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Wang S, Wang H, Su X, Liu B, Wang L, Yan H, Mao S, Huang H, Huang C, Cheng M, Wu G. β-adrenergic activation may promote myosin light chain kinase degradation through calpain in pressure overload-induced cardiac hypertrophy: β-adrenergic activation results in MLCK degradation. Biomed Pharmacother 2020; 129:110438. [PMID: 32768940 DOI: 10.1016/j.biopha.2020.110438] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/16/2020] [Revised: 06/13/2020] [Accepted: 06/17/2020] [Indexed: 12/17/2022] Open
Abstract
BACKGROUND β-adrenergic activation is able to exacerbate cardiac hypertrophy. Myosin light chain kinase (MLCK) and its phosphorylated substrate, phospho-myosin light chain 2 (p-MLC2), play vital roles in regulating cardiac hypertrophy. However, it is not yet clear whether there is a relationship between β-adrenergic activation and MLCK in the progression of cardiac hypertrophy. Therefore, we explored this relationship and the underlying mechanisms in this work. METHODS Cardiac hypertrophy and cardiomyocyte hypertrophy were induced by pressure overload and isoproterenol (ISO) stimulation, respectively. Echocardiography, histological analysis, immunofluorescence and qRT-PCR were used to confirm the successful establishment of the models. A β-blocker (metoprolol) and a calpain inhibitor (calpeptin) were administered to inhibit β-adrenergic activity in rats and calpain in cardiomyocytes, respectively. The protein expression levels of MLCK, myosin light chain 2 (MLC2), p-MLC2, myosin phosphatase 2 (MYPT2), calmodulin (CaM) and calpain were measured using western blotting. A cleavage assay was performed to assess the degradation of recombinant human MLCK by recombinant human calpain. RESULTS The β-blocker alleviated cardiac hypertrophy and dysfunction, increased MLCK and MLC2 phosphorylation and decreased calpain expression in pressure overload-induced cardiac hypertrophy. Additionally, the calpain inhibitor calpeptin attenuated cardiomyocyte hypertrophy, upregulated MLCK and p-MLC2 and reduced MLCK degradation in ISO-induced cardiomyocyte hypertrophy. Recombinant human calpain degraded recombinant human MLCK in vitro in concentration- and time-dependent manners, and this degradation was inhibited by the calpain inhibitor calpeptin. CONCLUSION Our study suggested that β-adrenergic activation may promote the degradation of MLCK through calpain in pressure overload-induced cardiac hypertrophy.
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Affiliation(s)
- Shun Wang
- Department of Cardiology, Renmin Hospital of Wuhan University, Cardiovascular Research Institute, Hubei Key Laboratory of Cardiology, Wuhan, 430060, China
| | - Haixiong Wang
- Department of Cardiology, Shanxi Cardiovascular Hospital, Taiyuan, 030001, China
| | - Xiaoling Su
- Department of Cardiology, Qinghai Provincial People's Hospital, Xining, 810007, China
| | - Beilei Liu
- Department of Cardiology, Renmin Hospital of Wuhan University, Cardiovascular Research Institute, Hubei Key Laboratory of Cardiology, Wuhan, 430060, China
| | - Le Wang
- Department of Cardiology, Renmin Hospital of Wuhan University, Cardiovascular Research Institute, Hubei Key Laboratory of Cardiology, Wuhan, 430060, China
| | - Hui Yan
- Department of Cardiology, Renmin Hospital of Wuhan University, Cardiovascular Research Institute, Hubei Key Laboratory of Cardiology, Wuhan, 430060, China
| | - Shuai Mao
- Department of Cardiology, Renmin Hospital of Wuhan University, Cardiovascular Research Institute, Hubei Key Laboratory of Cardiology, Wuhan, 430060, China
| | - He Huang
- Department of Cardiology, Renmin Hospital of Wuhan University, Cardiovascular Research Institute, Hubei Key Laboratory of Cardiology, Wuhan, 430060, China
| | - Congxin Huang
- Department of Cardiology, Renmin Hospital of Wuhan University, Cardiovascular Research Institute, Hubei Key Laboratory of Cardiology, Wuhan, 430060, China
| | - Mian Cheng
- Department of Geriatrics, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, 430074, China.
| | - Gang Wu
- Department of Cardiology, Renmin Hospital of Wuhan University, Cardiovascular Research Institute, Hubei Key Laboratory of Cardiology, Wuhan, 430060, China; Department of Cardiology, Ezhou Hospital, Renmin Hospital of Wuhan University, Ezhou, 436000, China.
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Ahmad S, Masjoan Juncos JX, Ahmad A, Zaky A, Wei CC, Bradley WE, Zafar I, Powell P, Mariappan N, Vetal N, Louch WE, Ford DA, Doran SF, Matalon S, Dell'Italia LJ. Bromine inhalation mimics ischemia-reperfusion cardiomyocyte injury and calpain activation in rats. Am J Physiol Heart Circ Physiol 2018; 316:H212-H223. [PMID: 30379573 DOI: 10.1152/ajpheart.00652.2017] [Citation(s) in RCA: 20] [Impact Index Per Article: 2.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 01/13/2023]
Abstract
Halogens are widely used, highly toxic chemicals that pose a potential threat to humans because of their abundance. Halogens such as bromine (Br2) cause severe pulmonary and systemic injuries; however, the mechanisms of their toxicity are largely unknown. Here, we demonstrated that Br2 and reactive brominated species produced in the lung and released in blood reach the heart and cause acute cardiac ultrastructural damage and dysfunction in rats. Br2-induced cardiac damage was demonstrated by acute (3-24 h) increases in circulating troponin I, heart-type fatty acid-binding protein, and NH2-terminal pro-brain natriuretic peptide. Transmission electron microscopy demonstrated acute (3-24 h) cardiac contraction band necrosis, disruption of z-disks, and mitochondrial swelling and disorganization. Echocardiography and hemodynamic analysis revealed left ventricular (LV) systolic and diastolic dysfunction at 7 days. Plasma and LV tissue had increased levels of brominated fatty acids. 2-Bromohexadecanal (Br-HDA) injected into the LV cavity of a normal rat caused acute LV enlargement with extensive disruption of the sarcomeric architecture and mitochondrial damage. There was extensive infiltration of neutrophils and increased myeloperoxidase levels in the hearts of Br2- or Br2 reactant-exposed rats. Increased bromination of sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) and increased phosphalamban after Br2 inhalation decreased cardiac SERCA activity by 70%. SERCA inactivation was accompanied by increased Ca2+-sensitive LV calpain activity. The calpain-specific inhibitor MDL28170 administered within 1 h after exposure significantly decreased calpain activity and acute mortality. Bromine inhalation and formation of reactive brominated species caused acute cardiac injury and myocardial damage that can lead to heart failure. NEW & NOTEWORTHY The present study defines left ventricular systolic and diastolic dysfunction due to cardiac injury after bromine (Br2) inhalation. A calpain-dependent mechanism was identified as a potential mediator of cardiac ultrastructure damage. This study not only highlights the importance of monitoring acute cardiac symptoms in victims of Br2 exposure but also defines calpains as a potential target to treat Br2-induced toxicity.
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Affiliation(s)
- Shama Ahmad
- Department of Anesthesiology and Perioperative Medicine, University of Alabama at Birmingham , Birmingham, Alabama
| | - Juan Xavier Masjoan Juncos
- Department of Anesthesiology and Perioperative Medicine, University of Alabama at Birmingham , Birmingham, Alabama
| | - Aftab Ahmad
- Department of Anesthesiology and Perioperative Medicine, University of Alabama at Birmingham , Birmingham, Alabama
| | - Ahmed Zaky
- Department of Anesthesiology and Perioperative Medicine, University of Alabama at Birmingham , Birmingham, Alabama
| | - Chih-Chang Wei
- Division of Cardiovascular Disease, Department of Medicine, University of Alabama at Birmingham , Birmingham, Alabama.,Department of Veterans Affairs Medical Center , Birmingham, Alabama
| | - Wayne E Bradley
- Division of Cardiovascular Disease, Department of Medicine, University of Alabama at Birmingham , Birmingham, Alabama.,Department of Veterans Affairs Medical Center , Birmingham, Alabama
| | - Iram Zafar
- Department of Anesthesiology and Perioperative Medicine, University of Alabama at Birmingham , Birmingham, Alabama
| | - Pamela Powell
- Division of Cardiovascular Disease, Department of Medicine, University of Alabama at Birmingham , Birmingham, Alabama.,Department of Veterans Affairs Medical Center , Birmingham, Alabama
| | - Nithya Mariappan
- Department of Anesthesiology and Perioperative Medicine, University of Alabama at Birmingham , Birmingham, Alabama
| | - Nilam Vetal
- Department of Anesthesiology and Perioperative Medicine, University of Alabama at Birmingham , Birmingham, Alabama
| | - William E Louch
- Institute for Experimental Medical Research, Oslo University Hospital and University of Oslo , Oslo , Norway.,KG Jebsen Cardiac Research Center and Center for Heart Failure Research, University of Oslo, Oslo, Norway
| | - David A Ford
- Department of Biochemistry and Molecular Biology and Center for Cardiovascular Research, Saint Louis University , St. Louis, Missouri
| | - Stephen F Doran
- Department of Anesthesiology and Perioperative Medicine, University of Alabama at Birmingham , Birmingham, Alabama
| | - Sadis Matalon
- Department of Anesthesiology and Perioperative Medicine, University of Alabama at Birmingham , Birmingham, Alabama
| | - Louis J Dell'Italia
- Division of Cardiovascular Disease, Department of Medicine, University of Alabama at Birmingham , Birmingham, Alabama.,Department of Veterans Affairs Medical Center , Birmingham, Alabama
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Kumar M, Kasala ER, Bodduluru LN, Kumar V, Lahkar M. Molecular and biochemical evidence on the protective effects of quercetin in isoproterenol-induced acute myocardial injury in rats. J Biochem Mol Toxicol 2016; 31:1-8. [DOI: 10.1002/jbt.21832] [Citation(s) in RCA: 42] [Impact Index Per Article: 4.7] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/24/2016] [Revised: 07/14/2016] [Accepted: 07/21/2016] [Indexed: 01/22/2023]
Affiliation(s)
- Mukesh Kumar
- Department of Pharmacology & Toxicology; National Institute of Pharmaceutical Education and Research; Guwahati 781 032 Assam India
| | - Eshvendar Reddy Kasala
- Department of Pharmacology & Toxicology; National Institute of Pharmaceutical Education and Research; Guwahati 781 032 Assam India
| | - Lakshmi Narendra Bodduluru
- Department of Pharmacology & Toxicology; National Institute of Pharmaceutical Education and Research; Guwahati 781 032 Assam India
| | - Vikas Kumar
- Neuropharmacology Research Laboratory, Department of Pharmaceutics; Indian Institute of Technology (Banaras Hindu University); Varanasi 221 005 Uttar Pradesh India
| | - Mangala Lahkar
- Department of Pharmacology & Toxicology; National Institute of Pharmaceutical Education and Research; Guwahati 781 032 Assam India
- Department of Pharmacology; Gauhati Medical College; Guwahati 781 032 India
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Neuhof C, Neuhof H. Calpain system and its involvement in myocardial ischemia and reperfusion injury. World J Cardiol 2014; 6:638-652. [PMID: 25068024 PMCID: PMC4110612 DOI: 10.4330/wjc.v6.i7.638] [Citation(s) in RCA: 61] [Impact Index Per Article: 5.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 12/26/2013] [Revised: 01/26/2014] [Accepted: 05/29/2014] [Indexed: 02/06/2023] Open
Abstract
Calpains are ubiquitous non-lysosomal Ca2+-dependent cysteine proteases also present in myocardial cytosol and mitochondria. Numerous experimental studies reveal an essential role of the calpain system in myocardial injury during ischemia, reperfusion and postischemic structural remodelling. The increasing Ca2+-content and Ca2+-overload in myocardial cytosol and mitochondria during ischemia and reperfusion causes an activation of calpains. Upon activation they are able to injure the contractile apparatus and impair the energy production by cleaving structural and functional proteins of myocytes and mitochondria. Besides their causal involvement in acute myocardial dysfunction they are also involved in structural remodelling after myocardial infarction by the generation and release of proapoptotic factors from mitochondria. Calpain inhibition can prevent or attenuate myocardial injury during ischemia, reperfusion, and in later stages of myocardial infarction.
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Effect of cortisol on calpains in the C2C12 and 3T3-L1 cells. Appl Biochem Biotechnol 2014; 172:3153-62. [PMID: 24497045 DOI: 10.1007/s12010-014-0753-1] [Citation(s) in RCA: 17] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/17/2013] [Accepted: 01/20/2014] [Indexed: 10/25/2022]
Abstract
The present study was carried out to understand the effect of cortisol on calpain system in the C2C12 and 3T3-L1 adipocyte cells under co-culture system. Cells were co-cultured by using transwell inserts with a 0.4 μm porous membrane to separate C2C12 and 3T3-L1 preadipocyte cells. Each cell type was grown independently on the transwell plates. Following cell differentiation, inserts containing 3T3-L1 cells were transferred to C2C12 plates. Ten microgram per milliliter of cortisol was added to the medium. Following treatment for 3 days, the cells in the lower well were harvested for analysis. Calpains such as μ-calpain, m-calpain, and calpastatin were selected for the analysis. RT-PCR results indicated the significant increase in the mRNA expression of μ-calpain, m-calpain, and calpastatin. In addition, the confocal microscopical investigation indicated the cortisol treatment increases calpain expression in the C2C12 and 3T3-L1 cells. Taking all these together, cortisol treatment with co-culture system shows most reliable status of calpains expression in the cells, which is quite distinct from one-dimensional monocultured cells.
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Pandurangan M, Moorthy H, Sambandam R, Jeyaraman V, Irisappan G, Kothandam R. Effects of stress hormone cortisol on the mRNA expression of myogenenin, MyoD, Myf5, PAX3 and PAX7. Cytotechnology 2013; 66:839-44. [PMID: 24113918 DOI: 10.1007/s10616-013-9635-6] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/21/2013] [Accepted: 08/17/2013] [Indexed: 10/26/2022] Open
Abstract
The present investigation was carried out to evaluate the effect of stress hormone cortisol on the myogenic markers in the C2C12 cells co-cultured with 3T3-L1 preadipocytes. Co-culturing was achieved by transwell inserts with a 0.4 μm porous membrane. C2C12 and 3T3-L1 cells were grown independently on the transwell plates. After differentiation, inserts containing 3T3-L1 cells were transferred to C2C12 plates for co-culturing. 10 μg/μl of cortisol was added to the medium. After 72 h of treatment, C2C12 cells which were in the lower well were harvested for analysis. RT-PCR analysis of myogenic markers such as of myogenin, MyoD, Myf5, PAX3 and PAX7 showed a significant reduction in the mRNA expression of these myogenic markers. In addition, cortisol increased calpain activity, which led to accelerated protein degradation, which in turn reduced the myogenic rate. In conclusion, cortisol treatment reduced mRNA expression of myogenic markers in the co-cultured C2C12 cells, which is quite distinct from one dimensional mono-cultured C2C12 cells.
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Affiliation(s)
- Muthuraman Pandurangan
- Department of Food Science and Nutrition, Catholic University of Daegu, Daegu, South Korea,
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Patterson C, Portbury A, Schisler JC, Willis MS. Tear me down: role of calpain in the development of cardiac ventricular hypertrophy. Circ Res 2011; 109:453-62. [PMID: 21817165 PMCID: PMC3151485 DOI: 10.1161/circresaha.110.239749] [Citation(s) in RCA: 53] [Impact Index Per Article: 3.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 01/21/2023]
Abstract
Cardiac hypertrophy develops most commonly in response to hypertension and is an independent risk factor for the development of heart failure. The mechanisms by which cardiac hypertrophy may be reversed to reduce this risk have not been fully determined to the point where mechanism-specific therapies have been developed. Recently, proteases in the calpain family have been implicated in the regulation of the development of cardiac hypertrophy in preclinical animal models. In this review, we summarize the molecular mechanisms by which calpain inhibition has been shown to modulate the development of cardiac (specifically ventricular) hypertrophy. The context within which calpain inhibition might be developed for therapeutic intervention of cardiac hypertrophy is then discussed.
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Affiliation(s)
- Cam Patterson
- McAllister Heart Institute, University of North Carolina, Chapel Hill, NC, USA
- Departments of Medicine, Pharmacology, Cell and Developmental Biology, University of North Carolina, Chapel Hill, NC, USA
| | - Andrea Portbury
- McAllister Heart Institute, University of North Carolina, Chapel Hill, NC, USA
| | | | - Monte S. Willis
- McAllister Heart Institute, University of North Carolina, Chapel Hill, NC, USA
- Department of Pathology and Laboratory Medicine, University of North Carolina, Chapel Hill, NC, USA
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8
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Bhuiyan MS, Shioda N, Fukunaga K. Chronic beta-AR activation-induced calpain activation and impaired eNOS-Akt signaling mediates cardiac injury in ovariectomized female rats. Expert Opin Ther Targets 2009; 13:275-86. [PMID: 19236150 DOI: 10.1517/14728220902721312] [Citation(s) in RCA: 15] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/25/2023]
Abstract
OBJECTIVE To address the pathophysiological relevance of ovarian hormones in chronic beta-adrenergic stimulation-induced myocardial injury, we assessed impairments of Ca(2+)-mediated cell signaling in the left ventricle of ovariectomized female rats. RESEARCH DESIGN/METHODS Female Wistar rats were subjected to bilateral ovariectomy and sham operation. Six weeks after ovariectomy (OVX), both OVX and sham rats were treated with isoproterenol (5mg/kg, intraperitoneally), a nonselective beta-adrenergic agonist, once a day for 28 days. RESULTS We found that chronic beta-adrenergic stimulation caused enhanced breakdown of sarcolemmal proteins such as dystrophin and utrophin in OVX rats compared to sham-operated rats. Generation of calpain-mediated 150 kDa-breakdown product of spectrin confirmed calpain activation following isoproterenol treatment. Marked breakdown of endogenous calpain inhibitor, calpastatin, in OVX rats was consistent with the calpain activation following chronic beta-adrenergic stimulation. In addition to calpain activation, we also found marked reduction of endothelial nitric oxide synthase (eNOS) activity with concomitant deregulation by heat shock proteins 90 kDa and caveolin 3, both of which are eNOS-associated proteins. Finally, we documented decreased Akt phosphorylation with concomitant increased glycogen synthase kinase 3beta phosphorylation underlying cell injury following chronic beta-adrenergic stimulation. CONCLUSION Taken together chronic beta-adrenergic stimulation caused severe cardiac injury in OVX rats through calpain activation and impairments of Akt and eNOS signaling pathways.
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Affiliation(s)
- Md Shenuarin Bhuiyan
- Department of Pharmacology, Tohoku University, Aramaki-Aoba Aoba-ku, Sendai 980 8578, Japan
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9
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Nakajima-Takenaka C, Sakata S, Kato S, Ohga Y, Murata KY, Taniguchi S, Takaki M. Detrimental effects after dobutamine infusion on rat left ventricular function: mechanical work and energetics. Exp Physiol 2005; 90:635-44. [PMID: 15849228 DOI: 10.1113/expphysiol.2005.030460] [Citation(s) in RCA: 11] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/08/2022]
Abstract
We have previously reported that continuous infusion of dobutamine into the coronary artery induces positive inotropic effects but induces no detrimental effects in cross-circulated, excised normal rat hearts and even in Ca2+ overload-induced contractile failing rat hearts. However, we hypothesized that some detrimental effects on left ventricular (LV) function are induced after continuous dobutamine infusion and the following clearance of blood dobutamine, as is the case after beta-adrenergic receptor stimulation. To test this hypothesis, we investigated LV mechanical work and energetics in the same type of preparations that underwent continuous dobutamine infusion and clearance of blood dobutamine. We found that both mean end-systolic pressure and systolic pressure-volume area (PVA; a measure of total mechanical energy per beat) at midrange LV volume were significantly (P < 0.01) decreased. The mean myocardial oxygen consumption per beat intercept, which is composed of for the total Ca2+ handling in excitation-contraction coupling and basal metabolism, of the and PVA linear relation was also significantly (P < 0.05) decreased (n=8). The mean slope of the linear relation was unchanged in such hearts. Post-dobutamine basal metabolism was unchanged (n = 5 of the 8 hearts). The moderate proteolysis of a cytoskeleton protein, alpha-fodrin was identified (n = 7 of the 8 hearts with the decreased intercept), after clearance of blood dobutamine. In agreement with our hypothesis, the detrimental effect of the post-beta-adrenergic receptor stimulation was induced by a moderate concentration of dobutamine; we found systolic dysfunction due to the impairment of Ca2+ handling in excitation-contraction coupling in the rat LV and proteolysis of a cytoskeleton protein, alpha-fodrin.
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Affiliation(s)
- Chikako Nakajima-Takenaka
- Department of Physiology II, Nara Medical University School of Medicine, 840 Shijo-cho, Kashihara, Nara 634-8521, Japan
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Kawada T, Masui F, Kumagai H, Koshimizu M, Nakazawa M, Toyo-Oka T. A novel paradigm for therapeutic basis of advanced heart failure--assessment by gene therapy. Pharmacol Ther 2005; 107:31-43. [PMID: 15963350 DOI: 10.1016/j.pharmthera.2004.12.006] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 12/27/2004] [Indexed: 11/19/2022]
Abstract
The precise mechanism(s) of the progression of advanced heart failure (HF) should be determined to establish strategies for its treatment or prevention. Based on pathological, molecular, and physiological findings in 3 animal models and human cases, we propose a novel scheme that a vicious cycle formed by increased sarcolemma (SL) permeability, preferential activation of calpain over calpastatin, and translocation and cleavage of dystrophin (Dys) commonly lead to advanced HF. The aim of this article was to assess our recent paradigm that disruption of myocardial Dys is a final common pathway to advanced HF, irrespective of its hereditary or acquired origin, but not intended to provide a comprehensive overview of the various factors that may be involved in the course of HF in different clinical settings. In addition, each component of Dys-associated proteins (DAP) was heterogeneously degraded in vivo and in vitro, i.e. Dys and alpha-sarcoglycan (SG) were markedly destroyed using isolated calpain 2, while delta-SG was not degraded at all. The up-regulation of calpain 2 was confirmed through previously published data that remain insufficient for precise evaluation, supporting our new scheme that the activation of calpain(s) is involved in the steady process of Dys cleavage. In addition, somatic gene therapy is discussed as a potential option to ameliorate the physiological/metabolic indices and to improve the prognosis.
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Affiliation(s)
- Tomie Kawada
- Division of Pharmacy, Niigata University of Medical and Dental Hospital, Niigata 951-8520, Japan
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Enns D, Karmazyn M, Mair J, Lercher A, Kountchev J, Belcastro A. Calpain, calpastatin activities and ratios during myocardial ischemia-reperfusion. Mol Cell Biochem 2002; 241:29-35. [PMID: 12482022 DOI: 10.1023/a:1020861120368] [Citation(s) in RCA: 15] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/12/2022]
Abstract
The purpose of this study was to test the hypothesis that myocardial ischemia-reperfusion (I/R) is accompanied by an early burst in calpain activity, resulting in decreased calpastatin activity and an increased calpain/calpastatin ratio, thereby promoting increased protein release. To determine the possibility of a 'calpain burst' impacting cardiac calpastatin inhibitory activity, rat hearts were subjected (Langendorff) to either 45 or 60 min of ischemia followed by 30 min of reperfusion with and without pre-administration (s.c.) of a cysteine protease inhibitor (E-64c). Myocardial function, calpain activities (casein release assay), calpastatin inhibitory activity and release of CK, LDH, cTnI and cTnT were determined (n = 8 for all groups). No detectable changes in calpain activities were observed following I/R with and without E-64c (p > 0.05). Both I/R conditions reduced calpastatin activity (p < 0.05) while E-64c pre-treatment was without effect, implicating a non-proteolytic event underlying the calpastatin changes. A similar result was noted for calpain-calpastatin ratios and the release of all marker proteins (p < 0.05). In regard to cardiac function, E-64c resulted in transient improvements (15 min) for left ventricular developed pressure (LVDP) and rate of pressure development (p < 0.05). E-64c had no effect on end diastolic pressure (LVEDP) or coronary pressure (CP) during I/R. These findings demonstrate that restricting the putative early burst in calpain activity, suggested for I/R, by pre-treatment of rats with E-64c does not prevent downregulation of calpastatin inhibitory activity and/or protein release despite a transient improvement in cardiac function. It is concluded that increases in calpain isoform activities are not a primary feature of l/R changes, although the role of calpastatin downregulation remains to be elucidated.
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Affiliation(s)
- D Enns
- School of Kinesiology, Faculty of Health Sciences, Faculty of Medicine and Dentistry, University of Western Ontario, London, Ontario, Canada
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12
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Ertbjerg P, Lawson MA, Purslow PP. Epinephrine upregulates calpain activity in cultured C2C12 muscle cells. Biochimie 2000; 82:197-201. [PMID: 10863002 DOI: 10.1016/s0300-9084(00)00207-8] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/25/2022]
Abstract
C2C12 cells were grown to confluence at 37 degrees C under a continuous 5% CO(2) stream and myotube formation was stimulated. The cultures were then incubated with or without 2 microg/mL epinephrine for 18 h prior to harvesting and calpain extraction. Epinephrine treatment resulted in a three-fold increase in extractable mu-calpain activity (P < 0.05), a three-fold increase in extractable m-calpain activity (P < 0.05), a 36% increase in calpastatin activity (P < 0.001), and a 16% decrease (P < 0.05) in the total protein content in the C2C12 cell homogenate. These results suggest that calpains may play a role in protein metabolism and that the hormone epinephrine may be directly involved in the regulation of their cellular expression.
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Affiliation(s)
- P Ertbjerg
- Department of Dairy and Food Science, The Royal Veterinary and Agricultural University, Frederiksberg, Denmark.
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13
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Turman MA, Bates CM, Mathews A, Haun SE. Effect of extracellular calcium on survival of human proximal tubular cells exposed to hypoxia. Ren Fail 1995; 17:421-35. [PMID: 7569113 DOI: 10.3109/08860229509037606] [Citation(s) in RCA: 13] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/26/2023] Open
Abstract
Removal of extracellular calcium has been demonstrated to improve membrane integrity of rodent myocytes, astrocytes, and renal tubular cells injured by hypoxia. In this study, the effect of extracellular calcium on long-term survival of cultured human proximal tubular epithelial cells (PTEC) subjected to hypoxia was evaluated. In addition, the effect of extracellular calcium on release of arachidonic acid metabolites (AAM) was assessed during and after hypoxia. To induce hypoxic injury, PTEC were incubated in an anaerobic chamber in glucose-free buffer (combined oxygen/glucose deprivation, COGD). Long-term survival was assessed by measuring lactate dehydrogenase (LDH) efflux during COGD and after an additional 24-h "recovery" period (in routine culture medium in 95% air/5% CO2). To determine if extracellular calcium influenced AAM release from membrane phospholipids, cells were preincubated with [3H]arachidonic acid and the release of AAM was measured during COGD and recovery. With this model system, PTEC exhibited minimal LDH efflux during < or = 12 h COGD, but LDH efflux increased to 73.9 +/- 4.7% by 24 h COGD. With 12-18 h of COGD, the extent of LDH efflux was greater during recovery than during COGD, indicating that, for human PTEC, the extent of membrane damage does not become fully evident by LDH efflux for hours after hypoxia. PTEC exposed to 24 h of COGD in the absence of extracellular calcium exhibited strikingly less LDH efflux during COGD than cells incubated in the presence of extracellular calcium, suggesting that extracellular calcium contributes to membrane damage during COGD. However, upon reexposure of PTEC to extracellular calcium, LDH efflux rapidly increased to control levels. Furthermore, despite allowing cells to recover in oxygen or oxygen and glucose before exposure to calcium-containing medium, a rapid increase in LDH efflux could not be avoided. These results suggest that COGD induces an irreversible injury that ultimately leads to loss of membrane integrity whether or not extracellular calcium is present; however, extracellular calcium accelerates the loss of membrane integrity caused by hypoxia. Extracellular calcium did not alter AAM release, indicating that the effect of extracellular calcium on membrane damage (as indicated by LDH efflux) was not mediated by an increased activity of phospholipases (such as phospholipase A2) that are involved in the release of AAM.
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Affiliation(s)
- M A Turman
- Wexner Institute for Pediatric Research, Department of Pediatrics, Children's Hospital, Ohio State University, Columbus 43205, USA
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14
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Yoshida K, Yamasaki Y, Kawashima S. Calpain activity alters in rat myocardial subfractions after ischemia or reperfusion. BIOCHIMICA ET BIOPHYSICA ACTA 1993; 1182:215-20. [PMID: 8357852 DOI: 10.1016/0925-4439(93)90143-o] [Citation(s) in RCA: 49] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 01/30/2023]
Abstract
To examine whether calpain is activated during ischemic or reperfusion injury, we measured calpain activity of the subfractions of rat myocardia after global ischemia for 60 min or the ischemia followed by 30 min reperfusion by the Langendorff procedure. The myocardial homogenate was fractionated into 600 x g, 10,000 x g and 100,000 x g pellet fractions as well as 10,000 x g supernatant fraction. The supernatant fraction was further subjected to DEAE cellulose and phenyl-Sepharose chromatographies to separate mu- and m-calpains. The m-calpain activity of the DEAE fractions after global ischemia for 60 min was higher but that after ischemia-reperfusion was lower than that of the control. On the other hand, the ischemia-reperfusion but not ischemia by itself raised the calpain activity of the phenyl-Sepharose fraction (mu-calpain) and the 10,000 x g pellet measured at 100 microM and 5 mM Ca2+. Treatment with verapamil but not with ryanodine during ischemia attenuated the increase in m-calpain activity. A dot-blotting analysis of calpain antigenicity showed a decrease in soluble but no change in the particulate fractions after ischemia-reperfusion. An immunoblotting technique did not detect proteolysis of the calpain 80-kDa subunit. These observations suggest that calpain is activated by Ca2+ influx during ischemia and reperfusion without gross changes in its amount. Some unknown processes other than translocation or autolysis are thought to be involved in the alterations.
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Affiliation(s)
- K Yoshida
- Department of Legal Medicine, Yamaguchi University School of Medicine, Japan
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