Accepted Manuscript Carvedilol promotes mitochondrial biogenesis by regulating the PGC-1/TFAM pathway in Human umbilical vein endothelial cells (HUVECs) Kai Yao, Wayne W. Zhang, Luyu Yao, Shu Yang, Wanpin Nie, Feizhou Huang PII:
S0006-291X(16)30089-4
DOI:
10.1016/j.bbrc.2016.01.089
Reference:
YBBRC 35206
To appear in:
Biochemical and Biophysical Research Communications
Received Date: 25 December 2015 Accepted Date: 15 January 2016
Please cite this article as: K. Yao, W.W. Zhang, L. Yao, S. Yang, W. Nie, F. Huang, Carvedilol promotes mitochondrial biogenesis by regulating the PGC-1/TFAM pathway in Human umbilical vein endothelial cells (HUVECs), Biochemical and Biophysical Research Communications (2016), doi: 10.1016/ j.bbrc.2016.01.089. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT
Title: Carvedilol promotes mitochondrial biogenesis by regulating the PGC1/TFAM pathway in Human umbilical vein endothelial cells (HUVECs)
RI PT
Authors Kai Yao1*, Wayne W. Zhang2*, Luyu Yao2, Shu Yang2，Wanpin Nie1, Feizhou1 Huang.
SC
Affiliations:
1. Department of General Surgery, The Third Xiang Ya Hospital of central south
M AN U
University, Changsha 410013 China;
2. Vascular and Endovascular Surgery, Louisiana State University Health Sciences Center-Shreveport, 71103 USA.
Corresponding to: Feizhou Huang
TE D
* Both authors contributed equally to this work.
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Department of General Surgery, The Third Xiang Ya Hospital of central south University, 138Tongzipo Road, Changsha 410013 China.
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Tel/Fax: +86073188618686
Email: [email protected]
ACCEPTED MANUSCRIPT
Abstract
RI PT
Carvedilol, a third-generation and nonselective β-adrenoceptor antagonist, is a licensed drug for treating patients suffering from heart failure in clinics. It has been shown that Carvedilol protects cells against mitochondrial dysfunction. However, it’s unknown whether Carvedilol affects mitochondrial biogenesis. In this study,
SC
we found that treatment with Carvedilol in HUVECs resulted in a significant increase of PGC-1α, NRF1, and TFAM. Notably, Carvedilol significantly
M AN U
increased mtDNA contents and the two mitochondrial proteins, cytochrome C and COX IV. In addition, MitoTracker Red staining results indicated that treatment with Carvedilol increased mitochondria mass. Mechanistically, we found that the effect of Carvedilol on the expression of PGC-1α is mediated by the PKA-CREB pathway. Importantly, our results revealed that stimulation of mitochondrial
TE D
biogenesis by carvedilol resulted in functional gain of the mitochondria by showing increased oxygen consumption and mitochondrial respiratory rate. The increased expression of PGC-1α and mitochondrial biogenesis induced by
in heart failure.
EP
Carvedilol might suggest a new mechanism of the therapeutic effects of Carvedilol
AC C
Key words: Carvedilol; mitochondria; PGC-1α; endothelium
ACCEPTED MANUSCRIPT
1. Introduction
RI PT
As the multifunctional organelles in cells, mitochondria are highly dynamic in eukaryotic cells [1]. In endothelial cells (ECs), the potential physiological role of mitochondria has attracted more and more attentions [2]. In order to maintain mitochondrial function, it has been considered to be imperative to identify an
SC
effective intervention to manipulate mitochondrial networks in the endothelium. Mitochondrial biogenesis is a complex process to initial the replication of
M AN U
mitochondrial DNA (mtDNA) and the expression of mitochondrial proteins encoded by both nuclear and mitochondrial genomes. Therefore, mitochondrial biogenesis plays a pivotal role in optimizing cellular mitochondrial function [3]. Peroxisome proliferator-activated receptor (PPAR) gamma coactivator-1 (PGC-1α and PGC-1β) has been considered as a central regulator of mitochondrial
TE D
biogenesis [4.]. PGC-1α transactivates nuclear respiratory factor 1 (NRF-1) which, in turn, activates mtDNA transcription factor A (TFAM) that regulates mtDNA transcription and replication [5].
EP
Carvedilol, a third-generation and nonselective β-adrenoceptor antagonist, is a licensed drug for treating patients suffering from heart failure in clinics [6]. Several
AC C
previous studies have demonstrated that Carvedilol displays its multiple pharmacological properties, including antioxidant, anti-inflammatory, antiapoptotic, and antifibrotic properties [7]. Notably, increasing evidence has shown that Carvedilol protects mitochondria against various stress. Oliveira and colleagues have recently reported that Carvedilol has a positive impact on cardiac mitochondria during in vitro, ex-vivo and in vivo models of cardiac dysfunction [8.]. A recent study also showed that Carvedilol inhibits mitochondrial complex I and
ACCEPTED MANUSCRIPT
induces resistance to H2O2 -mediated oxidative insult in H9C2 myocardial cells. Carvedilol is also found to inhibit the calcium-dependent augmentation of mVO(2) and ROS production upon complex I injury in mitochondria [9.]. Carvedilol also
RI PT
protects ischemic mitochondria by preventing oxidative mitochondrial damage and inhibiting the formation of the mitochondrial permeability transition (MPT) pore [10]. However, it’s unknown whether Carvedilol affects mitochondrial biogenesis
2. Materials and methods
M AN U
2.1. Cell culture, treatment and transfection
SC
and relevant signaling pathways.
Human umbilical vein endothelial cells (HUVECs) were from Lonza, USA. Cells were cultured in EGM™ BulletKit media containing supplemental growth factors. Cells were treated by 10 µM Carvedilol with or without 10 µM PKA inhibitor H89
TE D
(Sigma, USA). 2.2. MitoTracker red Staining
Upon the completion of indicated treatment, HUVECs were washed with
EP
phosphate buffer saline (PBS) for 3 times. Then cells were loaded with the mitochondria dye MitoTracker red (20 nM) and incubated for 30 min at 37℃ in
AC C
darkness. Nuclei were counterstained with the VECTASHIELD® Mounting Media. Fluorescence signals were investigated by 100×oil immersion using Zeiss fluorescence microscope. 100 cells were randomly selected/group. The average IOD analyzed with Image-Pro Plus software was used to index mitochondrial mass. 2.3 Real-time Polymerase Chain Reaction (PCR) Total intracellular RNA was isolated from HUVECs using Qiazol reagent (Qiagene, USA) according to the manufacturer’s protocol. Concentration and
ACCEPTED MANUSCRIPT
quality of RNA were determined by NanoDrop 1000. Equal amount of total RNA (2 µg) was then used as template for reverse transcription PCR to synthesize cDNA. The synthesized cDNA was used for quantitative real-time PCR analysis
RI PT
using SYBR Green qPCR Master Mix (Roche). Gene expression of target genes was normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) using the ∆∆Ct
method.
SC
2.4 Western Blot Analysis
Proteins were extracted from HUVECs using a commercial RIPA buffer (Sigma-
M AN U
Aldrich, USA) with a cocktail of protease inhibitor (Roche, USA). Protein concentration was determined by using the bicinchoninic acid assay (BCA assay) (Thermo Scientific, USA). Subsequently, equal amount of protein (20 µg) was subject to 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDSPAGE). Separate proteins were then transferred to a high-quality polyvinylidene
TE D
difluoride (PVDF) membrane. The membranes were blocked with a blocking buffer containing 5% powdered milk in TBS–Tween 20 for 1 h at RT. The membranes were then sequentially probed with primary antibodies overnight at
EP
4 °C and horseradish peroxidase (HRP)-conjugated secondary antibody for 2 h at RT. Blots were then developed using the Pierce ECL Plus Western Blotting
AC C
Substrate. Densitometric analysis was performed with the ImageJ software (NIH, USA).
2.5. Mitochondrial DNA (mtDNA) copy number Determination mtDNA copy number was determined by real-time PCR method as mentioned above. Total intracellular DNA was extracted from HUVECs using QIAamp DNA mini kit (QIAGEN, Germantown, MD, USA) according to the manufacturer’s instruction. Concentration and quality of DNA were determined by NanoDrop
ACCEPTED MANUSCRIPT
1000. ND1 was used to represent mtDNA and 18S was used to represent nuclear DNA. The following premiers were used for the real time PCR assay. ND1: forward, 5’-ATGGCCAACCTCCTACTCCT-3’; reverse: 5’-
ACGGACCAGAGCGAAAGCA-30and reverse, 5’GACATCTAAGGGCATCACAGAC-3’.
RI PT
GCGGTGATGTAGAGGGTGAT-3’; 18S, were: forward, 5’-
SC
2.6 Measurements of mitochondrial respiration and complex activities
In order to determine whether Carvedilol restores mitochondrial function, a
M AN U
respirometer (Oxygraph, Oroboros Instrument) equipped with a Peltier thermostat and electromagnetic stirrer was used to measure O2 consumption. Briefly, medium containing 5×106 HUVECs were put in a glass chamber equilibrated in ambient room air with continuous stirring (750 rpm) for 10 min. The oxygen consumption
of the O2 consumption. 2.7 Statistics
TE D
was recorded at 2 second intervals and the recording was stopped after stabilization
Experimental results are shown as mean±standard deviation. Statistical
EP
significance was determined using one-way analysis of variance (ANOVA) followed by Tukey's post hoc test. Differences between means were considered
AC C
significant at p
S0006-291X(16)30089-4
DOI:
10.1016/j.bbrc.2016.01.089
Reference:
YBBRC 35206
To appear in:
Biochemical and Biophysical Research Communications
Received Date: 25 December 2015 Accepted Date: 15 January 2016
Please cite this article as: K. Yao, W.W. Zhang, L. Yao, S. Yang, W. Nie, F. Huang, Carvedilol promotes mitochondrial biogenesis by regulating the PGC-1/TFAM pathway in Human umbilical vein endothelial cells (HUVECs), Biochemical and Biophysical Research Communications (2016), doi: 10.1016/ j.bbrc.2016.01.089. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT
Title: Carvedilol promotes mitochondrial biogenesis by regulating the PGC1/TFAM pathway in Human umbilical vein endothelial cells (HUVECs)
RI PT
Authors Kai Yao1*, Wayne W. Zhang2*, Luyu Yao2, Shu Yang2，Wanpin Nie1, Feizhou1 Huang.
SC
Affiliations:
1. Department of General Surgery, The Third Xiang Ya Hospital of central south
M AN U
University, Changsha 410013 China;
2. Vascular and Endovascular Surgery, Louisiana State University Health Sciences Center-Shreveport, 71103 USA.
Corresponding to: Feizhou Huang
TE D
* Both authors contributed equally to this work.
EP
Department of General Surgery, The Third Xiang Ya Hospital of central south University, 138Tongzipo Road, Changsha 410013 China.
AC C
Tel/Fax: +86073188618686
Email: [email protected]
ACCEPTED MANUSCRIPT
Abstract
RI PT
Carvedilol, a third-generation and nonselective β-adrenoceptor antagonist, is a licensed drug for treating patients suffering from heart failure in clinics. It has been shown that Carvedilol protects cells against mitochondrial dysfunction. However, it’s unknown whether Carvedilol affects mitochondrial biogenesis. In this study,
SC
we found that treatment with Carvedilol in HUVECs resulted in a significant increase of PGC-1α, NRF1, and TFAM. Notably, Carvedilol significantly
M AN U
increased mtDNA contents and the two mitochondrial proteins, cytochrome C and COX IV. In addition, MitoTracker Red staining results indicated that treatment with Carvedilol increased mitochondria mass. Mechanistically, we found that the effect of Carvedilol on the expression of PGC-1α is mediated by the PKA-CREB pathway. Importantly, our results revealed that stimulation of mitochondrial
TE D
biogenesis by carvedilol resulted in functional gain of the mitochondria by showing increased oxygen consumption and mitochondrial respiratory rate. The increased expression of PGC-1α and mitochondrial biogenesis induced by
in heart failure.
EP
Carvedilol might suggest a new mechanism of the therapeutic effects of Carvedilol
AC C
Key words: Carvedilol; mitochondria; PGC-1α; endothelium
ACCEPTED MANUSCRIPT
1. Introduction
RI PT
As the multifunctional organelles in cells, mitochondria are highly dynamic in eukaryotic cells [1]. In endothelial cells (ECs), the potential physiological role of mitochondria has attracted more and more attentions [2]. In order to maintain mitochondrial function, it has been considered to be imperative to identify an
SC
effective intervention to manipulate mitochondrial networks in the endothelium. Mitochondrial biogenesis is a complex process to initial the replication of
M AN U
mitochondrial DNA (mtDNA) and the expression of mitochondrial proteins encoded by both nuclear and mitochondrial genomes. Therefore, mitochondrial biogenesis plays a pivotal role in optimizing cellular mitochondrial function [3]. Peroxisome proliferator-activated receptor (PPAR) gamma coactivator-1 (PGC-1α and PGC-1β) has been considered as a central regulator of mitochondrial
TE D
biogenesis [4.]. PGC-1α transactivates nuclear respiratory factor 1 (NRF-1) which, in turn, activates mtDNA transcription factor A (TFAM) that regulates mtDNA transcription and replication [5].
EP
Carvedilol, a third-generation and nonselective β-adrenoceptor antagonist, is a licensed drug for treating patients suffering from heart failure in clinics [6]. Several
AC C
previous studies have demonstrated that Carvedilol displays its multiple pharmacological properties, including antioxidant, anti-inflammatory, antiapoptotic, and antifibrotic properties [7]. Notably, increasing evidence has shown that Carvedilol protects mitochondria against various stress. Oliveira and colleagues have recently reported that Carvedilol has a positive impact on cardiac mitochondria during in vitro, ex-vivo and in vivo models of cardiac dysfunction [8.]. A recent study also showed that Carvedilol inhibits mitochondrial complex I and
ACCEPTED MANUSCRIPT
induces resistance to H2O2 -mediated oxidative insult in H9C2 myocardial cells. Carvedilol is also found to inhibit the calcium-dependent augmentation of mVO(2) and ROS production upon complex I injury in mitochondria [9.]. Carvedilol also
RI PT
protects ischemic mitochondria by preventing oxidative mitochondrial damage and inhibiting the formation of the mitochondrial permeability transition (MPT) pore [10]. However, it’s unknown whether Carvedilol affects mitochondrial biogenesis
2. Materials and methods
M AN U
2.1. Cell culture, treatment and transfection
SC
and relevant signaling pathways.
Human umbilical vein endothelial cells (HUVECs) were from Lonza, USA. Cells were cultured in EGM™ BulletKit media containing supplemental growth factors. Cells were treated by 10 µM Carvedilol with or without 10 µM PKA inhibitor H89
TE D
(Sigma, USA). 2.2. MitoTracker red Staining
Upon the completion of indicated treatment, HUVECs were washed with
EP
phosphate buffer saline (PBS) for 3 times. Then cells were loaded with the mitochondria dye MitoTracker red (20 nM) and incubated for 30 min at 37℃ in
AC C
darkness. Nuclei were counterstained with the VECTASHIELD® Mounting Media. Fluorescence signals were investigated by 100×oil immersion using Zeiss fluorescence microscope. 100 cells were randomly selected/group. The average IOD analyzed with Image-Pro Plus software was used to index mitochondrial mass. 2.3 Real-time Polymerase Chain Reaction (PCR) Total intracellular RNA was isolated from HUVECs using Qiazol reagent (Qiagene, USA) according to the manufacturer’s protocol. Concentration and
ACCEPTED MANUSCRIPT
quality of RNA were determined by NanoDrop 1000. Equal amount of total RNA (2 µg) was then used as template for reverse transcription PCR to synthesize cDNA. The synthesized cDNA was used for quantitative real-time PCR analysis
RI PT
using SYBR Green qPCR Master Mix (Roche). Gene expression of target genes was normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) using the ∆∆Ct
method.
SC
2.4 Western Blot Analysis
Proteins were extracted from HUVECs using a commercial RIPA buffer (Sigma-
M AN U
Aldrich, USA) with a cocktail of protease inhibitor (Roche, USA). Protein concentration was determined by using the bicinchoninic acid assay (BCA assay) (Thermo Scientific, USA). Subsequently, equal amount of protein (20 µg) was subject to 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDSPAGE). Separate proteins were then transferred to a high-quality polyvinylidene
TE D
difluoride (PVDF) membrane. The membranes were blocked with a blocking buffer containing 5% powdered milk in TBS–Tween 20 for 1 h at RT. The membranes were then sequentially probed with primary antibodies overnight at
EP
4 °C and horseradish peroxidase (HRP)-conjugated secondary antibody for 2 h at RT. Blots were then developed using the Pierce ECL Plus Western Blotting
AC C
Substrate. Densitometric analysis was performed with the ImageJ software (NIH, USA).
2.5. Mitochondrial DNA (mtDNA) copy number Determination mtDNA copy number was determined by real-time PCR method as mentioned above. Total intracellular DNA was extracted from HUVECs using QIAamp DNA mini kit (QIAGEN, Germantown, MD, USA) according to the manufacturer’s instruction. Concentration and quality of DNA were determined by NanoDrop
ACCEPTED MANUSCRIPT
1000. ND1 was used to represent mtDNA and 18S was used to represent nuclear DNA. The following premiers were used for the real time PCR assay. ND1: forward, 5’-ATGGCCAACCTCCTACTCCT-3’; reverse: 5’-
ACGGACCAGAGCGAAAGCA-30and reverse, 5’GACATCTAAGGGCATCACAGAC-3’.
RI PT
GCGGTGATGTAGAGGGTGAT-3’; 18S, were: forward, 5’-
SC
2.6 Measurements of mitochondrial respiration and complex activities
In order to determine whether Carvedilol restores mitochondrial function, a
M AN U
respirometer (Oxygraph, Oroboros Instrument) equipped with a Peltier thermostat and electromagnetic stirrer was used to measure O2 consumption. Briefly, medium containing 5×106 HUVECs were put in a glass chamber equilibrated in ambient room air with continuous stirring (750 rpm) for 10 min. The oxygen consumption
of the O2 consumption. 2.7 Statistics
TE D
was recorded at 2 second intervals and the recording was stopped after stabilization
Experimental results are shown as mean±standard deviation. Statistical
EP
significance was determined using one-way analysis of variance (ANOVA) followed by Tukey's post hoc test. Differences between means were considered
AC C
significant at p
