Yunzhe Bai

Disruption of Type 5 Adenylyl Cyclase Enhances Desensitization of Cyclic Adenosine Monophosphate Signal and Increases Akt Signal With Chronic Catecholamine Stress

Background— Desensitization of the cyclic adenosine monophosphate signal protects cardiac myocytes against catecholamine stress, thus preventing the development of apoptosis. Molecular mechanisms of desensitization have been well studied at the level of adrenergic receptors but less so at the level of the effector enzyme, adenylyl cyclase (AC). Methods and Results— When the effects of long-term (1 to 2 weeks) isoproterenol infusion were compared between type 5 AC-null mice (AC5KO) and wild-type controls, we found that the subsequent responses of left ventricular ejection fraction to sudden intravenous isoproterenol challenge were reduced in AC5KO compared with wild-type mice (ie, physiological desensitization was more effective in AC5KO), consistent with enhanced downregulation of AC catalytic activity in AC5KO. One mechanism for the less effective desensitization in wild-type mice was paradoxical upregulation of type 5 AC protein expression. The number of apoptotic myocytes was similar at baseline but was significantly less in AC5KO after infusion. This was accompanied by a 4-fold greater increase in Bcl-2 and a 3-fold greater increase in phospho-Akt in AC5KO. The latter is most likely mediated by increased membrane localization of phosphoinositide-dependent protein kinase 1, which is known to be inhibited by the cyclic adenosine monophosphate signal. Conclusions— The absence of type 5 AC results in more effective desensitization after long-term catecholamine stress and protects against the development of myocyte apoptosis and deterioration of cardiac function, potentially elucidating a novel approach to the therapy of heart failure.

Epac1-dependent phospholamban phosphorylation mediates the cardiac response to stresses

PKA phosphorylates multiple molecules involved in calcium (Ca2+) handling in cardiac myocytes and is considered to be the predominant regulator of β-adrenergic receptor-mediated enhancement of cardiac contractility; however, recent identification of exchange protein activated by cAMP (EPAC), which is independently activated by cAMP, has challenged this paradigm. Mice lacking Epac1 (Epac1 KO) exhibited decreased cardiac contractility with reduced phospholamban (PLN) phosphorylation at serine-16, the major PKA-mediated phosphorylation site. In Epac1 KO mice, intracellular Ca2+ storage and the magnitude of Ca2+ movement were decreased; however, PKA expression remained unchanged, and activation of PKA with isoproterenol improved cardiac contractility. In contrast, direct activation of EPAC in cardiomyocytes led to increased PLN phosphorylation at serine-16, which was dependent on PLC and PKCε. Importantly, Epac1 deletion protected the heart from various stresses, while Epac2 deletion was not protective. Compared with WT mice, aortic banding induced a similar degree of cardiac hypertrophy in Epac1 KO; however, lack of Epac1 prevented subsequent cardiac dysfunction as a result of decreased cardiac myocyte apoptosis and fibrosis. Similarly, Epac1 KO animals showed resistance to isoproterenol- and aging-induced cardiomyopathy and attenuation of arrhythmogenic activity. These data support Epac1 as an important regulator of PKA-independent PLN phosphorylation and indicate that Epac1 regulates cardiac responsiveness to various stresses.

Cardiac-Specific Activation of Angiotensin II Type 1 Receptor–Associated Protein Completely Suppresses Cardiac Hypertrophy in Chronic Angiotensin II–Infused Mice

We cloned a novel molecule interacting with angiotensin II type 1 receptor, which we named ATRAP (for angiotensin II type 1 receptor–associated protein). Previous in vitro studies showed that ATRAP significantly promotes constitutive internalization of the angiotensin II type 1 receptor and further attenuates angiotensin II–mediated hypertrophic responses in cardiomyocytes. The present study was designed to investigate the putative functional role of ATRAP in cardiac hypertrophy by angiotensin II infusion in vivo. We first examined the effect of angiotensin II infusion on endogenous ATRAP expression in the heart of C57BL/6J wild-type mice. The angiotensin II treatment promoted cardiac hypertrophy, concomitant with a significant decrease in cardiac ATRAP expression, but without significant change in cardiac angiotensin II type 1 receptor expression. We hypothesized that a downregulation of the cardiac ATRAP to angiotensin II type 1 receptor ratio is involved in the pathogenesis of cardiac hypertrophy. To examine this hypothesis, we next generated transgenic mice expressing ATRAP specifically in cardiomyocytes under control of the &agr;-myosin heavy chain promoter. In cardiac-specific ATRAP transgenic mice, the development of cardiac hypertrophy, activation of p38 mitogen-activated protein kinase, and expression of hypertrophy-related genes in the context of angiotensin II treatment were completely suppressed, in spite of there being no significant difference in blood pressure on radiotelemetry between the transgenic mice and littermate control mice. These results demonstrate that cardiomyocyte-specific overexpression of ATRAP in vivo abolishes the cardiac hypertrophy provoked by chronic angiotensin II infusion, thereby suggesting ATRAP to be a novel therapeutic target in cardiac hypertrophy.

Intrarenal suppression of angiotensin II type 1 receptor binding molecule in angiotensin II-infused mice

ATRAP [ANG II type 1 receptor (AT1R)-associated protein] is a molecule which directly interacts with AT1R and inhibits AT1R signaling. The aim of this study was to examine the effects of continuous ANG II infusion on the intrarenal expression and distribution of ATRAP and to determine the role of AT1R signaling in mediating these effects. C57BL/6 male mice were subjected to vehicle or ANG II infusions at doses of 200, 1,000, or 2,500 ng·kg(-1)·min(-1) for 14 days. ANG II infusion caused significant suppression of ATRAP expression in the kidney but did not affect ATRAP expression in the testis or liver. Although only the highest ANG II dose (2,500 ng·kg(-1)·min(-1)) provoked renal pathological responses, such as an increase in the mRNA expression of angiotensinogen and the α-subunit of the epithelial sodium channel, ANG II-induced decreases in ATRAP were observed even at the lowest dose (200 ng·kg(-1)·min(-1)), particularly in the outer medulla of the kidney, based on immunohistochemical staining and Western blot analysis. The decrease in renal ATRAP expression by ANG II infusion was prevented by treatment with the AT1R-specific blocker olmesartan. In addition, the ANG II-mediated decrease in renal ATRAP expression through AT1R signaling occurred without an ANG II-induced decrease in plasma membrane AT1R expression in the kidney. On the other hand, a transgenic model increase in renal ATRAP expression beyond baseline was accompanied by a constitutive reduction of renal plasma membrane AT1R expression and by the promotion of renal AT1R internalization as well as the decreased induction of angiotensinogen gene expression in response to ANG II. These results suggest that the plasma membrane AT1R level in the kidney is modulated by intrarenal ATRAP expression under physiological and pathophysiological conditions in vivo.

Identification of Transcription Factor E3 (TFE3) as a Receptor-independent Activator of Gα16 GENE REGULATION BY NUCLEAR Gα SUBUNIT AND ITS ACTIVATOR

Receptor-independent G-protein regulators provide diverse mechanisms for signal input to G-protein-based signaling systems, revealing unexpected functional roles for G-proteins. As part of a broader effort to identify disease-specific regulators for heterotrimeric G-proteins, we screened for such proteins in cardiac hypertrophy using a yeast-based functional screen of mammalian cDNAs as a discovery platform. We report the identification of three transcription factors belonging to the same family, transcription factor E3 (TFE3), microphthalmia-associated transcription factor, and transcription factor EB, as novel receptor-independent activators of G-protein signaling selective for Gα16. TFE3 and Gα16 were both up-regulated in cardiac hypertrophy initiated by transverse aortic constriction. In protein interaction studies in vitro, TFE3 formed a complex with Gα16 but not with Gαi3 or Gαs. Although increased expression of TFE3 in heterologous systems had no influence on receptor-mediated Gα16 signaling at the plasma membrane, TFE3 actually translocated Gα16 to the nucleus, leading to the induction of claudin 14 expression, a key component of membrane structure in cardiomyocytes. The induction of claudin 14 was dependent on both the accumulation and activation of Gα16 by TFE3 in the nucleus. These findings indicate that TFE3 and Gα16 are up-regulated under pathologic conditions and are involved in a novel mechanism of transcriptional regulation via the relocalization and activation of Gα16.

Sarcalumenin is essential for maintaining cardiac function during endurance exercise training

Sarcalumenin (SAR), a Ca(2+)-binding protein located in the longitudinal sarcoplasmic reticulum (SR), regulates Ca(2+) reuptake into the SR by interacting with cardiac sarco(endo)plasmic reticulum Ca(2+)-ATPase 2a (SERCA2a). We have previously demonstrated that SAR deficiency induced progressive heart failure in response to pressure overload, despite mild cardiac dysfunction in sham-operated SAR knockout (SARKO) mice (26). Since responses to physiological stresses often differ from those to pathological stresses, we examined the effects of endurance exercise on cardiac function in SARKO mice. Wild-type (WT) and SARKO mice were subjected to endurance treadmill exercise training ( approximately 65% of maximal exercise ability for 60 min/day) for 12 wk. After exercise training, maximal exercise ability was significantly increased by 5% in WT mice (n = 6), whereas it was significantly decreased by 37% in SARKO mice (n = 5). Cardiac function assessed by echocardiographic examination was significantly decreased in accordance with upregulation of biomarkers of cardiac stress in SARKO mice after training. After training, expression levels of SERCA2a protein were significantly downregulated by 30% in SARKO hearts, whereas they were significantly upregulated by 59% in WT hearts. Consequently, SERCA2 activity was significantly decreased in SARKO hearts after training. Furthermore, the expression levels of other Ca(2+)-handling proteins, including phospholamban, ryanodine receptor 2, calsequestrin 2, and sodium/calcium exchanger 1, were significantly decreased in SARKO hearts after training. These results indicate that SAR plays a critical role in maintaining cardiac function under physiological stresses, such as endurance exercise, by regulating Ca(2+) transport activity into the SR. SAR may be a primary target for exercise-related adaptation of the Ca(2+) storage system in the SR to preserve cardiac function.

Post‐transcriptional downregulation of sarcolipin mRNA by triiodothyronine in the atrial myocardium

Thyroid hormone‐mediated positive cardiotropic effects are differently regulated between the atria and ventricles. This regulation is, at least in part, dependent on sarcoplasmic reticulum (SR) proteins. Sarcolipin, a homologue of phospholamban, has been recently identified as an atrium‐specific SR protein. The expression of sarcolipin mRNA was significantly decreased in the atria of mice with hyperthyroidism and in 3,5,3′‐triiodo‐l‐thyronine‐treated neonatal rat atrial myocytes. Promoter activity and mRNA stability analyses revealed that thyroid hormone post‐transcriptionally downregulated the expression of sarcolipin mRNA. The atrium‐specific effect of thyroid hormone may occur in part through the regulation of atrial sarcolipin gene expression.

Type 5 adenylyl cyclase plays a major role in stabilizing heart rate in response to microgravity induced by parabolic flight

It is well known that autonomic nervous activity is altered under microgravity, leading to disturbed regulation of cardiac function, such as heart rate. Autonomic regulation of the heart is mostly determined by beta-adrenergic receptors/cAMP signal, which is produced by adenylyl cyclase, in cardiac myocytes. To examine a hypothesis that a major cardiac isoform, type 5 adenylyl cyclase (AC5), plays an important role in regulating heart rate during parabolic flights, we used transgenic mouse models with either disrupted (AC5KO) or overexpressed AC5 in the heart (AC5TG) and analyzed heart rate variability. Heart rate had a tendency to decrease gradually in later phases within one parabola in each genotype group, but the magnitude of decrease was smaller in AC5KO than that in the other groups. The inverse of heart rate, i.e., the R-R interval, was much more variable in AC5KO and less variable in AC5TG than that in wild-type controls. The standard deviation of normal R-R intervals, a marker of total autonomic variability, was significantly greater in microgravity phase in each genotype group, but the magnitude of increase was much greater in AC5KO than that in the other groups, suggesting that heart rate regulation became unstable in the absence of AC5. In all, AC5 plays a major role in stabilizing heat rate under microgravity.