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Background: Cardiac hypertrophy is a key biological response to injurious stresses such as pressure overload and, when excessive, can lead to heart failure. Innate immune activation by danger signals, through intracellular pattern recognition receptors such as nucleotide-binding oligomerization domain 1 (Nod1) and its adaptor receptor-interacting protein 2 (RIP2), might play a major role in cardiac remodeling and progression to heart failure. We hypothesize that Nod1/RIP2 are major contributors to cardiac hypertrophy, but may not be sufficient to fully express the phenotype alone. Methods: To elucidate the contribution of Nod1/RIP2 signaling to cardiac hypertrophy, we randomized Nod1 –/– , RIP2 –/– , or wild-type mice to transverse aortic constriction or sham operations. Cardiac hypertrophy, fibrosis, and cardiac function were examined in these mice. Results: Nod1 and RIP2 proteins were upregulated in the heart after transverse aortic constriction, and this was paralleled by increased expression of mitochondrial proteins, including mitochondrial antiviral signaling protein (MAVS). Nod1 –/– and RIP2 –/– mice subjected to transverse aortic constriction exhibited better survival, improved cardiac function, and decreased cardiac hypertrophy. Downstream signal transduction pathways that regulate inflammation and fibrosis, including NF (nuclear factor) κB and MAPK (mitogen-activated protein kinase)-GATA4/p300, were reduced in both Nod1 –/– and RIP2 –/– mice after transverse aortic constriction compared with wild-type mice. Coimmunoprecipitation of extracted cardiac proteins and confocal immunofluorescence microscopy showed that Nod1/RIP2 interaction was robust and that this complex also included MAVS as an essential component. Suppression of MAVS expression attenuated the complex formation, NF κB signaling, and myocyte hypertrophy. Interrogation of mitochondrial function compared in the presence or ablation of MAVS revealed that MAVS serves to suppress mitochondrial energy output and mediate fission/fusion related dynamic changes. The latter is possibly linked to mitophagy during cardiomyocytes stress, which may provide an intriguing link between innate immune activation and mitochondrial energy balance under stress or injury conditions. Conclusions: We have identified that innate immune Nod1/RIP2 signaling is a major contributor to cardiac remodeling after stress. This process is critically joined by and regulated through the mitochondrial danger signal adapter MAVS. This novel complex coordinates remodeling, inflammatory response, and mitochondrial energy metabolism in stressed cardiomyocytes. Thus, Nod1/RIP2/MAVS signaling complex may represent an attractive new therapeutic approach toward heart failure.

Objectives Our objective was to test the hypothesis that there is a significant diurnal variation for the therapeutic benefit of angiotensin-converting enzyme (ACE) inhibitors on pressure-overload cardiovascular hypertrophy. Background Physiological and molecular processes exhibit diurnal rhythms that may affect efficacy of disease treatment (chronotherapy). Evidence suggests that the heart primarily remodels during sleep. Although a growing body of clinical and epidemiological evidence suggests that the timing of therapy, such as ACE inhibition, alters diurnal blood pressure patterns in patients with hypertension, the benefits of chronotherapy on myocardial and vascular remodeling have not been studied. Methods We examined the effects of the short-acting ACE inhibitor, captopril, on the structure and function of cardiovascular tissue subjected to pressure overload by transverse aortic constriction (TAC) in mice. Captopril (15 mg/kg intraperitoneally) or placebo was administered at either murine sleep time or wake time for 8 weeks starting 1 week after surgery. Results TAC mice given captopril at sleep time had improved cardiac function and significantly decreased heart: body weight ratios, myocyte cross-sectional areas, intramyocardial vascular medial wall thickness, and perivascular collagen versus TAC mice given captopril or placebo during wake time. Captopril induced similar drops in blood pressure at sleep or wake time, suggesting that time-of-day differences were not attributable to blood pressure changes. These beneficial effects of captopril were correlated with diurnal changes in ACE mRNA expression in the heart. Conclusions The ACE inhibitor captopril benefited cardiovascular remodeling only when administered during sleep; wake-time captopril ACE inhibition was identical to that of placebo. These studies support the hypothesis that the heart (and vessels) remodel during sleep time and also illustrate the importance of diurnal timing for some cardiovascular therapies.

Aims Cathepsin-L (CTSL) is a member of the lysozomal cysteine protease family, which participates in remodelling of various tissues. Herein, we sought to examine the potential regulation of CTSL in cardiac remodelling post-infarction. Methods and results Experimental myocardial infarction (MI) was created in CTSL-deficient ( Ctsl –/–) mice (B6 × FSB/GnEi a/a Ctslfs/J) and wild-type littermates ( Ctsl+/+ ) by left coronary artery ligation. At days 3, 7, 14, and 28 post-MI, we monitored survival rate and evaluated cardiac function, morphology, and molecular endpoints of repair and remodelling. Survival was 56% in Ctsl –/– mice in contrast to 80% ( P < 0.05) in Ctsl+/+ mice post-MI by day 28. The Ctsl –/– mice exhibited greater scar dilatation, wall thinning, and worse cardiac dysfunction when compared with Ctsl+/+ mice. Cardiac matrix metallopeptidase-9 (MMP-9) activity was also diminished, and c-kit -positive cells, natural killer cells, fibrocytes, and monocytes mobilized to peripheral blood and deposited to the infarcted myocardium were significantly decreased in Ctsl –/– mice. Furthermore, the local inflammatory response, and granulocyte-colony stimulating factor, stem cell factor (SCF), and stromal cell-derived factor-1 (SDF-1α) expression, as well as cell proliferation, revascularization, and myofibroblast deposition were significantly decreased in Ctsl –/– mice compared with Ctsl+/+ mice. Conclusion Our data indicate that CTSL regulates cardiac repair and remodelling post-MI through a mechanism with multiple pathways.

Background— The innate immune system greatly contributes to the inflammatory process after myocardial infarction (MI). Interleukin-1 receptor-associated kinase-4 (IRAK-4), downstream of Toll/interleukin-1 receptor signaling, has an essential role in regulating the innate immune response. The present study was designed to determine the mechanism by which IRAK-4 is responsible for the cardiac inflammatory process, which consequently affects left ventricular remodeling after MI. Methods and Results— Experimental MI was created in IRAK-4 −/− and wild-type mice by left coronary ligation. Mice with a targeted deletion of IRAK-4 had an improved survival rate at 4 weeks after MI. IRAK-4 −/− mice also demonstrated attenuated cardiac dilation and decreased inflammation in the infarcted myocardium, which was associated with less proinflammatory and Th1 cytokine expression mediated by suppression of nuclear factor-κB and c-Jun N-terminal kinase activation. IRAK-4 −/− mice had fewer infiltrations of CD45 + leukocytes and CD11c + dendritic cells, inhibition of apoptosis, and reduced fibrosis and nitric oxide production. Cardiac dendritic cells in IRAK-4 −/− mice were relatively immature or functionally naïve after MI in that they demonstrated less cytokine and costimulatory molecule gene expression. Furthermore, IRAK-4 −/− dendritic cells have less mobilization capacity. Transfer of wild type-derived bone marrow dendritic cells into IRAK-4 −/− mice for functional dendritic cell reconstitution negated the survival advantage and reduced the cardiac dilation observed with IRAK-4 −/− mice at 28 days after MI. Conclusions— Deletion of IRAK-4 has favorable effects on survival and left ventricular remodeling after MI through modification of the host inflammatory process by blunting the detrimental bone marrow dendritic cells mobilization after myocardial ischemia.

Gelsolin, a calcium-regulated actin severing and capping protein, is highly expressed in murine and human hearts after myocardial infarction and is associated with progression of heart failure in humans. The biological role of gelsolin in cardiac remodeling and heart failure progression after injury is not defined. To elucidate the contribution of gelsolin in these processes, we randomly allocated gelsolin knockout mice (GSN −/− ) and wild-type littermates (GSN +/+ ) to left anterior descending coronary artery ligation or sham surgery. We found that GSN −/− mice have a surprisingly lower mortality, markedly reduced hypertrophy, smaller late infarct size, less interstitial fibrosis, and improved cardiac function when compared with GSN +/+ mice. Gene expression and protein analysis identified significantly lower levels of deoxyribonuclease (DNase) I and reduced nuclear translocation and biological activity of DNase I in GSN −/− mice. Absence of gelsolin markedly reduced DNase I–induced apoptosis. The association of hypoxia-inducible factor (HIF)-1α with gelsolin and actin filaments cleaved by gelsolin may contribute to the higher activation of DNase. The expression pattern of HIF-1α was similar to that of gelsolin, and HIF-1α was detected in the gelsolin complex by coprecipitation and HIF-1α bound to the promoter of DNase I in both gel-shift and promoter activity assays. Furthermore, the phosphorylation of Akt at Ser473 and expression of Bcl-2 were significantly increased in GSN −/− mice, suggesting that gelsolin downregulates prosurvival factors. Our investigation concludes that gelsolin is an important contributor to heart failure progression through novel mechanisms of HIF-1α and DNase I activation and downregulation of antiapoptotic survival factors. Gelsolin inhibition may form a novel target for heart failure therapy.

Myocardial infarction (MI) and subsequent adverse remodeling cause heart failure. Previously we demonstrated a role for Kit ligand (KL) in improving cardiac function post-MI. KL has two major isoforms; KL-1 is secreted whereas KL-2 is predominantly membrane bound. We demonstrate here first that KL-2-deficient mice have worse survival and an increased heart/bodyweight ratio post-MI compared to mice with reduced c-Kit receptor expression. Next we synthesized recombinant lentiviral vectors (LVs) that engineered functional expression of murine KL-1 and KL-2. For in vivo analyses, we directly injected these LVs into the left ventricle of membrane-bound KL-deficient Sl/Sl(d) or wild-type (WT) mice undergoing MI. Control LV/enGFP injection led to measurable reporter gene expression in hearts. Injection of LV/KL-2 attenuated adverse left ventricular remodeling and dramatically improved survival post-MI in both Sl/Sl(d) and WT mice (from 12 to 71% and 35 to 73%, respectively, versus controls). With regard toward beginning to understand the possible salutary mechanisms involved in this effect, differential staining patterns of Sca-1 and Ly49 on peripheral blood (PB) cells from therapeutically treated animals was found. Our data show that LV/KL-2 gene therapy is a promising treatment for MI.

Day/night rhythms are recognized as important to normal cardiovascular physiology and timing of adverse cardiovascular events; however, their significance in disease has not been determined. We demonstrate that day/night rhythms play a critical role in compensatory remodeling of cardiovascular tissue, and disruption exacerbates disease pathophysiology. We use a murine model of pressure overload cardiac hypertrophy (transverse aortic constriction) in a rhythm-disruptive 20-hour versus 24-hour environment. Echocardiography reveals increased left ventricular end-systolic and -diastolic dimensions and reduced contractility in rhythm-disturbed transverse aortic constriction animals. Furthermore, cardiomyocytes and vascular smooth muscle cells exhibit reduced hypertrophy, despite increased pressure load. Microarray and real-time PCR demonstrate altered gene cycling in transverse aortic constriction myocardium and hypothalamic suprachiasmatic nucleus. With rhythm disturbance, there is a consequent altered cellular clock mechanism ( per2 and bmal ), whereas key genes in hypertrophic pathways ( ANF, BNP, ACE , and collagen ) are downregulated paradoxical to the increased pressure. Phenotypic rescue, including reversal/attenuation of abnormal pathology and genes, only occurs when the external rhythm is allowed to correspond with the animals’ innate 24-hour internal rhythm. Our study establishes the importance of diurnal rhythm as a vital determinant in heart disease. Disrupted rhythms contribute to progression of organ dysfunction; restoration of normal diurnal schedules appears to be important for effective treatment of disease.

Background— Pressure overload is accompanied by cardiac myocyte apoptosis, hypertrophy, and inflammatory/fibrogenic responses that lead to ventricular remodeling and heart failure. Despite incomplete understanding of how this process is regulated, the upregulation of tumor necrosis factor (TNF)-α after aortic banding in the myocardium is known. In the present study, we tested our hypothesis that TNF-α regulates the cardiac inflammatory response, extracellular matrix homeostasis, and ventricular hypertrophy in response to mechanical overload and contributes to ventricular dysfunction. Methods and Results— C57/BL wild-type mice and TNF-knockout (TNF −/− ) mice underwent descending aortic banding or sham operation. Compared with sham-operated mice, wild-type mice with aortic banding showed a significant increase in cardiac TNF-α levels, which coincided with myocyte apoptosis, inflammatory response, and cardiac hypertrophy in week 2 and a significant elevation in matrix metalloproteinase-9 activity and impaired cardiac function in weeks 2 and 6. Compared with wild-type mice with aortic banding, TNF −/− mice with aortic banding showed attenuated cardiac apoptosis, hypertrophy, inflammatory response, and reparative fibrosis. These mice also showed reduced cardiac matrix metalloproteinase-9 activity and improved cardiac function. Conclusions— Findings from the present study have suggested that TNF-α contributes to adverse left ventricular remodeling during pressure overload through regulation of cardiac repair and remodeling, leading to ventricular dysfunction.

Inappropriate cardiac remodeling and repair after myocardial infarction (MI) predisposes to heart failure. Studies have reported on the potential for lineage negative, steel factor positive ( c - kit + ) bone marrow-derived hematopoetic stem∕progenitor cells (HSPCs) to repair damaged myocardium through neovascularization and myogenesis. However, the precise contribution of the c-kit signaling pathway to the cardiac repair process has yet to be determined. In this study, we sought to directly elucidate the mechanistic contributions of c-kit + bone marrow-derived hematopoetic stem∕progenitor cells in the maintenance and repair of damaged myocardium after MI. Using c-kit -deficient mice, we demonstrate the importance of c-kit signaling in preventing ventricular dilation and hypertrophy, and the maintenance of cardiac function after MI in c-kit -deficient mice. Furthermore, we show phenotypic rescue of cardiac repair after MI of c-kit -deficient mice by bone marrow transplantation of wild-type HSPCs. The transplanted group also had reduced apoptosis and collagen deposition, along with an increase in neovascularization. To better understand the mechanisms underlying this phenotypic rescue, we investigated the gene expression pattern within the infarcted region by using microarray analysis. This analysis suggested activation of inflammatory pathways, specifically natural killer (NK) cell-mediated mobilization after MI in rescued hearts. This finding was confirmed by immunohistology and by using an NK blocker. Thus, our investigation revealed a previously uncharacterized role for c-kit signaling after infarction by mediating bone marrow-derived NK and angiogenic cell mobilization, which contributes to improved remodeling and cardiac function after MI.

BackgroundWe have recently demonstrated that remote ischemic preconditioning reduces ischemia-reperfusion injury in animal models. The mechanisms by which the remote ischemic preconditioning stimulus exerts its effect remain to be fully defined, and its effect on myocardial gene expression is unknown. We tested the hypothesis that remote ischemic preconditioning modifies myocardial gene expression immediately after the remote ischemic preconditioning stimulus (early phase) and 24 hours later (late phase).MethodsTwenty male (C57BL/6) 10- to 12-week-old mice were randomized into 4 groups: group 1 (control, early phase; n = 5), group 2 (remote ischemic preconditioning, early phase; n = 5), group 3 (control, late phase; n = 5), and group 4 (remote ischemic preconditioning, late phase; n = 5). Groups 2 and 4 underwent remote ischemic preconditioning induced by 6 cycles of 4 minutes of occlusion and 4 minutes of reperfusion of the femoral artery. Groups 1 and 2 were killed 15 minutes after completion of sham procedure or remote ischemic preconditioning, and the hearts were removed and frozen in liquid nitrogen. Groups 3 and 4 were killed 24 hours after remote ischemic preconditioning, and the hearts were harvested in the same fashion. Gene expression was assessed by using the Affymetrix MG-430A chip (Affymetrix, Santa Clara, Calif).ResultsData filtering (P < .05, analysis of variance) and hierarchic 2-way clustering identified significant differences in gene expression among the 4 groups. Genes involved in protection against oxidative stress (eg, Hadhsc, Prdx4, and Fabp4) and cytoprotection (Hsp73) were upregulated, whereas many proinflammatory genes (eg, Egr-1 and Dusp 1 and 6) were suppressed.ConclusionA simple remote ischemic preconditioning stimulus modifies myocardial gene expression by upregulating cardioprotective genes and suppressing genes potentially involved in the pathogenesis of ischemia-reperfusion injury.

Elevated serine elastase activity after myocardial infarction can contribute to remodeling associated with left ventricular dilatation and dysfunction. We therefore assessed the effects of overexpressing the selective serine elastase inhibitor elafin in transgenic mice in which a myocardial infarction was caused by ligation of the left anterior descending coronary artery (LAD). Elevated serine elastase activity was observed in nontransgenic littermates as early as 6 h after LAD ligation and persisted at 4 and 7 days but not in sham-operated or elafin-overexpressing transgenic mice. Myeloperoxidase activity (index of inflammatory cells) and matrix metalloproteinase 2 were also increased but only at 4 and 7 days and only in nontransgenic mice ( P < 0.05 for both comparisons), and this increase correlated with inflammatory cell infiltration. Echocardiographic study at 4 days revealed indexes of diastolic dysfunction in nontransgenic versus elafin-overexpressing mice ( P < 0.05). Morphometric and biochemical analyses at 28 days indicated impairment in cardiac performance, with greater scar thinning and infarct expansion in nontransgenic versus elafin transgenic littermates ( P < 0.05 for all comparisons). Thus serine elastase inhibition appears to suppress inflammation, cardiac dilatation, and dysfunction after myocardial infarct.

Background— Integrins are involved in structural remodeling and tissue repair. This study aimed to elucidate the role of the β-integrins in cardiac remodeling after myocardial infarction (MI). Methods and Results— The MI model was created by ligation of the left anterior descending coronary artery in rats. We detected cardiac integrins β1 and β3 gene expression (quantitative in situ hybridization) and protein production (Western blot and immunohistochemistry) and potential regulation by tumor necrosis factor (TNF) using neonatal ventricular myocytes and TNF −/− knockout mice. Integrins β1 and β3 gene expression and protein production were low in sham-operated hearts. After MI, the β1 and β3 mRNA and proteins were significantly increased at the site of MI at day 3, reached a peak at day 7, and gradually declined thereafter. Integrin β1A localized primarily in fibroblasts and inflammatory cells, β1D localized in myocytes, and integrin β3 was associated primarily with endothelial and smooth muscle cells in peri-infarct vessels. In cultured myocytes, there was isoform transition from the adult β1D to the fetal β1A on exposure to TNF-α. This was confirmed in vivo in the peri-infarct myocytes, but the transition was voided in TNF −/− -knockout mice. Conclusions— Integrins β1 and β3 are significantly activated in the infarcted myocardium. Integrin β1 is active particularly at sites of inflammation and fibrosis, whereas integrin β3 localizes to vessels in the peri-infarct zone in a temporally coordinated manner. Integrin β1D to β1A isoform transition in myocytes is regulated by TNF-α.

Background Endoplasmic reticulum ( ER ) resident protein 44 ( ER p44) is a member of the protein disulfide isomerase family, is induced during ER stress, and may be involved in regulating Ca 2+ homeostasis. However, the role of ER p44 in cardiac development and function is unknown. The aim of this study was to investigate the role of ER p44 in cardiac development and function in mice, zebrafish, and embryonic stem cell ( ESC )‐derived cardiomyocytes to determine the underlying role of ER p44. Methods and Results We generated and characterized ER p44 −/− mice, ER p44 morphant zebrafish embryos, and ER p44 −/− ESC ‐derived cardiomyocytes. Deletion of ER p44 in mouse and zebrafish caused significant embryonic lethality, abnormal heart development, altered Ca 2+ dynamics, reactive oxygen species generation, activated ER stress gene profiles, and apoptotic cell death. We also determined the cardiac phenotype in pressure overloaded, aortic‐banded ER p44 +/− mice: enhanced ER stress activation and increased mortality, as well as diastolic cardiac dysfunction with a significantly lower fractional shortening. Confocal and LacZ histochemical staining showed a significant transmural gradient for ER p44 in the adult heart, in which high expression of ER p44 was observed in the outer subepicardial region of the myocardium. Conclusions ER p44 plays a critical role in embryonic heart development and is crucial in regulating cardiac cell Ca 2+ signaling, ER stress, ROS ‐induced oxidative stress, and activation of the intrinsic mitochondrial apoptosis pathway.

Background —Estrogen may increase the long-term survival of women who have suffered from a myocardial infarction (MI). We examined the acute and chronic influence of estrogen on MI in the rat left coronary artery ligation model. Methods and Results —Female Sprague-Dawley rats (10 to 12 weeks, n=93), divided into 3 groups (rats with intact ovaries, ovariectomized rats administered 17β-estradiol [17β-E 2 ] replacement, and ovariectomized rats administered placebo 2 weeks before MI), were randomized to left coronary artery ligation (n=66) or sham-operated (n=27) groups. Ten to 11 weeks after MI, rats were randomly assigned to either (1) assessment of left ventricular (LV) function and morphometric analysis or (2) measurement of cardiopulmonary mRNA expression of preproendothelin-1 and endothelin A and B receptors. Acutely, estrogen was associated with a trend toward increased mortality. Infarct size was increased in the 17β-E 2 group compared with the placebo group (42±2% versus 26±3%, respectively; P =0.01). Chronically, wall tension was normalized through a reduction in LV cavity size with estrogen treatment (419±41 mm Hg/mm for 17β-E 2 versus 946±300 mm Hg/mm for placebo, P =0.039). In the LV, there was a 2.5-fold increase in endothelin B mRNA expression after MI in placebo-treated rats ( P =0.004 versus sham-operated rats) that was prevented in the 17β-E 2 group ( P =NS versus sham-operated rats). Conclusions —These results suggest that estrogen is detrimental at the time of MI or early post-MI period, resulting in an increased size of infarct or infarct expansion, but chronically, it can normalize wall tension and inhibit LV dilatation, which may in turn lead to increased long-term survival. Regulation of the endothelin system, particularly the expression of the endothelin B receptor, may contribute to these estrogenic effects.

Infections are thought to be important in the pathogenesis of many heart diseases. Coxsackievirus B3 (CVB3) has been linked to chronic dilated cardiomyopathy, a common cause of progressive heart disease, heart failure and sudden death. We show here that the sarcoma (Src) family kinase Lck (p56lck) is required for efficient CVB3 replication in T-cell lines and for viral replication and persistence in vivo. Whereas infection of wild-type mice with human pathogenic CVB3 caused acute and very severe myocarditis, meningitis, hepatitis, pancreatitis and dilated cardiomyopathy, mice lacking the p56lck gene were completely protected from CVB3-induced acute pathogenicity and chronic heart disease. These data identify a previously unknown function of Src family kinases and indicate that p56lck is the essential host factor that controls the replication and pathogenicity of CVB3.

Abstract —Viral myocarditis is an important cause of heart failure and dilated cardiomyopathy. T lymphocytes are implicated in myocardial damage in murine models of coxsackievirus B3 (CVB3) myocarditis. We used knockout mice lacking CD4 (CD4 −/− ), CD8 (CD8 −/− ), both coreceptors (CD4 −/− CD8 −/− ), or the T-cell receptor β chain (TCRβ −/− ) to address the contribution of T-cell subpopulations to host susceptibility to CVB3 myocarditis. Severity of disease was magnified in CD8 −/− mice but attenuated in CD4 −/− mice, consistent with a pathogenic role for CD4 + lymphocytes. Elimination of both CD4 and CD8 molecules from T lymphocytes by genetic knockout better protected mice from myocarditis, demonstrating that both CD4 + and CD8 + T cells contribute to host susceptibility. The same benefit occurred in TCRβ −/− mice, with prolonged survival and minimal myocardial disease observed after CVB3 infection. Elevated interferon-γ and decreased tumor necrosis factor-α expression are associated with attenuated myocardial damage in CD4 −/− CD8 −/− mice. These results show that the presence of TCRαβ + T cells enhances host susceptibility to myocarditis. The severity of myocardial damage and associated mortality are dependent on the predominant T-cell type available to respond to CVB3 infection. One mechanism by which CD4 + and CD8 + T-cell subsets influence the pathogenesis of myocarditis may involve specific cytokine expression patterns.

In viral myocarditis, inflammation and destruction of cardiac myocytes leads to fibrosis, causing progressive impairment in cardiac function. Here we show the etiologic importance of serine elastase activity in the pathophysiology of acute viral myocarditis and the therapeutic efficacy of an elastase inhibitor. In DBA/2 mice inoculated with the encephalomyocarditis virus, a more than 150% increase in myocardial serine elastase activity is observed. This is suppressed by a selective serine elastase inhibitor, ZD0892, which is biologically effective after oral administration. Mice treated with this compound had little evidence of microvascular constriction and obstruction associated with myocarditis-induced ischemia reperfusion injury, much less inflammation and necrosis, only mild fibrosis and myocardial collagen deposition, and normal ventricular function, compared with the infected nontreated group.

Background. The innate immune response is critical in mediating host protection from foreign antigen or stress. It is evident that multiple innate immune pathways also tightly regulate the remodeling process post-myocardial infarction (MI), however it is not clear how this process is mediated. In order to further elucidate the underlying mechanism of this process, we screened both mouse and human post-MI microarray databases and identified a common, significantly upregulated transcription factor of interest, interferon regulatory factor-3 (IRF3). To assess its function in vivo, we tested the hypothesis that activation of IRF3 post-MI coordinates maladaptive repair by promoting cardiomyocyte apoptosis. Methods/Results. We randomly allocated IRF-3 knockout (IRF-3-/-) mice and their wild-type (WT) counterparts to either left anterior descending (LAD) coronary artery ligation or sham operation. Four weeks post-MI, deficiency in IRF3 led to improved survival [88% (44/50) vs. 29% (14/49) in WT, p Conclusions. These data establish a novel link between the innate immune transcription factor IRF3 and maladaptive regulation of cardiomyocyte apoptosis through mitochondrial apoptotic pathway activation. As an acute responder coordinating maladaptive remodeling, IRF3 may prove to be an efficacious therapeutic target to improve outcome following MI.

Vasoconstriction occurs frequently following coronary angioplasty and is implicated in the pathogenesis of abrupt closure and restenosis. Control of vasomotor tone is regulated in part directly by smooth muscle cells and indirectly through the endothelium. To study the mechanisms underlying vasoconstriction, the effect of angioplasty and endothelial denudation on endothelium-dependent and -independent relaxation was examined in 15 mongrel dogs. Percutaneous transluminal angioplasty and endothelial denudation of the right femoral artery were performed. Endothelial injury was assessed by adhesion of indium-111-labeled platelets. Endothelium-dependent and -independent relaxation were assessed using acetylcholine and nitroglycerin, respectively. Vessels precontracted with potassium chloride and exposed to acetylcholine showed impaired relaxation in both the angioplasty and denuded groups (angioplasty = 14 +/- 5%, denuded = 0 +/- 0%, normal = 73 +/- 12%; P less than 0.05 for both angioplasty and denuded compared to normal). Precontraction with phenylephrine yielded similar results (angioplasty = 16 +/- 8%, denuded = 4 +/- 2%, normal = 39 +/- 10%; P less than 0.05 only for denuded segment compared to normal). Segments precontracted with phenylephrine and exposed to nitroglycerin did not demonstrate impaired relaxation (angioplasty = 73 +/- 9%, denuded = 68 +/- 9%, normal = 71 +/- 7%, P = ns). Mean indium-111 counts were similar in both the angioplasty and denuded segments (2820 +/- 1481 and 2963 +/- 1228 counts/min/g, respectively) compared to a lower count in the normal segment (1514 +/- 956 counts/min/g). Thus, angioplasty produces significant vascular injury and impairment of vasodilator function, comparable to that caused by endothelial denudation alone. This implies that vasoconstriction seen following coronary angioplasty may be due to endothelial injury and the resultant loss of control of vasomotor tone.

The pleiotropic cytokines, transforming growth factor beta1 (TGFbeta1), and tumor necrosis factor (TNF) play critical roles in tissue homeostasis in response to injury and are implicated in multiple human diseases and cancer. We reported that the loss of Timp3 (tissue inhibitor of metalloproteinase 3) leads to abnormal TNF signaling and cardiovascular function. Here we show that parallel deregulation of TGFbeta1 and TNF signaling in Timp3(-/-) mice amplifies their cross-talk at the onset of cardiac response to mechanical stress (pressure overload), resulting in fibrosis and early heart failure. Microarray analysis showed a distinct gene expression profile in Timp3(-/-) hearts, highlighting activation of TGFbeta1 signaling as a potential mechanism underlying fibrosis. Neonatal cardiomyocyte-cardiofibroblast co-cultures were established to measure fibrogenic response to agonists known to be induced following mechanical stress in vivo. A stronger response occurred in neonatal Timp3(-/-) co-cultures, as determined by increased Smad signaling and collagen expression, due to increased TNF processing and precocious proteolytic maturation of TGFbeta1 to its active form. The relationship between TGFbeta1 and TNF was dissected using genetic and pharmacological manipulations. Timp3(-/-)/Tnf(-/-) mice had lower TGFbeta1 than Timp3(-/-), and anti-TGFbeta1 antibody (1D11) negated the abnormal TNF response, indicating their reciprocal stimulatory effects, with each manipulation abolishing fibrosis and improving heart function. Thus, TIMP3 is a common innate regulator of TGFbeta1 and TNF in tissue response to injury. The matrix-bound TIMP3 balances the anti-inflammatory and proinflammatory processes toward constructive tissue remodeling.

Chromatin remodeling, particularly histone acetylation, plays a critical role in the progression of pathological cardiac hypertrophy and heart failure. We hypothesized that curcumin, a natural polyphenolic compound abundant in the spice turmeric and a known suppressor of histone acetylation, would suppress cardiac hypertrophy through the disruption of p300 histone acetyltransferase-dependent (p300-HAT-dependent) transcriptional activation. We tested this hypothesis using primary cultured rat cardiac myocytes and fibroblasts as well as two well-established mouse models of cardiac hypertrophy. Curcumin blocked phenylephrin-induced (PE-induced) cardiac hypertrophy in vitro in a dose-dependent manner. Furthermore, curcumin both prevented and reversed mouse cardiac hypertrophy induced by aortic banding (AB) and PE infusion, as assessed by heart weight/BW and lung weight/BW ratios, echocardiographic parameters, and gene expression of hypertrophic markers. Further investigation demonstrated that curcumin abrogated histone acetylation, GATA4 acetylation, and DNA-binding activity through blocking p300-HAT activity. Curcumin also blocked AB-induced inflammation and fibrosis through disrupting p300-HAT-dependent signaling pathways. Our results indicate that curcumin has the potential to protect against cardiac hypertrophy, inflammation, and fibrosis through suppression of p300-HAT activity and downstream GATA4, NF-kappaB, and TGF-beta-Smad signaling pathways.

Aims The maladaptive response to biomechanical stress is a fundamental response in heart disease. Loss of the 3′-lipid phosphatase, phosphatase and tensin homolog deleted on chromosome ten (PTEN), is associated with increased phosphorylation of Akt/protein kinase B and glycogen synthase kinase-β. We hypothesize that these key changes will halt the development of pathological hypertrophy and the progression to heart failure in response to pressure overload. Methods and results In mice, muscle-specific knockout of PTEN, mckCRE-PTENflox/flox (PTEN KO), resulted in basal hypertrophy and mild reduction in left ventricular (LV) systolic function. Male mice were subjected to aortic banding (AB) or sham operation. In contrast to mckCRE-PTEN+/+ control mice, pressure overload in PTEN KO mice resulted in reduced pathological hypertrophy, less interstitial fibrosis, and reduced apoptosis with a marked preservation of LV function. Western blot analysis of mitogen-activated protein kinase (MAPK) signalling showed equivalent phosphorylation of extracellular signal-regulated kinase (ERK)1 and ERK2 with markedly reduced phosphorylation of jun N-terminal kinase (JNK)1 and JNK2, and p38 in PTEN KO mice subjected to AB. Loss of PTEN was associated with increased expression of the proangiogenic factors, vascular endothelial growth factor-A and angiopoietin-2, with preservation of the myocardial capillary density in response to pressure overload. Moreover, banded PTEN KO mice maintained the expression of several key metabolic genes that are known to be dysregulated in heart failure. In contrast, a subpressor dose of the G protein-coupled receptor (GPCR) agonist angiotensin II (Ang II) leads to increased pathological hypertrophy and MAPK activation in PTEN KO mice. Conclusion Loss of PTEN prevents the development of maladaptive ventricular remodelling with preservation of angiogenesis and metabolic gene expression in response to pressure overload but not in response to the GPCR agonist, Ang II. Inhibition of PTEN signalling in the heart may represent a novel approach to slow the progression of heart failure in response to pathological biomechanical stress.

Fibrosis is the outcome of excess deposition of extracellular matrix components and underlies diastolic and systolic heart failure. Metalloproteinases (MMPs and ADAMs) and their inhibitors (TIMPs) are critical regulators of ECM turnover and fibrosis. TIMP3 is a potent inhibitor of MMPs and ADAMs, whereby it regulates tissue proteolysis and cytokine bioavailability. TIMP3 levels are reduced in human heart disease, and we found that mice lacking Timp3 (KO) exhibit severe dilated cardiomyopathy (DCM), extensive interstitial fibrosis and early heart failure (HF) following pressure overload compared to wild type (WT) mice. We showed upregulated TNF signaling within 6 hrs of aortic banding (AB) which when blocked, significantly improved DCM and prevented HF. We then investigated the mechanism underlying the extensive fibrosis despite the increased MMP activity in the TIMP3KO mice. Comparative microarray analyses show a significant upregulation of the TGFβ1 signaling pathway within 6 hrs of AB in KO vs WT hearts. In vivo treatment of KO-AB mice with a TGFβ1-neutralizing antibody (1D11) completely abolishes fibrosis, markedly improves left ventricular dilation, systolic and diastolic heart function (LVEDD(mm): 4.3 ± 0.1 in WT, 5.4 ± 0.1 in KO, 4.7 ± 0.1 in KO+ 1D11; fractional shortening(%): 40.2 ± 1.7 in WT, 19.6 ± 2.2 in KO, 36.2 ± 0.7 in KO+ 1D11; E/Ac ratio: 2.5 ± 0.3 in WT, 1.8 ± 0.3 in KO, 2.4 ± 0.2 in KO + 1D11; n = 22, P < 0.05). Interestingly, blocking TGFβ1 also prevented the early rise in TNF in KO hearts. Using primary neonatal culture systems, we found that cardiomyocyte-fibroblast interaction is essential for a fibrotic response to agonists (Ang II, PE). Further, KO co-cultures generate a 5-fold greater fibrosis (P < 0.05) through activation of Smad2/3 pathway compared to WT. TGFβ also regulates TNF transcription more potently in KO cells since a 3-fold higher TNF induction occurred in response to recombinant TGFβ1 in KO vs WT co-cultures (P < 0.05). In conclusion, TIMP3 is a critical regulator of TGFβ1 and TNF, and its deficiency induces parallel dysregulations in the signaling of these cytokines very early after pressure overload, leading to severe interstitial fibrosis and DCM, hence, TIMP3-based therapies can simultaneously impact fibrosis and DCM.

Apelin constitutes a novel endogenous peptide system suggested to be involved in a broad range of physiological functions, including cardiovascular function, heart development, control of fluid homeostasis, and obesity. Apelin is also a catalytic substrate for angiotensin-converting enzyme 2, the key severe acute respiratory syndrome receptor. The in vivo physiological role of Apelin is still elusive. Here we report the generation of Apelin gene–targeted mice. Apelin mutant mice are viable and fertile, appear healthy, and exhibit normal body weight, water and food intake, heart rates, and heart morphology. Intriguingly, aged Apelin knockout mice developed progressive impairment of cardiac contractility associated with systolic dysfunction in the absence of histological abnormalities. We also report that pressure overload induces upregulation of Apelin expression in the heart. Importantly, in pressure overload–induced heart failure, loss of Apelin did not significantly affect the hypertrophy response, but Apelin mutant mice developed progressive heart failure. Global gene expression arrays and hierarchical clustering of differentially expressed genes in hearts of banded Apelin −/y and Apelin +/y mice showed concerted upregulation of genes involved in extracellular matrix remodeling and muscle contraction. These genetic data show that the endogenous peptide Apelin is crucial to maintain cardiac contractility in pressure overload and aging.

Background Recombinant lentiviral vectors (LVs) offer the possibility of stable, long-term expression of transgenes even in non-dividing cells. In the present study this vector system was applied to a clinically relevant cardiovascular problem. Methods and Results Fabry disease results from deficient activity of α-galactosidase A (α-gal A) and cardiac abnormalities are a common and an important cause of death in patients with the disease. A therapeutic LV that delivers the α-gal A cDNA has been synthesized. In vitro studies established efficient transduction of the H9c2 rat cardiomyocytes and showed overexpression of enGFP (control) and α-gal A. In in vivo studies, the enGFP cDNA was transferred into C57BL/6 mouse hearts by direct intraventricular injection. Next, in a mouse model of Fabry disease, the recombinant therapeutic construct was delivered analogously. In cardiac tissue, α-gal A activity rose to 23% of normal levels at day 7 after LV injection, which is encouraging because levels of correction approximating 5% of normal may be curative for this disorder. There was also a corresponding reduction in globotriaosylceramide accumulation. Other organs assayed showed no detectable changes in α-gal A activity levels in injected animals. Conclusion A localized benefit of directly injecting a therapeutic LV into the heart has been shown, confirming the utility of this delivery system for research and therapy for a variety of cardiovascular disorders. (Circ J 2006; 70: 1503 - 1508)

Cytokine and extracellular matrix (ECM) homeostasis are distinct systems that are each dysregulated in heart failure. Here we show that tissue inhibitor of metalloproteinase (TIMP)-3 is a critical regulator of both systems in a mouse model of left ventricular (LV) dilation and dysfunction. Timp-3 −/− mice develop precipitous LV dilation and dysfunction reminiscent of dilated cardiomyopathy (DCM), culminating in early onset of heart failure by 6 weeks, compared with wild-type aortic-banding (AB). Timp-3 deficiency resulted in increased TNFα converting enzyme (TACE) activity within 6 hours after AB leading to enhanced tumor necrosis factor-α (TNFα) processing. In addition, TNFα production increased in timp-3 −/− -AB myocardium. A significant elevation in gelatinase and collagenase activities was observed 1 week after AB, with localized ECM degradation in timp-3 −/− -AB myocardium. Timp-3 −/− /tnfα −/− mice were generated and subjected to AB for comparative analyses with timp-3 −/− -AB mice. This revealed the critical role of TNFα in the early phase of LV remodeling, de novo expression of Matrix metalloproteinases (MMP)-8 in the absence of TNFα, and highlighted the importance of interstitial collagenases (MMP-2, MMP-13, and MT1-MMP) for cardiac ECM degradation. Ablation of TNFα, or limiting MMP activity with a synthetic MMP inhibitor (PD166793), each partially attenuated LV dilation and cardiac dysfunction in timp-3 −/− -AB mice. Notably, combining TNFα ablation with MMP inhibition completely rescued heart disease in timp-3 −/− -AB mice. This study provides a basis for anti-TNFα and MMP inhibitor combination therapy in heart disease.

Background— S100B, a 20-kDa, Ca 2+ -binding dimer, is a putative intrinsic negative regulator of myocardial hypertrophy expressed after myocardial infarction. S100B-overexpressing transgenic (TG) and S100B-knockout (KO) mice have been generated to assess the consequences of S100B expression and altered hypertrophy after infarction. Methods and Results— We compared 21 wild-type (WT), 20 TG, and 24 KO mice over 35 days after experimental myocardial infarction with sham-operated controls (n=56). Of those, 4 WT-infarcted mice, 7 TG-infarcted mice, and 1 KO-infarcted mouse and no sham-operated mice died during the observation period. Among survivors, echocardiography, hemodynamic studies, and postmortem examination indicated that the WT and KO groups of infarcted mice mounted a hypertrophic response that was augmented in KO mice. The S100B-overexpressing TG group did not develop hypertrophy but demonstrated increased apoptosis. The postinfarct end-diastolic pressure was lower in KO mice than in WT mice, in accordance with other structural, hemodynamic, and functional parameters, which suggests that abrogation of S100B expression augmented hypertrophy, decreased apoptosis, and was beneficial to preservation of cardiac function within this time frame. Conclusions— S100B regulates the hypertrophic response and remodeling in the early postinfarct period and represents a potential novel therapeutic target.

Background— We investigated the potential contributions of tumor necrosis factor-α (TNF-α) on the incidence of acute myocardial rupture and subsequent chronic cardiac dysfunction after myocardial infarction (MI) in TNF knockout (TNF −/− ) mice compared with C57/BL wild-type (WT) mice. Methods and Results— Animals were randomized to left anterior descending ligation or sham operation and killed on days 3, 7, 14, and 28. We monitored cardiac rupture rate, cardiac function, inflammatory response, collagen degradation, and net collagen formation. We found the following: (1) within 1 week after MI, 53.3% (n=120) of WT mice died of cardiac rupture, in contrast to 2.5% (n=80) of TNF −/− mice; (2) inflammatory cell infiltration and cytokine expression were significantly higher in the infarct zone in WT than TNF −/− mice on day 3; (3) matrix metalloproteinase-9 and -2 activity in the infarcted myocardium was significantly higher in WT than in TNF −/− mice on day 3; (4) on day 28 after MI compared with sham, there was a significant decrease in LV developed pressure (74%) and ±dP/dt max (68.3%/65.3%) in WT mice but a less significant decrease in ±dP/dt max (25.8%/28.8%) in TNF −/− mice; (5) cardiac collagen volume fraction was lower in WT than in TNF −/− mice on days 3 and 7 but higher on day 28 compared with TNF −/− mice; and (6) a reduction in myocyte apoptosis in TNF −/− mice occurred on day 28 compared with WT mice. Conclusions— Elevated local TNF-α in the infarcted myocardium contributes to acute myocardial rupture and chronic left ventricle dysfunction by inducing exuberant local inflammatory response, matrix and collagen degradation, increased matrix metalloproteinase activity, and apoptosis.

Under conditions of iron overload, which are now reaching epidemic proportions worldwide, iron-overload cardiomyopathy is the most important prognostic factor in patient survival. We hypothesize that in iron-overload disorders, iron accumulation in the heart depends on ferrous iron (Fe2+) permeation through the L-type voltage-dependent Ca2+ channel (LVDCC), a promiscuous divalent cation transporter. Iron overload in mice was associated with increased mortality, systolic and diastolic dysfunction, bradycardia, hypotension, increased myocardial fibrosis and elevated oxidative stress. Treatment with LVDCC blockers (CCBs; amlodipine and verapamil) at therapeutic levels inhibited the LVDCC current in cardiomyocytes, attenuated myocardial iron accumulation and oxidative stress, improved survival, prevented hypotension and preserved heart structure and function. Consistent with the role of LVDCCs in myocardial iron uptake, iron-overloaded transgenic mice with cardiac-specific overexpression of the LVDCC alpha1-subunit had twofold higher myocardial iron and oxidative stress levels, as well as greater impairment in cardiac function, compared with littermate controls; LVDCC blockade was again protective. Our results indicate that cardiac LVDCCs are key transporters of iron into cardiomyocytes under iron-overloaded conditions, and potentially represent a new therapeutic target to reduce the cardiovascular burden from iron overload.

Objectives: To determine the relationship between the total chronic dose of iron administered, ex-vivo cardiac function and the concentrations of cytotoxic aldehydes in heart tissue of a murine model. Methods: In the first experiment, 34 male B6D2F1 mice were randomized to receive intraperitoneal injections of 5, 10 or 20 mg of iron dextran for three weeks, or a placebo control. The mice were subsequently randomized to undergo ex-vivo assessment of cardiac function. In the second experiment, free radical generation, quantified by the presence of 20 separate cytotoxic aldehydes, was assessed in heart tissue of 40 mice that were randomized to receive chronic treatment with various concentrations of iron dextran (100 mg to 300 mg total chronic dose administered), placebo treatment with saline, or no treatment at all (baseline). Results: Iron-loaded groups displayed dose-dependent depressions of heart rate, systolic pressure, developed pressure, coronary pressure, −d P /d t and +d P /d t , and increases in diastolic pressure. Monotonic dose-dependent increases in total heart aldehydes were observed in the iron-treated groups ( r =0.97, p

Continued adverse remodeling of myocardium after infarction may lead to progressive ventricular dilation and heart failure. We tested the hypothesis that exercise training in a healed myocardial infarction-dysfunction rat model can favorably modify the adverse effects of ventricular remodeling including attenuation of abnormal myosin gene expression. Sprague-Dawley rats were subjected to either proximal LAD ligation or sham operation. At 5 wk after the operation, animals were randomly assigned to sedentary conditions or 6 wk of graduated swim training, creating four experimental groups: infarct sedentary (IS), infarct exercise (IE), sham sedentary (SS), and sham exercise (SE). At 11 wk all rats were sacrificed and analyzed. Compared to sedentary infarct controls, exercise training attenuated left ventricular (LV) dilation and allowed more hypertrophy of the non infarct wall. The exercise-trained hearts also showed a reduction in the estimated peak wall tension. Northern blot analysis showed an increase in beta-myosin heavy chain expression in the hearts of the sedentary infarction group soon after infarction when compared to sham controls. However, with exercise training, there was a significant attenuation of the beta-myosin heavy chain expression in the myocardium. Exercise training in a model of left ventricular dysfunction after healed myocardial infarction can improve the adverse remodeling process by attenuating ventricular dilation and reducing wall tension. The abnormal beta-myosin expression was also attenuated in the exercise trained group. This is evidence that abnormal gene expression following severe myocardial infarction dysfunction can be favorably modified by an intervention.

Myocardial fatty acid metabolism may be impaired in adriamycin cardiomyopathy. In order to determine the extent of fatty acid metabolism alterations, we measured steady state [14C]palmitate oxidation and the incorporation of [14C]palmitate into the neutral lipid pool in a rat model of adriamycin cardiomyopathy. Isolated hearts from control rats and rats treated with adriamycin were perfused with 1.2 mmol/l of [14C]palmitate for 30 min to achieve steady state oxidation measured as [14C]O2 production: then perfused with 1.2 mmol/l of unlabelled palmitate. Hearts were killed early (0-5 min) or late (10-30 min) after the [14C]palmitate perfusion, to determine incorporation into the neutral lipid pool, and neutral lipid pool utilization. In the control group steady state oxidation was reached in 10 min ([14C]O2 production = 580 ± 61 nmol/min/g dry wt) of perfusion. In the adriamycin treated group, mean CO2 production was significantly reduced at 10 min (329 ± 44 nmol/min/g dry wt, P < 0.01 v control). At 30 min, [14C]O2 production in the treated group was not significantly different than controls (521 ± 65 nmol/min/g dry wt v 617 ± 36 nmol/min/g dry wt, P = N.S.). The incorporation of [14C]palmitate into the neutral lipid pool measured in the early subgroup was significantly reduced for adriamycin treated hearts v controls (7.2 ± 0.6 v 12.0 ± 1.4 μmol/g dry wt respectively, P < 0.01). In the control group 14C labelled neutral lipid reduced with time to 8.4 ± 1.1 μmol/g dry wt (P < 0.05) in the late group. The adriamycin group demonstrated no significant change between early and late measurements. In conclusion, in adriamycin cardiomyopathy: (1) there is significant delay in achieving steady state palmitate oxidation, although the steady state rate is near normal; (2) palmitate incorporation into the neutral lipid pool is reduced; (3) neutral lipid pool utilization may also be reduced. These data suggest impaired uptake of palmitate into the cell in adriamycin cardiomyopathy, with a relatively maintained capacity for oxidative metabolism.

To investigate the role of cell viability and metabolism on the myocardial kinetics of a new tracer, technetium-99m-methoxyisobutyl isonitrile (Tc-99m-MIBI), 250 microCi/l Tc-99m-MIBI was infused in isolated rat hearts under constant flow conditions. The hearts were studied after inducing irreversible damage by cytochrome c oxidase inhibitor sodium cyanide (n = 8) or sarcolemmal membrane detergent Triton X-100 (n = 8). The control hearts (n = 6) received no toxins. Mean Tc-99m-MIBI peak accumulation activity was significantly reduced after cyanide (51.1 +/- 44.2% of control, p less than 0.01) and Triton (13.8 +/- 2.7% of control, p less than 0.001) administration. Kinetic studies also showed marked reduction in accumulation rates and marked increase in clearance rates for cyanide (p less than 0.01) and Triton (p less than 0.01) groups compared with controls. Potential changes in regional flow distribution were assessed using microspheres. When peak accumulation activity was corrected for these changes, there remained significant differences between the groups. In the cyanide and Triton groups, irreversible cell injury was confirmed by creatine kinase and lactate dehydrogenase release, triphenyl tetrazolium chloride staining, and electron microscopy. All the cells were viable in the control group. We conclude that the accumulation and clearance kinetics of Tc-99m-MIBI are significantly affected by cell viability. Tc-99m-MIBI kinetics appear to be dependent on sarcolemmal integrity and to a lesser extent on aerobic metabolism.

c-Kit Receptor is Essential for Functional Cardiac Repair Post-Myocardial Infarction Bilal Ayach, Makoto Yoshimitsu, Christopher Siatskas, Paul Lee, Hilda Lim, Fayez Dawood, Manyin Chen, Jeffrey A. Medin, Peter P. Liu; Medicine, The Heart and Stroke/Richard Lewar Centre of Excellence, Toronto, ON, Canada; Ontario Cancer Institute of the University Health Network, Toronto, ON, Canada; The University of Toronto, Toronto, ON, Canada