SEM. The unpaired, 2-tailed t test
was used for comparisons between 2 groups. For multiple comparisons,
ANOVA or repeated ANOVA followed by the LSD post hoc test was
used with GraphPad Prism® version 7.0 software. A P value
< 0.05 was considered as statistically significant.
Results
3.1 Compound pinocembrin
significantly improved the cardiac function and reduced infarct size
after I/R ex vivo
We perfused isolated rat hearts to explore the cardioprotective effects
of pinocembrin against I/R injury, 10 to 100 µM pinocembrin were
delivered during the first 5 min of reperfusion (fig 1A). During 45 min
reperfusion following 30min ischemia, the contractile function of left
ventricular (LV), including LV developed pressure (LVDP), LV
end-diastolic pressure (LVEDP), maximal rates of pressure development
over time (+dP/dtmax) and pressure decay over time (−dP/dtmax) were
significantly suppressed (fig 2 A-D). Pinocembrin itself does not affect
the heart rate during reperfusion (Supplementary fig 1), while it
dose-dependently improved the postischemic myocardial performance from
10 to 100 µM (fig 2 A-D).
Next, we explore whether pinocembrin improves cell survival during I/R
via examining lactate dehydrogenase (LDH) release, an indicator of
myocardial injury. Little LDH release was detected in the coronary
efflux before ischemia, while LDH release was obviously induced at the
end of reperfusion, while pinocembrin significantly inhibited the
release of LDH from 10 to 100 µM (fig 2E). Consistently, the I/R-induced
infarction after 2 h of reperfusion was significantly attenuated by
pinocembrin at the concentration of 30 µM (fig 2F). These data
demonstrated that pinocembrin exhibits beneficial effects on cardiac I/R
injury ex vivo .
Compound pinocembrin protects hearts from myocardial I/R injuryin vivo
To explore the cardioprotective effects of pinocembrin on myocardial I/R
injury in vivo , pinocembrin was intravenously injected into
wild-type (WT) mice 5 minutes before the end of sustained ischemia
(i.v., 5 mg/kg and 10 mg/kg), followed by reperfusion for 24 hours (fig
1B). Evans-blue/TTC dye method was used to determine the infarct size.
After reperfusion for 24 hours, there was no difference of area at risk
(AAR) between each group (Figure 3A and 3B). Nevertheless, compared with
I/R group, pinocembrin i.v. treatment significantly reduced the infarct
size by 20% (fig 3A and 3C). Besides, plasma levels of cTnI and LDH
activity were markedly elevated during myocardial I/R, which were both
suppressed with pinocembrin i.v. treatment (fig 3 D-E). Furthermore, the
echocardiographic results showed that pinocembrin can significantly
improve I/R-suppressed the ejection fraction (EF%) and fractional
shortening (FS%) (fig 3F).
3.3 Protective actions of pinocembrin against cardiomyocyte injury
responses in vitro
Next, we examined the direct effects of pinocembrin on cardiomyocytes.
To determine whether pinocembrin confers cardioprotective effects
through its direct action on the cardiomyocytes, we subjected the
isolated neonatal rat and mouse cardiomyocytes to H/R and applied 30 µM
pinocembrin to cells during the onset of reperfusion. In line with the
effects of pinocembrin on the myocardial I/R injury, simulated
I/R-reduced cell viability was significantly improved by pinocembrin
(data not shown). Moreover, our data demonstrated that pinocembrin
rescued cardiac troponin I (cTnI) release and LDH release post-simulated
I/R in vitro , in both rat and mouse cardiomyocytes (fig 4 A-D).
Pinocembrin increases glycolysis in
cardiomyocyte
During myocardial ischemia, enhanced glycolytic metabolism is essential
for maintaining homeostasis of cardiomyocytes. In addition, previously
studies reported that pinocembrin is involved in regulating glucose
uptake in cancer cells (Liu et al., 2018). Subsequently, we explored the
effects of pinocembrin on cellular bioenergetics with the Seahorse
extracellular flux analyzer and performed glycolysis stress tests to
measure glycolysis and glycolytic capacity both in intact rat and mouse
cardiomyocytes. We observed that brief pretreatment with pinocembrin
versus the control increased glycolysis by 21.4% (extracellular
acidification rate [ECAR]). Pinocembrin also increased glycolytic
capacity in cardiomyocyte by 23.7% (fig 5 A-B).
To further explore the underlying mechanism of pinocembrin promoting
myocardial glycolysis, mRNA expressions of a panel of glycolysis-related
genes was determined with qRT-PCR. As shown in Fig 5C, pinocembrin
significantly increased the expression of glycolysis-related genes,
especially PFKFB3 gene.
3.5 PFKFB3 inhibition alters glycolysis and abolished
pinocembrin-induced cardioprotection in cardiomyocytes
Next, we explored whether blockade of PFKFB3 inhibits cardiomyocyte
glycolysis and impairs pinocembrin-afforded cardioprotective effects. As
shown in Fig 6 A-D, exposure of cardiomyocyte to 10 µM PFKFB3 inhibitor,
3PO remarkably reversed pinocembrin-enhanced glycolysis. What’s more,
inhibition of PFKFB3 resulted in a significant increase of cTnI release
and LDH release post-H/R in vitro (fig 6 E-H), suggesting that
disruption of glucose metabolism leads to impaired cardioprotection of
pinocembrin during H/R.
3.6 PFKFB3 deficiency in normal mice using AAV9 abolished pinocembrin
improved cardiac injury and dysfunction after MI/R.
Since that pinocembrin alleviated H/R-induced cardiomyocytes death by
upregulating glycolysis via PFKFB3, we further evaluated the roles of
PFKFB3 on myocardial I/R injury in vivo . To determine whether
PFKFB3 regulation is directly involved in pinocembrin-related MI/R
injury improvement, the WT mice were injected with AAV9 encoding PFKFB3
shRNA to knockdown endogenous PFKFB3. Intravenous injection of
AAV9-shPFKFB3 on mice has successfully reduced protein level of PFKFB3
in heart (Supplementary fig 2).
Subsequently, we exposed the mice to in situ myocardial
ischemia–reperfusion to assess the functional role of
myocardial-specific PFKFB3 in pinocembrin-afforded cardioprotection. We
measured myocardial injury by infarct size area, serum cTnT levels and
LDH activity. Exposure of mice infected with AAV9-shPFKFB3 to myocardial
ischemia followed by reperfusion revealed larger infarct sizes and
elevated levels of the myocardial necrosis marker troponin I and LDH
release (fig 7 A–C). More importantly, delete myocardial PFKFB3
abolished pinocembrin-conferred protective effects on those indexes.
Furthermore, the pinocembrin-improved EF and FS were also abolished by
PFKFB3 deficiency with AAV9-shPFKFB3 (fig 7D). Collectively, these data
support that pinocembrin confers cardioprotective effects by enhanced
cardiomyocytes glycolysis through
activation of PFKFB3.