Activation and accumulation of cardiac fibroblasts, which result in excessive extracellular matrix deposition and consequent mechanical stiffness, myocyte uncoupling, and ischemia, are key contributors to heart failure progression. Recently, endothelial-to-mesenchymal transition (EndoMT) and the recruitment of circulating hematopoietic progenitors to the heart have been reported to generate substantial numbers of cardiac fibroblasts in response to pressure overload-induced injury; therefore, these processes are widely considered to be promising therapeutic targets. Here, using multiple independent murine Cre lines and a collagen1a1-GFP fusion reporter, which specifically labels fibroblasts, we found that following pressure overload, fibroblasts were not derived from hematopoietic cells, EndoMT, or epicardial epithelial-to-mesenchymal transition. Instead, pressure overload promoted comparable proliferation and activation of two resident fibroblast lineages, including a previously described epicardial population and a population of endothelial origin. Together, these data present a paradigm for the origins of cardiac fibroblasts during development and in fibrosis. Furthermore, these data indicate that therapeutic strategies for reducing pathogenic cardiac fibroblasts should shift from targeting presumptive EndoMT or infiltrating hematopoietically derived fibroblasts, toward common pathways upregulated in two endogenous fibroblast populations.

Abstract-Integrins link the extracellular matrix to the cellular cytoskeleton and serve important roles in cell growth, differentiation, migration, and survival. Ablation of ␤1 integrin in all murine tissues results in peri-implantation embryonic lethality. To investigate the role of ␤1 integrin in the myocardium, we used Cre-LoxP technology to inactivate the ␤1 integrin gene exclusively in ventricular cardiac myocytes. Animals with homozygous ventricular myocyte ␤1 integrin gene excision were born in appropriate numbers and grew into adulthood. These animals had 18% of control levels of ␤1D integrin protein in the heart and displayed myocardial fibrosis. High-fidelity micromanometer-tipped catheterization of the intact 5-week-old ␤1 integrin knockout mice showed depressed left ventricular basal and dobutamine-stimulated contractility and relaxation (LV dP/dt max and LV dP/dt min ) as compared with control groups (nϭ8 to 10 of each, PϽ0.01). Hemodynamic loading imposed by 7 days of transverse aortic constriction showed that the ␤1 integrin knockout mice were intolerant of this stress as they had 53% survival versus 88% in controls (nϭ15 each). Key Words: extracellular matrix Ⅲ homologous recombination Ⅲ Cre recombinase Ⅲ heart Ⅲ positron emission tomography I ntegrins are a large family of heterodimeric cell surface receptors composed of ␣ and ␤ subunits. They function in cell-extracellular matrix (ECM) adhesion and cell-cell adhesion, and signal bidirectionally across the cell membrane. 1,2 Further, they serve as mechanotransducers, converting mechanical signals to biochemical ones. 3 This combination of properties allows integrins to play important roles in cell growth, differentiation, migration, and survival 4 and also makes them attractive candidates for essential roles in the developing and postnatal heart.Our previous work has shown that ␤1 integrins are linked to the hypertrophic response of cultured ventricular myocytes and also that dominant-negative disruption of integrin function in transgenic mice resulted in cardiac fibrosis and abnormal cardiac function. 5-7 Ablation of ␤1 integrin expression in all murine tissues resulted in gastrulation defects and death by E5.5 of the 21-day gestation period. 8,9 Chimeric mice as well as embryoid bodies constructed from ␤1 integrin-null cells showed delayed development and differentiation of ␤1-deficient cells along the cardiac lineage, as well as abnormal sarcomerogenesis of these cardiac-like cells. 10 Although a few ␤1 integrin-null cells were detected in the myocardium of chimeric mice, cellular debris was always detected along with the null cells. These null cells were completely lost from the myocardium of the chimeric mouse heart by 6 months of age.To more specifically evaluate the role of ␤1 integrin in the myocardium, we used a Cre-loxP gene targeting approach. Cre recombinase expression driven by the myosin light chain-2 ventricular (MLC-2v) promoter caused ␤1 integrin gene excision exclusively in ventricular cardiac myocytes. 11 Our results in these ...

Mechanistic target of rapamycin (MTOR) plays a critical role in the regulation of cell growth and in the response to energy state changes. Drugs inhibiting MTOR are increasingly used in antineoplastic therapies. Myocardial MTOR activity changes during hypertrophy and heart failure (HF). However, whether MTOR exerts a positive or a negative effect on myocardial function remains to be fully elucidated. Here, we show that ablation of Mtor in the adult mouse myocardium results in a fatal, dilated cardiomyopathy that is characterized by apoptosis, autophagy, altered mitochondrial structure, and accumulation of eukaryotic translation initiation factor 4E-binding protein 1 (4E-BP1). 4E-BP1 is an MTOR-containing multiprotein complex-1 (MTORC1) substrate that inhibits translation initiation. When subjected to pressure overload, Mtor-ablated mice demonstrated an impaired hypertrophic response and accelerated HF progression. When the gene encoding 4E-BP1 was ablated together with Mtor, marked improvements were observed in apoptosis, heart function, and survival. Our results demonstrate a role for the MTORC1 signaling network in the myocardial response to stress. In particular, they highlight the role of 4E-BP1 in regulating cardiomyocyte viability and in HF. Because the effects of reduced MTOR activity were mediated through increased 4E-BP1 inhibitory activity, blunting this mechanism may represent a novel therapeutic strategy for improving cardiac function in clinical HF.

The prototypic second messenger cyclic AMP (cAMP) is essential for controlling cellular metabolism, including glucose and lipid homeostasis. In mammals, the majority of cAMP functions are mediated by cAMP-dependent protein kinase (PKA) and exchange proteins directly activated by cAMP (Epacs). To explore the physiological functions of Epac1, we generated Epac1 knockout mice. Here we report that Epac1 null mutants have reduced white adipose tissue and reduced plasma leptin levels but display heightened leptin sensitivity. Epac1-deficient mice are more resistant to high-fat diet-induced obesity, hyperleptinemia, and glucose intolerance. Furthermore, pharmacological inhibition of Epac by use of an Epac-specific inhibitor reduces plasma leptin levels in vivo and enhances leptin signaling in organotypic hypothalamic slices. Taken together, our results demonstrate that Epac1 plays an important role in regulating adiposity and energy balance. Obesity is a grave health problem, as it is closely related to the leading causes of morbidity and mortality, such as cardiovascular diseases, type 2 diabetes, hypertension, depression, and cancer (1). Over the last 2 decades, obesity has reached epidemic proportions in the United States: more than 35% of adults in the United States are obese, and more than two-thirds are overweight (2). Furthermore, 500 million people worldwide are obese, representing approximately 12% of the adult population on earth (3). Chronic excessive food/energy intake, mediated by leptin resistance, is a major factor contributing to obesity. To date, few effective treatment options are available for obesity (4). Therefore, a better understanding of the underlying molecular mechanisms of obesity development and effective, safe therapeutic interventions are urgently needed. Cyclic AMP (cAMP)-mediated signaling pathways are important for maintaining metabolic homeostasis and have been implicated in regulating leptin production and secretion (5-7). In mammals, the majority of cAMP functions are mediated by cAMP-dependent protein kinase (PKA) and exchange proteins directly activated by cAMP (Epacs) (8-10). A recent study revealed that activation of Epacs by an Epac-selective cAMP analog, 8-CPT-2=-O-Me-cAMP (11), interferes with leptin signaling in the hypothalamus, suggesting that Epacs may contribute to the pathophysiology of leptin resistance and represent a novel pharmacological target for treatment of obesity (12). To investigate the functional significance of Epac1 in leptin-mediated energy balance in vivo, we generated global loss-of-function mutants for Epac1. Analysis of these animals indicated resistance to high-fat diet (HFD)-induced obesity, heightened leptin signaling in the arcuate nucleus (AN), and improved glucose tolerance. These findings reveal an important role of Epac1 in metabolism and suggest that Epac1 may represent a novel therapeutic target for obesity. MATERIALS AND METHODS Mice.To construct an Epac1 targeting vector, two loxP sites were inserted into introns 2 and 5. A 3.8-kb upstream ...

Focal adhesion kinase (FAK) is a nonreceptor tyrosine kinase that plays an important role in integrin-mediated signal transduction.To explore the role and mechanisms of FAK in cardiac development, we inactivated FAK in embryonic cardiomyocytes by crossing the floxed FAK mice with myosin light chain-2a (MLC2a) Cre mice, which expressed Cre as early as embryonic day 9.5 in the heart. The majority of conditional FAK knockout mice generated from MLC2a-Cre (CFKO-2a) died in the embryonic stage with thin ventricular wall and ventricular septal defects. A small fraction of CFKO-2a mice survived to adulthood with spontaneous eccentric right ventricle hypertrophy. Transmission electron microscopy analysis displayed swelling in the rough endoplasmic reticulum in CFKO-2a embryonic cardiomyocytes. We found that decreased cell proliferation, but not increased cell apoptosis or differentiation, is the reason for the thin ventricular wall in CFKO-2a mice. Microarray analysis suggests that myocyte enhancer factor 2a (MEF2a) can be regulated by FAK and that inactivation of FAK in the embryonic heart compromised MEF2a expression. Last, we found that Src, but not PI3K, is important in mediating signal transduction for the regulation of MEF2a by FAK. Together, these results identified the role and mechanisms of FAK in embryonic cardiac development.cardiac development ͉ signal transduction T he heart is the first formed organ to begin functioning in a developing vertebrate embryo. Abnormalities of heart development induce congenital heart diseases, which are the most frequent form of birth defects in humans. Heart development and maturation depend on the interactions of integrins in cardiomyocyte with its surrounding extracellular matrix (ECM) (1). Integrins, which are cell surface receptors that mediate cellular adhesion to the ECM, are composed of noncovalently linked ␣-and ␤-subunits. ␤1 integrin knockout mice resulted in gastrulation defects and death by embryonic day 5.5 (E5.5) (2, 3). Chimeric mice constructed with a ␤1 integrin-null allele showed delayed development and differentiation of cardiac lineage, as well as abnormal sarcomergenesis of these cardiaclike cells (4). When ␤1 integrin was inactivated exclusively in ventricular cardiac myocytes, it resulted in myocardial fibrosis and cardiac failure (5). It is well established that focal adhesion kinase (FAK) is a key downstream kinase in the ECM-integrin signal transduction pathway (6-9). Therefore, these data suggest that FAK may play essential roles in heart development.Previous studies have shown that FAK gene inactivation in mice resulted in an embryonic lethal phenotype with major defects in the axial mesodermal tissues and cardiovascular system. Neither a normal heart nor fully developed blood vessels were present in the FAK-null embryos (10, 11). Despite the abundant knowledge of FAK interaction with other proteins and its roles in cell signaling in vitro, still relatively little is known about the in vivo functions of FAK in embryonic development or in the adult o...