Sa Shi

Anti-apoptotic action of hydrogen sulfide is associated with early JNK inhibition

The mechanism of action of Hydrogen sulfide (H2S) as a novel endogenous gaseous messenger and potential cardioprotectant is not fully understood. We therefore investigated the prevention of cardiomyocyte apoptosis by exogenous H2S and the signaling pathways leading to cardioprotection. Using a simulated ischemia–reperfusion (I/Re) model with primary cultured rat neonatal cardiomyocytes, I/Re induced a rapid, time‐dependent phosphorylation of c‐Jun N‐terminal kinase (JNK), with significant elevation at 0.25 h and a peak at 0.5 h during reperfusion. NaHS (H2S donor) significantly inhibited the early phosphorylation of JNK, especially at 0.5 h. Both NaHS and SP600125 (specific JNK inhibitor) decreased the number of apoptotic cells, lowered cytochrome C release and enhanced Bcl‐2 expression. When NaHS application was delayed 1 h after reperfusion, the inhibition of apoptosis by H2S was negated. In conclusion, this is novel evidence that early JNK inhibition during reperfusion is associated with H2S‐mediated protection against cardiomyocyte apoptosis.

Calhex231 Ameliorates Cardiac Hypertrophy by Inhibiting Cellular Autophagy in Vivo and in Vitro

Background/Aims: Intracellular calcium concentration ([Ca2+]i) homeostasis, an initial factor of cardiac hypertrophy, is regulated by the calcium-sensing receptor (CaSR) and is associated with the formation of autolysosomes. The aim of this study was to investigate the role of Calhex231, a CaSR inhibitor, on the hypertrophic response via autophagy modulation. Methods: Cardiac hypertrophy was induced by transverse aortic constriction (TAC) in 40 male Wistar rats, while 10 rats underwent a sham operation and served as controls. Cardiac function was monitored by transthoracic echocardiography, and the hypertrophy index was calculated. Cardiac tissue was stained with hematoxylin and eosin (H&E) or Massons trichrome reagent and examined by transmission electron microscopy. An angiotensin II (Ang II)-induced cardiomyocyte hypertrophy model was established and used to test the involvement of active molecules. Intracellular calcium concentration ([Ca2+]i) was determined by the introduction of Fluo-4/AM dye followed by confocal microscopy. The expression of various active proteins was analyzed by western blot. Results: The rats with TAC-induced hypertrophy had an increased heart size, ratio of heart weight to body weight, myocardial fibrosis, and CaSR and autophagy levels, which were suppressed by Calhex231. Experimental results using Ang II-induced hypertrophic cardiomyocytes confirmed that Calhex231 suppressed CaSR expression and downregulated autophagy by inhibiting the Ca2+/calmodulin-dependent-protein kinase-kinase-β (CaMKKβ)- AMP-activated protein kinase (AMPK)-mammalian target of rapamycin (mTOR) pathway to ameliorate cardiomyocyte hypertrophy. Conclusions: Calhex231 ameliorates myocardial hypertrophy induced by pressure-overload or Ang II via inhibiting CaSR expression and autophagy. Our results may support the notion that Calhex231 can become a new therapeutic agent for the treatment of cardiac hypertrophy.

Hematoporphyrin monomethyl ether-mediated photodynamic effects on THP-1 cell-derived macrophages

Photodynamic therapy (PDT) has been shown to attenuate atherosclerotic plaque progression and decrease macrophage-infiltration. The effectiveness of PDT depends strongly on the type of photosensitizers. Hematoporphyrin monomethyl ether (HMME) is a promising second-generation porphyrin-related photosensitizer for PDT. This study is designed to characterize effects of HMME-based PDT on THP-1 cell-derived macrophages and define the cell-death pathway. HMME was identified to accumulate in the macrophages by fluorescence microscopy and confocal scanning laser microscope. Our data demonstrated that the intensity of laser-induced HMME fluorescence in macrophages steadily increased with the increasing incubation concentration of HMME. The survival rate of macrophages determined by MTT assay decreased with the increasing HMME concentration and irradiation time. HMME-based PDT induced macrophage apoptosis via caspase-9 and caspase-3 activation pathway detected by caspase fluorescent assay kit and flow cytometer. The PDT increased the number of apoptotic macrophages by 14-fold at 12 h post irradiation by 9 J/cm(2) 635 nm diode laser. These results imply that photodynamic therapy with HMME may therefore be a useful clinical treatment for unstable atherosclerotic plaques.

Sonodynamic effect of an anti-inflammatory agent--emodin on macrophages.

Emodin has been used as an anti-inflammatory agent and inflammation is a crucial feature of atherosclerosis. Here, we investigated the sonodynamic effect of emodin on macrophages, the pivotal inflammatory cells in atherosclerotic plaque. THP-1 derived macrophages were cultured with emodin and exposed to ultrasound. Six hours later, unlike the cells treated for 5 and 10 min, the viability of cells treated for 15 min decreased significantly and the cells showed typical apoptotic chromatin fragmentation. The percentage of apoptotic and necrotic cells in the sonodynamic therapy (SDT) group was higher than that in the ultrasound group. Two hours after treatment for 15 min, the cytoskeleton lost its original features as the filaments dispersed and the cytoskeletal proteins aggregated. The percentage of cells with disturbed cytoskeletal filaments in the SDT group was higher than that in the ultrasound group. These results suggest emodin has a sonodynamic effect on macrophages and might be used as a novel sonosensitizer for SDT for atherosclerosis.

The efficacy and mechanism of apoptosis induction by hypericin-mediated sonodynamic therapy in THP-1 macrophages

Purpose To investigate the sonoactivity of hypericin (HY), together with its sonodynamic effect on THP-1 macrophages and the underlying mechanism. Materials and methods CCK-8 was used to examine cell viability. Confocal laser scanning microscopy was performed to assess the localization of HY in cells, reactive oxygen species (ROS) generation, and opening of the mitochondrial permeability transition pore (mPTP) after different treatments. Apoptosis was analyzed using Hoechst–propidium iodide and transmission electron microscopy. Mitochondrial membrane potential (ΔΨm) collapse was detected via fluorescence microscopy. Lipoprotein oxidation was determined in malondialdehyde (MDA) assays. Western blotting was conducted to determine the translocation of BAX and cytochrome C and the expression of apoptosis-related proteins. Results HY was sublocalized among the nuclei and the mitochondria, endoplasmic reticulum, Golgi apparatus, and lysosome in the cytosol of THP-1 macrophages. Under low-intensity ultrasound irradiation, HY significantly decreased cell viability and induced apoptosis. Furthermore, greater ROS generation, higher MDA levels, and greater ΔΨm loss were observed in the sonodynamic therapy (SDT) group. Both ROS generation and MDA levels were significantly reduced by the ROS scavenger N-acetyl cysteine (NAC) and the singlet oxygen scavenger sodium azide. Most of the loss of ΔΨm was inhibited by pretreatment with NAC, sodium azide, and the mPTP inhibitor cyclosporin A (CsA). mPTP opening was induced upon SDT but was reduced by pretreatment with bongkrekic acid, 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid disodium, CsA, and NAC. Western blot analyses revealed translocation of BAX and cytochrome C, downregulated expression of Bcl-2, and upregulated expression of cleaved caspase-9, cleaved caspase-3, and cleaved poly(ADP-ribose) polymerase in the SDT group, which were reversed by NAC. Conclusion HY mediated SDT-induced apoptosis in THP-1 macrophages via ROS generation. Then, the proapoptotic factor BAX translocated from the cytosol to the mitochondria, increasing the ratio of BAX/Bcl-2, and the mPTP opened to release cytochrome C. This study demonstrated the great potential of HY-mediated SDT for treating atherosclerosis.

DOPAMINE D2 RECEPTOR STIMULATION INHIBITS ANGIOTENSIN II‐INDUCED HYPERTROPHY IN CULTURED NEONATAL RAT VENTRICULAR MYOCYTES

1 Myocardial hypertrophy is a common pathological change that accompanies cardiovascular disease. Dopamine D2 receptors have been demonstrated in cardiovascular tissues. However, the pathophysiological involvement of D2 receptors in myocardial hypertrophy is unclear. Therefore, the effects of the D2 receptor agonist bromocriptine and the D2 receptor antagonist haloperidol on angiotensin (Ang) II‐ or endothelin (ET)‐1‐induced hypertrophy of cultured neonatal rat ventricular myocytes were investigated in the present study. 2 Protein content and protein synthesis, determined by examining [3H]‐leucine uptake, were used as estimates of cardiomyocyte hypertrophy. The expression of D2 receptor protein in neonatal rat ventricular myocytes was determined using western blotting. Changes in [Ca2+]i in cardiomyocytes were observed by laser scanning confocal microscopy. 3 Angiotensin II and ET‐1, both at 10 nmol/L, induced myocyte hypertrophy, as demonstrated by increased protein content and synthesis, [Ca2+]i levels, protein kinase C (PKC) activity and phosphorylation of extracellular signal‐regulated kinase, c‐Jun N‐terminal kinase and mitogen‐activated protein kinase (MAPK) p38 (p38). Concomitant treatment of cells with 10 nmol/L AngII plus 10 µmol/L bromocriptine significantly inhibited cardiomyocyte hypertrophy, MAPK phosphorylation and PKC activity in the membrane, as well as [Ca2+]i signalling pathways, compared with the effects of AngII alone. In addition, 10 µmol/L bromocriptine significantly inhibited cardiomyocyte hypertrophy induced by 10 nmol/L ET‐1. However, pretreatment with haloperidol (10 µmol/L) had no significant effects on cardiomyocyte hypertrophy induced by either AngII or ET‐1. 4 In conclusion, D2 receptor stimulation inhibits AngII‐induced hypertrophy of cultured neonatal rat ventricular myocytes via inhibition of MAPK, PKC and [Ca2+]i signalling pathways.

Real-time detection of intracellular reactive oxygen species and mitochondrial membrane potential in THP-1 macrophages during ultrasonic irradiation for optimal sonodynamic therapy.

Reactive oxygen species (ROS) elevation and mitochondrial membrane potential (MMP) loss have been proven recently to be involved in sonodynamic therapy (SDT)-induced macrophage apoptosis and necrosis. This study aims to develop an experimental system to monitor intracellular ROS and MMP in real-time during ultrasonic irradiation in order to achieve optimal effect in SDT. Cultured THP-1 derived macrophages were incubated with 5-aminolevulinic acid (ALA), and then sonicated at different intensities. Intracellular ROS elevation and MMP loss were detected in real-time by fluorospectrophotometer using fluorescence probe DCFH-DA and jc-1, respectively. Ultrasound at low intensities (less than 0.48W/cm(2)) had no influence on ROS and MMP in macrophages, whereas at an intensity of 0.48W/cm(2), ROS elevation and MMP loss were observed during ultrasonic irradiation. These effects were strongly enhanced in the presence of ALA. Quantitative analysis showed that ROS elevation and MMP loss monotonically increased with the rise of ultrasonic intensity between 0.48 and 1.16W/cm(2). SDT at 0.48 and 0.84W/cm(2) induced mainly apoptosis in THP-1 macrophages while SDT at 1.16W/cm(2) mainly cell necrosis. This study supports the validity and potential utility of real-time ROS and MMP detection as a dosimetric tool for the determination of optimal SDT.

Suppression of calcium‑sensing receptor ameliorates cardiac hypertrophy through inhibition of autophagy

The calcium-sensing receptor (CaSR) releases intracellular calcium ([Ca2+]i) by accumulating inositol phosphate. Changes in [Ca2+]i initiate myocardial hypertrophy. Furthermore, autophagy associated with [Ca2+]i. Autophagy has previously been demonstrated to participate in the hypertrophic process. The current study investigated whether suppression of CaSR affects the hypertrophic response via modulating autophagy. Isoproterenol (ISO) was used to induce cardiac hypertrophy in Wistar rats. Hypertrophic status was determined by echocardiographic assessment, hematoxylin and eosin, and Massons staining. The protein expression levels of CaSR and autophagy level were observed. Changes of hypertrophy and autophagy indicators were observed following intravenous injection of a CaSR inhibitor. An ISO-induced cardiomyocyte hypertrophy model was established and used determine the involvement of GdCl3. [Ca2+]i was determined using Fluo-4/AM dye followed by confocal microscopy. The expression levels of various active proteins were analyzed by western blotting. The size of the heart, expression levels of CaSR and autophagy level were markedly increased in hypertrophic myocardium. In addition, the present study demonstrated that the indicators of hypertrophy and autophagy were effectively suppressed by CaSR inhibitor. Furthermore, similar effects were demonstrated in neonatal rat hypertrophic cardiomyocytes treated with ISO. It was also observed that CaSR regulates the Ca2+/calmodulin-dependent protein kinase kinase β (CaMKKβ)-AMP-activated protein kinase (AMPK)-mammalian target of rapamycin (mTOR) signaling pathway induced by ISO in cardiomyocytes. Furthermore, the AMPK inhibition significantly reduced the autophagy level following CaSR stimulation (P<0.05). The results of the present demonstrated that inhibition of CaSR may ameliorate cardiac hypertrophy induced by ISO and the effect may be associated with the inhibition of autophagy and suppression of the CaMKKβ-AMPK-mTOR signaling pathway.

Spermine and spermidine reversed age-related cardiac deterioration in rats

Aging is the most important risk factor for cardiovascular disease (CVD). Slowing or reversing the physiological impact of heart aging may reduce morbidity and mortality associated with age-related CVD. The polyamines, spermine (SP) and spermidine (SPD) are essential for cell growth, differentiation and apoptosis, and levels of both decline with age. To explore the effects of these polyamines on heart aging, we administered SP or SPD intraperitoneally to 22- to 24-month-old rats for 6 weeks. Both treatments reversed and inhibited age-related myocardial morphology alterations, myocardial fibrosis, and cell apoptosis. Using combined proteomics and metabolomics analyses, we identified proteins and metabolites up- or downregulated by SP and SPD in aging rat hearts. SP upregulated 51 proteins and 28 metabolites while downregulating 80 proteins and 29 metabolites. SPD upregulated 44 proteins and 24 metabolites and downregulated 84 proteins and 176 metabolites. These molecules were mainly associated with immune responses, blood coagulation, lipid metabolism, and glutathione metabolism pathways. Our study provides novel molecular information on the cardioprotective effects of polyamines in the aging heart, and supports the notion that SP and SPD are potential clinical therapeutics targeting heart disease.Aging is the most important risk factor for cardiovascular disease (CVD). Slowing or reversing the physiological impact of heart aging may reduce morbidity and mortality associated with age-related CVD. The polyamines, spermine (SP) and spermidine (SPD) are essential for cell growth, differentiation and apoptosis, and levels of both decline with age. To explore the effects of these polyamines on heart aging, we administered SP or SPD intraperitoneally to 22- to 24-month-old rats for 6 weeks. Both treatments reversed and inhibited age-related myocardial morphology alterations, myocardial fibrosis, and cell apoptosis. Using combined proteomics and metabolomics analyses, we identified proteins and metabolites up- or downregulated by SP and SPD in aging rat hearts. SP upregulated 51 proteins and 28 metabolites while downregulating 80 proteins and 29 metabolites. SPD upregulated 44 proteins and 24 metabolites and downregulated 84 proteins and 176 metabolites. These molecules were mainly associated with immune responses, blood coagulation, lipid metabolism, and glutathione metabolism pathways. Our study provides novel molecular information on the cardioprotective effects of polyamines in the aging heart, and supports the notion that SP and SPD are potential clinical therapeutics targeting heart disease.

Thioredoxin 2 Offers Protection against Mitochondrial Oxidative Stress in H9c2 Cells and against Myocardial Hypertrophy Induced by Hyperglycemia

Mitochondrial oxidative stress is thought to be a key contributor towards the development of diabetic cardiomyopathy. Thioredoxin 2 (Trx2) is a mitochondrial antioxidant that, along with Trx reductase 2 (TrxR2) and peroxiredoxin 3 (Prx3), scavenges H2O2 and offers protection against oxidative stress. Our previous study showed that TrxR inhibitors resulted in Trx2 oxidation and increased ROS emission from mitochondria. In the present study, we observed that TrxR inhibition also impaired the contractile function of isolated heart. Our studies showed a decrease in the expression of Trx2 in the high glucose-treated H9c2 cardiac cells and myocardium of streptozotocin (STZ)-induced diabetic rats. Overexpression of Trx2 could significantly diminish high glucose-induced mitochondrial oxidative damage and improved ATP production in cultured H9c2 cells. Notably, Trx2 overexpression could suppress high glucose-induced atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) gene expression. Our studies suggest that high glucose-induced mitochondrial oxidative damage can be prevented by elevating Trx2 levels, thereby providing extensive protection to the diabetic heart.