International Journal of Cardiology 182 (2015) 399–402
Contents lists available at ScienceDirect
International Journal of Cardiology journal homepage: www.elsevier.com/locate/ijcard
Letter to the Editor
Alpha B-crystallin prevents ventricular arrhythmia by attenuating inflammation and oxidative stress in rat with autoimmune myocarditis☆ Hyewon Park a,1, Hyelim Park a,b,1, Hye Jin Hwang a, Hyun Seok Hwang c, Hyoeun Kim a, Bum-Rak Choi d, Hui-Nam Pak a, Moon-Hyoung Lee a, Ji Hyung Chung e, Boyoung Joung a,b,⁎ a
Division of Cardiology, Yonsei University College of Medicine, Seoul, Republic of Korea Brain Korea 21 PLUS Project for Medical Science, Yonsei University, Seoul, Republic of Korea The Department of Medicine, Vanderbilt University, Nashville, TN, USA d Cardiovascular Research Center, Rhode Island Hospital and Brown Medical School, Providence, RI, USA e The Department of Applied Bioscience, College of Life Science, CHA University, Seoul, Republic of Korea b c
a r t i c l e
i n f o
Article history: Received 22 December 2014 Accepted 29 December 2014 Available online 30 December 2014 Keywords: Alpha B-crystallin Myocarditis Arrhythmia Inflammation Oxidative stress
Myocarditis has various clinical courses from full recovery to serious sequels including permanent cardiac damage or sudden death by fatal arrhythmic events [1]. Alpha B-crystallin (CryAB), one of the small heat shock protein (HSP) 20 family, is abundantly expressed in cardiomyocytes and is induced by stressful conditions such as heat shock, inflammation, and oxidative stress [2]. Reactive oxygen species (ROS) is known to sustain Ca2 +/calmodulin-dependent protein kinase-II (CaMKII) activity by directly oxidizing the paired regulatory domain, methionine residues [3]. This study investigated whether systemic administration of exogenous CryAB reduced inflammation and
☆ Grant support: This study was supported in part by research grants from the Korean Heart Rhythm Society (2011–3), the Basic Science Research Program through the National Research Foundation of Korea funded by the Ministry of Education, Science and Technology (NRF-2010-0021993, NRF-2012R1A2A2A02045367), and a grant of the Korean Healthcare Technology R&D project funded by Ministry of Health & Welfare (HI12C1552). ⁎ Corresponding author at: Cardiology Division, Department of Internal Medicine, Yonsei University College of Medicine, 250 Seungsanno, Seodaemun-gu, Seoul 120-752, Republic of Korea. E-mail address: [email protected] (B. Joung). 1 Both authors contributed equally to this study.
http://dx.doi.org/10.1016/j.ijcard.2014.12.152 0167-5273/© 2015 Elsevier Ireland Ltd. All rights reserved.
arrhythmia by suppressing CaMKII activity in the experimental of autoimmune myocarditis (EAM). The CryAB fragment was amplified by PCR using the human cDNA library as a template and PCR primers 5′-CACCTAGAATTCATGGACATCG CCATCCAC-3′ (upstream primer, the underlining indicates the EcoRI site) and 5′-AAGAAACTCGAGCTATTTCTTGGGGGCTGC-3′ (downstream primer, the underlining indicates the XhoI site). The CryAB fragment was inserted into the EcoRI and XhoI sites of the pHis/TAT vector [4] for TAT-CryAB fusion protein expression. EAM was produced by a previously described method [5]. Rats were randomly divided into the following four groups. The control rats received injections of 0.2 ml of complete Freund's adjuvant in the same manner (control, n = 10). Six-week-old male Lewis rats were immunized by subcutaneous injection of 1 mg of purified cardiac myosin in each rear footpad on days 1 and 8 (EAM group, n = 10). In additional 14 EAM rats, CryAB (1 mg/kg) with TAT-protein transduction domain (EAM + CryAB group, n = 8) or GFP with TAT-protein transduction domain (EAM + GFP group, n = 6) were injected via intraperitoneum once a day for 2 weeks. On the 21st day after the initial immunization, optical mapping was performed using dual cameras (MiCAMUltima, BrainVision, Tokyo, Japan) as the previously described method [5]. Immunoblot for inflammatory cytokines and Ca2+ handling proteins was also performed as the previously described method [5]. Dynamic Ca2+ images were obtained using a confocal microscope (LSM700, Carl Zeiss) with Fluo-4 staining in rat neonatal cardiomyocytes. To simulate inflammation of cardiomyocytes, recombinant human TNF-α (Sigma Aldrich, Schnelldorf, Germany) was treated in neonatal cardiomyocytes. The animals received humane care in compliance with the ‘Principles of Laboratory Animal Care’ formulated by the National Society for Medical Research. The protocol was approved by the Institutional Animal Care and Use Committee of Yonsei University. TAT-CryAB suppressed oxidative stress and inflammation in EAM. The levels of high-mobility group protein B1, IL-6, TNF-α, cyclooxygenase-2 and iNOS increased significantly in EAM, but not in EAM + CryAB (Fig. 1A). CaMKII, autophosphorylation at CaMKII Thr287 (Thr287CaMKII) and CaMKII Thr306 (Thr306-CaMKII) increased significantly in EAM hearts (Fig. 1B). TAT-CryAB treatment reduced the levels of CaMKII and autophosphorylation at Thr287-CaMKII and Thr306-CaMKII.
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H. Park et al. / International Journal of Cardiology 182 (2015) 399–402
Fig. 1. TAT-CryAB attenuates inflammation, oxidative stress and the phosphorylation of Ca2+ handling protein in myocarditis. A, Representative western blots for HMGB1, IL-6, TNF-a, Cox2, iNOS, and GAPDH showing increased oxidative stress and inflammatory markers in EAM. B, Protein levels and autophosphorylation status of CaMKII at stimulatory (Thr287) and inhibitory (Thr306/Thr307) phosphorylation sites. Top panel, representative western blots for total CaMKII protein, Thr287-CaMKII phosphorylation, Thr306-CaMKII phosphorylation, and GAPDH expression. Bottom panel, quantification of total CaMKII expression and relative Thr287/Thr306 CaMKII phosphorylation levels. C, Phosphorylation of PLB. Top panel, representative Western blots for total PLB and Thr17-phosphorylated PLB in tissue homogenates. Bottom panel, quantification of total and relative Thr17 PLB phosphorylation levels. D, Phosphorylation of RyR. Left panel, representative western blots for total RyR2 and Ser2814-phosphorylated RyR2 in tissue homogenates. Right panel, total RyR2 expression and relative Ser2814 phosphorylation levels. *p b 0.05.
Moreover, the levels of Thr17-PLB and the ratio of Thr18 PLB to total PLB, which decreased in EAM hearts (n = 4), were restored by TAT-CryAB (n = 4) treatment (Fig. 1C). The expression levels of total RyR2 (127%, p = 0.001), RyR2 phosphorylation at Ser2808 (protein kinase A/CaMKII site, 174%, p = 0.001) and Ser2814-RyR2 phosphorylation (CaMKII site, 130%, p = 0.001) significantly increased in EAM hearts than control (n = 4). However, they were suppressed by TAT-CryAB treatment (Fig. 1D). CryAB-GFP (n = 4) did not reverse the increase of total and phosphorylated RyR and PLB induced by myocarditis. To investigate the effect of TAT-CryAB treatment on Vm and Cai transient, the dual optical mapping was performed. We observed prolongation of APD with an increase of Cai transient duration in EAM hearts (Fig. 2A and B). APD dispersion also increased in the groups of EAM (n = 5) and TAT-GFP (n = 5) treated hearts (Fig. 2B). Conduction velocity and conduction heterogeneity index were also improved by CryAB (Fig. 2C and D). Compared with the control group, the incidence of inducible VT or VF under rapid pacing protocol was higher in EAM (0% vs. 83%, p b 0.001) and EAM + TAT-GFP groups (0% vs. 100%, p = 0.007). However, TAT-CryAB treatment reduced inducible VT or VF (13%, p = 0.008 vs. EAM).
Rat neonatal cardiomyocytes were pretreated with CryAB followed by TNF-αchallenge. Ca2 + wave frequency and amplitude were increased in a dose dependent manner in cells treated with TNF-α (Fig. 3A, B and C). In contrast, Ca2 + wave frequency and amplitude were reduced in cells simultaneously treated with both TNF-α and CryAB. Compared to the TNF-α group, CryAB reduced Ca2+ wave frequency and amplitude by 3-fold (p b 0.001) and 2.5-fold (p b 0.001), respectively. The caffeine-induced rise of Ca2+ was increased from 1.0 ± 0.0 in control to 3.0 ± 0.2 ratio units (p b 0.001) in TNF-α treated cells. However, the caffeine-induced rise of Ca2+ was increased only to 1.2 ± 0.1 ratio units in cells treated with both TNF-α and CryAB (p = 1.00 vs. control) (Fig. 3D). In this study, we found that CryAB attenuated inflammation and oxidative stress in EAM, with the combination of the suppression of excessive CaMKII activity and the subsequent recovery of the Ca2 + homeostasis-related proteins. Our study suggests that CryAB might be beneficial to prevent arrhythmia in myocarditis. Human CryAB mutation, R120G CryAB overexpression, enhances anti-oxidative enzymatic and GSH recycling pathways [6]. In the ischemia/reperfusion model, in vitro perfused hearts from transgenic mice
H. Park et al. / International Journal of Cardiology 182 (2015) 399–402
401
Fig. 2. TAT-CryAB prevented APD dispersion and CV heterogeneity. A, Sample traces of Vm and Cai from four groups at pacing cycle length of 300 ms. B, APD, APD dispersion and Cai durations from four groups. Note that EAM group shows prolongation of APD and Cai durations. C, Upper panels, Conduction vector maps. Lower panel, Conduction heterogeneity index. Note the increase of heterogeneity index in EAM, but not in EAM + CryAB.
that ubiquitously overexpressed CryAB tolerated ischemia/reperfusion injury [7], implicating that CryAB may attenuate oxidative stress. In consistence, our study showed that CryAB may have anti-inflammatory role in rat myocarditis. Oxidative stress enhances CaMKII activity by directly oxidizing the paired regulatory domain, methionine residues (Met 281/ 282) in the absence of Ca2+/CaM [3,8]. In this study, ROS generation and phosphorylated CaMKII were significantly elevated in EAM and reversed by CryAB. Oxidative stress such as exposure to hydrogen peroxide and other agents is known to induce APD prolongation and promote after depolarization in cardiomyocytes via CaMKII activation [9,10]. Consistently, CryAB suppresses spontaneous Ca2+ release in rat neonatal myocytes treated with TNF-α in this study. Slow conduction with increased heterogeneity of CV was observed in EAM, but recovered by CryAB treatment. Sequentially, inducible or spontaneous ventricular arrhythmia was significantly reduced in CryAB treated hearts, leading to improved survival rate. In conclusion, CryAB improved survivals and suppressed arrhythmia with the attenuation of the inflammation, oxidative stress and the phosphorylation of Ca2+ handling proteins in EAM. CryAB might be a potential therapeutic modality for myocarditis.
Conflict of interest None.
References [1] U. Eriksson, J.M. Penninger, Autoimmune heart failure: new understandings of pathogenesis, Int. J. Biochem. Cell Biol. 37 (2005) 27–32. [2] G. Kappe, E. Franck, P. Verschuure, W.C. Boelens, J.A. Leunissen, W.W. de Jong, The human genome encodes 10 alpha-crystallin-related small heat shock proteins: HspB1-10, Cell Stress Chaperones 8 (2003) 53–61. [3] J.R. Erickson, M.L. Joiner, X. Guan, W. Kutschke, J. Yang, C.V. Oddis, et al., A dynamic pathway for calcium-independent activation of CaMKII by methionine oxidation, Cell 133 (2008) 462–474. [4] J.H. Kwon, J.B. Kim, K.H. Lee, S.M. Kang, N. Chung, Y. Jang, et al., Protective effect of heat shock protein 27 using protein transduction domain-mediated delivery on ischemia/reperfusion heart injury, Biochem. Biophys. Res. Commun. 363 (2007) 399–404. [5] H. Park, H. Park, D. Lee, S. Oh, J. Lim, H.J. Hwang, et al., Increased phosphorylation of Ca(2+) handling proteins as a proarrhythmic mechanism in myocarditis, Circ. J. 78 (2014) 2292–2301. [6] N.S. Rajasekaran, P. Connell, E.S. Christians, L.J. Yan, R.P. Taylor, A. Orosz, et al., Human alpha B-crystallin mutation causes oxido-reductive stress and protein aggregation cardiomyopathy in mice, Cell 130 (2007) 427–439. [7] P.S. Ray, J.L. Martin, E.A. Swanson, H. Otani, W.H. Dillmann, D.K. Das, Transgene overexpression of alphaB crystallin confers simultaneous protection against cardiomyocyte apoptosis and necrosis during myocardial ischemia and reperfusion, FASEB J. 15 (2001) 393–402. [8] M. Vila-Petroff, M.A. Salas, M. Said, C.A. Valverde, L. Sapia, E. Portiansky, et al., CaMKII inhibition protects against necrosis and apoptosis in irreversible ischemia– reperfusion injury, Cardiovasc. Res. 73 (2007) 689–698. [9] Y. Song, J.C. Shryock, S. Wagner, L.S. Maier, L. Belardinelli, Blocking late sodium current reduces hydrogen peroxide-induced arrhythmogenic activity and contractile dysfunction, J. Pharmacol. Exp. Ther. 318 (2006) 214–222. [10] L.H. Xie, F. Chen, H.S. Karagueuzian, J.N. Weiss, Oxidative-stress-induced afterdepolarizations and calmodulin kinase II signaling, Circ. Res. 104 (2009) 79–86.
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H. Park et al. / International Journal of Cardiology 182 (2015) 399–402
Fig. 3. CryAB suppresses spontaneous Ca2+ release in rat neonatal cardiomyocytes treated with TNF-α. A, Representative line scan images (red arrow) of spontaneous Ca2+ transients obtained from rat neonatal myocytes with TNF-α and TNF-α + CryAB (1 μM). B, The frequency of Ca2+ wave. C, Ca2+ amplitude for each group. CryAB suppresses frequency and amplitude of Ca2+ waves. D, Line scan and line plot examples of SR Ca2+ content evaluated by 10 mmol/L caffeine-evoked Ca2+ transient in control, TNF-α and TNF-α + CryAB. Bars represent mean ± SEM of values normalized by control values on each experimental day. Control (white, n = 15), TNF-α (red, n = 10), and TNF-α + CryAB (blue, n = 10). **p b 0.001 vs. control.
Contents lists available at ScienceDirect
International Journal of Cardiology journal homepage: www.elsevier.com/locate/ijcard
Letter to the Editor
Alpha B-crystallin prevents ventricular arrhythmia by attenuating inflammation and oxidative stress in rat with autoimmune myocarditis☆ Hyewon Park a,1, Hyelim Park a,b,1, Hye Jin Hwang a, Hyun Seok Hwang c, Hyoeun Kim a, Bum-Rak Choi d, Hui-Nam Pak a, Moon-Hyoung Lee a, Ji Hyung Chung e, Boyoung Joung a,b,⁎ a
Division of Cardiology, Yonsei University College of Medicine, Seoul, Republic of Korea Brain Korea 21 PLUS Project for Medical Science, Yonsei University, Seoul, Republic of Korea The Department of Medicine, Vanderbilt University, Nashville, TN, USA d Cardiovascular Research Center, Rhode Island Hospital and Brown Medical School, Providence, RI, USA e The Department of Applied Bioscience, College of Life Science, CHA University, Seoul, Republic of Korea b c
a r t i c l e
i n f o
Article history: Received 22 December 2014 Accepted 29 December 2014 Available online 30 December 2014 Keywords: Alpha B-crystallin Myocarditis Arrhythmia Inflammation Oxidative stress
Myocarditis has various clinical courses from full recovery to serious sequels including permanent cardiac damage or sudden death by fatal arrhythmic events [1]. Alpha B-crystallin (CryAB), one of the small heat shock protein (HSP) 20 family, is abundantly expressed in cardiomyocytes and is induced by stressful conditions such as heat shock, inflammation, and oxidative stress [2]. Reactive oxygen species (ROS) is known to sustain Ca2 +/calmodulin-dependent protein kinase-II (CaMKII) activity by directly oxidizing the paired regulatory domain, methionine residues [3]. This study investigated whether systemic administration of exogenous CryAB reduced inflammation and
☆ Grant support: This study was supported in part by research grants from the Korean Heart Rhythm Society (2011–3), the Basic Science Research Program through the National Research Foundation of Korea funded by the Ministry of Education, Science and Technology (NRF-2010-0021993, NRF-2012R1A2A2A02045367), and a grant of the Korean Healthcare Technology R&D project funded by Ministry of Health & Welfare (HI12C1552). ⁎ Corresponding author at: Cardiology Division, Department of Internal Medicine, Yonsei University College of Medicine, 250 Seungsanno, Seodaemun-gu, Seoul 120-752, Republic of Korea. E-mail address: [email protected] (B. Joung). 1 Both authors contributed equally to this study.
http://dx.doi.org/10.1016/j.ijcard.2014.12.152 0167-5273/© 2015 Elsevier Ireland Ltd. All rights reserved.
arrhythmia by suppressing CaMKII activity in the experimental of autoimmune myocarditis (EAM). The CryAB fragment was amplified by PCR using the human cDNA library as a template and PCR primers 5′-CACCTAGAATTCATGGACATCG CCATCCAC-3′ (upstream primer, the underlining indicates the EcoRI site) and 5′-AAGAAACTCGAGCTATTTCTTGGGGGCTGC-3′ (downstream primer, the underlining indicates the XhoI site). The CryAB fragment was inserted into the EcoRI and XhoI sites of the pHis/TAT vector [4] for TAT-CryAB fusion protein expression. EAM was produced by a previously described method [5]. Rats were randomly divided into the following four groups. The control rats received injections of 0.2 ml of complete Freund's adjuvant in the same manner (control, n = 10). Six-week-old male Lewis rats were immunized by subcutaneous injection of 1 mg of purified cardiac myosin in each rear footpad on days 1 and 8 (EAM group, n = 10). In additional 14 EAM rats, CryAB (1 mg/kg) with TAT-protein transduction domain (EAM + CryAB group, n = 8) or GFP with TAT-protein transduction domain (EAM + GFP group, n = 6) were injected via intraperitoneum once a day for 2 weeks. On the 21st day after the initial immunization, optical mapping was performed using dual cameras (MiCAMUltima, BrainVision, Tokyo, Japan) as the previously described method [5]. Immunoblot for inflammatory cytokines and Ca2+ handling proteins was also performed as the previously described method [5]. Dynamic Ca2+ images were obtained using a confocal microscope (LSM700, Carl Zeiss) with Fluo-4 staining in rat neonatal cardiomyocytes. To simulate inflammation of cardiomyocytes, recombinant human TNF-α (Sigma Aldrich, Schnelldorf, Germany) was treated in neonatal cardiomyocytes. The animals received humane care in compliance with the ‘Principles of Laboratory Animal Care’ formulated by the National Society for Medical Research. The protocol was approved by the Institutional Animal Care and Use Committee of Yonsei University. TAT-CryAB suppressed oxidative stress and inflammation in EAM. The levels of high-mobility group protein B1, IL-6, TNF-α, cyclooxygenase-2 and iNOS increased significantly in EAM, but not in EAM + CryAB (Fig. 1A). CaMKII, autophosphorylation at CaMKII Thr287 (Thr287CaMKII) and CaMKII Thr306 (Thr306-CaMKII) increased significantly in EAM hearts (Fig. 1B). TAT-CryAB treatment reduced the levels of CaMKII and autophosphorylation at Thr287-CaMKII and Thr306-CaMKII.
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Fig. 1. TAT-CryAB attenuates inflammation, oxidative stress and the phosphorylation of Ca2+ handling protein in myocarditis. A, Representative western blots for HMGB1, IL-6, TNF-a, Cox2, iNOS, and GAPDH showing increased oxidative stress and inflammatory markers in EAM. B, Protein levels and autophosphorylation status of CaMKII at stimulatory (Thr287) and inhibitory (Thr306/Thr307) phosphorylation sites. Top panel, representative western blots for total CaMKII protein, Thr287-CaMKII phosphorylation, Thr306-CaMKII phosphorylation, and GAPDH expression. Bottom panel, quantification of total CaMKII expression and relative Thr287/Thr306 CaMKII phosphorylation levels. C, Phosphorylation of PLB. Top panel, representative Western blots for total PLB and Thr17-phosphorylated PLB in tissue homogenates. Bottom panel, quantification of total and relative Thr17 PLB phosphorylation levels. D, Phosphorylation of RyR. Left panel, representative western blots for total RyR2 and Ser2814-phosphorylated RyR2 in tissue homogenates. Right panel, total RyR2 expression and relative Ser2814 phosphorylation levels. *p b 0.05.
Moreover, the levels of Thr17-PLB and the ratio of Thr18 PLB to total PLB, which decreased in EAM hearts (n = 4), were restored by TAT-CryAB (n = 4) treatment (Fig. 1C). The expression levels of total RyR2 (127%, p = 0.001), RyR2 phosphorylation at Ser2808 (protein kinase A/CaMKII site, 174%, p = 0.001) and Ser2814-RyR2 phosphorylation (CaMKII site, 130%, p = 0.001) significantly increased in EAM hearts than control (n = 4). However, they were suppressed by TAT-CryAB treatment (Fig. 1D). CryAB-GFP (n = 4) did not reverse the increase of total and phosphorylated RyR and PLB induced by myocarditis. To investigate the effect of TAT-CryAB treatment on Vm and Cai transient, the dual optical mapping was performed. We observed prolongation of APD with an increase of Cai transient duration in EAM hearts (Fig. 2A and B). APD dispersion also increased in the groups of EAM (n = 5) and TAT-GFP (n = 5) treated hearts (Fig. 2B). Conduction velocity and conduction heterogeneity index were also improved by CryAB (Fig. 2C and D). Compared with the control group, the incidence of inducible VT or VF under rapid pacing protocol was higher in EAM (0% vs. 83%, p b 0.001) and EAM + TAT-GFP groups (0% vs. 100%, p = 0.007). However, TAT-CryAB treatment reduced inducible VT or VF (13%, p = 0.008 vs. EAM).
Rat neonatal cardiomyocytes were pretreated with CryAB followed by TNF-αchallenge. Ca2 + wave frequency and amplitude were increased in a dose dependent manner in cells treated with TNF-α (Fig. 3A, B and C). In contrast, Ca2 + wave frequency and amplitude were reduced in cells simultaneously treated with both TNF-α and CryAB. Compared to the TNF-α group, CryAB reduced Ca2+ wave frequency and amplitude by 3-fold (p b 0.001) and 2.5-fold (p b 0.001), respectively. The caffeine-induced rise of Ca2+ was increased from 1.0 ± 0.0 in control to 3.0 ± 0.2 ratio units (p b 0.001) in TNF-α treated cells. However, the caffeine-induced rise of Ca2+ was increased only to 1.2 ± 0.1 ratio units in cells treated with both TNF-α and CryAB (p = 1.00 vs. control) (Fig. 3D). In this study, we found that CryAB attenuated inflammation and oxidative stress in EAM, with the combination of the suppression of excessive CaMKII activity and the subsequent recovery of the Ca2 + homeostasis-related proteins. Our study suggests that CryAB might be beneficial to prevent arrhythmia in myocarditis. Human CryAB mutation, R120G CryAB overexpression, enhances anti-oxidative enzymatic and GSH recycling pathways [6]. In the ischemia/reperfusion model, in vitro perfused hearts from transgenic mice
H. Park et al. / International Journal of Cardiology 182 (2015) 399–402
401
Fig. 2. TAT-CryAB prevented APD dispersion and CV heterogeneity. A, Sample traces of Vm and Cai from four groups at pacing cycle length of 300 ms. B, APD, APD dispersion and Cai durations from four groups. Note that EAM group shows prolongation of APD and Cai durations. C, Upper panels, Conduction vector maps. Lower panel, Conduction heterogeneity index. Note the increase of heterogeneity index in EAM, but not in EAM + CryAB.
that ubiquitously overexpressed CryAB tolerated ischemia/reperfusion injury [7], implicating that CryAB may attenuate oxidative stress. In consistence, our study showed that CryAB may have anti-inflammatory role in rat myocarditis. Oxidative stress enhances CaMKII activity by directly oxidizing the paired regulatory domain, methionine residues (Met 281/ 282) in the absence of Ca2+/CaM [3,8]. In this study, ROS generation and phosphorylated CaMKII were significantly elevated in EAM and reversed by CryAB. Oxidative stress such as exposure to hydrogen peroxide and other agents is known to induce APD prolongation and promote after depolarization in cardiomyocytes via CaMKII activation [9,10]. Consistently, CryAB suppresses spontaneous Ca2+ release in rat neonatal myocytes treated with TNF-α in this study. Slow conduction with increased heterogeneity of CV was observed in EAM, but recovered by CryAB treatment. Sequentially, inducible or spontaneous ventricular arrhythmia was significantly reduced in CryAB treated hearts, leading to improved survival rate. In conclusion, CryAB improved survivals and suppressed arrhythmia with the attenuation of the inflammation, oxidative stress and the phosphorylation of Ca2+ handling proteins in EAM. CryAB might be a potential therapeutic modality for myocarditis.
Conflict of interest None.
References [1] U. Eriksson, J.M. Penninger, Autoimmune heart failure: new understandings of pathogenesis, Int. J. Biochem. Cell Biol. 37 (2005) 27–32. [2] G. Kappe, E. Franck, P. Verschuure, W.C. Boelens, J.A. Leunissen, W.W. de Jong, The human genome encodes 10 alpha-crystallin-related small heat shock proteins: HspB1-10, Cell Stress Chaperones 8 (2003) 53–61. [3] J.R. Erickson, M.L. Joiner, X. Guan, W. Kutschke, J. Yang, C.V. Oddis, et al., A dynamic pathway for calcium-independent activation of CaMKII by methionine oxidation, Cell 133 (2008) 462–474. [4] J.H. Kwon, J.B. Kim, K.H. Lee, S.M. Kang, N. Chung, Y. Jang, et al., Protective effect of heat shock protein 27 using protein transduction domain-mediated delivery on ischemia/reperfusion heart injury, Biochem. Biophys. Res. Commun. 363 (2007) 399–404. [5] H. Park, H. Park, D. Lee, S. Oh, J. Lim, H.J. Hwang, et al., Increased phosphorylation of Ca(2+) handling proteins as a proarrhythmic mechanism in myocarditis, Circ. J. 78 (2014) 2292–2301. [6] N.S. Rajasekaran, P. Connell, E.S. Christians, L.J. Yan, R.P. Taylor, A. Orosz, et al., Human alpha B-crystallin mutation causes oxido-reductive stress and protein aggregation cardiomyopathy in mice, Cell 130 (2007) 427–439. [7] P.S. Ray, J.L. Martin, E.A. Swanson, H. Otani, W.H. Dillmann, D.K. Das, Transgene overexpression of alphaB crystallin confers simultaneous protection against cardiomyocyte apoptosis and necrosis during myocardial ischemia and reperfusion, FASEB J. 15 (2001) 393–402. [8] M. Vila-Petroff, M.A. Salas, M. Said, C.A. Valverde, L. Sapia, E. Portiansky, et al., CaMKII inhibition protects against necrosis and apoptosis in irreversible ischemia– reperfusion injury, Cardiovasc. Res. 73 (2007) 689–698. [9] Y. Song, J.C. Shryock, S. Wagner, L.S. Maier, L. Belardinelli, Blocking late sodium current reduces hydrogen peroxide-induced arrhythmogenic activity and contractile dysfunction, J. Pharmacol. Exp. Ther. 318 (2006) 214–222. [10] L.H. Xie, F. Chen, H.S. Karagueuzian, J.N. Weiss, Oxidative-stress-induced afterdepolarizations and calmodulin kinase II signaling, Circ. Res. 104 (2009) 79–86.
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Fig. 3. CryAB suppresses spontaneous Ca2+ release in rat neonatal cardiomyocytes treated with TNF-α. A, Representative line scan images (red arrow) of spontaneous Ca2+ transients obtained from rat neonatal myocytes with TNF-α and TNF-α + CryAB (1 μM). B, The frequency of Ca2+ wave. C, Ca2+ amplitude for each group. CryAB suppresses frequency and amplitude of Ca2+ waves. D, Line scan and line plot examples of SR Ca2+ content evaluated by 10 mmol/L caffeine-evoked Ca2+ transient in control, TNF-α and TNF-α + CryAB. Bars represent mean ± SEM of values normalized by control values on each experimental day. Control (white, n = 15), TNF-α (red, n = 10), and TNF-α + CryAB (blue, n = 10). **p b 0.001 vs. control.
