Fayez Dawood

L-type Ca2+ channels provide a major pathway for iron entry into cardiomyocytes in iron-overload cardiomyopathy.

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 α1-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.

Tumor Necrosis Factor-α Mediates Cardiac Remodeling and Ventricular Dysfunction After Pressure Overload State

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)-&agr; after aortic banding in the myocardium is known. In the present study, we tested our hypothesis that TNF-&agr; 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-&agr; 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-&agr; contributes to adverse left ventricular remodeling during pressure overload through regulation of cardiac repair and remodeling, leading to ventricular dysfunction.

Excessive Tumor Necrosis Factor Activation After Infarction Contributes to Susceptibility of Myocardial Rupture and Left Ventricular Dysfunction

Background—We investigated the potential contributions of tumor necrosis factor-&agr; (TNF-&agr;) 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/dtmax (68.3%/65.3%) in WT mice but a less significant decrease in ±dP/dtmax (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-&agr; 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.

Impaired Heart Contractility in Apelin Gene–Deficient Mice Associated With Aging and Pressure Overload

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.

Combination of Tumor Necrosis Factor-α Ablation and Matrix Metalloproteinase Inhibition Prevents Heart Failure After Pressure Overload in Tissue Inhibitor of Metalloproteinase-3 Knock-Out Mice

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.

Disturbed Diurnal Rhythm Alters Gene Expression and Exacerbates Cardiovascular Disease With Rescue by Resynchronization

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.

Curcumin prevents and reverses murine cardiac hypertrophy

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.

Favorable left ventricular remodeling following large myocardial infarction by exercise training. Effect on ventricular morphology and gene expression.

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.

The tyrosine kinase p56lck is essential in coxsackievirus B3-mediated heart disease.

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.

Temporal Response and Localization of Integrins β1 and β3 in the Heart After Myocardial Infarction: Regulation by Cytokines

Background—Integrins are involved in structural remodeling and tissue repair. This study aimed to elucidate the role of the &bgr;-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 &bgr;1 and &bgr;3 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 &bgr;1 and &bgr;3 gene expression and protein production were low in sham-operated hearts. After MI, the &bgr;1 and &bgr;3 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 &bgr;1A localized primarily in fibroblasts and inflammatory cells, &bgr;1D localized in myocytes, and integrin &bgr;3 was associated primarily with endothelial and smooth muscle cells in peri-infarct vessels. In cultured myocytes, there was isoform transition from the adult &bgr;1D to the fetal &bgr;1A on exposure to TNF-&agr;. This was confirmed in vivo in the peri-infarct myocytes, but the transition was voided in TNF−/−-knockout mice. Conclusions—Integrins &bgr;1 and &bgr;3 are significantly activated in the infarcted myocardium. Integrin &bgr;1 is active particularly at sites of inflammation and fibrosis, whereas integrin &bgr;3 localizes to vessels in the peri-infarct zone in a temporally coordinated manner. Integrin &bgr;1D to &bgr;1A isoform transition in myocytes is regulated by TNF-&agr;.