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Naturally found chrysosplenol-C (49,5,6-trihydroxy-3,39,7-trimethoxyflavone) increases the contractility of cardiac myocytes independent of b-adrenergic signaling. We investigated the cellular mechanism for chrysosplenol-C-induced positive inotropy. Global and local Ca2+ signals, L-type Ca2+ current (ICa), and contraction were measured from adult rat ventricular myocytes using two-dimensional confocal Ca2+ imaging, the whole-cell patch clamp technique, and video-edge detection, respectively. Application of chrysosplenol-C reversibly increased Ca2+ transient magnitude with a maximal increase of ~55% within 2-3-min-exposures (EC50 =~21 mM). This chemical did not alter ICa and slightly increased diastolic Ca2+ level. The frequency and size of resting Ca2+ sparks were increased by chrysosplenol-C. Chrysosplenol-C significantly increased sarcoplasmic reticulum (SR) Ca2+ content but not fractional release. Pretreatment of protein kinase C (PKC) inhibitor, but not Ca2+/calmodulin-dependent protein kinase II (CaMKII) inhibitor, abolished the stimulatory effects of chrysosplenol-C on Ca2+ transients and Ca2+ sparks. Chrysosplenol-C-induced positive inotropy was removed by the inhibition of PKC, but not CaMKII or phospholipase C. Western blotting assessment revealed that PKC-δ protein level in the membrane fractions significantly increase within 2 min after chrysosplenol-C exposure with a delayed (5 min) increase in PKC-α levels in insoluble membrane. These results suggest that chrysosplenol-C enhances contractility via PKC (most likely PKC-δ)-dependent enhancement of SR Ca2+ releases in ventricular myocytes. Significance Statement We show that chrysosplenol-C, a natural flavone showing a positive inotropic effect, increases sarcoplasmic reticulum (SR) Ca2+ releases on depolarizations and Ca2+ sparks with an increase of SR Ca2+ loading, but not L-type Ca2+ current, in ventricular myocytes. Chrysosplenol-C-induced enhancement in contraction is eliminated by protein kinase C (PKC) inhibition, and it is associated with redistributions of PKC to the membrane. These indicate that chrysosplenol-C enhances contraction via PKC-dependent augmentations of SR Ca2+ release and Ca2+ loading during action potentials.

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.

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.

TWIK-related two-pore domain K+ channels (TREKs) are activated by acidic intracellular pH (pHi), membrane stretch, temperature, and arachidonic acid (AA). Phosphatidylinositol 4,5-bisphosphate (PIP2) exerts concentration-dependent biphasic regulations, which have been observed: inhibition by high PIP2, activation by partial decrease of PIP2, and inhibition by depletion of PIP2. Consistently, the stimulation of voltage-sensitive PIP2 phosphatase (Dr-VSP) induces initial activation and subsequent inhibition of TREKs. Lys in the proximal C-terminus (pCt) is responsible for the inhibition by high PIP2, which is generated by phosphatidylinositol kinases with ATP; its neutralizing mutation [K330A of human TREK-2 (hTREK-2)] induces tonic high activity, irrespective of ATP. Here we focus on triple successive Arg in pCt (R3-pCt) as a candidate region for the stimulatory regulation by lower PIP2. Their neutralized mutant (R3A-pCt; RRR340-2A and RRR355-7A in hTREK-1 and -2, respectively) showed negligible basal current and was not affected by ATP removal or by Dr-VSP activation. Phosphatidic acid, a phospholipid agonist of TREKs, did not activate R3A-pCt. In contrast, acidic pHi, AA, and high temperature activated R3A-pCt normally, whereas activation by membrane stretch was attenuated. In hTREK-2, combined neutralizations of the inhibitory K330 and R3-pCt (K330A/RRR355-7A) did not recover the suppressed current. In contrast, combined neutralization of pHi-sensing Glu (E332A/R355-7A) induced tonic high current and no further activation by pHi. Interestingly, when the Gly between K330/E332 and R3-pCt was mutated (G334A), hTREK-2 was tonic activated with reversed responses to ATP and acidic pHi. Therefore, we propose that the PIP2-dependent converse regulation of TREKs by Lys and R3-pCt with Gly implies structural flexibility.

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

TWIK-related two-pore domain K+ channels (TREKs) are regulated by intracellular pH (pHi) and Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2). Previously, Glu306 in proximal C-terminal (pCt) of mouse TREK-1 was identified as the pHi-sensing residue. The direction of PI(4,5)P2 sensitivity is controversial, and we have recently shown that TREKs are inhibited by intracellular ATP via endogenous PI(4,5)P2 formation. Here we investigate the anionic and cationic residues of pCt for the pHi and ATP-sensitivity in human TREK-2 (hTREK-2). In inside-out patch clamp recordings (ITREK-2,i-o), acidic pHi-induced activation was absent in E332A and was partly attenuated in E335A. Neutralization of cationic Lys (K330A) also eliminated the acidic pHi sensitivity of ITREK-2,i-o. Unlike the inhibition of wild-type (WT) ITREK-2,i-o by intracellular ATP, neither E332A nor K330A was sensitive to ATP. Nevertheless, exogenous PI(4,5)P2 (10 μM) abolished ITREK-2 i-o in all the above mutants as well as in WT, indicating unspecific inhibition by exogenous PI(4,5)P2. In whole-cell recordings of TREK-2 (ITREK-2,w-c), K330A and E332A showed higher or fully active basal activity, showing attenuated or insignificant activation by 2-APB, arachidonic acid, or acidic pHe 6.9. ITREK-1,w-c of WT is largely suppressed by pHe 6.9, and the inhibition is slightly attenuated in K312A and E315A. The results show concerted roles of the oppositely charged Lys and Glu in pCt for the ATP-dependent low basal activity and pHi sensitivity.

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.

TWIK-related two-pore domain K(+) channels 1 and 2 (TREKs) are activated under various physicochemical conditions. However, the directions in which they are regulated by PI(4,5)P2 and intracellular ATP are not clearly presented yet. In this study, we investigated the effects of ATP and PI(4,5)P2 on overexpressed TREKs (HEK293T and COS-7) and endogenously expressed TREK-2 (mouse astrocytes and WEHI-231 B cells). In all of these cells, both TREK-1 and TREK-2 currents were spontaneously increased by dialysis with ATP-free pipette solution for whole-cell recording (ITREK-1,w-c and ITREK-2w-c) or by membrane excision for inside-out patch clamping without ATP (ITREK-1,i-o and ITREK-2,i-o). Steady state ITREK-2,i-o was reversibly decreased by 3 mM ATP applied to the cytoplasmic side, and this reduction was prevented by wortmannin, a PI-kinase inhibitor. An exogenous application of PI(4,5)P2 inhibited the spontaneously increased ITREKs,i-o, suggesting that intrinsic PI(4,5)P2 maintained by intracellular ATP and PI kinase may set the basal activity of TREKs in the intact cells. The inhibition of intrinsic TREK-2 by ATP was more prominent in WEHI-231 cells than astrocytes. Interestingly, unspecific screening of negative charges by poly-L-lysine also inhibited ITREK-2,i-o. Application of PI(4,5)P2 after the poly-L-lysine treatment showed dose-dependent dual effects, initial activation and subsequent inhibition of ITREK-2,i-o at low and high concentrations, respectively. In HEK293T cells coexpressing TREK-2 and a voltage-sensitive PI(4,5)P2 phosphatase, sustained depolarization increased ITREK-2,w-c initially (

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

Pulmonary arteries (PAs) have high compliance, buffering the wide ranges of blood flow. Here, we addressed a hypothesis that PA smooth muscle cells (PASMCs) express nitric oxide synthases (NOS) that might be activated by mechanical stress and vasoactive agonists. In the myograph study of endothelium-denuded rat PAs, NOS inhibition (L-NAME) induced strong contraction (96 % of 80 mM KCl-induced contraction (80K)) in the presence of 5 nM U46619 (thromboxane A2 (TXA2) analogue) with relatively high basal stretch (2.94 mN, S(+)). With lower basal stretch (0.98 mN, S(-)), however, L-NAME application following U46619 (TXA2/L-NAME) induced weak contraction (27 % of 80K). Inhibitors of nNOS and iNOS had no such effect in S(+) PAs. In endothelium-denuded S(+) mesenteric and renal arteries, TXA2/L-NAME-induced contraction was only 18 and 21 % of 80K, respectively. Expression of endothelial-type NOS (eNOS) in rat PASMCs was confirmed by RT-PCR and immunohistochemistry. Even in S(-) PAs, pretreatment with H2O2 (0.1-10 μM) effectively increased the sensitivity to TXA2/L-NAME (105 % of 80K). Vice versa, NADPH oxidase inhibitors, reactive oxygen species scavengers, or an Akt inhibitor (SC-66) suppressed TXA2/L-NAME-induced contraction in S(+) PAs. In a human PASMC line, immunoblot analysis showed the following: (1) eNOS expression, (2) Ser(1177) phosphorylation by U46619 and H2O2, and (3) Akt activation (Ser(473) phosphorylation) by U46619. In the cell-attached patch clamp study, H2O2 facilitated membrane stretch-activated cation channels in rat PASMCs. Taken together, the muscular eNOS in PAs can be activated by TXA2 and mechanical stress via H2O2 and Akt-mediated signaling, which may counterbalance the contractile signals from TXA2 and mechanical stimuli.

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.

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.

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 cardiac Na+-Ca2+ exchanger (NCX) is an important regulator of intracellular ion homeostasis and cardiac function. Gaining insight into modulation of the NCX is therefore important in order to understand ion handling in the heart under physiological and pathological conditions. Typically, the functional contribution of the NCX is often regarded as "secondary" to the changes in luminal Na+ and Ca2+. Whilst it is well accepted that the NCX can be regulated by various factors, including the concentrations of transported ions, direct receptor-mediated modulation of the cardiac NCX is more controversial. Evidence from several different laboratories supports the notion that the cardiac NCX is a direct target of neurotransmitters and hormones and their downstream signalling pathways; however, the issue remains unresolved due to conflicting data showing a lack of direct modulation. The present review summarizes overall findings regarding the modulation of the cardiac NCX, in particular on molecular mechanisms of direct phosphorylation of NCX by beta-adrenergic/adenylate cyclase/protein kinase A and (for comparative purposes) on endothelin-1/protein kinase C signalling pathways. It also aims to consider whether it is currently possible to reconcile discrepancies between studies in the interpretation of the regulation of the cardiac NCX by agents stimulating the beta-adrenoceptor/PKA pathway.

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.

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.

With increasing use of genetically modified mice to study endothelial nitric oxide (NO) biology, methods for reliable quantification of vascular NO production by mouse tissues are crucial. We describe a technique based on electron paramagnetic resonance (EPR) spectroscopy, using colloid iron (II) diethyldithiocarbamate [Fe(DETC)2], to trap NO. A signal was seen from C57BL/6 mice aortas incubated with Fe(DETC)2, that increased 4.7-fold on stimulation with calcium ionophore A23187 [3.45 ± 0.13 vs 0.73 ± 0.13 au (arbitrary units)]. The signal increased linearly with incubation time (r2=0.93), but was abolished by addition of NG-nitro- l -arginine methyl ester ( l -NAME) or endothelial removal. Stimulated aortas from eNOS knockout mice had virtually undetectable signals (0.14 ± 0.06 vs 3.17 ± 0.21 au in littermate controls). However, the signal was doubled from mice with transgenic eNOS overexpression (7.17 ± 0.76 vs 3.37 ± 0.43 au in littermate controls). We conclude that EPR is a useful tool for direct NO quantification in mouse vessels.

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.

The aim of this study was to investigate whether or not the activity of the cardiac Na(+)-Ca(2+) exchanger might be directly sensitive to external K(+) concentration ([K(+)](e)). Measurements of whole-cell exchanger current (I(NaCa)) were made at 37 degrees C from guinea-pig isolated ventricular myocytes, using whole-cell patch clamp recording with major interfering conductances blocked. Changing [K(+)](e) from 0 to 5mM significantly reduced both outward and inward exchange currents in a time-dependent manner. Various [K(+)](e) between 1 and 15 mM were tested and the inhibitory effect was observed to be concentration-dependent. At steady-state, 5mM [K(+)](e) decreased the density of Ni(2+)-sensitive current by 52.8+/-4.3% (mean+/-S.E.M., n=6) and of 0Na0Ca-sensitive current by 39.0+/-4.4% (n=5). The possibility that the inhibitory effect of external K(+) on I(NaCa) might wholly or in part be secondary to activation of the sarcolemmal Na(+)-K(+) pump was investigated by testing the effect of K(+) addition in the presence of a high concentration of strophanthidin (500 microM). Ni(2+)-sensitive I(NaCa) was still observed to be sensitive to external K(+) (I(NaCa) decreased by 39.4+/-9.4%, n=4), suggesting that the inhibitory effect could occur independently of activation of the Na(+)-K(+) pump. The effect of external K(+) on I(NaCa) was verified using a baby hamster kidney (BHK) cell line stably expressing the cardiac Na(+)-Ca(2+) exchanger isoform, NCX1. Similar to native I(NaCa), NCX1 current was also suppressed by [K(+)](e). However, [K(+)](e) did not alter current amplitude in untransfected BHK cells. The effect of [K(+)](e) on I(NaCa) could not be attributed to simply adding any monovalent cation back to the external solution, since it was not reproduced by application of equimolar Li(+), Cs(+) and TEA(+). Rb(+), however, could mimic the effect of K(+). Collectively, these data suggest that external K(+) at physiologically and pathologically relevant concentrations might be able to modulate directly the activity of the cardiac Na(+)-Ca(2+) exchanger.

Purpose To explore the influence of S100 calcium binding protein A4 (S100A4) knockout (KO) on methionine-choline-deficient (MCD) diet-induced non-alcoholic fatty liver disease (NAFLD) in mice. Materials and methods S100A4 KO mice (n=20) and their wild-type (WT) counterparts (n=20) were randomly divided into KO/MCD, Ko/methionine-choline-sufficient (MCS), WT/MCD, and WT/MCS groups. After 8 weeks of feeding, blood lipid and liver function-related indexes were measured. HE, Oil Red O, and Masson stainings were used to observe the changes of liver histopathology. Additionally, expressions of S100A4 and proinflammatory and profibrogenic cytokines were detected by qRT-PCR and Western blot, while hepatocyte apoptosis was revealed by TUNEL staining. Results Serum levels of aminotransferase, aspartate aminotransferase, triglyceride, and total cholesterol in mice were increased after 8-week MCD feeding, and hepatocytes performed varying balloon-like changes with increased inflammatory cell infiltration and collagen fibers; however, these effects were improved in mice of KO/MCD group. Meanwhile, total NAFLD activity scores and fibrosis were lower compared to WT+MCD group. Compared to WT/MCS group, S100A4 expression in liver tissue of WT/MCD group was enhanced. The expression of proinflammatory (TNF-α, IL-1β, IL-6) and profibrogenic cytokines (TGF-β1, COL1A1, α-SMA) in MCD-induced NAFLD mice were increased, as well as apoptotic index (AI). For MCD group, the expressions of proinflammatory and profibrogenic cytokines and AI in KO mice were lower than those of WT mice. Conclusion S100A4 was detected to be upregulated in NAFLD, while S100A4 KO alleviated liver fibrosis and inflammation, in addition to inhibiting hepatocyte apoptosis.

Angiotensin II (Ang II), a potent precursor of hypertrophy and heart failure, upregulates neuronal nitric oxide synthase (nNOS or NOS1) in the myocardium. Here, we investigate the involvement of type 1 and 2 angiotensin receptors (AT1R and AT2R) and molecular mechanisms mediating Ang II-upregulation of nNOS. Our results showed that pre-treatment of left ventricular (LV) myocytes with antagonists of AT1R or AT2R (losartan, PD123319) and ROS scavengers (apocynin, tiron or PEG-catalase) blocked Ang II-upregulation of nNOS. Surface biotinylation or immunocytochemistry experiments demonstrated that AT1R expression in plasma membrane was progressively decreased (internalization), whereas AT2R was increased (membrane trafficking) by Ang II. Inhibition of AT1R or ROS scavengers prevented Ang II-induced translocation of AT2R to plasma membrane, suggesting an alignment of AT1R-ROS-AT2R. Furthermore, Ang II increased eNOS-Ser(1177) but decreased eNOS-Thr(495), indicating concomitant activation of eNOS. Intriguingly, ROS scavengers but not AT2R antagonist prevented Ang II-activation of eNOS. NOS inhibitor (L-NG-Nitroarginine Methyl Ester, L-NAME) or eNOS gene deletion (eNOS(-/-)) abolished Ang II-induced membrane trafficking of AT2R, nNOS protein expression and activity. Mechanistically, S-nitrosation of AT2R was increased by sodium nitroprusside (SNP), a NO donor. Site-specific mutagenesis analysis reveals that C-terminal cysteine 349 in AT2R is essential in AT2R translocation to plasma membrane. Taken together, we demonstrate, for the first time, that Ang II upregulates nNOS protein expression and activity via AT1R/ROS/eNOS-dependent S-nitrosation and membrane translocation of AT2R. Our results suggest a novel crosstalk between AT1R and AT2R in regulating nNOS via eNOS in the myocardium under pathogenic stimuli.

Arterioles and small arteries change their tone in response to transmural pressure changes, called myogenic tone (MT). In comparison to the branches of cerebral arteries (CA) showing prominent MT, the third branches of mesenteric arteries (MA) with similar diameters show weaker MT. Here, we aimed to analyze the electrophysiological differences responsible for the weaker MT in MA (MT MA ) than MT in CA (MT CA ). We measured ionic current using patch clamp in isolated MA smooth muscle cells (MASMCs) and CA smooth muscle cells (CASMCs) of rats. MT was analyzed using video analysis of pressurized small arteries. Quantitative‐PCR (q‐PCR) and immunofluorescence confocal microscopy were used to compare the mRNA and protein expression level of big‐conductance Ca 2+ ‐activated K + channel (BK C a ) subunits (Slo1 α and Slo β 1). Whole‐cell patch clamp study revealed higher density of voltage‐operated Ca 2+ channel current (I C aV ) in the MASMCs than in CASMCs. Although voltage‐gated K + channel current (I K v ) was also higher in MASMCs, treatment with Kv inhibitor (4‐aminopyridine) did not affect MT MA . Interestingly, BK C a current density and the frequency of spontaneous transient outward currents (STOCs) were consistently higher in MASMCs than in CASMCs. Inside‐out patch clamp showed that the Ca 2+ ‐sensitivity of BK C a is higher in MASMCs than in CASMCs. Iberiotoxin, a selective BK C a inhibitor, augmented MT MA by a larger extent than MT CA . Although q‐PCR analysis did not reveal a significant difference of mRNAs for Slo1 α and Slo β 1, immunofluorescence image suggested higher expression of Slo1 α in MASMCs than in CASMCs. Despite the large I C aV density, the high activities of BK C a including the more frequent STOCs in MASMCs veils the potentially strong MT MA .