Noritaka Yasuda

Cardiac side population cells have a potential to migrate and differentiate into cardiomyocytes in vitro and in vivo

Side population (SP) cells, which can be identified by their ability to exclude Hoechst 33342 dye, are one of the candidates for somatic stem cells. Although bone marrow SP cells are known to be long-term repopulating hematopoietic stem cells, there is little information about the characteristics of cardiac SP cells (CSPs). When cultured CSPs from neonatal rat hearts were treated with oxytocin or trichostatin A, some CSPs expressed cardiac-specific genes and proteins and showed spontaneous beating. When green fluorescent protein–positive CSPs were intravenously infused into adult rats, many more (∼12-fold) CSPs were migrated and homed in injured heart than in normal heart. CSPs in injured heart differentiated into cardiomyocytes, endothelial cells, or smooth muscle cells (4.4%, 6.7%, and 29% of total CSP-derived cells, respectively). These results suggest that CSPs are intrinsic cardiac stem cells and involved in the regeneration of diseased hearts.

Conformational switch of angiotensin II type 1 receptor underlying mechanical stress-induced activation

The angiotensin II type 1 (AT1) receptor is a G protein‐coupled receptor that has a crucial role in the development of load‐induced cardiac hypertrophy. Here, we show that cell stretch leads to activation of the AT1 receptor, which undergoes an anticlockwise rotation and a shift of transmembrane (TM) 7 into the ligand‐binding pocket. As an inverse agonist, candesartan suppressed the stretch‐induced helical movement of TM7 through the bindings of the carboxyl group of candesartan to the specific residues of the receptor. A molecular model proposes that the tight binding of candesartan to the AT1 receptor stabilizes the receptor in the inactive conformation, preventing its shift to the active conformation. Our results show that the AT1 receptor undergoes a conformational switch that couples mechanical stress‐induced activation and inverse agonist‐induced inactivation.

Excessive cardiac insulin signaling exacerbates systolic dysfunction induced by pressure overload in rodents

Although many animal studies indicate insulin has cardioprotective effects, clinical studies suggest a link between insulin resistance (hyperinsulinemia) and heart failure (HF). Here we have demonstrated that excessive cardiac insulin signaling exacerbates systolic dysfunction induced by pressure overload in rodents. Chronic pressure overload induced hepatic insulin resistance and plasma insulin level elevation. In contrast, cardiac insulin signaling was upregulated by chronic pressure overload because of mechanical stretch-induced activation of cardiomyocyte insulin receptors and upregulation of insulin receptor and Irs1 expression. Chronic pressure overload increased the mismatch between cardiomyocyte size and vascularity, thereby inducing myocardial hypoxia and cardiomyocyte death. Inhibition of hyperinsulinemia substantially improved pressure overload-induced cardiac dysfunction, improving myocardial hypoxia and decreasing cardiomyocyte death. Likewise, the cardiomyocyte-specific reduction of insulin receptor expression prevented cardiac ischemia and hypertrophy and attenuated systolic dysfunction due to pressure overload. Conversely, treatment of type 1 diabetic mice with insulin improved hyperglycemia during pressure overload, but increased myocardial ischemia and cardiomyocyte death, thereby inducing HF. Promoting angiogenesis restored the cardiac dysfunction induced by insulin treatment. We therefore suggest that the use of insulin to control hyperglycemia could be harmful in the setting of pressure overload and that modulation of insulin signaling is crucial for the treatment of HF.

Cardiac mast cells cause atrial fibrillation through PDGF-A–mediated fibrosis in pressure-overloaded mouse hearts

Atrial fibrillation (AF) is a common arrhythmia that increases the risk of stroke and heart failure. Here, we have shown that mast cells, key mediators of allergic and immune responses, are critically involved in AF pathogenesis in stressed mouse hearts. Pressure overload induced mast cell infiltration and fibrosis in the atrium and enhanced AF susceptibility following atrial burst stimulation. Both atrial fibrosis and AF inducibility were attenuated by stabilization of mast cells with cromolyn and by BM reconstitution from mast cell-deficient WBB6F1-KitW/W-v mice. When cocultured with cardiac myocytes or fibroblasts, BM-derived mouse mast cells increased platelet-derived growth factor A (PDGF-A) synthesis and promoted cell proliferation and collagen expression in cardiac fibroblasts. These changes were abolished by treatment with a neutralizing antibody specific for PDGF alpha-receptor (PDGFR-alpha). Consistent with these data, upregulation of atrial Pdgfa expression in pressure-overloaded hearts was suppressed by BM reconstitution from WBB6F1-KitW/W-v mice. Furthermore, injection of the neutralizing PDGFR-alpha-specific antibody attenuated atrial fibrosis and AF inducibility in pressure-overloaded hearts, whereas administration of homodimer of PDGF-A (PDGF-AA) promoted atrial fibrosis and enhanced AF susceptibility in normal hearts. Our results suggest a crucial role for mast cells in AF and highlight a potential application of controlling the mast cell/PDGF-A axis to achieve upstream prevention of AF in stressed hearts.

A novel mechanism of mechanical stress-induced angiotensin II type 1–receptor activation without the involvement of angiotensin II

The angiotensin II (AngII) type 1 (AT1) receptor is a seven transmembrane-spanning G-protein-coupled receptor, and the activation of AT1 receptor plays an important role in the development of load-induced cardiac hypertrophy. Locally generated AngII was believed to trigger cardiac hypertrophy by an autocrine or paracrine mechanism. However, we found that mechanical stress can activate AT1 receptor independently of AngII. Without the involvement of AngII, mechanical stress not only activates extracellular signal-regulated kinases in vitro, but also induces cardiac hypertrophy in vivo. All of these events are inhibited by candesartan as an inverse agonist for AT1 receptor. It is conceptually novel that AT1 receptor directly mediates mechanical stress-induced cellular responses, and inverse-agonist activity emerges as an important pharmacological parameter for AT1-receptor blockers that determines their efficacy in preventing organ damage in cardiovascular diseases.

PDK1 coordinates survival pathways and β-adrenergic response in the heart

The 3-phosphoinositide-dependent kinase-1 (PDK1) plays an important role in the regulation of cellular responses in multiple organs by mediating the phosphoinositide 3-kinase (PI3-K) signaling pathway through activating AGC kinases. Here we defined the role of PDK1 in controlling cardiac homeostasis. Cardiac expression of PDK1 was significantly decreased in murine models of heart failure. Tamoxifen-inducible and heart-specific disruption of Pdk1 in adult mice caused severe and lethal heart failure, which was associated with apoptotic death of cardiomyocytes and β1-adrenergic receptor (AR) down-regulation. Overexpression of Bcl-2 protein prevented cardiomyocyte apoptosis and improved cardiac function. In addition, PDK1-deficient hearts showed enhanced activity of PI3-Kγ, leading to robust β1-AR internalization by forming complex with β-AR kinase 1 (βARK1). Interference of βARK1/PI3-Kγ complex formation by transgenic overexpression of phosphoinositide kinase domain normalized β1-AR trafficking and improved cardiac function. Taken together, these results suggest that PDK1 plays a critical role in cardiac homeostasis in vivo by serving as a dual effector for cell survival and β-adrenergic response.

Mechanisms and functions of agonist-independent activation in the angiotensin II type 1 receptor.

The angiotensin II (AngII) type 1 (AT(1)) receptor is a seven-transmembrane G protein-coupled receptor, and is involved in regulating the physiological and pathological process of the cardiovascular system. Systemically and locally generated AngII has agonistic action on AT(1) receptor, but recent studies have demonstrated that AT(1) receptor inherently shows spontaneous activity even in the absence of AngII. Furthermore, mechanical stress can activate AT(1) receptor by inducing conformational switch without the involvement of AngII, and induce cardiac hypertrophy in vivo. These agonist-independent activities of AT(1) receptor can be inhibited by inverse agonists, but not by neutral antagonists. Considerable attention has been directed to molecular mechanisms and clinical implications of agonist-independent AT(1) receptor activation, and inverse agonist activity emerges as an important pharmacological parameter for AT(1) receptor blockers that will improve efficacy and expand therapeutic potentials in cardiovascular medicine.

Agonist-Independent Constitutive Activity of Angiotensin II Receptor Promotes Cardiac Remodeling in Mice

The angiotensin II (Ang II) type 1 (AT1) receptor mainly mediates the physiological and pathological actions of Ang II, but recent studies have suggested that AT1 receptor inherently shows spontaneous constitutive activity even in the absence of Ang II in culture cells. To elucidate the role of Ang II–independent AT1 receptor activation in the pathogenesis of cardiac remodeling, we generated transgenic mice overexpressing AT1 receptor under the control of &agr;-myosin heavy chain promoter in angiotensinogen-knockout background (AT1Tg-AgtKO mice). In AT1Tg-AgtKO hearts, redistributions of the G&agr;q11 subunit into cytosol and phosphorylation of extracellular signal-regulated kinases were significantly increased, compared with angiotensinogen-knockout mice hearts, suggesting that the AT1 receptor is constitutively activated independent of Ang II. As a consequence, AT1Tg-AgtKO mice showed spontaneous systolic dysfunction and chamber dilatation, accompanied by severe interstitial fibrosis. Progression of cardiac remodeling in AT1Tg-AgtKO mice was prevented by treatment with candesartan, an inverse agonist for the AT1 receptor, but not by its derivative candesartan-7H, deficient of inverse agonism attributed to a lack of the carboxyl group at the benzimidazole ring. Our results demonstrate that constitutive activity of the AT1 receptor under basal conditions contributes to the cardiac remodeling even in the absence of Ang II, when the AT1 receptor is upregulated in the heart.

Angiotensin II Type 1 Receptor Signaling Regulates Feeding Behavior through Anorexigenic Corticotropin-releasing Hormone in Hypothalamus

The activation of renin-angiotensin system contributes to the development of metabolic syndrome and diabetes as well as hypertension. However, it remains undetermined how renin-angiotensin system is implicated in feeding behavior. Here, we show that angiotensin II type 1 (AT1) receptor signaling regulates the hypothalamic neurocircuit that is involved in the control of food intake. Compared with wild-type Agtr1a+/+ mice, AT1 receptor knock-out (Agtr1a−/−) mice were hyperphagic and obese with increased adiposity on an ad libitum diet, whereas Agtr1a−/− mice were lean with decreased adiposity on a pair-fed diet. In the hypothalamus, mRNA levels of anorexigenic neuropeptide corticotropin-releasing hormone (Crh) were lower in Agtr1a−/− mice than in Agtr1a+/+ mice both on an ad libitum and pair-fed diet. Furthermore, intracerebroventricular administration of CRH suppressed food intake both in Agtr1a+/+ and Agtr1a−/− mice. In addition, the Crh gene promoter was significantly transactivated via the cAMP-responsive element by angiotensin II stimulation. These results thus demonstrate that central AT1 receptor signaling plays a homeostatic role in the regulation of food intake by maintaining gene expression of Crh in hypothalamus and suggest a therapeutic potential of central AT1 receptor blockade in feeding disorders.

Multivalent ligand-receptor interactions elicit inverse agonist activity of AT(1) receptor blockers against stretch-induced AT(1) receptor activation.

Type 1 angiotensin II (AT1) receptor has a critical role in the development of load-induced cardiac hypertrophy. Recently, we showed that mechanical stretching of cells activates the AT1 receptor without the involvement of angiotensin II (AngII) and that this AngII-independent activation is inhibited by the inverse agonistic activity of the AT1 receptor blocker (ARB), candesartan. Although the inverse agonist activity of ARBs has been studied in terms of their action on constitutively active AT1 receptors, the structure–function relationship of the inverse agonism they exert against stretch-induced AT1 receptor activation has not been fully elucidated. Assays evaluating c-fos gene expression and phosphorylated extracellular signal-regulated protein kinases (ERKs) have shown that olmesartan has strong inverse agonist activities against the constitutively active AT1 receptor and the stretch-induced activation of AT1 receptor, respectively. Ternary drug–receptor interactions, which occur between the hydroxyl group of olmesartan and Tyr113 and between the carboxyl group of olmesartan and Lys199 and His256, were essential for the potent inverse agonist action olmesartan exerts against stretch-induced ERK activation and the constitutive activity of the AT1-N111G mutant receptor. Furthermore, the inverse agonist activity olmesartan exerts against stretch-induced ERK activation requires an additional drug–receptor interaction involving the tetrazole group of olmesartan and Gln257 of the AT1 receptor. These results suggest that multivalent interactions between an inverse agonist and the AT1 receptor are required to stabilize the receptor in an inactive conformation in response to the distinct processes that lead to an AngII-independent activation of the AT1 receptor.