Cardioprotection by CaMKII-deltaB is mediated by phosphorylation of heat shock factor 1 and subsequent expression of inducible heat shock protein 70.
ABSTRACT: RATIONALE:Ca2+/calmodulin-dependent protein kinase (CaMK)II is a multifunctional kinase involved in vital cellular processes such as Ca(2+) handling and cell fate regulation. In mammalian heart, 2 primary CaMKII isoforms, deltaB and deltaC, localize in nuclear and cytosolic compartments, respectively. Although previous studies have established an essential role of CaMKII-deltaC in cardiomyocyte apoptosis, the functional role of the more abundant isoform, CaMKII-deltaB, remains elusive. OBJECTIVE:Here, we determined the potential role of CaMKII-deltaB in regulating cardiomyocyte viability and explored the underlying mechanism. METHODS AND RESULTS:In cultured neonatal rat cardiomyocytes, the expression of CaMKII-deltaB and CaMKII-deltaC was inversely regulated in response to H2O2-induced oxidative stress with a profound reduction of the former and an increase of the later. Similarly, in vivo ischemia/reperfusion (IR) led to an opposite regulation of these CaMKII isoforms in a rat myocardial IR model. Notably, overexpression of CaMKII-deltaB protected cardiomyocytes against oxidative stress-, hypoxia-, and angiotensin II-induced apoptosis, whereas overexpression of its cytosolic counterpart promoted apoptosis. Using cDNA microarray, real-time PCR and Western blotting, we demonstrated that overexpression of CaMKII-deltaB but not CaMKII-deltaC elevated expression of heat shock protein (HSP)70 family members, including inducible (i)HSP70 and its homolog (Hst70). Moreover, overexpression of CaMKII-deltaB led to phosphorylation and activation of heat shock factor (HSF)1, the primary transcription factor responsible for HSP70 gene regulation. Importantly, gene silencing of iHSP70, but not Hst70, abolished CaMKII-deltaB-mediated protective effect, indicating that only iHSP70 was required for CaMKII-deltaB elicited antiapoptotic signaling. CONCLUSIONS:We conclude that cardiac CaMKII-deltaB and CaMKII-deltaC were inversely regulated in response to oxidative stress and IR injury, and that in contrast to CaMKII-deltaC, CaMKII-deltaB serves as a potent suppressor of cardiomyocyte apoptosis triggered by multiple death-inducing stimuli via phosphorylation of HSF1 and subsequent induction of iHSP70, marking both CaMKII-delta isoforms as promising therapeutic targets for the treatment of ischemic heart disease.
Project description:Hsp70's are highly conserved essential protein chaperones that assist protein folding and prevent protein aggregation. They have modular structures consisting of ATPase, substrate-binding, and C-terminal domains. Substrate binding and release is regulated by ATP hydrolysis and nucleotide exchange, which in turn are regulated by cochaperones. Eukaryotes have constitutive (Hsc70) and stress-inducible (iHsp70) isoforms, but their functions have not been systematically compared. Using a yeast system to evaluate heterologous Hsp70's we find that primate Hsc70 supported growth but iHsp70 did not. Plant Hsc70 and iHsp70 counterparts behaved similarly, implying evolutionary conservation of this distinction. Swapping yeast and primate Hsp70 domains showed that (i) the Hsc70-iHsp70 distinction resided in the ATPase domain, (ii) substrate-binding domains of Hsp70's within and across species functioned similarly regarding growth, (iii) C-terminal domain function was important for growth, and (iv) Hsp70 functions important for cell growth and prion propagation were separable. Enzymatic analysis uncovered a correlation between substrate affinity and prion phenotype and showed that ATPase and protein-folding activities were generally similar. Our data support a view that intrinsic activities of Hsp70 isoforms are comparable, and functional differences in vivo lie mainly in complex interactions of Hsp70 with cochaperones.
Project description:Calcium/calmodulin-dependent protein kinase II (CaMKII) plays a central role in cardiac contractility and heart disease. However, the specific role of alternatively spliced variants of CaMKII in cardiac disease and apoptosis remains poorly explored. Here we report that the deltaB subunit of CaMKII (CaMKIIdeltaB), which is the predominant nuclear isoform of calcium/calmodulin-dependent protein kinases in heart muscle, acts as an anti-apoptotic factor and is a novel target of the antineoplastic and cardiomyopathic drug doxorubicin (Dox (adriamycin)). Hearts of rats that develop cardiomyopathy following chronic treatment with Dox also show down-regulation of CaMKIIdeltaB mRNA, which correlates with decreased cardiac function in vivo, reduced expression of sarcomeric proteins, and increased tissue damage associated with Dox cardiotoxicity. Overexpression of CaMKIIdeltaB in primary cardiac cells inhibits Dox-mediated apoptosis and prevents the loss of the anti-apoptotic protein Bcl-2. Specific silencing of CaMKIIdeltaB by small interfering RNA prevents the formation of organized sarcomeres and decreases the expression of Bcl-2, which all mimic the effect of Dox. CaMKIIdeltaB is required for GATA-4-mediated co-activation and binding to the Bcl-2 promoter. These results reveal that CaMKIIdeltaB plays an essential role in cardiomyocyte survival and provide a mechanism for the protective role of CaMKIIdeltaB. These results suggest that selective targeting of CaMKII in the nuclear compartment might represent a strategy to regulate cardiac apoptosis and to reduce Dox-mediated cardiotoxicity.
Project description:Kaposi's sarcoma-associated herpesvirus (KSHV) is an oncogenic herpesvirus associated with various AIDS-related malignancies. Like other herpesviruses, multiple processes required for KSHV lytic replication, including viral transcription, viral DNA synthesis and capsid assembly occur in virus-induced intranuclear structures, termed replication and transcription compartments (RTCs). Here we utilised a novel methodology, combining subcellular fractionation and quantitative proteomics, to identify cellular proteins which are recruited to KSHV-induced RTCs and thus play a key role in KSHV lytic replication. We show that several isoforms of the HSP70 chaperone family, Hsc70 and iHsp70, are redistributed from the cytoplasm into the nucleus coinciding with the initial formation of KSHV-induced RTCs. We demonstrate that nuclear chaperone foci are dynamic, initially forming adjacent to newly formed KSHV RTCs, however during later time points the chaperones move within KSHV RTCs and completely co-localise with actively replicating viral DNA. The functional significance of Hsp70 isoforms recruitment into KSHV RTCs was also examined using the specific Hsp70 isoform small molecule inhibitor, VER-155008. Intriguingly, results highlight an essential role of Hsp70 isoforms in the KSHV replication cycle independent of protein stability and maturation. Notably, inhibition of Hsp70 isoforms precluded KSHV RTC formation and RNA polymerase II (RNAPII) relocalisation to the viral genome leading to the abolishment of global KSHV transcription and subsequent viral protein synthesis and DNA replication. These new findings have revealed novel mechanisms that regulate KSHV lytic replication and highlight the potential of HSP70 inhibitors as novel antiviral agents.
Project description:Stress-induced cardiomyocyte apoptosis plays an important role in the pathogenesis of a variety of cardiovascular diseases. Our early studies showed that HSP70 effectively inhibited apoptosis, but the underlying mechanism remained unclear. Fas-associated factor 1 (FAF1) is a member of the Fas death-inducing signaling complex (Fas-DISC) that acts upstream of caspase-8. We investigated the interactions among FAF1, HSP70, and FAS in stressed cardiomyocytes to elucidate the protective mechanism of HSP70. FAS and caspase-3/8 activity was higher in cardiomyocytes undergoing stress-induced apoptosis in restraint-stressed rats compared with cardiomyocytes in non-stressed rats, which indicated that the Fas signaling pathway was activated after restraint stress. Geranylgeranylacetone (GGA) induced an increase in HSP70 expression, which reduced stress-induced apoptosis. Additionally, overexpression of HSP70 via transfection with the pEGFP-rHSP70 plasmid attenuated norepinephrine (NE)-induced apoptosis. FAF1 expression increased during stress-induced apoptosis, and overexpression of FAF1 exacerbated NE-induced apoptosis. We also found that HSP70 interacted with FAF1. Overexpression of HSP70 inhibited the binding of FAF1 to FAS in H9C2 cells, which indicated that HSP70 suppressed NE-induced apoptosis by competitively binding to FAF1. An N-terminal deletion mutant of HSP70 (HSP70-?N) was unable to interact with FAF1. After HSP70-?N was transfected into H9C2 cells, the cells were unable to attenuate the NE-induced increases in caspase-8 and apoptosis. These results indicate that the 1-120 sequence of HSP70 binds to FAF1, which alters the interactions between FAS and FAF1 and inhibits the activation of the Fas signaling pathway and apoptosis.
Project description:Ca<sup>2+</sup>/calmodulin-dependent protein kinase II (CaMKII) and nuclear factor-kappa B (NF-?B) play crucial roles in pathogenesis of doxorubicin (DOX)-induced cardiomyopathy. Their activities are regulated by intracellular Ca<sup>2+</sup>. We hypothesized that blockade of L-type Ca<sup>2+</sup> channel (LTCC) could attenuate DOX-induced cardiomyopathy by regulating CaMKII and NF-?B. DOX activated CaMKII and NF-?B through their phosphorylation and increased cleaved caspase 3 in cardiomyocytes. Pharmacological blockade or gene knockdown of LTCC by nifedipine or small interfering RNA, respectively, suppressed DOX-induced phosphorylation of CaMKII and NF-?B and apoptosis in cardiomyocytes, accompanied by decreasing intracellular Ca<sup>2+</sup> concentration. Autocamtide 2-related inhibitory peptide (AIP), a selective CaMKII inhibitor, inhibited DOX-induced phosphorylation of NF-?B and cardiomyocyte apoptosis. Inhibition of NF-?B activity by ammonium pyrrolidinedithiocarbamate (PDTC) suppressed DOX-induced cardiomyocyte apoptosis. DOX-treatment (18?mg/kg via intravenous 3 injections over 1 week) increased phosphorylation of CaMKII and NF-?B in mouse hearts. Nifedipine (10?mg/kg/day) significantly suppressed DOX-induced phosphorylation of CaMKII and NF-?B and cardiomyocyte injury and apoptosis in mouse hearts. Moreover, it attenuated DOX-induced left ventricular dysfunction and dilatation. Our findings suggest that blockade of LTCC attenuates DOX-induced cardiomyocyte apoptosis via suppressing intracellular Ca<sup>2+</sup> elevation and activation of CaMKII-NF-?B pathway. LTCC blockers might be potential therapeutic agents against DOX-induced cardiomyopathy.
Project description:Pathological remodeling of the myocardium is an integral part of the events that lead to heart failure (HF), which involves altered gene expression, disturbed signaling pathways and altered Ca2+ homeostasis and the players involved in this process. Of particular interest is the chronic activation of Ca2+/calmodulin-dependent protein kinase II (CaMKII) isoforms in heart, which further aggravate the injury to myocardium. Expression and activity of CaMKII have been found to be elevated in various conditions of stressed myocardium and in different heart diseases in both animal models as well as heart patients. CaMKII is a signaling molecule that regulates many cellular pathways by phosphorylating several proteins involved in excitation-contraction coupling and relaxation events in heart, cardiomyocyte apoptosis, transcriptional activation of genes related to cardiac hypertrophy, inflammation, and arrhythmias. CaMKII is activated by reactive oxygen species (ROS), which are elevated under conditions of ischemia-reperfusion injury and in a cyclical manner, CaMKII in turn elevates ROS production. Both ROS and activated CaMKII increase Ca-induced Ca release from sarcoplasmic reticulum, which leads to cardiomyocyte membrane depolarization and arrhythmias. These CaMKII-mediated changes in heart ultimately culminate in dysfunctional myocardium and HF. Genetic studies in animal models clearly demonstrated that inactivation of CaMKII is protective against a variety of stress induced cardiac dysfunctions. Despite significant leaps in understanding the structural details of CaMKII, which is a very complicated and multimeric modular protein, currently there is no specific and potent inhibitor of this enzyme, that can be developed for therapeutic purposes.
Project description:Vascular smooth muscle (VSM) expresses calcium/calmodulin-dependent protein kinase II (CaMKII)-? and -? isoforms. CaMKII? promotes VSM proliferation and vascular remodeling. We tested CaMKII? function in vascular remodeling after injury. CaMKII? protein decreased 90% 14 d after balloon injury in rat carotid artery. Intraluminal transduction of adenovirus encoding CaMKII?C rescued expression to 35% of uninjured controls, inhibited neointima formation (>70%), inhibited VSM proliferation (>60%), and increased expression of the cell-cycle inhibitor p21 (>2-fold). Comparable doses of CaMKII?2 adenovirus had no effect. Similar dynamics in CaMKII? mRNA and protein expression were observed in ligated mouse carotid arteries, correlating closely with expression of VSM differentiation markers. Targeted deletion of CaMKII? in smooth muscle resulted in a 20-fold increase in neointimal area, with a 3-fold increase in the cell proliferation index, no change in apoptosis, and a 60% decrease in p21 expression. In cultured VSM, CaMKII? overexpression induced p53 mRNA (1.7 fold) and protein (1.8-fold) expression; induced the p53 target gene p21 (3-fold); decreased VSM cell proliferation (>50%); and had no effect on expression of apoptosis markers. We conclude that regulated CaMKII isoform composition is an important determinant of the injury-induced vasculoproliferative response and that CaMKII? and -? isoforms have nonequivalent, opposing functions.
Project description:Background:The Chinese medicine Huoxue Wentong Formula (HXWTF) was used to treat thoracic obstruction and angina pectoris in clinic, which has not been investigated in myocardial ischemia-induced apoptosis and angiogenic function. Here we aimed to investigate the roles of HXWTF in rats with myocardial ischemia-induced apoptosis and angiogenesis disorders, as well as to reveal the potential mechanisms. Methods:Male SD rats were subjected to coronary artery ligation followed by HXWTF (420, 840 and 1680 mg/kg/day, p.o.) or isosorbide mononitrate (6.3 mg/kg/day, p.o.) treatment for 4 weeks. Electrocardiogram (ECG) and Echocardiography (ECHO) were used to measure cardiac function. Hematoxylin and eosin (H&E) staining and CD34/?-SMA immunohistochemical staining were performed to observe the ischemic heart sections pathological changes and angiogenesis. Then, the effects on cardiomyocyte apoptosis of H9c2 and tube formation of HCMECs were observed, as well as the changes in the levels of total calmodulin dependent protein kinase II (t-CaMKII), phosphorylated CaMKII (p-CaMKII), oxidized CaMKII (ox-CaMKII), CD34, and Bcl-2/Bax ratio were detected. Results:Rats with coronary artery ligation exhibited abnormal cardiac function, enlarged myocardial space, disorderly arranged myocardial fibers, inflammatory cells infiltrated, and aggravated myocardial cell apoptosis, along with angiogenesis dysfunction. The expressions of CD34, p-CaMKII, and ox-CaMKII were elevated and Bcl-2/Bax ratio was diminished in ischemic hearts and H/SD-treated H9c2 or HCMECs, while HXWTF treatment completely rescued angiogenic dysfunction, inhibited cardiomyocyte apoptosis, and down-regulated cardiac CaMKII oxidation and phosphorylation activities. Conclusion:Our study demonstrates that HXWTF improves myocardial infarction possibly through inhibiting CaMKII oxidation and phosphorylation levels, facilitating angiogenic function and alleviating cardiomyocyte apoptosis. Thus, therapeutics targeting CaMKII activities may be a promising strategy for rescuing ischemic cardiomyopathy.
Project description:CaMKII was suggested to mediate ischemic myocardial injury and adverse cardiac remodeling. Here, we investigated the roles of different CaMKII isoforms and splice variants in ischemia/reperfusion (I/R) injury by the use of new genetic CaMKII mouse models. Although CaMKII?C was upregulated 1 day after I/R injury, cardiac damage 1 day after I/R was neither affected in CaMKII?-deficient mice, CaMKII?-deficient mice in which the splice variants CaMKII?B and C were re-expressed, nor in cardiomyocyte-specific CaMKII?/? double knockout mice (DKO). In contrast, 5 weeks after I/R, DKO mice were protected against extensive scar formation and cardiac dysfunction, which was associated with reduced leukocyte infiltration and attenuated expression of members of the chemokine (C-C motif) ligand family, in particular CCL3 (macrophage inflammatory protein-1?, MIP-1?). Intriguingly, CaMKII was sufficient and required to induce CCL3 expression in isolated cardiomyocytes, indicating a cardiomyocyte autonomous effect. We propose that CaMKII-dependent chemoattractant signaling explains the effects on post-I/R remodeling. Taken together, we demonstrate that CaMKII is not critically involved in acute I/R-induced damage but in the process of post-infarct remodeling and inflammatory processes.
Project description:Following myocardial infarction, nonischemic myocyte death results in infarct expansion, myocardial loss, and ventricular dysfunction. Here, we demonstrate that a specific proapoptotic gene, Bnip3, minimizes ventricular remodeling in the mouse, despite having no effect on early or late infarct size. We evaluated the effects of ablating Bnip3 on cardiomyocyte death, infarct size, and ventricular remodeling after surgical ischemia/reperfusion (IR) injury in mice. Immediately following IR, no significant differences were observed between Bnip3(-/-) and WT mice. However, at 2 days after IR, apoptosis was diminished in Bnip3(-/-) periinfarct and remote myocardium, and at 3 weeks after IR, Bnip3(-/-) mice exhibited preserved LV systolic performance, diminished LV dilation, and decreased ventricular sphericalization. These results suggest myocardial salvage by inhibition of apoptosis. Forced cardiac expression of Bnip3 increased cardiomyocyte apoptosis in unstressed mice, causing progressive LV dilation and diminished systolic function. Conditional Bnip3 overexpression prior to coronary ligation increased apoptosis and infarct size. These studies identify postischemic apoptosis by myocardial Bnip3 as a major determinant of ventricular remodeling in the infarcted heart, suggesting that Bnip3 may be an attractive therapeutic target.