The crucial relationship between miRNA-27 and CSE/H2S, and the mechanism of action of GLP-1 in myocardial hypertrophy.

Cardiac hypertrophy is the most prevalent compensatory heart disease that ultimately leads to spontaneous heart failure. Mounting evidence suggests that microRNAs (miRs) and endogenous hydrogen sulfide (H2S) play a crucial role in the regulation of cardiac hypertrophy. In this study, we aimed to investigate whether inhibition of miR-27a could protect against cardiac hypertrophy by modulating H2S signaling. We established a model of cardiac hypertrophy by obtaining hypertrophic tissue from mice subjected to transverse aortic constriction (TAC) and from cells treated with angiotensin-II. Molecular alterations in the myocardium were quantified using quantitative real time PCR (qRT-PCR), Western blotting, and ELISA. Morphological changes were characterized by hematoxylin and eosin (HE) staining and Masson's trichrome staining. Functional myocardial changes were assessed using echocardiography. Our results demonstrated that miR-27a levels were elevated, while H2S levels were reduced in TAC mice and myocardial hypertrophy. Further luciferase and target scan assays confirmed that cystathionine-γ-lyase (CSE) was a direct target of miR-27a and was negatively regulated by it. Notably, enhancement of H2S expression in the heart was observed in mice injected with recombinant adeno-associated virus vector 9 (rAAV9)-anti-miR-27a and in cells transfected with a miR-27a inhibitor during cardiac hypertrophy. However, this effect was abolished by co-transfection with CSE siRNA and the miR-27a inhibitor. Conversely, injecting rAAV9-miR-27a yielded opposite results. Interestingly, our findings demonstrated that glucagon-like peptide-1 (GLP-1) agonists could mitigate myocardial damage by down-regulating miR-27a and up-regulating CSE. In summary, our study suggests that inhibition of miR-27a holds therapeutic promise for the treatment of cardiac hypertrophy by increasing H2S levels. Furthermore, our findings unveil a novel mechanism of GLP-1 agonists involving the miR-27a/H2S pathway in the management of cardiac hypertrophy.

Piezo1 channel activation facilitates baroreflex afferent neurotransmission with subsequent blood pressure reduction in control and hypertension rats.

Mechanosensitive cation channels such as Piezo1 and Piezo2 are activated by mechanical force like a starched wall of the aorta while blood pressure (BP) rising, which helps to elucidate the underlying mechanism of mechanotransduction of baroreceptor endings. In this study we investigated how Piezo1 channel activation-mediated gender- and afferent-specific BP regulation in rats. We established high-fat diet and fructose drink-induced hypertension model rats (HFD-HTN) and deoxycorticosterone (DOCA)-sensitive hypertension model rats. We showed that the expression levels of Piezo1 and Piezo2 were significantly up-regulated in left ventricle of HFD and DOCA hypertensive rats, whereas the down-regulation of Piezo1 was likely to be compensated by Piezo2 up-regulation in the aorta. Likewise, down-regulated Piezo1 was observed in the nodose ganglion (NG), while up-regulated Piezo2 was found in the nucleus tractus solitarius (NTS), which might synergistically reduce the excitatory neurotransmitter release from the presynaptic membrane. Notably, microinjection of Yoda1 (0.025-2.5 mg/ml) into the NG concentration-dependently reduced BP in both hypertensive rat models as well as in control rats with similar EC50; the effect of Yoda1 was abolished by microinjection of a Piezo1 antagonist GsMTx4 (1.0 µM). Functional analysis in an in vitro aortic arch preparation showed that instantaneous firing frequency of single Ah-fiber of aortic depressor nerve was dramatically increased by Yoda1 (0.03-1.0 µM) and blocked by GsMTx4 (1.0 µM). Moreover, spontaneous synaptic currents recorded from identified 2nd-order Ah-type baroreceptive neurons in the NTS was also facilitated over 100% by Yoda1 (1.0 µM) and completely blocked by GsMTx4 (3.0 µM). These results demonstrate that Piezo1 expressed on Ah-type baroreceptor and baroreceptive neurons in the NG and NTS plays a key role in a sexual-dimorphic BP regulation under physiological and hypertensive condition through facilitation of baroreflex afferent neurotransmission, which is presumably collaborated by Piezo2 expression at different level of baroreflex afferent pathway via compensatory and synergistic mechanisms.

Anticancer peptides from induced tumor-suppressing cells for inhibiting osteosarcoma cells.

Osteosarcoma (OS) is the most frequent primary bone cancer, which is mainly suffered by children and young adults. While the current surgical treatment combined with chemotherapy is effective for the early stage of OS, advanced OS preferentially metastasizes to the lung and is difficult to treat. Here, we examined the efficacy of ten anti-OS peptide candidates from a trypsin-digested conditioned medium that was derived from the secretome of induced tumor-suppressing cells (iTSCs). Using OS cell lines, the antitumor capabilities of the peptide candidates were evaluated by assaying the alterations in metabolic activities, proliferation, motility, and invasion of OS cells. Among ten candidates, peptide P05 (ADDGRPFPQVIK), a fragment of aldolase A (ALDOA), presented the most potent OS-suppressing capabilities. Its efficacy was additive with standard-of-care chemotherapeutic agents such as cisplatin and doxorubicin, and it downregulated oncoproteins such as epidermal growth factor receptor (EGFR), Snail, and Src in OS cells. Interestingly, P05 did not present inhibitory effects on non-OS skeletal cells such as mesenchymal stem cells and osteoblast cells. Collectively, this study demonstrated that iTSC-derived secretomes may provide a source for identifying anticancer peptides, and P05 may warrant further evaluations for the treatment of OS.

Ah-type Baroreceptor Neurons Expressing Estrogen Dependent mGluR7 Mediate Descending Inhibition of Cardiac Nociception.

Silent myocardial infarction (MI) is critical for clinical practice with increasing risk for women and the cause remains a medical mystery. Upon the discovery of female-specific Ah-type baroreceptor neurons (BRNs), we hypothesize that glutamate mediates depressor response through afferent-specific expression of particular glutamate receptors (mGluRs) leading descending inhibition of cardiac nociception. In vivo, tail-flick reflex and electromyography were assessed to evaluate glutamate-mediated blood pressure regulation, peripheral and cardiac nociception. The results showed that glutamate decreased mean arterial pressure (MAP) and increased peripheral nociception. Interestingly, glutamate-mediated capsaicin-induced cardiac nociception was strongly reduced in female rats compared with males. Furthermore, Nodose (NG) microinjection of mGluR7 agonist significantly increased MAP in males and slightly decreased that in females. Even though mGluR8 direct activation intensified baroreceptor activation, the sensitivity was similar between sexes. In vitro, the expression profiles of mGluRs were investigated using Western blot and identified BRNs using single-cell qRT-PCR under ischemic conditions. Glutamate in serum, NG and nucleus tractus solitary (NTS) was raised significantly in the model rats of both sexes vs. sham-controls. Female-specific expression of mGluR7 in the baroreflex afferent pathway, especially higher expression in Ah-type BRNs, contributes significantly to cardiac analgesia, which may explain that the pathogenesis of silent MI occurs mainly in female patients. Therefore, higher expression of mGluR7 in female-specific subpopulation of Ah-type BRNs plays a critical role in cardiac analgesia and peripheral nociception.

Estrogen-dependent depressor response of melatonin via baroreflex afferent function and intensification of PKC-mediated Nav1.9 activation.

Recent studies suggest that melatonin (Mel) plays an important role in the regulation of blood pressure (BP) via the aortic baroreflex pathway. In this study, we investigated the interaction between the baroreflex afferent pathway and Mel-mediated BP regulation in rats under physiological and hypertensive conditions. Mel (0.1, 0.3, and 1.0 mg/mL) was microinjected into the nodose ganglia (NG) of rats. We showed that Mel-induced reduction of mean arterial pressure in female rats was significantly greater than that in male and in ovariectomized rats under physiological condition. Consistently, the expression of Mel receptors (MTNRs) in the NG of female rats was significantly higher than that of males. In L-NAME-induced hypertensive and spontaneously hypertensive rat models, MTNRs were upregulated in males but downregulated in female models. Interestingly, Mel-induced BP reduction was found in male hypertensive models. In whole-cell recording from identified baroreceptor neurons (BRNs) in female rats, we found that Mel (0.1 µM) significantly increased the excitability of a female-specific subpopulation of Ah-type BRNs by increasing the Nav1.9 current density via a PKC-mediated pathway. Similar results were observed in baroreceptive neurons of the nucleus tractus solitarius, showing the facilitation of spontaneous and evoked excitatory post-synaptic currents in Ah-type neurons. Collectively, this study reveals the estrogen-dependent effect of Mel/MTNRs under physiological and hypertensive conditions is mainly mediated by Ah-type BRNs, which may provide new theoretical basis and strategies for the gender-specific anti-hypertensive treatment in clinical practice.

Bradykinin-mediated estrogen-dependent depressor response by direct activation of female-specific distribution of myelinated Ah-type baroreceptor neurons in rats.

AIM: To understand the direct impact of bradykinin in autonomic control of circulation through baroreflex afferent pathway. METHODS: The mean arterial pressure (MAP) was monitored while bradykinin and its agonists were applied via nodose (NG) microinjection, the expression of bradykinin receptors (BRs) in the NG (1st -order) and nucleus tractus solitarius (NTS, 2nd -order) were tested in adult male, age-matched female, and ovariectomized rats under physiological and hypertensive conditions. Additionally, bradykinin-induced depolarization was also tested in identified baroreceptor and baroreceptive neurons using whole-cell patch-clamp technique. RESULTS: Under physiological condition, bradykinin-induced dose- and estrogen-dependent reductions of MAP with lower estimated EC50 in females. B2 R agonist mediated more dramatic MAP reduction with long-lasting effect compared with B1 R activation. These functional observations were consistent with the molecular and immunostaining evidences. However, under hypertensive condition, the MAP reduction was significantly less dramatic in N' -Nitro-L-Arginine-methyl ester (L-NAME) induced secondary and spontaneous hypertension rats in males compared with female rats. Electrophysiological data showed that bradykinin-elicited concentration-dependent membrane depolarization with discharges during initial phase in identified myelinated Ah-types baroreceptor neurons, not myelinated A-types; while, higher concentration of bradykinin was required for depolarization of unmyelinated C-types without initial discharges. CONCLUSION: These datasets have demonstrated for the first time that bradykinin mediates direct activation of baroreflex afferent function to trigger estrogen-dependent depressor response, which is due mainly to the direct activation/neuroexcitation of female-specific myelinated Ah-type baroreceptor neurons leading to a sexual dimorphism in parasympathetic domination of blood pressure regulation via activation of B2 R/B1 R expression in baroreflex afferent pathway.

Estrogen-dependent KCa1.1 modulation is essential for retaining neuroexcitation of female-specific subpopulation of myelinated Ah-type baroreceptor neurons in rats.

Female-specific subpopulation of myelinated Ah-type baroreceptor neurons (BRNs) in nodose ganglia is the neuroanatomical base of sexual-dimorphic autonomic control of blood pressure regulation, and KCa1.1 is a key player in modulating the neuroexcitation in nodose ganglia. In this study we investigated the exact mechanisms underlying KCa1.1-mediated neuroexcitation of myelinated Ah-type BRNs in the presence or absence of estrogen. BRNs were isolated from adult ovary intact (OVI) or ovariectomized (OVX) female rats, and identified electrophysiologically and fluorescently. Action potential (AP) and potassium currents were recorded using whole-cell recording. Consistently, myelinated Ah-type BRNs displayed a characteristic discharge pattern and significantly reduced excitability after OVX with narrowed AP duration and faster repolarization largely due to an upregulated iberiotoxin (IbTX)-sensitive component; the changes in AP waveform and repetitive discharge of Ah-types from OVX female rats were reversed by G1 (a selective agonist for estrogen membrane receptor GPR30, 100 nM) and/or IbTX (100 nM). In addition, the effect of G1 on repetitive discharge could be completely blocked by G15 (a selective antagonist for estrogen membrane receptor GPR30, 3 µM). These data suggest that estrogen deficiency by removing ovaries upregulates KCa1.1 channel protein in Ah-type BRNs, and subsequently increases AP repolarization and blunts neuroexcitation through estrogen membrane receptor signaling. Intriguingly, this upregulated KCa1.1 predicted electrophysiologically was confirmed by increased mean fluorescent intensity that was abolished by estrogen treatment. These electrophysiological findings combined with immunostaining and pharmacological manipulations reveal the crucial role of KCa1.1 in modulation of neuroexcitation especially in female-specific subpopulation of myelinated Ah-type BRNs and extend our current understanding of sexual dimorphism of neurocontrol of BP regulation.

Parkinson-like early autonomic dysfunction induced by vagal application of DOPAL in rats.

AIM: To understand why autonomic failures, a common non-motor symptom of Parkinson's disease (PD), occur earlier than typical motor disorders. METHODS: Vagal application of DOPAL (3,4-dihydroxyphenylacetaldehyde) to simulate PD-like autonomic dysfunction and understand the connection between PD and cardiovascular dysfunction. Molecular and morphological approaches were employed to test the time-dependent alternation of α-synuclein aggregation and the ultrastructure changes in the heart and nodose (NG)/nucleus tractus solitarius (NTS). RESULTS: Blood pressure (BP) and baroreflex sensitivity of DOPAL-treated rats were significantly reduced accompanied with a time-dependent change in orthostatic BP, consistent with altered echocardiography and cardiomyocyte mitochondrial ultrastructure. Notably, time-dependent and collaborated changes in Mon-/Tri-α-synuclein were paralleled with morphological alternation in the NG and NTS. CONCLUSION: These all demonstrate that early autonomic dysfunction mediated by vagal application of DOPAL highly suggests the plausible etiology of PD initiated from peripheral, rather than central site. It will provide a scientific basis for the prevention and early diagnosis of PD.

Estrogen-dependent MicroRNA-504 Expression and Related Baroreflex Afferent Neuroexcitation via Negative Regulation on KCNMB4 and KCa1.1 ß4-subunit Expression.

Large conductance of Ca2+-activated K+ channel (KCa1.1) plays an inhibitory role in neuroexcitation. However, the expression of KCNMB4/ß4-subunit in the nodose ganglia (NG) and nucleus tractus solitarius (NTS), and its effect and regulation on baroreflex afferent function at post-transcriptional level of female rats remains unknown. Here, we demonstrated that the expression of ß4-subunit encoded by KCNMB4 was significantly lower in females vs. males and ovariectomized (OVX) rats in the NG. Although all baroreceptor neurons (BRNs) expressed ß4-subunit, altered discharge characteristics were only observed in Ah-type neurons after ovariectomy. Notably, the decreased excitability of Ah-types was restored by paxilline and further enhanced by iberiotoxin. The consistent changes were observed in excitatory post-synaptic currents. The level of miR-504 was higher in females, which was predicted to bind to the 3'UTR of KCNMB4. In consistent, an inverse expression pattern between miR-504 and KCNMB4 was observed in baroreflex afferents. The paxilline-sensitive ß4-subunits is less in Ah-types and up-regulated by ovariectomy. These data indicated that KCa1.1 ß4-subunit is the key regulator in neuroexcitation of Ah-types and sexual-dimorphism in baroreflex afferent function through estrogen-dependent inhibition of KCNMB4 expression via miR-504.