Qibin Jiao

Prostaglandin E2-activated Epac Promotes Neointimal Formation of the Rat Ductus Arteriosus by a Process Distinct from That of cAMP-dependent Protein Kinase A

We have demonstrated that chronic stimulation of the prostaglandin E2-cAMP-dependent protein kinase A (PKA) signal pathway plays a critical role in intimal cushion formation in perinatal ductus arteriosus (DA) through promoting synthesis of hyaluronan. We hypothesized that Epac, a newly identified effector of cAMP, may play a role in intimal cushion formation (ICF) in the DA distinct from that of PKA. In the present study, we found that the levels of Epac1 and Epac2 mRNAs were significantly up-regulated in the rat DA during the perinatal period. A specific EP4 agonist, ONO-AE1-329, increased Rap1 activity in the presence of a PKA inhibitor, PKI-(14-22)-amide, in DA smooth muscle cells. 8-pCPT-2′-O-Me-cAMP (O-Me-cAMP), a cAMP analog selective to Epac activator, promoted migration of DA smooth muscle cells (SMC) in a dose-dependent manner. Adenovirus-mediated Epac1 or Epac2 gene transfer further enhanced O-Me-cAMP-induced cell migration, although the effect of Epac1 overexpression on cell migration was stronger than that of Epac2. In addition, transfection of small interfering RNAs for Epac1, but not Epac2, significantly inhibited serum-mediated migration of DA SMCs. In the presence of O-Me-cAMP, actin stress fibers were well organized with enhanced focal adhesion, and cell shape was widely expanded. Adenovirus-mediated Epac1, but not Epac2 gene transfer, induced prominent ICF in the rat DA explants when compared with those with green fluorescent protein gene transfer. The thickness of intimal cushion became significantly greater (1.98-fold) in Epac1-overexpressed DA. O-Me-cAMP did not change hyaluronan production, although it decreased proliferation of DA SMCs. The present study demonstrated that Epac, especially Epac1, plays an important role in promoting SMC migration and thereby ICF in the rat DA.

Activator of G Protein Signaling 8 (AGS8) Is Required for Hypoxia-induced Apoptosis of Cardiomyocytes ROLE OF Gβγ AND CONNEXIN 43 (CX43)

Ischemic injury of the heart is associated with activation of multiple signal transduction systems including the heterotrimeric G-protein system. Here, we report a role of the ischemia-inducible regulator of Gβγ subunit, AGS8, in survival of cardiomyocytes under hypoxia. Cultured rat neonatal cardiomyocytes (NCM) were exposed to hypoxia or hypoxia/reoxygenation following transfection of AGS8siRNA or pcDNA::AGS8. Hypoxia-induced apoptosis of NCM was completely blocked by AGS8siRNA, whereas overexpression of AGS8 increased apoptosis. AGS8 formed complexes with G-proteins and channel protein connexin 43 (CX43), which regulates the permeability of small molecules under hypoxic stress. AGS8 initiated CX43 phosphorylation in a Gβγ-dependent manner by providing a scaffold composed of Gβγ and CX43. AGS8siRNA blocked internalization of CX43 following exposure of NCM to repetitive hypoxia; however it did not influence epidermal growth factor-mediated internalization of CX43. The decreased dye flux through CX43 that occurred with hypoxic stress was also prevented by AGS8siRNA. Interestingly, the Gβγ inhibitor Gallein mimicked the effect of AGS8 knockdown on both the CX43 internalization and the changes in cell permeability elicited by hypoxic stress. These data indicate that AGS8 is required for hypoxia-induced apoptosis of NCM, and that AGS8-Gβγ signal input increased the sensitivity of cells to hypoxic stress by influencing CX43 regulation and associated cell permeability. Under hypoxic stress, this unrecognized response program plays a critical role in the fate of NCM.

T-type Ca2+ channels promote oxygenation-induced closure of the rat ductus arteriosus not only by vasoconstriction but also by neointima formation

The ductus arteriosus (DA), an essential vascular shunt for fetal circulation, begins to close immediately after birth. Although Ca2+ influx through several membrane Ca2+ channels is known to regulate vasoconstriction of the DA, the role of the T-type voltage-dependent Ca2+ channel (VDCC) in DA closure remains unclear. Here we found that the expression of α1G, a T-type isoform that is known to exhibit a tissue-restricted expression pattern in the rat neonatal DA, was significantly up-regulated in oxygenated rat DA tissues and smooth muscle cells (SMCs). Immunohistological analysis revealed that α1G was localized predominantly in the central core of neonatal DA at birth. DA SMC migration was significantly increased by α1G overexpression. Moreover, it was decreased by adding α1G-specific small interfering RNAs or using R(−)-efonidipine, a highly selective T-type VDCC blocker. Furthermore, an oxygenation-mediated increase in an intracellular Ca2+ concentration of DA SMCs was significantly decreased by adding α1G-specific siRNAs or using R(−)-efonidipine. Although a prostaglandin E receptor EP4 agonist potently promoted intimal thickening of the DA explants, R(−)-efonidipine (10−6 m) significantly inhibited EP4-promoted intimal thickening by 40% using DA tissues at preterm in organ culture. Moreover, R(−)-efonidipine (10−6 m) significantly attenuated oxygenation-induced vasoconstriction by ∼27% using a vascular ring of fetal DA at term. Finally, R(−)-efonidipine significantly delayed the closure of in vivo DA in neonatal rats. These results indicate that T-type VDCC, especially α1G, which is predominantly expressed in neonatal DA, plays a unique role in DA closure, implying that T-type VDCC is an alternative therapeutic target to regulate the patency of DA.

DNA microarray profiling identified a new role of growth hormone in vascular remodeling of rat ductus arteriosus

The ductus arteriosus (DA), a fetal arterial connection between the pulmonary artery and the aorta, has a character distinct from the adjacent arteries. We compared the transcriptional profiles of the DA and the aorta of Wistar rat fetuses on embryonic day 19 (preterm) and day 21 (near-term) using DNA microarray analyses. We found that 39 genes were expressed 2.5-fold greater in the DA than in the aorta. Growth hormone (GH) receptor (GHR) exhibited the most significant difference in expression. Then, we found that GH significantly promoted migration of DA smooth muscle cells (SMCs), thus enhancing the intimal cushion formation of the DA explants. GH also regulated the expression of cytoskeletal genes in DA SMCs, which may retain a synthetic phenotype in the smooth muscle-specific cytoskeletal genes. Thus, the present study revealed that GH-GHR signal played a role in the vascular remodeling of the DA.

Acupuncture ameliorated skeletal muscle atrophy induced by hindlimb suspension in mice

Preventing skeletal muscle atrophy is critical for maintaining quality of life, but it is often a challenging goal for the elderly and patients with severe conditions. We hypothesized that acupuncture in place of exercise training is an alternative non-pharmacological intervention that can help to prevent muscle atrophy. To elucidate the effects of acupuncture on skeletal muscle atrophy caused by hindlimb suspension (HS), we performed acupuncture on mice according to two different methods: acupuncture with electrical stimulation (EA: electroacupuncture) and without electrical stimulation (MA: manual acupuncture). A needle was retained in the gastrocnemius muscle for 30 min every day for 2 weeks in the EA and MA groups. In the EA group, 30 min of repetitive electrical stimulation (1 Hz, 1 ms pulse width, 6.5 mA intensity) was also applied. HS significantly reduced muscle mass and the cross-sectional area of the soleus muscles. This HS-induced reduction was significantly improved in the EA group, although the level of improvement remained insufficient when compared with the control group. We found that the mRNA expression levels of atrogin-1 and MuRF1, which play a principal role in muscle-specific degradation as E3 ubiquitin ligases, were significantly increased in the HS group compared to the control group. EA and MA reduced the HS-induced upregulation of atrogin-1 (p<0.01 in EA and MA) and MuRF1 (p<0.01 in EA) mRNAs. We also found that the expression levels of PI3K, Akt1, TRPV4, adenosine A1 receptor, myostatin, and SIRT1 mRNAs tended to be increased by HS. EA and MA further increased the HS-induced upregulation of Akt1 (p<0.05 in MA) and TRPV4 (p<0.05 in MA) mRNAs. We concluded that acupuncture partially prevented skeletal muscle atrophy. This effect might be due to an increase in protein synthesis and a decrease in protein degradation.

Sarcalumenin plays a critical role in age-related cardiac dysfunction due to decreases in SERCA2a expression and activity

Impaired Ca(2+) reuptake into the sarcoplasmic reticulum (SR) underlies a primary pathogenesis of heart failure in the aging heart. Sarcalumenin (SAR), a Ca(2+)-binding glycoprotein located in the longitudinal SR, regulates Ca(2+) reuptake by interacting with SR Ca(2+)-ATPase (SERCA). Here we found that the expression levels of both SAR and SERCA2 proteins were significantly downregulated in senescent wild-type mice (18-month old) and that downregulation of SAR protein preceded downregulation of SERCA2 protein. The downregulation of SERCA2 protein was greater in senescent SARKO mice than in age-matched senescent wild-type mice, which was at least in part due to progressive degradation of SERCA2 protein in SARKO mice. Senescent SARKO mice exhibited typical findings of heart failure such as increased sympathetic activity, impaired exercise tolerance, and upregulation of biomarkers of cardiac stress. Consequently, cardiac function was progressively decreased in senescent SARKO. We also found that the expression levels of endoplasmic reticulum (ER) stress-related genes such as x-box binding protein 1 (XBP1) were significantly increased in senescent SARKO mice, indicating that senescent SARKO mice exhibited ER stress. Thus we uncovered the important role of SAR in maintaining Ca(2+) transport activity of SERCA2a and cardiac function in the senescent population.

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.

αB-Crystallin R120G variant causes cardiac arrhythmias and alterations in the expression of Ca2+-handling proteins and endoplasmic reticulum stress in mice

Mutations of αB‐crystallin (CryαB), a small heat shock protein abundantly expressed in cardiac and skeletal muscles, are known to cause desmin‐related myopathies. The CryαB R120G allele has been linked to a familial desminopathy and, in transgenic mice, causes a sudden death at about 28 weeks of age. To investigate the mechanisms of the sudden cardiac arrest of CryαB R120G transgenic mice, we prepared protein samples from left ventricular tissues of two different age groups (10 and 28 weeks) and examined Ca2+‐handling proteins. Expression of sarcoplasmic/endoplasmic reticulum calcium ATPase (SERCA) 2, phospholamban, ryanodine receptor 2 and calsequestrin 2 was significantly decreased in 28‐ versus 10‐week‐old CryαB R120G transgenic mice. In addition, low heart rate variability, including heart rate, total power and low frequency, was observed and continuous electrocardiogram monitoring revealed cardiac arrhythmias, such as ventricular tachycardia, atrioventricular block and atrial flutter, in 28‐week‐old CryαB R120G transgenic mice. In contrast, expression of endoplasmic reticulum (ER) degradation enhancing α‐mannosidase‐like protein, inositol requirement 1 and X‐box binding protein 1 were increased significantly in 28‐ versus 10‐week‐old CryαBR120G transgenic mice, suggesting that the CryαBR120G transgenic mice exhibit increased ER stress compared with wild‐type mice. Together, the data suggest that the CryαB R120G dominant variant induces ER stress and impairs Ca2+ regulation, leading to ageing‐related cardiac dysfunction, arrhythmias and decreased autonomic tone with shortened lifespan.

Metabolomic profiling analysis reveals chamber-dependent metabolite patterns in the mouse heart

Energy of the cardiac muscle largely depends on fatty acid oxidation. It is known that the atrium and ventricle have chamber-specific functions, structures, gene expressions, and pathologies. The left ventricle works as a high-pressure chamber to pump blood toward the body, and its muscle wall is thicker than those of the other chambers, suggesting that energy utilization in each of the chambers should be different. However, a chamber-specific pattern of metabolism remains incompletely understood. Recently, innovative techniques have enabled the comprehensive analysis of metabolites. Therefore, we aimed to clarify differences in metabolic patterns among the chambers. Male C57BL6 mice at 6 wk old were subject to a comprehensive measurement of metabolites in the atria and ventricles by capillary electrophoresis and mass spectrometry. We found that overall metabolic profiles, including nucleotides and amino acids, were similar between the right and left ventricles. On the other hand, the atria exhibited a distinct metabolic pattern from those of the ventricles. Importantly, the high-energy phosphate pool (the total concentration of ATP, ADP, and AMP) was higher in both ventricles. In addition, the levels of lactate, acetyl CoA, and tricarboxylic acid cycle contents were higher in the ventricles. Accordingly, the activities and/or expression levels of key enzymes were higher in the ventricles to produce more energy. The present study provides a basis for understanding the chamber-specific metabolism underlining pathophysiology in the heart.

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.