Overexpressing superoxide dismutase 2 induces a supernormal cardiac function by enhancing redox-dependent mitochondrial function and metabolic dilation.

During heightened cardiac work, O2 consumption by the heart benefits energy production via mitochondria. However, some electrons leak from the respiratory chain and yield superoxide, which is rapidly metabolized into H2O2 by SOD2. To understand the systemic effects of the metabolic dilator, H2O2, we studied mice with cardiac-specific SOD2 overexpression (SOD2-tg), which increases the H2O2 produced by cardiac mitochondria. Contrast echocardiography was employed to evaluate cardiac function, indicating that SOD2-tg had a significantly greater ejection fraction and a lower mean arterial pressure (MAP) that was partially normalized by intravenous injection of catalase. Norepinephrine-mediated myocardial blood flow (MBF) was significantly enhanced in SOD2-tg mice. Coupling of MBF to the double product (Heart Rate×MAP) was increased in SOD2-tg mice, indicating that the metabolic dilator, "spilled" over, inducing systemic vasodilation. The hypothesis that SOD2 overexpression effectively enhances mitochondrial function was further evaluated. Mitochondria of SOD2-tg mice had a decreased state 3 oxygen consumption rate, but maintained the same ATP production flux under the basal and L-NAME treatment conditions, indicating a higher bioenergetic efficiency. SOD2-tg mitochondria produced less superoxide, and had lower redox activity in converting cyclic hydroxylamine to stable nitroxide, and a lower GSSG concentration. EPR analysis of the isolated mitochondria showed a significant decrease in semiquinones at the SOD2-tg Qi site. These results support a more reductive physiological setting in the SOD2-tg murine heart. Cardiac mitochondria exhibited no significant differences in the respiratory control index between WT and SOD2-tg. We conclude that SOD2 overexpression in myocytes enhances mitochondrial function and metabolic vasodilation, leading to a phenotype of supernormal cardiac function.

Hyperglycemia enhances function and differentiation of adult rat cardiac fibroblasts.

Diabetes is an independent risk factor for cardiovascular disease that can eventually cause cardiomyopathy and heart failure. Cardiac fibroblasts (CF) are the critical mediators of physiological and pathological cardiac remodeling; however, the effects of hyperglycemia on cardiac fibroblast function and differentiation is not well known. Here, we performed a comprehensive investigation on the effects of hyperglycemia on cardiac fibroblasts and show that hyperglycemia enhances cardiac fibroblast function and differentiation. We found that high glucose treatment increased collagen I, III, and VI gene expression in rat adult cardiac fibroblasts. Interestingly, hyperglycemia increased CF migration and proliferation that is augmented by collagen I and III. Surprisingly, we found that short term hyperglycemia transiently inhibited ERK1/2 activation but increased AKT phosphorylation. Finally, high glucose treatment increased spontaneous differentiation of cardiac fibroblasts to myofibroblasts with increasing passage compared with low glucose. Taken together, these findings suggest that hyperglycemia induces cardiac fibrosis by modulating collagen expression, migration, proliferation, and differentiation of cardiac fibroblasts.

Injury models to study cardiac remodeling in the mouse: myocardial infarction and ischemia-reperfusion.

Deep tissue wound healing requires a complex sequence of several factors working in unison to repair the organ at risk. Myocardial infarction (MI) is particularly complex due to several local and systemic factors mediating the repair process within the heart. The wound healing process during this time is critical-the cardiac myocytes are at risk of apoptotic cell death, autophagy, and necrosis. During the early remodeling period, the fibroblasts and myofibroblasts play critical roles in infarct scar formation, a process that is greatly influenced by a robust inflammatory response. Construction of the infarct scar is a "necessary evil" that helps to limit expansion of the infarction; however, the collagen and matrix deposition will often spread to the healthy areas of the heart, causing reactive fibrosis in areas remote from the original damage. This chapter outlines in detail the procedures for two myocardial infarction injury models as well as how to quantify the size of the experimentally induced injury. These procedures are critical to the development of in vivo approaches to study myocardial injury, particularly for use in knockout and transgenic mice.

TRPV4 channels mediate cardiac fibroblast differentiation by integrating mechanical and soluble signals.

The phenotypic switch underlying the differentiation of cardiac fibroblasts into hypersecretory myofibroblasts is critical for cardiac remodeling following myocardial infarction. Myofibroblasts facilitate wound repair in the myocardium by secreting and organizing extracellular matrix (ECM) during the wound healing process. However, the molecular mechanisms involved in myofibroblast differentiation are not well known. TGF-ß has been shown to promote differentiation and this, combined with the robust mechanical environment in the heart, lead us to hypothesize that the mechanotransduction and TGF-ß signaling pathways play active roles in the differentiation of cardiac fibroblasts to myofibroblasts. Here, we show that the mechanosensitve ion channel TRPV4 is required for TGF-ß1-induced differentiation of cardiac fibroblasts into myofibroblasts. We found that the TRPV4-specific antagonist AB159908 and siRNA knockdown of TRPV4 significantly inhibited TGFß1-induced differentiation as measured by incorporation of α-SMA into stress fibers. Further, we found that TGF-ß1-induced myofibroblast differentiation was dependent on ECM stiffness, a response that was attenuated by TRPV4 blockade. Finally, TGF-ß1 treated fibroblasts exhibited enhanced TRPV4 expression and TRPV4-mediated calcium influx compared to untreated controls. Taken together these results suggest for the first time that the mechanosensitive ion channel, TRPV4, regulates cardiac fibroblast differentiation to myofibroblasts by integrating signals from TGF-ß1 and mechanical factors.

Absence of type VI collagen paradoxically improves cardiac function, structure, and remodeling after myocardial infarction.

RATIONALE: We previously reported that type VI collagen deposition increases in the infarcted myocardium in vivo. To date, a specific role for this nonfibrillar collagen has not been explored in the setting of myocardial infarction (MI). OBJECTIVE: To determine whether deletion of type VI collagen in an in vivo model of post-MI wound healing would alter cardiac function and remodeling in the days to weeks after injury. METHODS AND RESULTS: Wild-type and Col6a1(-/-) mice were subjected to MI, followed by serial echocardiographic and histological assessments. At 8 weeks after MI, infarct size was significantly reduced, ejection fraction was significantly preserved (43.9% ± 3.3% versus 29.1% ± 4.3% for wild-type), and left ventricular chamber dilation was attenuated in the Col6a1(-/-) MI group (25.8% ± 7.9% increase versus 62.6% ± 16.5% for wild-type). The improvement in cardiac remodeling was evident as early as 10 days after MI in the Col6a1(-/-) mice. Myocyte apoptosis within the infarcted zones was initially greater in the Col6a1(-/-) group 3 days after MI, but by day 14 this was significantly reduced. Collagen deposition also was reduced in the infarcted and remote areas of the Col6a1(-/-) hearts. The reductions in chronic myocyte apoptosis and fibrosis are critical events leading to improved long-term remodeling and functional outcomes. CONCLUSIONS: These unexpected results demonstrate for the first time that deletion of type VI collagen in this knockout model plays a critical protective role after MI by limiting infarct size, chronic apoptosis, aberrant remodeling, and fibrosis, leading to preservation of cardiac function.

Chemopreventive doses of resveratrol do not produce cardiotoxicity in a rodent model of hepatocellular carcinoma.

Hepatocellular carcinoma (HCC), one of the most lethal cancers, results in more than one million fatalities worldwide every year. In view of the limited therapeutic alternatives and poor prognosis of liver cancer, preventive control approaches, notably chemoprevention, have been considered to be the best strategy in lowering the present prevalence of the disease. Resveratrol, a naturally occurring antioxidant and antiinflammatory agent found in grapes and red wine, inhibits carcinogenesis with a pleiotropic mode of action. Recently, we have reported that dietary resveratrol significantly prevents chemically-induced liver tumorigenesis in rats. One of the mechanisms of resveratrol-mediated chemoprevention of hepatocarcinogenesis could be related to its antiinflammatory action through hepatic cyclooxygenase (COX-2) inhibition. Although several COX-2 inhibitors are known to exert chemopreventive efficacy, not all are considered ideal candidates for chemoprevention due to the risk of adverse cardiovascular events. Accordingly, the objective of the present study was to evaluate the role of resveratrol on cardiac performance during experimental hepatocarcinogenesis initiated with diethylnitrosamine and promoted by phenobarbital. Rats had free access to diet supplemented with resveratrol four weeks before the carcinogen injection and 14 weeks thereafter. The cardiotoxicity of resveratrol was assessed by monitoring the cardiac function using transthoracic echocardiography as well as Western blot analysis of cardiac tissue. Long-term dietary administration of resveratrol dose-dependently suppressed hepatic tumor multiplicity, the principal endpoint for evaluating the chemopreventive potential of a candidate agent. The chemopreventive effects of resveratrol were also reflected in histopathological assessment of hepatic tissues. Resveratrol did not exhibit any cardiotoxicity but rather improved the cardiac function in a dose-responsive fashion. Our results indicate that resveratrol-mediated chemoprevention of rat liver carcinogenesis is devoid of any adverse cardiovascular events. Resveratrol may be developed as a chemopreventive as well as therapeutic drug for human HCC.

Impact of type 1 diabetes on cardiac fibroblast activation: enhanced cell cycle progression and reduced myofibroblast content in diabetic myocardium.

Diabetic patients are prone to developing myocardial fibrosis and suffer from decreased wound healing capabilities. The purpose of this study was to determine whether diabetes alters cardiac fibroblast activity in the myocardium in a 6-wk streptozotocin-induced type 1 diabetic model. In vivo echocardiography indicated significant dilation of the left ventricle (LV) in the diabetic animals, while cardiac function was comparable to that in the normal group. We isolated cardiac fibroblasts from diabetic and control hearts and observed increased proliferation of the diabetic fibroblasts. Microarray analysis using mRNA collected from whole LVs revealed downregulation of known inhibitors of proliferation, p53 and p21, in the diabetic group, consistent with our proliferation data. Western blot analysis confirmed a reduction in p53 protein expression in the diabetic hearts compared with control. We explored the potential signaling underlying the downregulation of these cell cycle mediators and determined that activated Akt, a signal that inhibits p53, was elevated in the diabetic group. Surprisingly, the hearts from the diabetic group contained lower levels of the myofibroblast marker α-smooth muscle actin (α-SMA) and higher levels of desmin and platelet endothelial cell adhesion molecule (PECAM). The isolated fibroblasts from the diabetic group also contained significantly less α-SMA. These data suggest that early-stage diabetic hearts contain highly proliferative fibroblasts, which predisposes the diabetic myocardium to fibrosis, but have fewer myofibroblasts, which may compromise wound healing.

Beta-adrenoceptor stimulation of alveolar fluid clearance is increased in rats with heart failure.

The alveolar epithelium plays a critical role in resolving pulmonary edema. We thus hypothesized that its function might be upregulated in rats with heart failure, a condition that severely challenges the lung's ability to maintain fluid balance. Heart failure was induced by left coronary artery ligation. Echocardiographic and cardiovascular hemodynamics confirmed its development at 16 wk postligation. At that time, alveolar fluid clearance was measured by an increase in protein concentration over 1 h of a 5% albumin solution instilled into the lungs. Baseline alveolar fluid clearance was similar in heart failure and age-matched control rats. Terbutaline was added to the instillate to determine whether heart failure rats responded to beta-adrenoceptor stimulation. Alveolar fluid clearance in heart failure rats was increased by 194% after terbutaline stimulation compared with a 153% increase by terbutaline in control rats. To determine the mechanisms responsible for this accelerated alveolar fluid clearance, we measured ion transporter expression (ENaC, Na-K- ATPase, CFTR). No significant upregulation was observed for these ion transporters in the heart failure rats. Lung morphology showed significant alveolar epithelial type II cell hyperplasia in heart failure rats. Thus, alveolar epithelial type II cell hyperplasia is the likely explanation for the increased terbutaline-stimulated alveolar fluid clearance in heart failure rats. These data provide evidence for previously unrecognized mechanisms that can protect against or hasten resolution of alveolar edema in heart failure.

Cardiac myofibroblast differentiation is attenuated by alpha(3) integrin blockade: potential role in post-MI remodeling.

Cardiac fibroblasts and myofibroblasts are responsible for post-MI remodeling which occurs via regulation of extracellular matrix (ECM). Accelerated post-MI remodeling leads to excessive ECM deposition and fibrosis, contributing to impaired contractile function, arrhythmias, and heart failure. We have previously reported that type VI collagen induces myofibroblast differentiation in cultured cardiac fibroblasts, and that type VI collagen and myofibroblast content were both elevated in the myocardium 20 weeks post-MI. The purpose of this study was to determine the expression patterns of type VI collagen and myofibroblast content in early post-myocardial infarction (MI) remodeling to gain insight into whether type VI collagen induces in vivo myofibroblast differentiation via specific matrix-receptor interactions. Adult male Sprague-Dawley rats were anesthetized and left coronary arteries were permanently ligated. Histological tissue sections and whole tissue protein lysates were obtained from infarcted and non-infarcted areas of MI hearts and sham operated controls. At 3 days post-MI, we observed a significant increase in alpha(3) integrin expression (2.02+/-0.18 fold); at 7 days post-infarction both type VI collagen (2.27+/-0.18 fold) and myofibroblast (4.65+/-0.6 fold) content increased. By 14 days myofibroblast content returned to sham control levels, although type VI collagen (2.42+/-0.11 fold) was still elevated. In vitro cross-linking confirmed that the alpha(3) integrin interacts with type VI collagen, and alpha(3) integrin function blocking antibodies inhibited the differentiation of isolated cardiac fibroblasts. Collectively, our in vitro results indicate that the alpha(3) integrin receptor interacts with type VI collagen to promote myofibroblast differentiation, and that this interaction may impact in vivo post-MI remodeling.