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We aimed to analyze whether opisthenar microvessel area (OMA, measured with Optical Coherence Tomography (OCT) angiography) was associated with blood pressure (BP), arterial stiffness and whether OMA can predict arterial stiffness in hypertensive (HTN) patients. Results from 90 participants showed that BP, brachial-ankle pulse wave velocity (baPWV) and ankle brachial index (ABI) were significantly higher but OMA (in control, with cold- and warm-stimulation, NT, CST, HST and the differences, CSD, HSD) were significantly reduced in HTN group (n = 36) compared to non-HTN (n = 54). NT, CST, HST and HSD showed negative correlations with baPWV and ABI in all participants, female (n = 47) and male group (n = 43), but the correlation was absent when the participants were divided into HTN and non-HTN. Logistic Regression analysis showed that only baPWV was a significant risk factor for HSD (OR 19.7, 95%CI 4.959–78.733, p p > 0.05). Receiver Operating Characteristics analysis for HSD was 0.781, 0.804, 0.770, respectively. HSD 2 predicted high BP and arterial stiffness (95% CI in all participants: baPWV, 0.681–0.881, SBP, 0.709–0.900, DBP, 0.672–0.867, p

Nitric oxide (NO) affects mitochondrial activity through its interactions with complexes. Here, we investigated regulations of complex I (C-I) and complex II (C-II) by neuronal NO synthase (nNOS) in the presence of fatty acid supplementation and the impact on left ventricular (LV) mitochondrial activity from sham and angiotensin II (Ang-II)–induced hypertensive (HTN) rats. Our results showed that nNOS protein was expressed in sham and HTN LV mitochondrial enriched fraction. In sham, oxygen consumption rate (OCR) and intracellular ATP were increased by palmitic acid (PA) or palmitoyl-carnitine (PC). nNOS inhibitor, S-methyl-l-thiocitrulline (SMTC), did not affect OCR or cellular ATP increment by PA or PC. However, SMTC increased OCR with PA + malonate (a C-II inhibitor), but not with PA + rotenone (a C-I inhibitor), indicating that nNOS attenuates C-I with fatty acid supplementation. Indeed, SMTC increased C-I activity but not that of C-II. Conversely, nNOS-derived NO was increased by rotenone + PA in LV myocytes. In HTN, PC increased the activity of C-I but reduced that of C-II, consequently OCR was reduced. SMTC increased both C-I and C-II activities with PC, resulted in OCR enhancement in the mitochondria. Notably, SMTC increased OCR only with rotenone, suggesting that nNOS modulates C-II-mediated OCR in HTN. nNOS-derived NO was partially reduced by malonate + PA. Taken together, nNOS attenuates C-I-mediated mitochondrial OCR in the presence of fatty acid in sham and C-I modulates nNOS activity. In HTN, nNOS attenuates C-I and C-II activities whereas interactions between nNOS and C-II maintain mitochondrial activity.

Dilated cardiomyopathy (DCM) is clinically characterized by dilated ventricular cavities and reduced ejection fraction, leading to heart failure and increased thromboembolic risk. Mutations in thin-filament regulatory proteins can cause DCM and have been shown in vitro to reduce contractility and myofilament Ca2+-affinity. In this work we have studied the functional consequences of mutations in cardiac troponin T (R131W), cardiac troponin I (K36Q) and α-tropomyosin (E40K) using adenovirally transduced isolated guinea pig left ventricular cardiomyocytes. We find significantly reduced fractional shortening with reduced systolic Ca2+. Contraction and Ca2+ reuptake times were slowed, which contrast with some findings in murine models of myofilament Ca2+ desensitization. We also observe increased sarcoplasmic reticulum (SR) Ca2+ load and smaller fractional SR Ca2+ release. This corresponds to a reduction in SR Ca2+-ATPase activity and increase in sodium-calcium exchanger activity. We also observe dephosphorylation and nuclear translocation of the nuclear factor of activated T cells (NFAT), with concordant RAC-α-serine/threonine protein kinase (Akt) phosphorylation but no change to extracellular signal-regulated kinase activation in chronically paced cardiomyocytes expressing DCM mutations. These changes in Ca2+ handling and signaling are common to all three mutations, indicating an analogous pathway of disease pathogenesis in thin-filament sarcomeric DCM. Previous work has shown that changes to myofilament Ca2+ sensitivity caused by DCM mutations are qualitatively opposite from hypertrophic cardiomyopathy (HCM) mutations in the same genes. However, we find several common pathways such as increased relaxation times and NFAT activation that are also hallmarks of HCM. This suggests more complex intracellular signaling underpinning DCM, driven by the primary mutation.NEW & NOTEWORTHY Dilated cardiomyopathy (DCM) is a frequently occurring cardiac disorder with a degree of genetic inheritance. We have found that DCM mutations in proteins that regulate the contractile machinery cause alterations to contraction, calcium-handling, and some new signaling pathways that provide stimuli for disease development. We have used guinea pig cells that recapitulate human calcium-handling and introduced the mutations using adenovirus gene transduction to look at the initial triggers of disease before remodeling.

Background and aims: Hepatitis B virus-associated acute-on-chronic liver failure (HBV-ACLF) is a complicated syndrome with extremely high short-term mortality. Whether plasma exchange (PE) improves HBV-ACLF outcomes remains controversial. Here, PE-based non-bioartificial liver support system (NB-ALSS) effects on short-term HBV-ACLF patient outcomes were investigated. Materials and methods: HBV-ACLF patients from Chinese Acute-on-chronic Liver Failure (CATCH-LIFE) cohort receiving standard medical therapy (SMT) alone or PE-based NB-ALSS in addition to SMT were allocated to SMT and SMT+PE groups, respectively; propensity score matching (PSM) was used to eliminate confounding bias. Short-term (28/90-day and 1-year) survival rates were calculated (Kaplan-Meier). Results: In total, 524 patients with HBV-ACLF were enrolled in this study; 358 received SMT alone (SMT group), and the remaining 166 received PE-based NB-ALSS in addition to SMT (SMT+PE group). PSM generated 166 pairs of cases. In the SMT+PE group, 28-day, 90-day, and 1-year survival rates were 11.90, 8.00, and 10.90%, respectively, higher than those in the SMT group. Subgroup analysis revealed that PE-based NB-ALSS had the best efficacy in patients with ACLF grade 2 or MELD scores of 30–40 (MELD grade 3). In MELD grade 3 patients who received SMT+PE, 28-day, 90-day, and 1-year survival rates were improved by 18.60, 14.20, and 20.10%, respectively. According to multivariate Cox regression analysis, PE-based NB-ALSS was the only independent protective factor for HBV-ACLF patient prognosis at 28 days, 90 days, and 1 year (28 days, HR = 0.516, p = 0.001; 90 days, HR = 0.663, p = 0.010; 1 year, HR = 0.610, p = 0.051). For those who received SMT+PE therapy, PE-based NB-ALSS therapy frequency was the only independent protective factor for short-term prognosis (28-day, HR = 0.597, p = 0.001; 90-day, HR = 0.772, p = 0.018). Conclusions: This multicenter prospective study showed that the addition of PE-based NB-ALSS to SMT improves short-term (28/90 days and 1-year) outcomes in patients with HBV-ACLF, especially in MELD grade 3 patients. Optimization of PE-based NB-ALSS may improve prognosis or even save lives among HBV-ACLF patients.

Recently, neutrophil‐to‐lymphocyte ratio (NLR) and monocyte‐to‐lymphocyte ratio (MLR) are frequently used to evaluate disease progression and outcome. Here, we aim to analyze the associations between NLR or MLR and kidney function in undiagnosed hypertensive individuals from general population during routine health checkup. Liver function was analyzed for comparison. From 2011 to 2016, 53 939 examiners have registered for health checkup in Yanbian University Hospital, Yanbian, China. Among 15 219 participants who have complete datasets, 4997 individuals were hypertensive (HTN, SBP/DBP: ≥ 140/90 mm Hg). NLR, glucose, lipids (Chol, TG, LDL), kidney (CREA, BUN), and liver (AST, ALT, GGT, ALB, TBIL) functional parameters were significantly higher in HTN. Pearman correlation analysis showed that NLR was positively correlated with SBP and CREA only in HTN. MLR was associated with CREA in both HTN and non‐HTN. NLR or MLR was associated with liver functions similarly in HTN and non‐HTN. The authors then divided NLR or MLR into tertiles (NLR: 0‐1.7276, 1.7276‐3, >3; MLR: 0‐0.1845, 0.1845‐0.3, >0.3). NLR was positively associated with BUN at NLR >1.7276 and with CREA at all tertiles in HTN. MLR was correlated with CREA and BUN at high MLR in non‐HTN. Further analysis showed that age or gender did not affect the associations of NLR and MLR with kidney function in HTN, but strong association was observed in male or aged (>65 years old) non‐HTN group. These results showed that NLR could be used as a cost‐effective predictor of kidney abnormality in HTN patients even in a general population.

Atrophic limbs exhibit decreased blood flow and histological changes in the arteries perfusing muscles. However, the effect of atrophy on vascular smooth muscle function is poorly understood. Here, we investigated the effect of unilateral sciatic denervation on the myogenic response (MR) and the ionic currents in deep femoral artery (DFA) smooth muscles from Sprague-Dawley rats. Because denervated rats were capable of treadmill exercise (20 m/min, 30 min, 3 times/wk), the impact of exercise training on these effects was also assessed. Skeletal arteries were harvested 3 or 5 wk after surgery. Then skeletal arteries or myocytes were subjected to video analysis of pressurized artery, myography, whole-cell patch clamp, and real-time quantitative PCR to determine the effect of hindlimb paralysis in the presence/absence of exercise training on MR, contractility, ionic currents, and channel transcription, respectively. In sedentary rats, atrophy was associated with loss of MR in the DFA at 5 wk. The contralateral DFA had a normal MR. At 5 wk after surgery, DFA myocytes from the atrophic limbs exhibited depressed L-type Ca2+currents, GTPγS-induced transient receptor potential cation channel (TRPC)-like currents, 80 mM KCl-induced vasoconstriction, TRPC6 mRNA, and voltage-gated K+and inwardly rectifying K+currents. Exercise training abrogated the differences in all of these functions between atrophic side and contralateral side DFA myocytes. These results suggest that a probable increase in hemodynamic stimuli in skeletal artery smooth muscle plays an important role in maintaining MR and ionic currents in skeletal artery smooth muscle. This may also explain the observed benefits of exercise in patients with limb paralysis.NEW & NOTEWORTHY Myogenic responses (MRs) in rat skeletal arteries feeding the unilateral atrophic hindlimb were impaired. In addition, the L-type Ca2+channel current, the TRPC6-like current, and TRPC6 mRNA levels in the corresponding myocytes decreased. Voltage-gated K+channel currents and inwardly rectifying K+channel currents were also attenuated in atrophic side myocytes. Exercise training effectively abrogated electrophysiological dysfunction of atrophic side myocytes and prevented loss of the MR.

Mitochondrial dysfunction is a feature of type I and type II diabetes, but there is a lack of consistency between reports and links to disease development. We aimed to investigate if mitochondrial structure–function remodelling occurs in the early stages of diabetes by employing a mouse model (GENA348) of Maturity Onset Diabetes in the Young, exhibiting hyperglycemia, but not hyperinsulinemia, with mild left ventricular dysfunction. Employing 3-D electron microscopy (SBF-SEM) we determined that compared to wild-type, WT, the GENA348 subsarcolemma mitochondria (SSM) are ~ 2-fold larger, consistent with up-regulation of fusion proteins Mfn1, Mfn2 and Opa1. Further, in comparison, GENA348 mitochondria are more irregular in shape, have more tubular projections with SSM projections being longer and wider. Mitochondrial density is also increased in the GENA348 myocardium consistent with up-regulation of PGC1-α and stalled mitophagy (down-regulation of PINK1, Parkin and Miro1). GENA348 mitochondria have more irregular cristae arrangements but cristae dimensions and density are similar to WT. GENA348 Complex activity (I, II, IV, V) activity is decreased but the OCR is increased, potentially linked to a shift towards fatty acid oxidation due to impaired glycolysis. These novel data reveal that dysregulated mitochondrial morphology, dynamics and function develop in the early stages of diabetes.

An increasing number of epidemiological studies have suggested that birth weight (BW) may be a determinant of bone health later in life, although the underlying genetic mechanism remains unclear. Here, we applied a pleiotropic conditional false discovery rate (cFDR) approach to the genome-wide association study (GWAS) summary statistics for lumbar spine bone mineral density (LS BMD) and BW, aiming to identify novel susceptibility variants shared between these two traits. We detected 5 novel potential pleiotropic loci which are located at or near 7 different genes (NTAN1, PDXDC1, CACNA1G, JAG1, FAT1P1, CCDC170, ESR1), among which PDXDC1 and FAT1P1 have not previously been linked to these phenotypes. To partially validate the findings, we demonstrated that the expression of PDXDC1 was dramatically reduced in ovariectomized (OVX) mice in comparison with sham-operated (SHAM) mice in both the growth plate and trabecula bone. Furthermore, immunohistochemistry assay with serial sections showed that both osteoclasts and osteoblasts express PDXDC1, supporting its potential role in bone metabolism. In conclusion, our study provides insights into some shared genetic mechanisms for BMD and BW as well as a novel potential therapeutic target for the prevention of OP in the early stages of the disease development.

Context Although metabolic profiles appear to play an important role in menopausal bone loss, the functional mechanisms by which metabolites influence bone mineral density (BMD) during menopause are largely unknown. Objective We aimed to systematically identify metabolites associated with BMD variation and their potential functional mechanisms in peri- and postmenopausal women. Design and Methods We performed serum metabolomic profiling and whole-genome sequencing for 517 perimenopausal (16%) and early postmenopausal (84%) women aged 41 to 64 years in this cross-sectional study. Partial least squares regression and general linear regression analysis were applied to identify BMD-associated metabolites, and weighted gene co-expression network analysis was performed to construct co-functional metabolite modules. Furthermore, we performed Mendelian randomization analysis to identify causal relationships between BMD-associated metabolites and BMD variation. Finally, we explored the effects of a novel prominent BMD-associated metabolite on bone metabolism through both in vivo/in vitro experiments. Results Twenty metabolites and a co-functional metabolite module (consisting of fatty acids) were significantly associated with BMD variation. We found dodecanoic acid (DA), within the identified module causally decreased total hip BMD. Subsequently, the in vivo experiments might support that dietary supplementation with DA could promote bone loss, as well as increase the osteoblast and osteoclast numbers in normal/ovariectomized mice. Dodecanoic acid treatment differentially promoted osteoblast and osteoclast differentiation, especially for osteoclast differentiation at higher concentrations in vitro (eg,10, 100 μM). Conclusions This study sheds light on metabolomic profiles associated with postmenopausal osteoporosis risk, highlighting the potential importance of fatty acids, as exemplified by DA, in regulating BMD.

Although gut microbiota influences osteoporosis risk, the individual species involved, and underlying mechanisms, are unknown. We performed integrative analyses in a Chinese cohort with metagenomics/targeted metabolomics/whole-genome sequencing. Bacteroides vulgatus was found negatively associated with bone mineral density (BMD), this association was validated in US Caucasians. Serum valeric acid was positively associated with BMD, and B.vulgatus causally downregulated it. Ovariectomized mice fed B.vulgatus had decreased bone formation and increased bone resorption, lower BMD and poorer bone micro-structure. Valeric acid suppressed NF-κB p65 protein production (pro-inflammatory), and enhanced IL-10 mRNA expression (anti-inflammatory), leading to suppressed maturation of osteoclast-like cells, and enhanced maturation of osteoblasts in vitro. B.vulgatus and valeric acid represent promising targets for osteoporosis prevention/treatment.

Oxidative stress under pathological conditions, such as ischemia/reperfusion and inflammation, results in the production of various reactive chemicals. Of these chemicals, 4-hydroxynonenal (4-HNE), a peroxidation product of ω6-polyunsaturated fatty acid, has garnered significant attention. However, the effect of 4-HNE on cardiac electrophysiology has not yet been reported. In the present study, we investigated the effects of 4-HNE on several cardiac ion channels, including human ether-a-go-go-related (hERG) channels, using the whole-cell patch clamp technique. Short-term exposure to 100 μM 4-HNE (4-HNE100S), which mimics local levels under oxidative stress, decreased the amplitudes of rapidly activating delayed rectifier K+ current (IKr) in guinea pig ventricular myocytes (GPVMs) and HEK293T cells overexpressing hERG (IhERG). MS analysis revealed the formation of 4-HNE-hERG adduct on specific amino acid residues, including C276, K595, H70, and H687. Long-term treatment (1–3 h) with 10 μM 4-HNE (4-HNE10L), suppressed IKr and IhERG, but not IKs and ICa,L. Action potential duration (APD) of GPVMs was prolonged by 37% and 64% by 4-HNE100S and 4-HNE10L, respectively. Western blot analysis using surface biotinylation revealed a reduction in mature membrane hERG protein after treatment with 4-HNE10L. Proteasomal degradation inhibitors, such as bortezomib, prevented the 4-HNE10L-induced decrease in mature hERG, suggesting a retrograde degradation of membrane hERG due to 4-HNE. Taken together, 4-HNE100S and 4-HNE10L suppressed IhERG via functional inhibition and downregulation of membrane expression of hERG, respectively. The exposure of 4-HNE under pathological oxidative stress may increase the risk of proarrhythmic events via APD prolongation. Keywords: Lipid peroxidant, 4-hydroxynonenal, hERG channel, Cardiac action potential prolongation

Cardiac neuronal nitric oxide synthase (nNOS) is an important molecule that regulates intracellular Ca2+ homeostasis and contractility of healthy and diseased hearts. Here, we examined the effects of nNOS on fatty acid (FA) regulation of left ventricular (LV) myocyte contraction in sham and angiotensin II (Ang II)-induced hypertensive (HTN) rats. Our results showed that palmitic acid (PA, 100 μM) increased the amplitudes of sarcomere shortening and intracellular ATP in sham but not in HTN despite oxygen consumption rate (OCR) was increased by PA in both groups. Carnitine palmitoyltransferase-1 inhibitor, etomoxir (ETO), reduced OCR and ATP with PA in sham and HTN but prevented PA potentiation of sarcomere shortening only in sham. PA increased nNOS-derived NO only in HTN. Inhibition of nNOS with S-methyl-l-thiocitrulline (SMTC) prevented PA-induced OCR and restored PA potentiation of myocyte contraction in HTN. Mechanistically, PA increased intracellular Ca2+ transient ([Ca2+]i) without changing Ca2+ influx via L-type Ca2+ channel (I-LTCC) and reduced myofilament Ca2+ sensitivity in sham. nNOS inhibition increased [Ca2+]i, I-LTCC and reduced myofilament Ca2+ sensitivity prior to PA supplementation; as such, normalized PA increment of [Ca2+]i. In HTN, PA reduced I-LTCC without affecting [Ca2+]i or myofilament Ca2+ sensitivity. However, PA increased I-LTCC, [Ca2+]i and reduced myofilament Ca2+ sensitivity following nNOS inhibition. Myocardial FA oxidation (18F-fluoro-6-thia-heptadecanoic acid, 18F-FTHA) was comparable between groups, but nNOS inhibition increased it only in HTN. Collectively, PA increases myocyte contraction through stimulating [Ca2+]i and mitochondrial activity in healthy hearts. PA-dependent cardiac inotropy was limited by nNOS in HTN, predominantly due to its modulatory effect on [Ca2+]i handling.

Background The combined effects of diabetes mellitus (DM), admission plasma glucose (APG), and glycated hemoglobin (HbA1c) levels on predicting long-term clinical outcomes in patients with ST-segment elevation myocardial infarction (STEMI) undergoing primary percutaneous coronary intervention (pPCI) are unknown. Therefore, we evaluated their combined effects on long-term clinical outcomes in STEMI patients treated with pPCI. Methods In total, 350 consecutive patients with STEMI undergoing pPCI were enrolled. Patients were divided into 3 groups according to DM history and APG and HbA1c levels. The cumulative rates of 24-month all-cause deaths and major adverse cardiac and cerebrovascular events (MACCEs) were calculated. Results Both the incidence of all-cause deaths and cumulative rates of MACCEs were significantly the lowest in patients without a DM history and admission HbA1c level 1 correlated with 24-month all-cause death; HbA1c levels on admission, DM history, APG levels, history of stroke, history of coronary heart disease, and TG levels on admission were significantly associated with MACCEs through the 24-month follow-up. The predictive effects of combining DM and APG and HbA1c levels were such that for STEMI patients undergoing pPCI, DM patients with poor glycemic control or with stress hyperglycemia on admission had worse prognosis than other patients. Conclusion Strict control of glycemic status may improve the survival of patients who have both DM and coronary heart diseases.

Recent evidence suggests that mitochondrial complex II is an essential mediator of myocardial ischemia-reperfusion injury. The present study aimed to investigate the effects of fatty acid supplementation or high-fat diet (HFD) on cardiac mitochondrial activity. The changes of complex I and complex II activities and mitochondrial oxygen consumption rate (OCR) following hypoxia and re-oxygenation under these conditions were studied. Our results have shown that OCR (mitochondrial activity) was significantly increased with palmitoylcarnitine supplementation in mitochondria-enriched fraction from C57BL/6 mice hearts. Mitochondrial complex I activity was unaffected by palmitoylcarnitine but complex II activity was enhanced. Re-oxygenation following 30-min hypoxia transiently increased OCR but such an effect on OCR was abolished by complex II inhibitor, malonate, but not by complex I inhibitor, rotenone, despite that complex I activity was significantly increased with re-oxygenation following hypoxia in the presence of palmitoylcarnitine. Furthermore, OCR and complex II activity were significantly increased in the mitochondria from high-fat diet mice heart compared with those of normal or low-fat diet mice. Re-oxygenation to mitochondria following 30-min hypoxia increased OCR in all three groups but significantly more in HFD. Malonate abolished re-oxygenation-induced OCR increment in all groups. Our results indicate that complex II activity and OCR are enhanced with palmitoylcarnitine or in HFD mice heart. Although re-oxygenation following hypoxia enhanced complex II and complex I activities, complex II plays an important role in increasing mitochondrial activity, which may be instrumental in myocardial injury following ischemic reperfusion.

Left ventricle (LV) and right ventricle (RV) have distinctive structural and functional characteristics as well as heterogeneous physiological properties. The RV is exposed to a relatively low pulmonary vasculature, which results in less mechanical afterload. Consistent with these physiological differences, the RV has a thinner free wall than the LV, and the movement of its contraction is geometrically different. Despite these definite differences, the studies of basic excitation-contraction coupling and calcium homeostasis of RV has been less studied than in LV. To establish the interventricular difference, we evaluated the basic electrophysiological and calcium-contractile properties of myocyte with or without β-adrenergic stimulation. Analyses of contraction and Ca 2+ signaling and action potential duration (APD) in isolated RV myocytes showed more prominent APD prolongation with less significant changes in sarcomere shortening and calcium transient, implying less efficient E-C coupling RV myocytes. To investigate the prolonged APD of RV, we measured transient outward potassium (Ito) current and L-type calcium channel (LTCC) current of two ventricles. Although the peak current of two ion channel was not different significantly in two ventricle, the half inactivation voltage of RV Ito was smaller than LV. Comparing with LV, RV myocytes showed round peak, slower early relaxation, and faster late relaxation, suggesting the difference of calcium sensitivity between two ventricles. To investigate the difference, we examined the expression level of calcium-binding proteins that regulate myofilament activities. Using immunoblotting from enriched protein of myofilament fraction, we found that the calcium binding proteins such as troponin I were lower in RV. Taken together, our results suggest that calcium binding proteins of RV was differ from that of LV, which induce the modified calcium sensitivity. We confirmed that reduced calcium binding proteins induced the decrease of calcium sensitivity and modified E-C coupling using Bers’ mathematical rat cardiomyocyte model. The calcium sensitivity of RV is a clue to explain the different physiological properties of the RV.

Angiotensin II (AngII) triggers a transient contraction of pulmonary arteries (PAs) followed by protracted desensitization. Based on the unconventional eNOS expression in PA smooth muscle cells (PASMCs), we hypothesized that activation of smooth muscle eNOS by AngII might be responsible for fast relaxation and tachyphylaxis. Using dual-wire myograph, mechanically endothelium-denuded rat PA [E(−)PA] showed AngII concentration–dependent transient contractions (ΔTAngII, 95% decay within 1 min), which were abolished by losartan (AT1R antagonist). Neither PD123319 (AT2R antagonist) nor A779 (MasR antagonist) affected ΔTAngII. When the vessels were pretreated with L-NAME (NOS inhibitor), ODQ (guanylate cyclase inhibitor), or KT5823 (PKG inhibitor), ΔTAngII of E(−)PA became larger and sustained, whereas nNOS or iNOS inhibitors had no such effect. Immunoblotting of human PASMCs (hPASMCs) also showed eNOS expression, and AngII treatment induced activating phosphorylations of Ser1177 in eNOS and of Ser473 in Akt (Ser/Thr protein kinase B), an upstream signal of eNOS phosphorylation. In addition, L-NAME co-treatment promoted AngII-induced Ser19 phosphorylation of myosin light chain. In hPASMCs, AngII abolished plasma membrane expression of AT1R, and recovery by washout took more than 1 h. Consistent with the data from hPASMCs, the second application of AngII to E(−)PA did not induce contraction, and significant recovery of ΔTAngII required prolonged washout (> 2 h) in the myography study. L-NAME treatment before the second application facilitated recovery of ΔTAngII. Muscular eNOS plays an auto-inhibitory role in ΔTAngII of PAs. The molecular changes investigated in hPASMCs revealed eNOS phosphorylation and internalization of AT1R by AngII. We propose that the rat PA smooth muscle eNOS-induced lusitropy and slow recovery of AT1R from tachyphylaxis might counterbalance the excessive contractile response to AngII, contributing to the distinctive low-pressure pulmonary circulation.

Fatty acid (FA)-dependent oxidation is the predominant process for energy supply in normal heart. Impaired FA metabolism and metabolic insufficiency underlie the failing of the myocardium. So far, FA metabolism in normal cardiac physiology and heart failure remains undetermined. Here, we evaluate the mechanisms of FA and major metabolic substrates (termed NF) on the contraction, relaxation, and Ca2+ handling in rat left ventricular (LV) myocytes. Our results showed that NF significantly increased myocyte contraction and facilitated relaxation. Moreover, NF increased the amplitudes of diastolic and systolic Ca2+ transients ([Ca2+]i), abbreviated time constant of [Ca2+]i decay (tau), and prolonged the peak duration of [Ca2+]i. Whole-cell patch-clamp experiments revealed that NF increased Ca2+ influx via L-type Ca2+ channels (LTCC, ICa-integral) and prolonged the action potential duration (APD). Further analysis revealed that NF shifted the relaxation phase of sarcomere lengthening vs. [Ca2+]i trajectory to the right and increased [Ca2+]i for 50 % of sarcomere relengthening (EC50), suggesting myofilament Ca2+ desensitization. Butanedione monoxime (BDM), a myosin ATPase inhibitor that reduces myofilament Ca2+ sensitivity, abolished the NF-induced enhancement of [Ca2+]i amplitude and the tau of [Ca2+]i decay, indicating the association of myofilament Ca2+ desensitization with the changes in [Ca2+]i profile in NF. NF reduced intracellular pH ([pHi]). Increasing [pH]i buffer capacity with HCO3/CO2 attenuated Δ [pH]i and reversed myofilament Ca2+ desensitization and Ca2+ handling in NF. Collectively, greater Ca2+ influx through LTCCs and myofilament Ca2+ desensitization, via reducing [pH]i, are likely responsible for the positive inotropic and lusitropic effects of NF. Computer simulation recapitulated the effects of NF. Electronic supplementary material The online version of this article (doi:10.1007/s00424-016-1892-8) contains supplementary material, which is available to authorized users.

The present study aimed to investigate the correlation between ultrasonographic features, basic fibroblast growth factor (bFGF), and the local invasiveness of papillary thyroid carcinoma (PTC). A total of 350 samples of thyroid nodules were collected. Routine ultrasonography was performed before the operation and routine pathological diagnosis and bFGF detection were performed after the operation.’ These 350 samples of thyroid nodules included 90 samples of nodular goiter, 36 samples of focal thyroiditis, and 224 samples of PTC. A total of 326 thyroid nodules were examined for bFGF. The results revealed that the difference in the expression of bFGF between the benign and malignant groups was statistically significant (P

Na(+)/Ca(2+) exchanger current (INCX) triggered by spontaneous Ca(2+) release from sarcoplasmic reticulum (SR) has been suggested as one of the cardiac pacemaker mechanisms ("Ca(2+) clock model"). In human embryonic stem cell-derived cardiomyocytes (hESC-CMs) showing spontaneous action potentials (APs), we found that substantial population (35 %) showed regular oscillation of inward currents (SICs) in nystatin-perforated voltage clamp between -40 and 40 mV (-80 ± 10.6 pA, at -20 mV). SICs were similarly observed between nodal, atrial, and ventricular hESC-CMs. Oscillations of [Ca(2+)]i synchronized with SICs were observed under voltage clamp. SICs were eliminated by lowering [Ca(2+)]e, L-type Ca(2+) channel (VOCCL) blocker (nifedipine, 10 µM), ryanodine receptor (RyR) agonist (caffeine, 10 mM), or NCX inhibitor (1 µM SN-6 and 10 µM KB-R7943). Plasma membrane expression of NCX1 was confirmed using immunofluorescence confocal microcopy. Both caffeine and SN-6 slowed the pacemaker potential but did not abolish the AP generation. The inhibitors of funny current (3 µM ivabradine) or voltage-gated K(+) channel currents (1 µM E4031 and 10 µM chromanol-293B) also did not abolish but slowed the pacemaker potential. In a computational model of cardiac pacemaker by Maltsev and Lakatta (2009), after modifying the spatial distribution of RyR, VOCCL, and NCX by using our multiparameter adjust algorithm, we could successfully reproduce spontaneous SR Ca(2+) release and SICs under voltage clamp. It was proposed that, under the membrane depolarization activating VOCCL, oscillatory Ca(2+) releases via RyR induce sharp increases in subsarcolemmal [Ca(2+)]i and inward INCX (SICs). Since the hESC-CMs without SICs still showed spontaneous APs, the putative "Ca(2+) clock" would provide a redundant pacemaker or augmenting mechanism in hESC-CMs.

Neuronal nitric oxide synthase (nNOS) is important in cardiac protection in diseased heart. Recently, we have reported that nNOS is associated with myofilament Ca(2+) desensitization in cardiac myocytes from hypertensive rats. So far, the effect of myofilament Ca(2+) desensitization or nNOS on L-type Ca(2+) channel activity (I(Ca)) in cardiac myocyte is unclear. Here, we examined nNOS regulation of I(Ca) in left ventricular (LV) myocytes from sham and angiotensin II (Ang II)-induced hypertensive rats. Our results showed that basal I(Ca) was not different between sham and hypertension (from -60 to +40 mV, 0.1 Hz). S-methyl-L-thiocitrulline (SMTC), a selective nNOS inhibitor, increased peak I(Ca) similarly in both groups. However, chelation of intracellular Ca(2+) [Ca(2+)]i with BAPTA increased I(Ca) and abolished SMTC-augmentation of I(Ca) only in hypertension. Myofilament Ca(2+) desensitization with butanedione monoxime (BDM), a myosin ATPase inhibitor, decreased I(Ca) in both groups but to a greater extent in hypertension. Intracellular BAPTA or nNOS inhibition reinstated I(Ca) in the presence of BDM to the basal level, suggesting Ca(2+)-dependent inactivation of I(Ca) by nNOS and greater vulnerability in hypertension. Increasing stimulation frequencies (2, 4 and 8 Hz) attenuated myofilament Ca(2+) sensitivity in sham and reduced peak ICa in both groups. Nevertheless, SMTC or BAPTA exerted no effect on I(Ca) at high frequencies in either group. These results suggest that nNOS attenuates I(Ca) via Ca(2+)-dependent mechanism and the vulnerability is greater in hypertension subject to myofilament Ca(2+) desensitization. nNOS or [Ca(2+)]i does not affect I(Ca) at high stimulation frequencies. The results were recapitulated with computer simulation.

Background: Neutrophil to lymphocyte ratio (NLR) in peripheral blood is established to correlate with the morbidity and mortality of heart disease patients. We aimed to define the severity of inflammation (NLR) by observing the association of NLR with cardiac functions or myocardial damage parameters in patients with acute myocardial infarction. Methods: Data from 715 patients who underwent percutaneous coronary intervention (PCI) within 72 hours of incidence in 2016 were analysed retrospectively. Results: The NLR ranges from 0.50 to 46 (medium ± SD, 2.76 ± 2.96) in 715 patients. NLR positively correlated with myocardial damage (NLR vs. CK-mB: p 2.76 compared to those of NLR 4. The correlation was diminished between NLR and CK-mB at NLR > 2.76 or at NLR > 4, but that of NLR and EF or FS was maintained in NLR > 2.76 and at NLR > 4. EF and FS were comparable between NLR > 2.76 and NLR > 4. But myocardial damage parameters increased significantly at NLR > 4 compared to those of NLR > 2.76. Conclusion: NLR is a strong predictor of myocardial damage in acute myocardial patients. High NLR are associated with myocardial dysfunction in all the patients. Severe inflammation (NLR) can predict the consequence of the heart in patients with coronary syndrome. Keywords: Neutrophil, Lymphocyte, Acute myocardial infarction, Myocardial damage, Dysfunction

Nitric oxide (NO) is an imperative regulator of the cardiovascular system and is a critical mechanism in preventing the pathogenesis and progression of the diseased heart. The scenario of bioavailable NO in the myocardium is complex: 1) NO is derived from both endogenous NO synthases (endothelial, neuronal, and/or inducible NOSs [eNOS, nNOS, and/or iNOS]) and exogenous sources (entero-salivary NO pathway) and the amount of NO from exogenous sources varies significantly; 2) NOSs are located at discrete compartments of cardiac myocytes and are regulated by distinctive mechanisms under stress; 3) NO regulates diverse target proteins through different modes of post-transcriptional modification (soluble guanylate cyclase [sGC]/cyclic guanosine monophosphate [cGMP]/protein kinase G [PKG]-dependent phosphorylation, S-nitrosylation, and transnitrosylation); 4) the downstream effectors of NO are multidimensional and vary from ion channels in the plasma membrane to signalling proteins and enzymes in the mitochondria, cytosol, nucleus, and myofilament; 5) NOS produces several radicals in addition to NO (e.g. superoxide, hydrogen peroxide, peroxynitrite, and different NO-related derivatives) and triggers redox-dependent responses. However, nNOS inhibits cardiac oxidases to reduce the sources of oxidative stress in diseased hearts. Recent consensus indicates the importance of nNOS protein in cardiac protection under pathological stress. In addition, a dietary regime with high nitrate intake from fruit and vegetables together with unsaturated fatty acids is strongly associated with reduced cardiovascular events. Collectively, NO-dependent mechanisms in healthy and diseased hearts are better understood and shed light on the therapeutic prospects for NO and NOSs in clinical applications for fatal human heart diseases.

Neuronal nitric oxide synthase (nNOS or NOS1) is the major endogenous source of myocardial nitric oxide (NO), which facilitates cardiac relaxation and modulates contraction. In the healthy heart it regulates intracellular Ca2+, signalling pathways and oxidative homeostasis and is upregulated from early phases upon pathogenic insult. nNOS plays pivotal roles in protecting the myocardium from increased oxidative stress, systolic/diastolic dysfunction, adverse structural remodelling and arrhythmias in the failing heart. Here, we show that the downstream target proteins of nNOS and underlying post-transcriptional modifications are shifted during disease progression from Ca2+-handling proteins [e.g. PKA-dependent phospholamban phosphorylation (PLN-Ser16)] in the healthy heart to cGMP/PKG-dependent PLN-Ser16 with acute angiotensin II (Ang II) treatment. In early hypertension, nNOS-derived NO is involved in increases of cGMP/PKG-dependent troponin I (TnI-Ser23/24) and cardiac myosin binding protein C (cMBP-C-Ser273). However, nNOS-derived NO is shown to increase S-nitrosylation of various Ca2+-handling proteins in failing myocardium. The spatial compartmentation of nNOS and its translocation for diverse binding partners in the diseased heart or various nNOS splicing variants and regulation in response to pathological stress may be responsible for varied underlying mechanisms and functions. In this review, we endeavour to outline recent advances in knowledge of the molecular mechanisms mediating the functions of nNOS in the myocardium in both normal and diseased hearts. Insights into nNOS gene regulation in various tissues are discussed. Overall, nNOS is an important cardiac protector in the diseased heart. The dynamic localization and various mediating mechanisms of nNOS ensure that it is able to regulate functions effectively in the heart under stress.

S-palmitoylation is an important post-translational modification that affects the translocation and the activity of target proteins in a variety of cell types including cardiomyocytes. Since endothelial nitric oxide synthase (eNOS) is known to be palmitoylated and the activity of eNOS is essential in fatty acid-dependent β-oxidation in muscle, we aimed to test whether palmitoylation of eNOS is involved in palmitic acid (PA) regulation of left ventricular (LV) myocyte contraction from healthy (sham) and hypertensive (HTN) rats. Our results showed that PA, a predominant metabolic substrate for cardiac β-oxidation, significantly increased contraction and oxygen consumption rate (OCR) in LV myocytes from sham. Nω-nitro-L-arginine methyl ester hydrochloride (L-NAME) or eNOS gene deletion prevented PA regulation of the myocyte contraction or OCR, indicating the pivotal role of eNOS in mediating the effects of PA in cardiac myocytes. PA increased the palmitoylation of eNOS in LV myocytes and depalmitoylation with 2-bromopalmitate (2BP; 100 μM) abolished the increment. Furthermore, although PA did not increase eNOS-Ser1177, 2BP reduced eNOS-Ser1177 with and without PA. Intriguingly, PA-induced increases in contraction and OCR were unaffected by 2BP treatment. In HTN, PA did not affect eNOS palmitoylation, eNOS-Ser1177, or myocyte contraction. However, 2BP diminished eNOS palmitoylation and eNOS-Ser1177 in the presence and absence of PA but did not change myocyte contraction. Collectively, our results confirm eNOS palmitoylation in LV myocytes from sham and HTN rats and its upregulation by PA in sham. However, such post-transcriptional modification plays negligible role in PA regulation of myocyte contraction and mitochondrial activity in sham and HTN.

Human ether-á-go-go-related gene (hERG) K(+) channel current (I hERG ) is inhibited by various compounds and genetic mutations, potentially resulting in cardiac arrhythmia. Here, we investigated effects of caffeic acid phenethyl ester (CAPE) and curcumin, two natural anti-inflammatory polyphenols, on I hERG in HEK-293 cells overexpressed with hERG. CAPE dose-dependently decreased repolarization tail current of hERG (I hERG,tail; IC50, 10.6 ± 0.5 μM). CAPE also shifted half-activation voltage (V 1/2) to the left (from -17.5 to -26.5 mV) and accelerated activation and inactivation kinetics. The CAPE inhibition of I hERG,tail was not attenuated in the pore-blocker site mutants of hERG (Y652A and F656A). A point mutation of Cys723 (C723S) mimicked the effects of CAPE and caused a left shift of V 1/2 and acceleration of I hERG,tail deactivation. However, I hERG,tail inhibition by CAPE was still observed in C723S. Taken together, CAPE inhibits hERG channel by class 3 mechanism, i.e., modification of gating, not by blocking the pore. Curcumin induced changes of I hERG similar to those of CAPE, while additional interaction with pore-blocking sites was suggested from attenuated I hERG,tail inhibition in Y652A and F656A. Interestingly, I hERG induced by human action potential voltage clamp was increased by CAPE while decreased by curcumin. Mathematical simulation of action potential derived from the experimental results of CAPE and curcumin supports that CAPE, but not curcumin, would induce shortening of AP duration by facilitation of I hERG . The above results revealed intriguing roles of Cys723 in hERG kinetics and suggested that conventional drug screening by using step pulse protocol for I hERG,tail would overlook the hERG kinetic modulations that could compensate the decrease of I hERG,tail.

Hypertension is a prevalent condition worldwide and is the key risk factor for fatal cardiovascular complications, such as stroke, sudden cardiac death and heart failure. Reduced bioavailability of nitric oxide (NO) in the endothelium is an important precursor for impaired vasodilation and hypertension. In the heart, NO deficiency deteriorates the adverse consequences of pressure-overload and causes cardiac hypertrophy, fibrosis and myocardial infarction which lead to fatal heart failure and sudden cardiac death. Recent consensus is that both endothelial and neuronal nitric oxide synthases (eNOS or NOS3 and nNOS or NOS1) are the constitutive sources of NO in the myocardium. Between the two, nNOS is the predominant isoform of NOS that controls intracellular Ca2+ homeostasis, myocyte contraction, relaxation and signaling pathways including nitroso-redox balance. Notably, our recent research indicates that cardiac eNOS protein is reduced but nNOS protein expression and activity are increased in hypertension. Furthermore, nNOS is induced by the interplay between angiotensin II (Ang II) type 1 receptor (AT1R) and Ang II type 2 receptor (AT2R), mediated by NADPH oxidase and reactive oxygen species (ROS)-dependent eNOS activity in cardiac myocytes. nNOS, in turn, protects the heart from pathogenesis via positive lusitropy in hypertension. Soluble guanylate cyclase (sGC)-cGMP/PKG-dependent phosphorylation of myofilament proteins are novel targets of nNOS in hypertensive myocardium. In this short review, we will endeavor to overview new findings of the up-stream and downstream regulation of cardiac nNOS in hypertension, shed light on the underlying mechanisms which may be of therapeutic value in hypertensive cardiomyopathy.

Heart failure and cardiac arrhythmias are the leading causes of mortality and morbidity worldwide. However, the mechanism of pathogenesis and myocardial malfunction in the diseased heart remains to be fully clarified. Recent compelling evidence demonstrates that changes in the myofilament Ca2+ sensitivity affect intracellular Ca2+ homeostasis and ion channel activities in cardiac myocytes, the essential mechanisms responsible for the cardiac action potential and contraction in healthy and diseased hearts. Indeed, activities of ion channels and transporters underlying cardiac action potentials (e.g., Na+, Ca2+ and K+ channels and the Na+-Ca2+ exchanger) and intracellular Ca2+ handling proteins (e.g., ryanodine receptors and Ca2+-ATPase in sarcoplasmic reticulum (SERCA2a) or phospholamban and its phosphorylation) are conventionally measured to evaluate the fundamental mechanisms of cardiac excitation-contraction (E-C) coupling. Both electrical activities in the membrane and intracellular Ca2+ changes are the trigger signals of E-C coupling, whereas myofilament is the functional unit of contraction and relaxation, and myofilament Ca2+ sensitivity is imperative in the implementation of myofibril performance. Nevertheless, few studies incorporate myofilament Ca2+ sensitivity into the functional analysis of the myocardium unless it is the focus of the study. Here, we describe a protocol that measures sarcomere shortening/re-lengthening and the intracellular Ca2+ level using Fura-2 AM (ratiometric detection) and evaluate the changes of myofilament Ca2+ sensitivity in cardiac myocytes from rat hearts. The main aim is to emphasize that myofilament Ca2+ sensitivity should be taken into consideration in E-C coupling for mechanistic analysis. Comprehensive investigation of ion channels, ion transporters, intracellular Ca2+ handling, and myofilament Ca2+ sensitivity that underlie myocyte contractility in healthy and diseased hearts will provide valuable information for designing more effective strategies of translational and therapeutic value.

Mathematical modeling of Ca2+dynamics in the heart has the potential to provide an integrated understanding of Ca2+-handling mechanisms. However, many previous published models used heterogeneous experimental data sources from a variety of animals and temperatures to characterize model parameters and motivate model equations. This methodology limits the direct comparison of these models with any particular experimental data set. To directly address this issue, in this study, we present a biophysically based model of Ca2+dynamics directly fitted to experimental data collected in left ventricular myocytes isolated from the C57BL/6 mouse, the most commonly used genetic background for genetically modified mice in studies of heart diseases. This Ca2+dynamics model was then integrated into an existing mouse cardiac electrophysiology model, which was reparameterized using experimental data recorded at consistent and physiological temperatures. The model was validated against the experimentally observed frequency response of Ca2+dynamics, action potential shape, dependence of action potential duration on cycle length, and electrical restitution. Using this framework, the implications of cardiac Na+/Ca2+exchanger (NCX) overexpression in transgenic mice were investigated. These simulations showed that heterozygous overexpression of the canine cardiac NCX increases intracellular Ca2+concentration transient magnitude and sarcoplasmic reticulum Ca2+loading, in agreement with experimental observations, whereas acute overexpression of the murine cardiac NCX results in a significant loss of Ca2+from the cell and, hence, depressed sarcoplasmic reticulum Ca2+load and intracellular Ca2+concentration transient magnitude. From this analysis, we conclude that these differences are primarily due to the presence of allosteric regulation in the canine cardiac NCX, which has not been observed experimentally in the wild-type mouse heart.

OBJECTIVE: Cardiac parasympathetic nerve activity is reduced in most cardiovascular disease states, and this may contribute to enhanced cardiac sympathetic responsiveness. Disruption of inhibitory G-proteins (Gi) ablates the cholinergic pathway and increases cardiac endothelial nitric oxide (NO) synthase (eNOS) expression, suggesting that NO may offset the impaired attenuation of beta-adrenergic regulation of supraventricular excitability. To test this, we investigated the role of endogenous NO production on beta-adrenergic regulation of rate (HR), contraction (CR) and calcium (Ca2+) handling in atria following blockade of Gi-coupled muscarinic receptors. METHODS: Mice were administered pertussis toxin (PTx, n=105) or saline (C, n=100) intraperitoneally. After 3 days, we measured CR, HR, and NOS protein levels in isolated atria. Intracellular calcium (Ca2+) transients and Ca2+ current density (I(Ca)) were also measured in atrial myocytes. RESULTS: PTx treatment increased atrial myocyte eNOS protein levels compared to C (P

Myocardial constitutive No production depends on the activity of both endothelial and neuronal NOS (eNOS and nNOS, respectively). Stimulation of myocardial β(3)-adrenergic receptor (β(3)-AR) produces a negative inotropic effect that is dependent on eNOS. We evaluated whether nNOS also plays a role in β(3)-AR signaling and found that the β(3)-AR-mediated reduction in cell shortening and [Ca(2+)](i) transient amplitude was abolished both in eNOS(-/-) and nNOS(-/-) left ventricular (LV) myocytes and in wild type LV myocytes after nNOS inhibition with S-methyl-l-thiocitrulline. LV superoxide (O(2)()) production was increased in nNOS(-/-) mice and reduced by l-N(ω)-nitroarginine methyl ester (l-NAME), indicating uncoupling of eNOS activity. eNOS S-glutathionylation and Ser-1177 phosphorylation were significantly increased in nNOS(-/-) myocytes, whereas myocardial tetrahydrobiopterin, eNOS Thr-495 phosphorylation, and arginase activity did not differ between genotypes. Although inhibitors of xanthine oxidoreductase (XOR) or NOX2 NADPH oxidase caused a similar reduction in myocardial O(2)(), only XOR inhibition reduced eNOS S-glutathionylation and Ser-1177 phosphorylation and restored both eNOS coupled activity and the negative inotropic and [Ca(2+)](i) transient response to β(3)-AR stimulation in nNOS(-/-) mice. In summary, our data show that increased O(2)() production by XOR selectively uncouples eNOS activity and abolishes the negative inotropic effect of β(3)-AR stimulation in nNOS(-/-) myocytes. These findings provide unequivocal evidence of a functional interaction between the myocardial constitutive NOS isoforms and indicate that aspects of the myocardial phenotype of nNOS(-/-) mice result from disruption of eNOS signaling.

Angiotensin II (Ang II) is critical in myocardial pathogenesis, mostly via stimulating NADPH oxidase. Neuronal nitric oxide synthase (nNOS) has recently been shown to play important roles in modulating myocardial oxidative stress and contractility. Here, we examine whether nNOS is regulated by Ang II and affects NADPH oxidase production of intracellular reactive oxygen species (ROS(i)) and contractile function in left ventricular (LV) myocytes. Our results showed that Ang II induced biphasic effects on ROS(i) and LV myocyte relaxation (TR(50)) without affecting the amplitude of sarcomere shortening and L-type Ca(2+) current density: TR(50) was prolonged at 30 min but was shortened after 3h (or after Ang II treatment in vivo). Correspondingly, ROS(i) was increased, followed by a reduction to control level. Quantitative RT-PCR and immunoblotting experiments showed that Ang II (3h) increased the mRNA and protein expression of nNOS and increased NO production (nitrite assay) in LV myocyte homogenates, suggesting that nNOS activity may be enhanced and involved in mediating the effects of Ang II. Indeed, n(omega)-nitro-l-arginine methyl ester (l-NAME) or a selective nNOS inhibitor, S-methyl-l-thiocitrulline (SMTC) increased NADPH oxidase production of superoxide/ROS(i) and abolished faster myocyte relaxation induced by Ang II. The positive lusitropic effect of Ang II was not mediated by PKA-, CaMKII-dependent signaling or peroxynitrite. Conversely, inhibition of cGMP/PKG pathway abolished the Ang II-induced faster relaxation by reducing phospholamban (PLN) Ser(16) phosphorylation. Taken together, these results clearly demonstrate that myocardial nNOS is up-regulated by Ang II and functions as an early adaptive mechanism to attenuate NADPH oxidase activity and facilitate myocardial relaxation.

Transient receptor potential vanilloid 1 (TRPV1) is a Ca2+-permeable cation channel activated by a variety of physicochemical stimuli. The effect of hypoxia ( $$ {\text{P}}_{{{\text{O}}_{2} }} $$ , 3%) on rat TRPV1 overexpressed in HEK293T has been studied. The basal TRPV1 current (I TRPV1) was partly activated by hypoxia, whereas capsaicin-induced TRPV1 (I TRPV1,Cap) was attenuated. Such changes were also suggested from hypoxia- and capsaicin-induced Ca2+ signals in TRPV1-expressing cells. Regarding plausible changes of reactive oxygen species (ROS) under hypoxia, the effects of antioxidants, vitamin C and tiron, as membrane-impermeable and -permeable, respectively, were tested. Both I TRPV1 and I TRPV1,Cap were increased by vitamin C, while only I TRPV1 was slightly increased by tiron. The hypoxic inhibition of I TRPV1,Cap was still persistent under hypoxia/vitamin C. Interestingly, hypoxia/tiron strongly inhibited both I TRPV1 and I TRPV1,Cap. Also, with vitamin C applied through a pipette solution, hypoxia inhibited I TRPV1 and I TRPV1,Cap. In contrast, hypoxia and hypoxia/tiron had no effect on the I TRPV1 induced by acid (pH 6.2, I TRPV1,Acid). Taken together, hypoxia partly activated TRPV1 while it decreased their sensitivity to capsaicin. Putative changes of ROS under hypoxia might underlie the side-specific effects of ROS on TRPV1: inhibitory at the extracellular and stimulatory at the intracellular side, respectively. The differential effects of hypoxia on I TRPV1,Cap and I TRPV1,Acid suggested that the intracellular ROS increase might attenuate the pharmacological potency of capsaicin.

Hypoxia-induced pulmonary vasoconstriction (HPV) is critical for matching of ventilation/perfusion in lungs. Although hypoxic inhibition of K+ channels has been a leading hypothesis for depolarization of pulmonary arterial smooth muscle cells (PASMCs) under hypoxia, pharmacological inhibition of K+ channels does not induce significant contraction in rat pulmonary arteries. Because a partial contraction by thromboxane A2 (TXA2) is required for induction of HPV, we hypothesize that TXA2 receptor (TP) stimulation might activate depolarizing nonselective cation channels (NSCs). Consistently, we found that 5–10 nM U46619, a stable agonist for TP, was indispensible for contraction of rat pulmonary arteries by 4-aminopyridine, a blocker of voltage-gated K+ channel (Kv). Whole cell voltage clamp with rat PASMC revealed that U46619 induced a NSC current ( INSC,TXA2) with weakly outward rectifying current-voltage relation. INSC,TXA2 was blocked by ruthenium red (RR), an antagonist of the transient receptor potential vanilloid-related channel (TRPV) subfamily. 2-Aminoethoxydiphenyl borate, an agonist for TRPV1–3, consistently activated NSC channels in PASMCs. In contrast, agonists for TRPV1 (capsaicin), TRPV3 (camphor), or TRPV4 (α-PDD) rarely induced an increase in the membrane conductance of PASMCs. RT-PCR analysis showed the expression of transcripts for TRPV2 and -4 in rat PASMCs. Finally, it was confirmed that pretreatment with RR largely inhibited HPV in the presence of U46619. The pretreatment with agonists for TRPV1 (capsaicin) and TRPV4 (α-PDD) was ineffective as pretone agents for HPV. Taken together, it is suggested that the concerted effects of INSC,TXA2 activation and Kv inhibition under hypoxia induce membrane depolarization sufficient for HPV. TRPV2 is carefully suggested as the TXA2-activated NSC in rat PASMC.

The polypeptide hormone atrial natriuretic peptide (ANP) plays vital roles in maintaining blood volume and arterial blood pressure. The recognition of clinical benefits of ANP both in healthy and diseased heart identifies ANP as a potential candidate for therapeutic strategy in the treatment of heart disease. ANP is synthesized and stored in cardiac myocytes and it is released through the exocytosis of ANP granules both constitutively and in response to stimuli. It is well known that mechanical stretch is the predominant stimulus for ANP secretion. However, the mechanistic link between mechanical stimuli and exocytosis of ANP vesicles in single atrial myocyte has not yet been demonstrated. Over the last decade, compelling evidence suggested that stretch-activated ion channels might function as mechanosensors. We showed previously that direct stretch of single atrial myocyte using two micro-electrodes activated a non-selective cation channel (SAC). So far it is not known whether activation of SAC is involved in stretch-induced ANP secretion. The present article aims to give an overview of the mechanism of mechanical stretch-stimulated ANP secretion and describes an innovative technique to detect ANP secretion from isolated rat atrial myocytes with high time-resolution. Combined with capacitance measurement and patch-clamp technique in conjunction with in situ ANP bioassay, we were able to demonstrate that SAC in rat atrial myocytes acts as a mechanosensor to transduce stretch signals into the ANP secretion pathway.

We previously reported that hypoxia augments α-adrenergic contraction (hypoxic vasoconstriction, HVC) of skeletal arteries in rats. The underlying mechanism may involve hypoxic inhibition of endothelial nitric oxide synthase (eNOS) expressed in skeletal arterial myocytes (16). To further explore the novel role of muscular eNOS in the skeletal artery, we compared HVC in femoral arteries (FAs) from eNOS knockout (KO) mice with that from wild-type (WT) and heterozygous (HZ) mice. Immunohistochemical assays revealed that, in addition to endothelia, eNOS is also expressed in the medial layer of FAs, albeit at a much lower level. However, the medial eNOS signal was not evident in HZ FAs, despite strong expression in the endothelium; similar observations were made in WT carotid arteries (CAs). The amplitude of contraction induced by 1 μM phenylephrine (PhE) was greater in HZ than in WT FAs. Hypoxia (3% Po2) significantly augmented PhE-induced contraction in WT FAs but not in HZ or KO FAs. No HVC was observed in PhE-pretreated WT CAs. The NOS inhibitor nitro-l-arginine methyl ester (0.1 mM) also augmented PhE contraction in endothelium-denuded WT FAs but not in WT CAs. Inhibitors specific to neuronal NOS and inducible NOS did not augment PhE-induced contraction of WT FAs. NADPH oxidase 4 (NOX4) inhibitor (GKT137831, 5 μM), but not NOX2 inhibitor (apocynin, 100 μM), suppressed HVC. Consistent with the role of reactive oxygen species (ROS), HVC was also inhibited by pretreatment with tiron or polyethylene glycol-catalase. Taken together, these data suggest that the eNOS expressed in smooth muscle cells in FAs attenuates α-adrenergic vasoconstriction; this suppression is alleviated under hypoxia, which potentiates vasoconstriction in a NOX4/ROS-dependent mechanism.

The proarrhythmic effects of new drugs have been assessed by measuring rapidly activating delayed-rectifier K+ current (I-Kr) antagonist potency. However, recent data suggest that even drugs thought to be highly specific I-Kr blockers can be arrhythmogenic via a separate, time-dependent pathway such as late Na+ current augmentation. Here, we report a mechanism for a quinolone antibiotic, sparfloxacin-induced action potential duration (APD) prolongation that involves increase in late L-type Ca2+ current (I-CaL) caused by a decrease in Ca2+-dependent inactivation (CDI). Acute exposure to sparfloxacin, an I-Kr blocker with prolongation of QT interval and torsades de pointes (TdP) produced a significant APD prolongation in rat ventricular myocytes, which lack I-Kr due to E4031 pretreatment. Sparfloxacin reduced peak I-CaL but increased late I-CaL by slowing its inactivation. In contrast, ketoconazole, an I-Kr blocker without prolongation of QT interval and TdP produced reduction of both peak and late I-CaL, suggesting the role of increased late I-CaL in arrhythmogenic effect. Further analysis showed that sparfloxacin reduced CDI. Consistently, replacement of extracellular Ca2+ with Ba2+ abolished the sparfloxacin effects on I-CaL. In addition, sparfloxacin modulated I-CaL in a use-dependent manner. Cardiomyocytes from adult mouse, which is lack of native I-Kr, demonstrated similar increase in late I-CaL and afterdepolarizations. The present findings show that sparfloxacin can prolong APD by augmenting late I-CaL. Thus, drugs that cause delayed I-CaL inactivation and I-Kr blockage may have more adverse effects than those that selectively block I-Kr. This mechanism may explain the reason for discrepancies between clinically reported proarrhythmic effects and I-Kr antagonist potencies.

4-Hydroxynonenal (4-HNE) is considered as a harmful signaling molecule. The accumulation of 4-HNE is implicated under oxidative stress such as ischemia/reperfusion injury. Previously we have report...

OBJECTIVE: The role of constitutive nitric oxide (NO) production in the regulation of beta-adrenergic and muscarinic responses remains controversial. Conflicting data in left ventricular (LV) myocytes from eNOS knockout mice (eNOS-/-) have been ascribed to inconsistent experimental conditions (i.e., differences in the choice of controls, age of the mice, myocytes' stimulation frequency, and in the level of beta-adrenergic stimulation); however, the recent identification of a neuronal-like NO synthase (nNOS) in the LV myocardium has raised the possibility that this isoform may be involved in the modulation of beta-adrenergic and muscarinic responses. METHODS: To address these issues we recorded sarcomere shortening at 35 degrees C under basal conditions, in the presence of isoproterenol (ISO, 10-100 nmol/L) and of ISO plus carbamylcholine (CCh, 1 micromol/L) in LV myocytes isolated from eNOS-/- and nNOS-/- mice, their wild type littermates (eNOS+/+ and nNOS+/+) or C57BL/6J mice. eNOS-/- and control myocytes were studied at 1 and 3 Hz, in the presence of 10 and 100 nmol/L ISO, and responses were compared between young (3 months) and old (> or =12 months) mice. RESULTS: Contraction did not differ between young eNOS-/- and eNOS+/+ mice at all stages of the experimental protocol, either at 1 or 3 Hz or in response to 10 or 100 nmol/L ISO. However, myocytes from old eNOS-/- mice showed a reduced inotropic response to ISO compared with age-matched eNOS+/+ mice (P = 0.02). Similarly, there was a significant difference in the ISO response between eNOS+/+ and C57BL/6J myocytes (P < 0.01), suggesting that experimental variables such as age and the choice of control animals may have contributed to the inconsistency in the results reported in the literature. In contrast, nNOS-/- myocytes showed greater contraction and slower relaxation at all stages of the experimental protocol (P = 0.0003 and P = 0.01 vs. nNOS+/+ myocytes). CONCLUSIONS: Constitutive eNOS expression in murine LV myocytes is not essential for the muscarinic-mediated inhibition of beta-adrenergic signalling and does not appear to play a significant role in the regulation of basal and beta-adrenergic myocardial contraction. Our data suggest that nNOS is the myocardial constitutive isoform responsible for the NO-mediated autocrine regulation of myocardial inotropy and relaxation.

4-Aminopyridine (4-AP) has been used extensively to study transient outward K+ current (ITO,1) in cardiac cells and tissues. We report here inhibition by 4-AP of HERG (the human ether-à-go-go-related gene) K+ channels expressed in a mammalian cell line, at concentrations relevant to those used to study ITO,1. Under voltage clamp, whole cell HERG current (IHERG) tails following commands to +30 mV were blocked with an IC50 of 4.4 +/- 0.5 mM. Development of block was contingent upon HERG channel gating, with a preference for activated over inactivated channels. Treatment with 5 mM 4-AP inhibited peak IHERG during an applied action potential clamp waveform by ~59 %. It also significantly prolonged action potentials and inhibited resurgent IK tails from guinea-pig isolated ventricular myocytes, which lack an ITO,1. We conclude that by blocking the alpha-subunit of the IKr channel, millimolar concentrations of 4-AP can modulate ventricular repolarisation independently of any action on ITO,1.

Insulin regulates cardiac metabolism and function by targeting metabolic proteins or voltage-gated ion channels. This study provides evidence for a novel, voltage-dependent, nonselective cation channel (NSCC) in the heart. Under voltage clamp at 37°C and with major known conductances blocked, insulin (1 nmol/L to 1 μmol/L) activated an outwardly rectifying current ( I insulin ) in guinea pig ventricular myocytes. I insulin could be carried by Cs + , K + , Li + , and Na + ions but not by NMDG + . It was inhibited by the NSCC blockers gadolinium and SKF96365 but not flufenamic acid. I insulin was largely blocked by the insulin receptor tyrosine kinase inhibitor HNMPA-(AM) 3 and by the phospholipase C inhibitor U73122 but not by its inactive analogue U73433. Staurosporine, a potent blocker of protein kinase C, did not prevent the activation of I insulin . Application of an analogue of diacylglycerol, 1-oleoyl-2-acetyl-sn-glycerol, mimicked the effect of insulin. This activated an outwardly rectifying NSCC that could be carried by Cs + , K + , Li + , or Na + and that was blocked by gadolinium but not by flufenamic acid or staurosporine. We conclude that the intracellular pathway leading to activation of this novel cardiac NSCC involves phospholipase C, is protein kinase C–independent, and may depend on direct channel activation by diacylglycerol.