Honokiol blocks and reverses cardiac hypertrophy in mice by activating mitochondrial SIRT3 methods
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HKL blocks hypertrophic response of cardiomyocytes in vitro
Based on previous reports showing that SIRT3 protects cardiomyocytes from hypertrophic stimuli, we asked whether HKL can also protect cardiomyocytes from developing hypertrophy. To this end, cardiomyocytes were treated with a hypertrophic agonist phenylephrine (PE) in the presence or absence of HKL. After 48 hrs of treatment cells were harvested and mitochondrial acetylation was analyzed by western blotting. PE-treated cardiomyocytes showed increased acetylation, whereas this acetylation was reduced in HKL-treated cardiomyocytes ( ). Consistent with this we also observed increased levels of SIRT3 in HKL-treated samples ( ). Hypertrophy of cardiomyocytes was evaluated by measuring incorporation of [ 3 H]-leucine into total cellular proteins, a marker of hypertrophy. HKL-treatment dose dependently attenuated PE-induced [ 3 H] leucine incorporation into total cellular proteins of card...
HKL protects mice from developing cardiac hypertrophy
We next tested the ability of HKL to block development of cardiac hypertrophy in vivo. Mice were subjected to TAC for 28 days. Second day after surgery, HKL-treatment was started (0.2 mg/kg/day) and it was maintained throughout the course of study. TAC-induction in control mice resulted in 25% cardiac hypertrophy as estimated by heart weight to body weight ratio (HW/BW) ( ). This was associated with increased ventricular wall thickness and activation of the fetal gene program (collagen, β-MHC and ANF). These changes were markedly reduced in HKL-treated mice (, ). HKL-treatment also reduced TAC-induced accumulation of fibrosis in the interstitial space, and increase in cardiomyocyte size as revealed by Masson's trichrome staining and WGA staining, respectively ( ). We also tested SIRT3 levels and its activity in mice underwent to TAC. SIRT3 levels were markedly reduced in...
HKL attenuates pre-established cardiac hypertrophy in mice
Knowing that HKL possess anti-hypertrophic activity, we next investigated if HKL can reverse pre-existing (post-banding) cardiac hypertrophy, which is a more clinically relevant situation. Mice were subjected to aortic banding to develop hypertrophy for 4 months. Once the hypertrophy was established, they were treated with HKL for 28 days. As shown in, HKL-treatment significantly reduced the HW/BW ratio in mice subjected to TAC. Consistent with this, HKL-treatment also decreased the ventricular wall thickness and improved the fractional shortening following TAC, compared to untreated mice ( ). Additionally, HKL-treatment significantly reduced the accumulation of interstitial fibrosis and activation of the fetal gene program ( ). We then analyzed the effect of HKL on the signaling program that is known to be activated during hypertrophy. Increased Akt activation is known to induce c...
HKL blocks proliferation and differentiation of cardiac fibroblasts
Interstitial fibrosis is one of the hallmarks of maladaptive cardiac hypertrophy. Since HKL-treatment reduced interstitial fibrosis in the TAC and isoproterenol models, we asked whether HKL can block proliferation and differentiation of cardiac fibroblasts to myofibroblasts, an essential marker of fibrosis. We measured fibroblasts proliferation by analyzing Brdu incorporation into cellular DNA by FACS analysis. HKL-treatment dose dependently reduced the proportion of S-phase cells, while increasing the proportion of G0-G1 cells, suggesting that HKL was capable of blocking cardiac fibroblasts proliferation in the G0-G1 phase ( ). We next determined the effect of HKL on transformation of cardiac fibroblasts into myofibroblasts, by using smooth muscle alpha actin (SMA), fibronectin and collagen-1 as critical determinants of myofibroblast differentiation. Stimulation of fibroblasts with...
HKL reduces ROS production and prevents cardiomyocyte death
We have previously shown that SIRT3 protects cardiomyocytes from oxidative and genotoxic stress by reducing ROS production. To get further support for the ability of HKL to activate SIRT3, we measured H 2 O 2 -induced ROS production in cardiomyocytes. Neonatal cardiomyocytes from wild type and SIRT3-KO mice were treated with H 2 O 2 in the presence or absence of HKL. As shown in, HKL-treatment contained H 2 O 2- induced ROS levels in wild-type cells, but not in SIRT3-KO cells ( ). To gather further support for these results, we also performed a cell death experiment. Consistent with our ROS results, HKL-treatment helped to rescue wild-type cells from H 2 O 2 induced cell death, but not SIRT3-KO cells, suggesting that the cytoprotective effect of HKL is mediated through activation of SIRT3 ( ). To further confirm antiapoptotic activity of HKL, we also tested PARP cleavage in cardio...
HKL directly binds to and activates SIRT3
To understand the mechanism through which HKL activates SIRT3, we asked whether HKL can directly bind to SIRT3 and enhance its enzymatic activity. Different amounts of HKL was incubated with the human SIRT3 (3 µM) and its binding to protein was measured by using fluorescence anisotropy. The results indicated reduced anisotropy values for SIRT3 with increasing amounts of HKL, suggesting a direct binding of HKL to SIRT3 ( ). We next investigated if HKL can enter into mitochondria in order to bind to SIRT3. Mitochondria were isolated and viability of preparation was evaluated by monitoring absorbance at 540nm following incubation with CaCl 2, as described in our previous studies ( ),. Viable mitochondria were then incubated with 10 µM HKL for increasing time duration ranging from 0, 15, 30, 60 and 120 min. After completion of incubation time mitochondria were pelleted, wash...
HKL directly binds to and activates SIRT3
Since in our initial experiments we had found increased protein levels of SIRT3 in HKL treated cells ( ), we tested whether HKL can also stabilize SIRT3 protein levels. Cells were treated with cycloheximide for indicated time points in the presence or absence of HKL, and the protein levels analyzed by western blotting. The presence of cycloheximide did not increase SIRT3 level in HKL-treated cells. These results thus excluded the possibility of protein stabilization as a cause of increased SIRT3 levels by HKL ( ). We then asked whether HKL can activate SIRT3 gene transcription leading to increased SIRT3 levels. Cardiomyocytes were treated with different doses of HKL (5 and 10 µM) for 6 hrs and then analyzed for SIRT3 mRNA levels by RT-PCR analysis. HKL-treatment dose dependently increased SIRT3 mRNA levels, 1.5 fold and 2 fold, respectively ( ). To further confirm these results...
Material and Methods
Primary cultures of cardiac myocytes were prepared from neonatal rat hearts. Briefly, hearts were removed from 1-3 day-old pups (Sprague-Dawley rats, either sex) and kept in cold DMEM. Ventricles were cut into 4 to 6 evenly sized pieces using small scissors and digested using collagenase type II (Worthington). The digested solution was collected with the cannula-syringe avoiding the tissue chunks and was added to one of the already aliquoted 10ml FBS (100%). These steps were repeated six to seven times till no tissue chunks are visible. Tissue digest was spun and pellet was dissolved in DMEM with 5%FBS. Cells were pre-plated for 1hr to remove fibroblasts and unattached cardiomyocytes in suspension were collected and plated in fibronectin-coated culture plates. Cardiomyocytes cultures were used after 24hrs of plating.
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Based on previous reports showing that SIRT3 protects cardiomyocytes from hypertrophic stimuli, we asked whether HKL can also protect cardiomyocytes from developing hypertrophy. To this end, cardiomyocytes were treated with a hypertrophic agonist phenylephrine (PE) in the presence or absence of HKL. After 48 hrs of treatment cells were harvested and mitochondrial acetylation was analyzed by western blotting. PE-treated cardiomyocytes showed increased acetylation, whereas this acetylation was reduced in HKL-treated cardiomyocytes ( ). Consistent with this we also observed increased levels of SIRT3 in HKL-treated samples ( ). Hypertrophy of cardiomyocytes was evaluated by measuring incorporation of [ 3 H]-leucine into total cellular proteins, a marker of hypertrophy. HKL-treatment dose dependently attenuated PE-induced [ 3 H] leucine incorporation into total cellular proteins of cardiomyocytes ( ). Hypertrophic stimuli are known to cause translocation of NFAT into the nucleus, resulting in activation of hypertrophic gene program. To investigate if HKL can block agonist-induced NFAT activation, cardiomyocytes were infected with an adenovirus vector expressing NFAT promoter-luc...
We next tested the ability of HKL to block development of cardiac hypertrophy in vivo. Mice were subjected to TAC for 28 days. Second day after surgery, HKL-treatment was started (0.2 mg/kg/day) and it was maintained throughout the course of study. TAC-induction in control mice resulted in 25% cardiac hypertrophy as estimated by heart weight to body weight ratio (HW/BW) ( ). This was associated with increased ventricular wall thickness and activation of the fetal gene program (collagen, β-MHC and ANF). These changes were markedly reduced in HKL-treated mice (, ). HKL-treatment also reduced TAC-induced accumulation of fibrosis in the interstitial space, and increase in cardiomyocyte size as revealed by Masson's trichrome staining and WGA staining, respectively ( ). We also tested SIRT3 levels and its activity in mice underwent to TAC. SIRT3 levels were markedly reduced in mice subjected to TAC, but were maintained to control levels in HKL-treated mice ( ). This increase in SIRT3 levels also correlated with the acetylation status of MnSOD. MnSOD was hyper-acetylated in mice subjected to TAC, whereas HKL-treatment restored it to control levels (, ). These results s...
Knowing that HKL possess anti-hypertrophic activity, we next investigated if HKL can reverse pre-existing (post-banding) cardiac hypertrophy, which is a more clinically relevant situation. Mice were subjected to aortic banding to develop hypertrophy for 4 months. Once the hypertrophy was established, they were treated with HKL for 28 days. As shown in, HKL-treatment significantly reduced the HW/BW ratio in mice subjected to TAC. Consistent with this, HKL-treatment also decreased the ventricular wall thickness and improved the fractional shortening following TAC, compared to untreated mice ( ). Additionally, HKL-treatment significantly reduced the accumulation of interstitial fibrosis and activation of the fetal gene program ( ). We then analyzed the effect of HKL on the signaling program that is known to be activated during hypertrophy. Increased Akt activation is known to induce cardiac hypertrophy in response to variety of stresses. We therefore examined the role of HKL in regulating the Akt signaling. Increased phosphorylation of Akt was observed in banded mice, and HKL-treatment helped to maintain it to control levels. Consistent with Akt, ERK1/2 was also activated in TA...
Interstitial fibrosis is one of the hallmarks of maladaptive cardiac hypertrophy. Since HKL-treatment reduced interstitial fibrosis in the TAC and isoproterenol models, we asked whether HKL can block proliferation and differentiation of cardiac fibroblasts to myofibroblasts, an essential marker of fibrosis. We measured fibroblasts proliferation by analyzing Brdu incorporation into cellular DNA by FACS analysis. HKL-treatment dose dependently reduced the proportion of S-phase cells, while increasing the proportion of G0-G1 cells, suggesting that HKL was capable of blocking cardiac fibroblasts proliferation in the G0-G1 phase ( ). We next determined the effect of HKL on transformation of cardiac fibroblasts into myofibroblasts, by using smooth muscle alpha actin (SMA), fibronectin and collagen-1 as critical determinants of myofibroblast differentiation. Stimulation of fibroblasts with the pro-fibrotic agent Ang-II resulted in marked increase in stress fiber formation and expression of SMA and fibronectin. These changes were blocked when cells were treated with HKL, suggesting that HKL attenuated fibroblasts differentiation into myofibroblasts (, and ). We also determined if inh...
We have previously shown that SIRT3 protects cardiomyocytes from oxidative and genotoxic stress by reducing ROS production. To get further support for the ability of HKL to activate SIRT3, we measured H 2 O 2 -induced ROS production in cardiomyocytes. Neonatal cardiomyocytes from wild type and SIRT3-KO mice were treated with H 2 O 2 in the presence or absence of HKL. As shown in, HKL-treatment contained H 2 O 2- induced ROS levels in wild-type cells, but not in SIRT3-KO cells ( ). To gather further support for these results, we also performed a cell death experiment. Consistent with our ROS results, HKL-treatment helped to rescue wild-type cells from H 2 O 2 induced cell death, but not SIRT3-KO cells, suggesting that the cytoprotective effect of HKL is mediated through activation of SIRT3 ( ). To further confirm antiapoptotic activity of HKL, we also tested PARP cleavage in cardiomyocytes following H 2 O 2 treatment. As shown in, H2O2-treatement increased levels of cleaved PARP in cardiomyocytes, which was restored back to control levels after HKL treatment, thus supporting anti-apoptotic activity of HKL in cardiomyocytes ( ). To gain additional evidence that the effects o...
To understand the mechanism through which HKL activates SIRT3, we asked whether HKL can directly bind to SIRT3 and enhance its enzymatic activity. Different amounts of HKL was incubated with the human SIRT3 (3 µM) and its binding to protein was measured by using fluorescence anisotropy. The results indicated reduced anisotropy values for SIRT3 with increasing amounts of HKL, suggesting a direct binding of HKL to SIRT3 ( ). We next investigated if HKL can enter into mitochondria in order to bind to SIRT3. Mitochondria were isolated and viability of preparation was evaluated by monitoring absorbance at 540nm following incubation with CaCl 2, as described in our previous studies ( ),. Viable mitochondria were then incubated with 10 µM HKL for increasing time duration ranging from 0, 15, 30, 60 and 120 min. After completion of incubation time mitochondria were pelleted, washed with the incubation buffer and extracted for analysis by liquid chromatography-high-resolution mass spectrometry. The presence of HKL in mitochondria was confirmed by ion-dissociation mass-spectrometry and co-elution with the authentic standard. Increased HKL levels were observed with increasing...
Since in our initial experiments we had found increased protein levels of SIRT3 in HKL treated cells ( ), we tested whether HKL can also stabilize SIRT3 protein levels. Cells were treated with cycloheximide for indicated time points in the presence or absence of HKL, and the protein levels analyzed by western blotting. The presence of cycloheximide did not increase SIRT3 level in HKL-treated cells. These results thus excluded the possibility of protein stabilization as a cause of increased SIRT3 levels by HKL ( ). We then asked whether HKL can activate SIRT3 gene transcription leading to increased SIRT3 levels. Cardiomyocytes were treated with different doses of HKL (5 and 10 µM) for 6 hrs and then analyzed for SIRT3 mRNA levels by RT-PCR analysis. HKL-treatment dose dependently increased SIRT3 mRNA levels, 1.5 fold and 2 fold, respectively ( ). To further confirm these results we treated cardiomyocytes with 10µm HKL for 3hr or 6hr and analyzed for SIRT3 mRNA levels. HKL treatment increased SIRT3 levels 1.5 fold at 3 hrs and nearly 2 fold at 6 hrs, thus confirming increased SIRT3 mRNA expression by HKL ( ). The expression of SIRT3 gene is shown to be regulated by th...
Primary cultures of cardiac myocytes were prepared from neonatal rat hearts. Briefly, hearts were removed from 1-3 day-old pups (Sprague-Dawley rats, either sex) and kept in cold DMEM. Ventricles were cut into 4 to 6 evenly sized pieces using small scissors and digested using collagenase type II (Worthington). The digested solution was collected with the cannula-syringe avoiding the tissue chunks and was added to one of the already aliquoted 10ml FBS (100%). These steps were repeated six to seven times till no tissue chunks are visible. Tissue digest was spun and pellet was dissolved in DMEM with 5%FBS. Cells were pre-plated for 1hr to remove fibroblasts and unattached cardiomyocytes in suspension were collected and plated in fibronectin-coated culture plates. Cardiomyocytes cultures were used after 24hrs of plating.
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