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Structural and molecular mechanisms of gap junction remodeling in epicardial border zone myocytes following myocardial infarction.

ABSTRACT: Lateralization of the ventricular gap junction protein connexin 43 (Cx43) occurs in epicardial border zone myocytes following myocardial infarction (MI) and is arrhythmogenic. Alterations in Cx43 protein partners have been hypothesized to play a role in lateralization although mechanisms by which this occurs are unknown. To examine potential mechanisms we did nuclear magnetic resonance, yeast 2-hybrid, and surface plasmon resonance studies and found that the SH3 domain of the tyrosine kinase c-Src binds to the Cx43 scaffolding protein zonula occludens-1 (ZO-1) with a higher affinity than does Cx43. This suggests c-Src outcompetes Cx43 for binding to ZO-1, thus acting as a chaperone for ZO-1 and causing unhooking from Cx43. To determine whether c-Src/ZO-1 interactions affect Cx43 lateralization within the epicardial border zone, we performed Western blot, immunoprecipitation, and immunolocalization for active c-Src (p-cSrc) post-MI using a canine model of coronary occlusion. We found that post-MI p-cSrc interacts with ZO-1 as Cx43 begins to decrease its interaction with ZO-1 and undergo initial loss of intercalated disk localization. This indicates that the molecular mechanisms by which Cx43 is lost from the intercalated disk following MI includes an interaction of p-cSrc with ZO-1 and subsequent loss of scaffolding of Cx43 leaving Cx43 free to diffuse in myocyte membranes from areas of high Cx43, as at the intercalated disk, to regions of lower Cx43 content, the lateral myocyte membrane. Therefore shifts in Cx43 protein partners may underlie, in part, arrhythmogenesis in the post-MI heart.

SUBMITTER: Kieken F

PROVIDER: S-EPMC2892879 | biostudies-literature | 2009 May

REPOSITORIES: biostudies-literature

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Structural and molecular mechanisms of gap junction remodeling in epicardial border zone myocytes following myocardial infarction.

Kieken Fabien F Mutsaers Nancy N Dolmatova Elena E Virgil Kelly K Wit Andrew L AL Kellezi Admir A Hirst-Jensen Bethany J BJ Duffy Heather S HS Sorgen Paul L PL

Circulation research 20090402 9

Lateralization of the ventricular gap junction protein connexin 43 (Cx43) occurs in epicardial border zone myocytes following myocardial infarction (MI) and is arrhythmogenic. Alterations in Cx43 protein partners have been hypothesized to play a role in lateralization although mechanisms by which this occurs are unknown. To examine potential mechanisms we did nuclear magnetic resonance, yeast 2-hybrid, and surface plasmon resonance studies and found that the SH3 domain of the tyrosine kinase c-S ...[more]

PMID: 19342602

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Project description:Dronedarone improves microvascular flow during atrial fibrillation and reduces the infarct size in acute models of myocardial infarction. However, dronedarone might be harmful in patients with recent decompensated heart failure and increases mortality in patients with permanent atrial fibrillation. A pathophysiological explanation for these discrepant data is lacking. This study investigated the effects of dronedarone on gene and protein expression in the infarcted area and border zone in pigs subjected to anterior ischemia/reperfusion myocardial infarction. The ischemia/reperfusion myocardial infarction was induced in 16 pigs. Eight pigs were treated with dronedarone for 28 days after myocardial infarction, the remaining pigs served as control. Microarray-based transcriptome profiling and 2D-DIGE-based proteome analysis were used to assess the effects of dronedarone on left ventricular gene expression in healthy (LV), infarcted (MI), and border zone tissue. Selected targets were validated by RT-qPCR or immunoblot analyses, with special emphasize given to the transcriptome/proteome overlap. Combined "omics" analysis was performed to identify most significant disease and function charts affected by dronedarone and to establish an integrated network. The levels of 879 (BZ) or 7 (MI) transcripts and 51 (LV) or 15 (BZ) proteins were significantly altered by dronedarone, pointing to a substantial efficacy of dronedarone in the border zone. Transcriptome and proteome data indicate that dronedarone influences post-infarction remodeling processes and identify matricellular proteins as major targets of dronedarone in this setting. This finding is fully supported by the disease and function charts as well as by the integrated network established by combined "omics". Dronedarone therapy alters myocardial gene expression after acute myocardial infarction with pronounced effects in the border zone. Dronedarone promotes infarct healing via regulation of periostin and might contribute to the limitation of its expansion as well as cardiac rupture. Thus, there are no experimental hints that dronedarone per se has direct harmful effects after MI in ventricular tissue. Impact statement Dronedarone reduced the infarct size in models of acute myocardial infarction (MI). Here, we show that dronedarone attenuates many of the substantial changes in gene expression that are provoked by acute myocardial infarction (AMI) in pigs. Dronedarone modifies the expression of gene panels related to post-infarction cardiac healing and remodeling processes and, most remarkably, this occurs predominantly in the infarction border-zone and much less so in the vital or infarcted myocardium. Combined "omics" identified matricellular proteins and ECM as major dronedarone-regulated targets and emphasizes their relevance for Disease Charts and Tox Function Charts associated with tissue remodeling and cellular movement. The results demonstrate dronedarone's capability of regulating cardiac repair and remodeling processes specifically in the infarction border zone and identify underlying mechanisms and pathways that might be employed in future therapeutic strategies to improve long-term cardiac tissue function and stability.

Project description:Effective therapeutic targets against post-myocardial infarction (MI) arrhythmias remain to be discovered. We aimed to investigate the role of macrophages in post-MI arrhythmias. Methods: Mononuclear cell accumulation, macrophage polarization from M0 to M1 subset, and gap junction formation were analyzed in MI patients and MI mice by flow cytometry, immunofluorescence and patch clamping. Differentially expressed genes were identified by RNA sequencing. Macrophages and cardiomyocytes were cocultured in vitro, and the effects of gap junction and KCa3.1 on electrophysiological properties were assessed by patch clamping. The effects of KCa3.1 inhibition on post-MI arrhythmias were assessed by intracardiac stimulation and ambulatory electrocardiograms in vivo. Results: Percentage of pro-inflammatory mononuclear cells were significantly elevated in patients with post-MI arrhythmias compared with MI patients without arrhythmias and healthy controls (p<0.001). Macrophages formed gap junction with cardiomyocytes in MI border zones of MI patient and mice, and pro-inflammatory macrophages were significantly increased 3 days post-MI (p<0.001). RNA sequencing identified Kcnn4 as the most differentially expressed gene encoding ion channel, and the upregulation is mainly attributed to macrophage accumulation and polarization into pro-inflammatory subset. In vitro coculture experiments demonstrated that connection with M0 macrophages via gap junction slightly shortened the action potential durations (APDs) of cardiomyocytes. However, the APD90 of cardiomyocytes connected with M1 macrophages were significantly prolonged (p<0.001), which were effectively attenuated by gap junction inhibition (p=0.002), KCa3.1 inhibition (p=0.008), KCa3.1 silencing (p<0.001) and store-operated Ca2+ channel inhibition (p=0.005). In vivo results demonstrated that KCa3.1 inhibition significantly decreased the QTc durations (p=0.031), intracardiac stimulation-induced ventricular arrhythmia durations (p=0.050) and incidence of premature ventricular contractions (p=0.030) in MI mice. Conclusion: Macrophage polarization leads to APD heterogeneity and post-MI arrhythmias via gap junction and KCa3.1 activation. The results provide evidences of a novel mechanism of post-MI heterogeneous repolarization and arrhythmias, rendering macrophages and KCa3.1 to be potential therapeutic targets.

Project description:PurposeNitric oxide (NO) is constitutively produced and released from the endothelium and several blood cell types by the isoform 3 of the NO synthase (NOS3). We have shown that NO protects against myocardial ischemia/reperfusion (I/R) injury and that depletion of circulating NOS3 increases within 24 h of ischemia/reperfusion the size of myocardial infarction (MI) in chimeric mice devoid of circulating NOS3. In the current study we hypothesized that circulating NOS3 also affects remodeling of the left ventricle following reperfused MI.MethodsTo analyze the role of circulating NOS3 we transplanted bone marrow of NOS3-/- and wild type (WT) mice into WT mice, producing chimerae expressing NOS3 only in vascular endothelium (BC-/EC+) or in both, blood cells and vascular endothelium (BC+/EC+). Both groups underwent 60 min of coronary occlusion in a closed-chest model of reperfused MI. During the 3 weeks post MI, structural and functional LV remodeling was serially assessed (24 h, 4 d, 1 w, 2 w and 3 w) by echocardiography. At 72 hours post MI, gene expression of several extracellular matrix (ECM) modifying molecules was determined by quantitative RT-PCR analysis. At 3 weeks post MI, hemodynamics were obtained by pressure catheter, scar size and collagen content were quantified post mortem by Gomori's One-step trichrome staining.ResultsThree weeks post MI, LV end-systolic (53.2±5.9 μl; ***p≤0.001; n = 5) and end-diastolic volumes (82.7±5.6 μl; *p<0.05; n = 5) were significantly increased in BC-/EC+, along with decreased LV developed pressure (67.5±1.8 mm Hg; n = 18; ***p≤0.001) and increased scar size/left ventricle (19.5±1.5%; n = 13; **p≤0.01) compared to BC+/EC+ (ESV: 35.6±2.2 μl; EDV: 69.1±2.6 μl n = 8; LVDP: 83.2±3.2 mm Hg; n = 24; scar size/LV13.8±0.7%; n = 16). Myocardial scar of BC-/EC+ was characterized by increased total collagen content (20.2±0.8%; n = 13; ***p≤0.001) compared to BC+/EC+ (15.9±0.5; n = 16), and increased collagen type I and III subtypes.ConclusionCirculating NOS3 ameliorates maladaptive left ventricular remodeling following reperfused myocardial infarction.

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Structural and molecular mechanisms of gap junction remodeling in epicardial border zone myocytes following myocardial infarction.

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Structural and molecular mechanisms of gap junction remodeling in epicardial border zone myocytes following myocardial infarction.

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