MMP9 inhibition increases autophagic flux in chronic heart failure.

Increased matrix metalloprotease 9 (MMP9) after myocardial infarction (MI) exacerbates ischemia-induced chronic heart failure (CHF). Autophagy is cardioprotective during CHF; however, whether increased MMP9 suppresses autophagic activity in CHF is unknown. This study aimed to determine whether increased MMP9 suppressed autophagic flux and MMP9 inhibition increased autophagic flux in the heart of rats with post-MI CHF. Sprague-Dawley rats underwent either sham surgery or coronary artery ligation 6-8 wk before being treated with MMP9 inhibitor for 7 days, followed by cardiac autophagic flux measurement with lysosomal inhibitor bafilomycin A1. Furthermore, autophagic flux was measured in vitro by treating H9c2 cardiomyocytes with two independent pharmacological MMP9 inhibitors, salvianolic acid B (SalB) and MMP9 inhibitor-I, and CRISPR/cas9-mediated MMP9 genetic ablation. CHF rats showed cardiac infarct, significantly increased left ventricular end-diastolic pressure (LVEDP), and increased MMP9 activity and fibrosis in the peri-infarct areas of left ventricular myocardium. Measurement of the autophagic markers LC3B-II and p62 with lysosomal inhibition showed decreased autophagic flux in the peri-infarct myocardium. Treatment with SalB for 7 days in CHF rats decreased MMP9 activity and cardiac fibrosis but increased autophagic flux in the peri-infarct myocardium. As an in vitro corollary study, measurement of autophagic flux in H9c2 cardiomyocytes and fibroblasts showed that pharmacological inhibition or genetic ablation of MMP9 upregulates autophagic flux. These data are consistent with our observations that MMP9 inhibition upregulates autophagic flux in the heart of rats with CHF. In conclusion, the results in this study suggest that the beneficial outcome of MMP9 inhibition in pathological cardiac remodeling is in part mediated by improved autophagic flux.NEW & NOTEWORTHY This study elucidates that the improved cardiac extracellular matrix (ECM) remodeling and cardioprotective effect of matrix metalloprotease 9 (MMP9) inhibition in chronic heart failure (CHF) are via increased autophagic flux. Autophagy is cardioprotective; however, the mechanism of autophagy suppression in CHF is unknown. We for the first time demonstrated here that increased MMP9 suppressed cardiac autophagy and ablation of MMP9 increased cardiac autophagic flux in CHF rats. Restoring the physiological level of autophagy in the failing heart is a challenge, and our study addressed this challenge. The novelty and highlights of this report are as follows: 1) MMP9 regulates cardiomyocyte and fibroblast autophagy, 2) MMP9 inhibition protects CHF after myocardial infarction (MI) via increased cardiac autophagic flux, 3) MMP9 inhibition increased cardiac autophagy via activation of AMP-activated protein kinase (AMPK)α, Beclin-1, Atg7 pathway and suppressed mechanistic target of rapamycin (mTOR) pathway.

GLP-1 mediated diuresis and natriuresis are blunted in heart failure and restored by selective afferent renal denervation.

BACKGROUND: Glucagon-like peptide-1 (GLP-1) induces diuresis and natriuresis. Previously we have shown that GLP-1 activates afferent renal nerve to increase efferent renal sympathetic nerve activity that negates the diuresis and natriuresis as a negative feedback mechanism in normal rats. However, renal effects of GLP-1 in heart failure (HF) has not been elucidated. The present study was designed to assess GLP-1-induced diuresis and natriuresis in rats with HF and its interactions with renal nerve activity. METHODS: HF was induced in rats by coronary artery ligation. The direct recording of afferent renal nerve activity (ARNA) with intrapelvic injection of GLP-1 and total renal sympathetic nerve activity (RSNA) with intravenous infusion of GLP-1 were performed. GLP-1 receptor expression in renal pelvis, densely innervated by afferent renal nerve, was assessed by real-time PCR and western blot analysis. In separate group of rats after coronary artery ligation selective afferent renal denervation (A-RDN) was performed by periaxonal application of capsaicin, then intravenous infusion of GLP-1-induced diuresis and natriuresis were evaluated. RESULTS: In HF, compared to sham-operated control; (1) response of increase in ARNA to intrapelvic injection of GLP-1 was enhanced (3.7 ± 0.4 vs. 2.0 ± 0.4 µV s), (2) GLP-1 receptor expression was increased in renal pelvis, (3) response of increase in RSNA to intravenous infusion of GLP-1 was enhanced (132 ± 30% vs. 70 ± 16% of the baseline level), and (4) diuretic and natriuretic responses to intravenous infusion of GLP-1 were blunted (urine flow 53.4 ± 4.3 vs. 78.6 ± 4.4 µl/min/gkw, sodium excretion 7.4 ± 0.8 vs. 10.9 ± 1.0 µEq/min/gkw). A-RDN induced significant increases in diuretic and natriuretic responses to GLP-1 in HF (urine flow 96.0 ± 1.9 vs. 53.4 ± 4.3 µl/min/gkw, sodium excretion 13.6 ± 1.4 vs. 7.4 ± 0.8 µEq/min/gkw). CONCLUSIONS: The excessive activation of neural circuitry involving afferent and efferent renal nerves suppresses diuretic and natriuretic responses to GLP-1 in HF. These pathophysiological responses to GLP-1 might be involved in the interaction between incretin-based medicines and established HF condition. RDN restores diuretic and natriuretic effects of GLP-1 and thus has potential beneficial therapeutic implication for diabetic HF patients.

Central angiotensin II-Protein inhibitor of neuronal nitric oxide synthase (PIN) axis contribute to neurogenic hypertension.

Activation of renin-angiotensin- system, nitric oxide (NO•) bioavailability and subsequent sympathoexcitation plays a pivotal role in the pathogenesis of many cardiovascular diseases, including hypertension. Previously we have shown increased protein expression of PIN (a protein inhibitor of nNOS: neuronal nitric oxide synthase, known to dissociate nNOS dimers into monomers) with concomitantly reduced levels of catalytically active dimers of nNOS in the PVN of rats with heart failure. To elucidate the molecular mechanism by which Angiotensin II (Ang II) increases PIN expression, we used Sprague-Dawley rats (250-300 g) subjected to intracerebroventricular infusion of Ang II (20 ng/min, 0.5 µl/h) or saline as vehicle (Veh) for 14 days through osmotic mini-pumps and NG108-15 hybrid neuronal cell line treated with Ang II as an in vitro model. Ang II infusion significantly increased baseline renal sympathetic nerve activity and mean arterial pressure. Ang II infusion increased the expression of PIN (1.24 ± 0.04* Ang II vs. 0.65 ± 0.07 Veh) with a concomitant 50% decrease in dimeric nNOS and PIN-Ub conjugates (0.73 ± 0.04* Ang II vs. 1.00 ± 0.03 Veh) in the PVN. Substrate-dependent ligase assay in cells transfected with pCMV-(HA-Ub)8 vector revealed a reduction of HA-Ub-PIN conjugates after Ang II and a proteasome inhibitor, Lactacystin (LC), treatment (4.5 ± 0.7* LC Ang II vs. 9.2 ± 2.5 LC). TUBE (Tandem Ubiquitin-Binding Entities) assay showed decrease PIN-Ub conjugates in Ang II-treated cells (0.82 ± 0.12* LC Ang II vs. 1.21 ± 0.06 LC) while AT1R blocker, Losartan (Los) treatment diminished the Ang II-mediated stabilization of PIN (1.21 ± 0.07 LC Los vs. 1.16 ± 0.04* LC Ang II Los). Taken together, our studies suggest that increased central levels of Ang II contribute to the enhanced expression of PIN leading to reduced expression of the dimeric form of nNOS, thus diminishing the inhibitory action of NO• on pre-autonomic neurons in the PVN resulting in increased sympathetic outflow.

Does glucagon-like peptide-1 induce diuresis and natriuresis by modulating afferent renal nerve activity?

Glucagon-like peptide-1 (GLP-1), an incretin hormone, has diuretic and natriuretic effects. The present study was designed to explore the possible underlying mechanisms for the diuretic and natriuretic effects of GLP-1 via renal nerves in rats. Immunohistochemistry revealed that GLP-1 receptors were avidly expressed in the pelvic wall, the wall being adjacent to afferent renal nerves immunoreactive to calcitonin gene-related peptide, which is the dominant neurotransmitter for renal afferents. GLP-1 (3 µM) infused into the left renal pelvis increased ipsilateral afferent renal nerve activity (110.0 ± 15.6% of basal value). Intravenous infusion of GLP-1 (1 µg·kg-1·min-1) for 30 min increased renal sympathetic nerve activity (RSNA). After the distal end of the renal nerve was cut to eliminate the afferent signal, the increase in efferent renal nerve activity during intravenous infusion of GLP-1 was diminished compared with the increase in total RSNA (17.0 ± 9.0% vs. 68.1 ± 20.0% of the basal value). Diuretic and natriuretic responses to intravenous infusion of GLP-1 were enhanced by total renal denervation (T-RDN) with acute surgical cutting of the renal nerves. Selective afferent renal nerve denervation (A-RDN) was performed by bilateral perivascular application of capsaicin on the renal nerves. Similar to T-RDN, A-RDN enhanced diuretic and natriuretic responses to GLP-1. Urine flow and Na+ excretion responses to GLP-1 were not significantly different between T-RDN and A-RDN groups. These results indicate that the diuretic and natriuretic effects of GLP-1 are partly governed via activation of afferent renal nerves by GLP-1 acting on sensory nerve fibers within the pelvis of the kidney.

Exercise training augments neuronal nitric oxide synthase dimerization in the paraventricular nucleus of rats with chronic heart failure.

Exercise training (ExT) is an established non-pharmacological therapy that improves the health and quality of life in patients with chronic heart failure (CHF). Exaggerated sympathetic drive characterizes CHF due to an imbalance of the autonomic nervous system. Neuronal nitric oxide synthase (nNOS) in the paraventricular nucleus (PVN) produce nitric oxide (NO•), which is known to regulate the sympathetic tone. Previously we have shown that during CHF, the catalytically active dimeric form of nNOS is significantly decreased with a concurrent increase in protein inhibitor of nNOS (PIN) expression, a protein that dissociates dimeric nNOS to monomers and facilitates its degradation. Dimerization of nNOS also requires (6R)-5,6,7,8-tetrahydrobiopterin (BH4) for stability and activity. Previously, we have shown that ExT improves NO-mediated sympathetic inhibition in the PVN; however, the molecular mechanism remains elusive. We hypothesized; ExT restores the sympathetic drive by increasing the levels and catalytically active form of nNOS by abrogating changes in the PIN in the PVN of CHF rats. CHF was induced in adult male Sprague-Dawley rats by coronary artery ligation, which reliably mimics CHF in patients with myocardial infarction. After 4 weeks of surgery, Sham and CHF rats were subjected to 3 weeks of progressive treadmill exercise. ExT significantly (p < 0.05) decreased PIN expression and increased dimer/monomer ratio of nNOS in the PVN of rats with CHF. Moreover, we found decreased GTP cyclohydrolase 1(GCH1) expression: a rate-limiting enzyme for BH4 biosynthesis in the PVN of CHF rats suggesting that perhaps reduced BH4 availability may also contribute to decreased nNOS dimers. Interestingly, CHF induced decrease in GCH1 expression was increased with ExT. Our findings revealed that ExT rectified decreased PIN and GCH1 expression and increased dimer/monomer ratio of nNOS in the PVN, which may lead to increase NO• bioavailability resulting in amelioration of activated sympathetic drive during CHF.

An outreach program with hands-on, physiology-based exercises generates questions about STEM career expectations.

Scientific advocacy and outreach programs are encouraged to increase public understanding of scientific knowledge and generate interest in science, technology, engineering, and mathematics (STEM) careers. However, evaluation of these events' effectiveness is difficult and somewhat rare. This study's purpose was to better understand how effective an established physiology-based outreach program was in generating interest in STEM careers, while simultaneously providing information that can be used to increase the effectiveness of future events. We partnered with a private school located in Omaha, Nebraska, where 64-80 students participated in 3 h of physiology-based activities presented by volunteers from the University of Nebraska Medical Center. The event included a brief presentation of the eye, sensory, heart, and lung systems, followed by hands-on demonstrations and activities. Each session concluded with 15 min of questions and answers (Q&A), where students were encouraged to engage the volunteers in inquiries about what they just learned, career-related questions, or any topic of their choosing. Each Q&A session was audio recorded and evaluated using thematic analysis to identify patterns in the Q&A data. Two major themes of questions were identified: 1) scientific content (animal circulatory systems and how organs are affected by disease or stimulus); and 2) career-related content, including typical day-to-day activities of a scientist and the volunteers' satisfaction with a scientific career. We conclude that hands-on physiology-based learning opportunities are effective in generating short-term interest in STEM content and careers. The results of this study will also facilitate informed modification of event content to better suit student's interests.

GABA-containing liposomes: neuroscience applications and translational perspectives for targeting neurological diseases.

There are multiple challenges for neuropharmacology in the future. Undoubtedly, one of the greatest challenges is the development of strategies for pharmacological targeting of specific brain regions for treatment of diseases. GABA is the main inhibitory neurotransmitter in the central nervous system, and dysfunction of GABAergic mechanisms is associated with different neurological conditions. Liposomes are lipid vesicles that are able to encapsulate chemical compounds and are used for chronic drug delivery. This short review reports our experience with the development of liposomes for encapsulation and chronic delivery of GABA to sites within the brain. Directions for future research regarding the efficacy and practical use of GABA-containing liposomes for extended periods of time as well as understanding and targeting neurological conditions are discussed.

A novel role for miR-133a in centrally mediated activation of the renin-angiotensin system in congestive heart failure.

An activated renin-angiotensin system (RAS) within the central nervous system has been implicated in sympathoexcitation during various disease conditions including congestive heart failure (CHF). In particular, activation of the RAS in the paraventricular nucleus (PVN) of the hypothalamus has been recognized to augment sympathoexcitation in CHF. We observed a 2.6-fold increase in angiotensinogen (AGT) in the PVN of CHF. To elucidate the molecular mechanism for increased expression of AGT, we performed in silico analysis of the 3'-untranslated region (3'-UTR) of AGT and found a potential binding site for microRNA (miR)-133a. We hypothesized that decreased miR-133a might contribute to increased AGT in the PVN of CHF rats. Overexpression of miR-133a in NG108 cells resulted in 1.4- and 1.5-fold decreases in AGT and angiotensin type II (ANG II) type 1 receptor (AT1R) mRNA levels, respectively. A luciferase reporter assay performed on NG108 cells confirmed miR-133a binding to the 3'-UTR of AGT. Consistent with these in vitro data, we observed a 1.9-fold decrease in miR-133a expression with a concomitant increase in AGT and AT1R expression within the PVN of CHF rats. Furthermore, restoring the levels of miR-133a within the PVN of CHF rats with viral transduction resulted in a significant reduction of AGT (1.4-fold) and AT1R (1.5-fold) levels with a concomitant decrease in basal renal sympathetic nerve activity (RSNA). Restoration of miR-133a also abrogated the enhanced RSNA responses to microinjected ANG II within the PVN of CHF rats. These results reveal a novel and potentially unique role for miR-133a in the regulation of ANG II within the PVN of CHF rats, which may potentially contribute to the commonly observed sympathoexcitation in CHF.NEW & NOTEWORTHY Angiotensinogen (AGT) expression is upregulated in the paraventricular nucleus of the hypothalamus through posttranscriptional mechanism interceded by microRNA-133a in heart failure. Understanding the mechanism of increased expression of AGT in pathological conditions leading to increased sympathoexcitation may provide the basis for the possible development of new therapeutic agents with enhanced specificity.

Renal denervation improves cardiac function in rats with chronic heart failure: Effects on expression of ß-adrenoceptors.

Chronic activation of the sympathetic drive contributes to cardiac remodeling and dysfunction during chronic heart failure (HF). The present study was undertaken to assess whether renal denervation (RDN) would abrogate the sympathoexcitation in HF and ameliorate the adrenergic dysfunction and cardiac damage. Ligation of the left coronary artery was used to induce HF in Sprague-Dawley rats. Four weeks after surgery, RDN was performed, 1 wk before the final measurements. At the end of the protocol, cardiac function was assessed by measuring ventricular hemodynamics. Rats with HF had an average infarct area >30% of the left ventricle and left ventricular end-diastolic pressure (LVEDP) >20 mmHg. ß1- and ß2-adrenoceptor proteins in the left ventricle were reduced by 37 and 49%, respectively, in the rats with HF. RDN lowered elevated levels of urinary excretion of norepinephrine and brain natriuretic peptide levels in the hearts of rats with HF. RDN also decreased LVEDP to 10 mmHg and improved basal dP/dt to within the normal range in rats with HF. RDN blunted loss of ß1-adrenoceptor (by 47%) and ß2-adrenoceptor (by 100%) protein expression and improved isoproterenol (0.5 µg/kg)-induced increase in +dP/dt (by 71%) and -dP/dt (by 62%) in rats with HF. RDN also attenuated the increase in collagen 1 expression in the left ventricles of rats with HF. These findings demonstrate that RDN initiated in chronic HF condition improves cardiac function mediated by adrenergic agonist and blunts ß-adrenoceptor expression loss, providing mechanistic insights for RDN-induced improvements in cardiac function in the HF condition.

Effects of exercise training on SFO-mediated sympathoexcitation during chronic heart failure.

Exercise training (ExT) has been shown to reduce sympathetic drive during heart failure (HF). The subfornical organ (SFO) is involved in the neural control of sympathetic drive. We hypothesized that an activated SFO contributes to enhanced sympathetic activity in HF. We also postulated that ExT would reduce the activation of the SFO and its contribution to the sympathetic drive during HF. Sprague-Dawley rats were subjected to coronary artery ligation to induce HF. Rats were assigned to ExT for 3-4 wk. Rats with HF had a 2.5-fold increase in FosB-positive cells in the SFO compared with sham-operated rats, and this was normalized by ExT. Microinjection of ANG II (100 pmol) into the SFO resulted in a greater increase in renal sympathetic nerve activity (RSNA), blood pressure, and heart rate in the HF group than in the sham-operated group. These responses were normalized after ExT (change in RSNA: 23 ± 3% vs. 8 ± 2%). ExT also abolished the decrease in RSNA in HF rats after the microinjection of losartan (200 pmol) into the SFO (-21 ± 4% vs. -2 ± 3%). Finally, there was elevated mRNA (5-fold) and protein expression (43%) of ANG II type 1 receptors in the SFO of rats with HF, which were reversed after ExT. These data suggest that the enhanced activity of the SFO by elevated tonic ANG II contributes to the enhanced sympathoexcitation exhibited in HF. The decrease in ANG II type 1 receptor expression in the SFO by ExT may be responsible for reversing the neuronal activation in the SFO and SFO-mediated sympathoexcitation in rats with HF.

Angiotensin II-mediated posttranslational modification of nNOS in the PVN of rats with CHF: role for PIN.

An increased sympathetic drive is an adverse characteristic in chronic heart failure (CHF). The protein expression of neuronal nitric oxide synthase (nNOS)- and hence nitric oxide (NO)-mediated sympathoinhibition is reduced in the paraventricular nucleus (PVN) of rats with CHF. However, the molecular mechanism(s) of nNOS downregulation remain(s) unclear. The aim of the study was to reveal the underlying molecular mechanism for the downregulation of nNOS in the PVN of CHF rats. Sprague-Dawley rats with CHF (6-8 wk after coronary artery ligation) demonstrated decreased nNOS dimer/monomer ratio (42%), with a concomitant increase in the expression of PIN (a protein inhibitor of nNOS known to dissociate nNOS dimers into monomers) by 47% in the PVN. Similarly, PIN expression is increased in a neuronal cell line (NG108) treated with angiotensin II (ANG II). Furthermore, there is an increased accumulation of high-molecular-weight nNOS-ubiquitin (nNOS-Ub) conjugates in the PVN of CHF rats (29%). ANG II treatment in NG108 cells in the presence of a proteasome inhibitor, lactacystin, also leads to a 69% increase in accumulation of nNOS-Ub conjugates immunoprecipitated by an antiubiquitin antibody. There is an ANG II-driven, PIN-mediated decrease in the dimeric catalytically active nNOS in the PVN, due to ubiquitin-dependent proteolytic degradation in CHF. Our results show a novel intermediary mechanism that leads to decreased levels of active nNOS in the PVN, involved in subsequent reduction in sympathoinhibition during CHF, offering a new target for the treatment of CHF and other cardiovascular diseases.

Pyruvate restores ß-adrenergic sensitivity of L-type Ca(2+) channels in failing rat heart: role of protein phosphatase.

Oxidative stress plays a major role in the pathogenesis of heart failure, where the contractile response to ß-adrenergic stimulation is profoundly depressed. This condition involves L-type Ca(2+) channels, but the mechanisms underlying their impaired adrenergic regulation are unclear. Thus the present study explored the basis for impaired adrenergic control of Ca(2+) channels in a rat infarction model of heart failure. Patch-clamp recordings of L-type Ca(2+) current (I(Ca,L)) from ventricular myocytes isolated from infarcted hearts showed a blunted response to intracellular cAMP that was reversed by treatment with exogenous pyruvate. Biochemical studies showed that basal and cAMP-stimulated protein kinase A activities were similar in infarcted and sham-operated hearts, whereas molecular analysis also found that binding of protein kinase A to the α(1C) subunit of voltage-gated Ca(2+) channel isoform 1.2 was not different between groups. By contrast, protein phosphatase 2A (PP2A) activity and binding to α(1C) were significantly less in infarcted hearts. The PP2A inhibitor okadaic acid markedly increased I(Ca,L) in sham-operated myocytes, but this response was significantly less in myocytes from infarcted hearts. However, pyruvate normalized I(Ca,L) stimulation by okadaic acid, and this effect was blocked by inhibitors of thioredoxin reductase, implicating a functional role for the redox-active thioredoxin system. Our data suggest that blunted ß-adrenergic stimulation of I(CaL) in failing hearts results from hyperphosphorylation of Ca(2+) channels secondary to oxidation-induced impairment of PP2A function. We propose that the redox state of Ca(2+) channels or PP2A is controlled by the thioredoxin system which plays a key role in Ca(2+) channel remodeling of the failing heart.

Nitric oxide inhibits the expression of AT1 receptors in neurons.

We have previously observed an increased of angiotensin II (ANG II) type 1 receptor (AT(1)R) with enhanced AT(1)R-mediated sympathetic outflow and concomitant downregulation of neuronal nitric oxide (NO) synthase (nNOS) with reduced NO-mediated inhibition from the paraventricular nucleus (PVN) in rats with heart failure. To test the hypothesis that NO exerts an inhibitory effect on AT(1)R expression in the PVN, we used primary cultured hypothalamic cells of neonatal rats and neuronal cell line NG108-15 as in vitro models. In hypothalamic primary culture, NO donor sodium nitroprusside (SNP) induced dose-dependent decreases in mRNA and protein of AT(1)R (10(-5) M SNP, AT(1)R protein was 10 ± 2% of control level) while NOS inhibitor N(G)-monomethyl-l-arginine (l-NMMA) induced dose-dependent increases in mRNA and protein levels of AT(1)R (10(-5) M l-NMMA, AT(1)R protein was 148 ± 8% of control level). Similar effects of SNP and l-NMMA on AT(1)R expression were also observed in NG108-15 cell line (10(-6) M SNP, AT(1)R protein was 30 ± 4% of control level while at the dose of 10(-6) M l-NMMA, AT(1)R protein was 171 ± 15% of the control level). Specific inhibition of nNOS, using antisense, caused an increase in AT(1)R expression while overexpression of nNOS, using adenoviral gene transfer (Ad.nNOS), caused an inhibition of AT(1)R expression in NG108 cells. Antisense nNOS transfection augmented the increase while Ad.nNOS infection blunted the increase in intracellular calcium concentration in response to ANG II treatment in NG108 cells. In addition, downregulation of AT(1)R mRNA as well as protein level in neuronal cell line in response to S-nitroso-N-acetyl pencillamine (SNAP) treatment was blocked by protein kinase G (PKG) inhibitor, while the peroxynitrite scavenger deforxamine had no effect. These results suggest that NO acts as an inhibitory regulator of AT(1)R expression and the activation of PKG is the required step in the regulation of AT(1)R gene expression via cGMP-dependent signaling pathway.

The non-canonical protein binding site at the monomer-monomer interface of yeast proliferating cell nuclear antigen (PCNA) regulates the Rev1-PCNA interaction and Polζ/Rev1-dependent translesion DNA synthesis.

Rev1 and DNA polymerase ζ (Polζ) are involved in the tolerance of DNA damage by translesion synthesis (TLS). The proliferating cell nuclear antigen (PCNA), the auxiliary factor of nuclear DNA polymerases, plays an important role in regulating the access of TLS polymerases to the primer terminus. Both Rev1 and Polζ lack the conserved hydrophobic motif that is used by many proteins for the interaction with PCNA at its interdomain connector loop. We have previously reported that the interaction of yeast Polζ with PCNA occurs at an unusual site near the monomer-monomer interface of the trimeric PCNA. Using GST pull-down assays, PCNA-coupled affinity beads pull-down and gel filtration chromatography, we show that the same region is required for the physical interaction of PCNA with the polymerase-associated domain (PAD) of Rev1. The interaction is disrupted by the pol30-113 mutation that results in a double amino acid substitution at the monomer-monomer interface of PCNA. Genetic analysis of the epistatic relationship of the pol30-113 mutation with an array of DNA repair and damage tolerance mutations indicated that PCNA-113 is specifically defective in the Rev1/Polζ-dependent TLS pathway. Taken together, the data suggest that Polζ and Rev1 are unique among PCNA-interacting proteins in using the novel binding site near the intermolecular interface of PCNA. The new mode of Rev1-PCNA binding described here suggests a mechanism by which Rev1 adopts a catalytically inactive configuration at the replication fork.

Exercise training normalizes enhanced sympathetic activation from the paraventricular nucleus in chronic heart failure: role of angiotensin II.

Exercise training (ExT) normalizes the increased sympathetic outflow in heart failure (HF), but the underlying mechanisms are not known. We hypothesized ExT would normalize the augmented activation of the paraventricular nucleus (PVN) via an angiotensinergic mechanism during HF. Four groups of rats used were the following: 1) sham-sedentary (Sed); 2) sham-ExT; 3) HF-Sed, and 4) HF-ExT. HF was induced by left coronary artery ligation. Four weeks after surgery, 3 wk of treadmill running was performed in ExT groups. The number of FosB-positive cells in the PVN was significantly increased in HF-Sed group compared with the sham-Sed group. ExT normalized (negated) this increase in the rats with HF. In anesthetized condition, the increases in renal sympathetic nerve activity (RSNA), mean arterial pressure (MAP), and heart rate (HR) in response to microinjection of angiotensin (ANG) II (50∼200 pmol) in the PVN of HF-Sed group were significantly greater than of the sham-Sed group. In the HF-ExT group the responses to microinjection of ANG II were not different from sham-Sed or sham-ExT groups. Blockade of ANG II type 1 (AT(1)) receptors with losartan in the PVN produced a significantly greater decrease in RSNA, MAP, and HR in HF-Sed group compared with sham-Sed group. ExT prevented the difference between HF and sham groups. AT(1) receptor protein expression was increased 50% in HF-Sed group compared with sham-Sed group. In the HF-ExT group, AT(1) receptor protein expression was not significantly different from sham-Sed or sham-ExT groups. In conclusion, one mechanism by which ExT alleviates elevated sympathetic outflow in HF may be through normalization of angiotensinergic mechanisms within the PVN.

Enhanced Expression and Function of Renal SGLT2 (Sodium-Glucose Cotransporter 2) in Heart Failure: Role of Renal Nerves.

BACKGROUND: Recent clinical studies demonstrate that SGLT2 (sodium-glucose cotransporter 2) inhibitors ameliorate heart failure (HF). The present study was conducted to assess the expression and function of renal SGLT2 and the influence of enhanced renal sympathetic tone in HF. METHODS: Four weeks after coronary artery ligation surgery to induce HF, surgical bilateral renal denervation (RDN) was performed in rats. Four groups of rats (Sham-operated control [Sham], Sham+RDN, HF and HF+RDN; n=6/group) were used. Immunohistochemistry and Western blot analysis were performed to evaluate the renal SGLT2 expression. One week after RDN (5 weeks after induction of HF), intravenous injection of SGLT2 inhibitor dapagliflozin were performed to assess renal excretory responses. In vitro, human embryonic kidney cells were used to investigate the fractionation of SGLT2 after norepinephrine treatment. RESULTS: In rats with HF, (1) SGLT2 expression in the proximal tubule of the kidney was increased; (2) the response of increases in urine flow, sodium excretion, and glucose excretion to dapagliflozin were greater; and (3) RDN attenuated renal SGLT2 expression and normalized renal functional responses to dapagliflozin. In vitro, norepinephrine promoted translocation of SGLT2 to the cell membrane. CONCLUSIONS: These results indicate that the enhanced tonic renal sympathetic nerve activation in HF increases the expression and functional activity of renal SGLT2. Potentiated trafficking of SGLT2 to cell surface in renal proximal tubules mediated by norepinephrine may contribute to this functional activation of SGLT2 in HF. These findings provide critical insight into the underlying mechanisms for the beneficial effects of SGLT2 inhibitors on HF reported in the clinical studies.

Central Ang II (Angiotensin II)-Mediated Sympathoexcitation: Role for HIF-1α (Hypoxia-Inducible Factor-1α) Facilitated Glutamatergic Tone in the Paraventricular Nucleus of the Hypothalamus.

Central infusion of Ang II (angiotensin II) has been associated with increased sympathetic outflow resulting in neurogenic hypertension. In the present study, we appraised whether the chronic increase in central Ang II activates the paraventricular nucleus of the hypothalamus (PVN) resulting in elevated sympathetic tone and altered baro- and chemoreflexes. Further, we evaluated the contribution of HIF-1α (hypoxia-inducible factor-1α), a transcription factor involved in enhancing the expression of N-methyl-D-aspartate receptors and thus glutamatergic-mediated sympathetic tone from the PVN. Ang II infusion (20 ng/minute, intracerebroventricular, 14 days) increased mean arterial pressure (126±9 versus 84±4 mm Hg), cardiac sympathetic tone (96±7 versus 75±6 bpm), and decreased cardiac parasympathetic tone (16±2 versus 36±3 versus bpm) compared with saline-infused controls in conscious rats. The Ang II-infused group also showed an impaired baroreflex control of heart rate (-1.50±0.1 versus -2.50±0.3 bpm/mm Hg), potentiation of the chemoreflex pressor response (53±7 versus 30±7 mm Hg) and increased number of FosB-labeled cells (53±3 versus 19±4) in the PVN. Concomitant with the activation of the PVN, there was an increased expression of HIF-1α and N-Methyl-D-Aspartate-type1 receptors in the PVN. Further, Ang II-infusion showed increased renal sympathetic nerve activity (20.5±2.3% versus 6.4±1.9% of Max) and 3-fold enhanced renal sympathetic nerve activity responses to microinjection of N-methyl-D-aspartate (200 pmol) into the PVN of anesthetized rats. Further, silencing of HIF-1α in NG108 cells abrogated the expression of N-methyl-D-aspartate-N-methyl-D-aspartate-type1 induced by Ang II. Taken together, our studies suggest a novel Ang II-HIF-1α-N-methyl-D-aspartate receptor-mediated activation of preautonomic neurons in the PVN, resulting in increased sympathetic outflow and alterations in baro- and chemoreflexes.

Chronic AT1 receptor blockade normalizes NMDA-mediated changes in renal sympathetic nerve activity and NR1 expression within the PVN in rats with heart failure.

Exercise training normalizes enhanced glutamatergic mechanisms within the paraventricular nucleus (PVN) concomitant with the normalization of increased plasma ANG II levels in rats with heart failure (HF). We tested whether ANG II type 1 (AT(1)) receptors are involved in the normalization of PVN glutamatergic mechanisms using chronic AT(1) receptor blockade with losartan (Los; 50 mg.kg(-1).day(-1) in drinking water for 3 wk). Left ventricular end-diastolic pressure was increased in both HF + vehicle (Veh) and HF + Los groups compared with sham-operated animals (Sham group), although it was significantly attenuated in the HF + Los group compared with the HF + Veh group. The effect of Los on cardiac function was similar to exercise training. At the highest dose of N-methyl-d-aspartate (NMDA; 200 pmol) injected into the PVN, the increase in renal sympathetic nerve activity was 93 +/- 13% in the HF + Veh group, which was significantly higher (P < 0.05) than the increase in the Sham + Veh (45 +/- 2%) and HF + Los (47 +/- 2%) groups. Relative NMDA receptor subunit NR(1) mRNA expression within the PVN was increased 120% in the HF + Veh group compared with the Sham + Veh group (P < 0.05) but was significantly attenuated in the HF + Los group compared with the HF + Veh group (P < 0.05). NR(1) protein expression increased 87% in the HF + Veh group compared with the Sham + Veh group but was significantly attenuated in the HF + Los group compared with the HF + Veh group (P < 0.05). Furthermore, in in vitro experiments using neuronal NG-108 cells, we found that ANG II treatment stimulated NR(1) protein expression and that Los significantly ameliorated the NR(1) expression induced by ANG II. These data are consistent with our hypothesis that chronic AT(1) receptor blockade normalizes glutamatergic mechanisms within the PVN in rats with HF.

Angiotensin-converting enzyme 2 activator, DIZE in the basolateral amygdala attenuates the tachycardic response to acute stress by modulating glutamatergic tone.

The basolateral amygdala (BLA) is critical in the control of the sympathetic output during stress. Studies demonstrated the involvement of the renin-angiotensin system components in the BLA. Angiotensin-(1-7) [Ang-(1-7)], acting through Mas receptors, reduces stress effects. Considering that angiotensin-converting enzyme 2 (ACE2) is the principal enzyme for the production of Ang-(1-7), here we evaluate the cardiovascular reactivity to acute stress after administration of the ACE2 activator, diminazene aceturate (DIZE) into the BLA. We also tested whether systemic treatment with DIZE could modify synaptic activity in the BLA and its effect directly on the expression of the N-methyl-d-aspartate receptors (NMDARs) in NG108 neurons in-vitro. Administration of DIZE into the BLA (200 pmol/100 nL) attenuated the tachycardia to stress (ΔHR, bpm: vehicle = 103 ± 17 vs DIZE = 49 ± 7 p = 0.018); this effect was inhibited by Ang-(1-7) antagonist, A-779 (ΔHR, bpm: DIZE = 49 ± 7 vs A-779 + DIZE = 100 ± 15 p = 0.04). Systemic treatment with DIZE attenuated the excitatory synaptic activity in the BLA (Frequency (Hz): vehicle = 2.9 ± 0.4 vs. DIZE =1.8 ± 0.3 p < 0.04). NG108 cells treated with DIZE demonstrated decreased expression of l subunit NMDAR-NR1 (NR1 expression (a.u): control = 0.534 ± 0.0593 vs. DIZE = 0.254 ± 0.0260) of NMDAR and increases of Mas receptors expression. These data demonstrate that DIZE attenuates the tachycardia evoked by acute stress. This effect results from a central action in the BLA involving activation of Mas receptors. The ACE2 activation via DIZE treatment attenuated the frequency of excitatory synaptic activity in the basolateral amygdala and this effect can be related with the decreases of the NMDAR-NR1 receptor expression.

Central Glucagon-like Peptide-1 Receptor Signaling via Brainstem Catecholamine Neurons Counteracts Hypertension in Spontaneously Hypertensive Rats.

Glucagon-like peptide-1 receptor (GLP-1R) agonists, widely used to treat type 2 diabetes, reduce blood pressure (BP) in hypertensive patients. Whether this action involves central mechanisms is unknown. We here report that repeated lateral ventricular (LV) injection of GLP-1R agonist, liraglutide, once daily for 15 days counteracted the development of hypertension in spontaneously hypertensive rats (SHR). In parallel, it suppressed urinary norepinephrine excretion, and induced c-Fos expressions in the area postrema (AP) and nucleus tractus solitarius (NTS) of brainstem including the NTS neurons immunoreactive to dopamine beta-hydroxylase (DBH). Acute administration of liraglutide into fourth ventricle, the area with easy access to the AP and NTS, transiently decreased BP in SHR and this effect was attenuated after lesion of NTS DBH neurons with anti-DBH conjugated to saporin (anti-DBH-SAP). In anti-DBH-SAP injected SHR, the antihypertensive effect of repeated LV injection of liraglutide for 14 days was also attenuated. These findings demonstrate that the central GLP-1R signaling via NTS DBH neurons counteracts the development of hypertension in SHR, accompanied by attenuated sympathetic nerve activity.