Patricia E. Shamhart

Angiotensin II-Induced Extracellular Signal-Regulated Kinase 1/2 Activation Is Mediated by Protein Kinase Cδ and Intracellular Calcium in Adult Rat Cardiac Fibroblasts

Angiotensin II (Ang II)–induced proliferation of cardiac fibroblasts is a major contributing factor to the pathogenesis of cardiac fibrosis. Ang II activates extracellular signal–regulated kinase (ERK) 1/2 to induce cardiac fibroblast proliferation, but the signaling pathways leading to ERK 1/2 activation have not been elucidated in these cells. The goal of the current study was to identify the intracellular mediators of Ang II–induced ERK 1/2 activation in adult rat cardiac fibroblasts. We determined that 100 nmol/L of Ang II–induced ERK 1/2 phosphorylation is inhibited by simultaneous chelation of cytosolic calcium and downregulation of protein kinase C (PKC) by phorbol ester or by the specific PKC&dgr; inhibitor rottlerin, as well as PKC&dgr; small interfering RNA, but not by inhibition of 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetate, phorbol ester, rottlerin, or PKC&dgr; small interfering RNA alone. We also found that Ang II does not transactivate the epidermal growth factor receptor in adult cardiac fibroblasts, because pretreatment with 1 &mgr;mol/L of AG 1478 did not significantly inhibit [3H]-thymidine incorporation or ERK 1/2 activation. In addition, immunoprecipitation of the epidermal growth factor receptor demonstrated no significant Ang II–induced phosphorylation of tyrosine residues. Inhibition of phosphatidylinositide 3-kinase, PKC&zgr;, and src tyrosine kinase had no effect on Ang II–induced ERK 1/2 activation. Collectively, these data demonstrate that Ang II does not transactivate the epidermal growth factor receptor in adult rat cardiac fibroblasts to activate ERK 1/2, a common pathway described in vascular smooth muscle and other cell types, but rather occurs via activation of distinct parallel signaling pathways mechanistically controlled by intracellular Ca2+ and PKC&dgr;.

Non-fibrillar collagens: key mediators of post-infarction cardiac remodeling?

Cardiac remodeling is accelerated during pathological conditions and several anabolic and catabolic regulators work in concert to repair the myocardium and maintain its functionality. The fibroblasts play a major role in this process via collagen deposition as well as supplying the degradative matrix metalloproteinases. During the more acute responses to a myocardial infarction (MI) the heart relies on a more aggressive wound healing sequence that includes the myofibroblasts, specialized secretory cells necessary for infarct scar formation and thus, rescue of the myocardium. The activated fibroblasts and myofibroblasts deposit large amounts of fibrillar collagen during the post-MI wound healing phase, type I and III collagen are the most abundant collagens in the heart and they maintain the structural integrity under normal and disease states. While collagen I and III have been the traditional focus of the myocardial matrix, recent studies have suggested that the non-fibrillar collagens (types IV and VI) are also deposited during pathological wound healing and may play key roles in myofibroblast differentiation and organization of the fibrillar collagen network. This review highlights the potential roles of the non-fibrillar collagens and how they work in concert with the fibrillar collagens in mediating myocardial remodeling.

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.

Cardiac myofibroblast differentiation is attenuated by α3 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.

Early upregulation of myocardial CXCR4 expression is critical for dimethyloxalylglycine-induced cardiac improvement in acute myocardial infarction

The stromal cell-derived factor-1 (SDF-1):CXCR4 is important in myocardial repair. In this study we tested the hypothesis that early upregulation of cardiomyocyte CXCR4 (CM-CXCR4) at a time of high myocardial SDF-1 expression could be a strategy to engage the SDF-1:CXCR4 axis and improve cardiac repair. The effects of the hypoxia inducible factor (HIF) hydroxylase inhibitor dimethyloxalylglycine (DMOG) on CXCR4 expression was tested on H9c2 cells. In mice a myocardial infarction (MI) was produced in CM-CXCR4 null and wild-type controls. Mice were randomized to receive injection of DMOG (DMOG group) or saline (Saline group) into the border zone after MI. Protein and mRNA expression of CM-CXCR4 were quantified. Echocardiography was used to assess cardiac function. During hypoxia, DMOG treatment increased CXCR4 expression of H9c2 cells by 29 and 42% at 15 and 24 h, respectively. In vivo DMOG treatment increased CM-CXCR4 expression at 15 h post-MI in control mice but not in CM-CXCR4 null mice. DMOG resulted in increased ejection fraction in control mice but not in CM-CXCR4 null mice 21 days after MI. Consistent with greater cardiomyocyte survival with DMOG treatment, we observed a significant increase in cardiac myosin-positive area within the infarct zone after DMOG treatment in control mice, but no increase in CM-CXCR4 null mice. Inhibition of cardiomyocyte death in MI through the stabilization of HIF-1α requires downstream CM-CXCR4 expression. These data suggest that engagement of the SDF-1:CXCR4 axis through the early upregulation of CM-CXCR4 is a strategy for improving cardiac repair after MI.

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.

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.