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Mitochondria transplantation emerges as an effective therapeutic strategy for ischemic-related diseases but the roles in the donor hearts for transplant remain unidentified. Here, we investigated whether the preservation of the donor heart with human platelet-derived mitochondria (pl-MT) could improve mitochondrial and cardiac function. Incubation with pl-MT resulted in the internalization of pl-MT and the enhancement of ATP production in primary cardiomyocytes. In addition, incubation of rat hearts with pl-MT ex vivo for 9 h clearly demonstrated pl-MT transfusion into the myocardium. Mitochondria isolated from the hearts incubated with pl-MT showed increased mitochondrial membrane potential and greater ATP synthase activity and citrate synthase activity. Importantly, the production of reactive oxygen species from cardiac mitochondria was not different with and without pl-MT incubation. Functionally, the heartbeat and the volume of coronary circulation perfusate were significantly increased in the Langendorff perfusion system and the viability of cardiomyocytes was increased from pl-MT hearts.Taken together, these results suggest that incubation with Pl-MT improves mitochondrial activity and maintains the cardiac function of rat hearts with prolonged preservation time. The study provides the proof of principle for pl-MT application as an enhancer of the donor heart.

Abstract 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. Key messages We investigated pleiotropy-informed enrichment between LS BMD and BW. We identified genetic variants related to both LS BMD and BW by utilizing a cFDR approach. PDXDC1 is a novel pleiotropic gene which may be related to both LS BMD and BW. Elevated expression of PDXDC1 is related to higher BMD and lower ratio n-6/n-3 PUFA indicating a bone protective effect of PDXDC1.

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

Background Pre-eclampsia (PE) is a pregnancy disorder that is related to an enhanced immune response. Immune cell characteristics such as neutrophil or monocyte to lymphocyte ratios (NLR, MLR) are known to be related to kidney and liver dysfunction in hypertensive patients. Here, we aimed to analyze the correlations between NLR, MLR and platelet to lymphocyte ratio (PLR) and liver, renal and coagulation functional parameters and the impacts of these immune cell profiles to the prognostic significance in PE patients. Methods Pre-delivery hematological and biochemical parameters of 320 first-time pregnant women registered at the Obstetrics Department of Yanbian University Hospital from 2016 to 2019 were analyzed retrospectively. Patients were divided into normal pregnancy (normal, n = 161), mild PE (mPE, n = 28) and severe PE (sPE, n = 131) groups according to diagnostic criteria. Pearson correlation analysis were performed and area under the curve (AUC) were conducted for the diagnostic values of NLR, MLR and PLR. Results were validated with data from the Department of Obstetrics and Gynecology of Seoul National University Hospital (SNUH). Results Kidney functional indexes were adversative in mPE and sPE and liver and coagulation indexes were worse in sPE compared to normal groups. Among immune cells, lymphocytes were increased in mPE and sPE patients, resulted in reduced NLR, MLR and PLR in PE groups, more significant difference were shown in sPE. NLR and PLR were associated with CREA and/or BUN negatively and positive associations were observed with total protein (TP) and albumin (ALB) in sPE. Only NLR showed positive associations with coagulation indexes (PT and APTT) in sPE. AUC analysis for NLR, MLR and PLR were 0.700, 0.656, 0.643, respectively, and NLR p Conclusion NLR could be used as an independent predictor of liver and coagulation dysfunction in PE patients. Our results may provide non-invasive and efficient way of the risk assessment among PE patients.

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.

OBJECTIVES The molecular basis supporting the superiority of the left internal thoracic artery (LITA) as a bypass conduit is limited. This study was conducted to compare the expression and localization of hydrogen sulphide synthesizing enzymes in LITA and radial artery (RA). METHODS Nineteen patients who underwent coronary artery bypass grafting using LITA and RA were enrolled. The remnant LITA and RA were collected to measure the expression levels of 3 hydrogen sulphide-producing enzymes: cystathionine β-synthase, cystathionine γ-lyase and 3-mercaptopyruvate sulphurtransferase using quantitative real-time polymerase chain reaction. Expression levels of these enzymes in the LITA and RA were compared in each subject. The expression and localization patterns of the enzymes were also analysed by immunohistochemistry. RESULTS The mRNA expression of the cystathionine β-synthase was greater in the LITA than in the RA (P = 0.033), whereas the expression levels of the other 2 enzymes did not significantly differ between the 2 arteries. The immunohistochemistry analysis demonstrated greater expression of the cystathionine β-synthase in the LITA than in the RA (P = 0.006). This protein was present in both tunica intima and tunica media of the LITA, although it was present only in the tunica media of the RA. Localization patterns of the other 2 enzymes were not different between LITA and RA. CONCLUSIONS Expression levels of the mRNA and protein of cystathionine β-synthase were significantly greater in LITA than in the RA. These findings might be a factor that affects the superior patency rate of LITA.

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.

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.

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.

Despite distinctive functional and anatomic differences, a precise understanding of the cardiac interventricular differences in excitation–contraction (E–C) coupling mechanisms is still lacking. Here, we directly compared rat right and left cardiomyocytes (RVCM and LVCM). Whole-cell patch clamp, the IonOptix system, and fura-2 fluorimetry were used to measure electrical properties (action potential and ionic currents), single-cell contractility, and cytosolic Ca2+ ([Ca2+]i), respectively. Myofilament proteins were analyzed by immunoblotting. RVCM showed significantly shorter action potential duration (APD) and higher density of transient outward K+ current (Ito). However, the triggered [Ca2+]i change (Ca2+ transient) was not different, while the decay rate of the Ca2+ transient was slower in RVCM. Although the relaxation speed was also slower, the sarcomere shortening amplitude (ΔSL) was smaller in RVCM. SERCA activity was ∼60% lower in RVCM, which is partly responsible for the slower decay of the Ca2+ transient. Immunoblot analysis revealed lower expression of the cardiac troponin complex (cTn) in RVCM, implying a smaller Ca2+ buffering capacity (κS), which was proved by in situ analysis. The introduction of these new levels of cTn, Ito, and SERCA into a mathematical model of rat LVCM reproduced the similar Ca2+ transient, slower Ca2+ decay, shorter APD, and smaller ΔSL of RVCM. Taken together, these data show reduced expression of cTn proteins in the RVCM, which provides an explanation for the interventricular difference in the E–C coupling kinetics.

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.

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. Listen to this article's corresponding podcast at https://ajpheart.podbean.com/e/dcm-mutations-alter-intracellular-ca2-and-signaling/.

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.

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.

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

TWIK-related two-pore domain K

The objective of this study was to compare the fee-for-service and set fee for diagnosis-related group systems with regard to quality of medical care and cost to appendectomy patients. We conducted a retrospective study of 208 inpatients (from 20 hospitals) who undergone appendectomy in Changsha, China during 2013. Data were obtained from databases of medical insurance information systems directly connected to the hospital information systems. We collected and compared patient ages, length of study, and total medical costs for impatient appendectomies between patients using fee-for-service and set fee for diagnosisrelated group systems. One hundred thirty-three patients used the fee for service system and 75 used the set fee diagnosis related group system. For those using the diagnosis-related group system, the mean length of hospitalization (6.2 days) and mean number of prescribed antimicrobials (2.4) per patient were significantly lower than those of the patients who used the fee-for-service system (7.3 days and 3.0, respectively; p = 0.018; p0.05) and were accompanied by lower medical costs and cost of antimicrobials (RMB 2,518 versus RMB 4,484 and RMB476 versus RMB1,108, respectively; p = 0.000, p = 0.000). There were no significant differences in post-surgical complications between the two systems. The diagnosis-related group system had significantly medical costs for appendectomy compared to the fee-for-service system, without sacrificing quality of medical care.

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.

Splice variant forms of neuronal nitric oxide synthase (nNOS or NOS1), nNOSα and nNOSμ, are well established to be functionally expressed in discrete compartments in cardiomyocytes (e.g. sarcoplasmic reticulum, SR, caveolae in plasma membrane or mitochondria). So far, whether nNOS is expressed in myofilament fraction of cardiomyocytes and the splice variant form of nNOS are unknown. Immunoblotting results using two nNOS specific antibodies (BD Transduction Laboratories aa 1095-1289 and Santa Cruz Biotechnology aa 2-300) clearly demonstrated that nNOS was abundantly expressed in myofilament-enriched fraction of cardiomyocytes. Whilst the molecular weight of nNOS in membrane/cytosol fractions was ∼165 kDa, nNOS in myofilament was below 140 kDa, suggesting that the predominant splice variant of nNOS in myofilament is nNOSβ. RT-PCR results confirmed the expressions of both nNOSα and nNOSβ mRNAs in rat cardiomyocytes. Similarly, immunoprecipitation experiments using myofilament lysates of cardiomyocytes identified nNOS with low molecular weight (M.W. ∼140 kDa), confirming nNOSβ. Intriguingly, all three splice variants of nNOS were undetectable in the lysates of cardiomyocytes (including myofilament fractions) from nNOS-/- mice (which lacks nNOSα/μ). Furthermore, nNOSβ expression in myofilament of cardiomyocytes was not different in hypertensive rats compared to the level expressed in sham. iTRAQ-based quantitative proteomics analysis revealed that nNOS regulates phosphorylations of ∼20 proteins in cardiac myofilaments. Collectively, we provide direct evidence that different splice variants of nNOS are expressed in myofilament and membrane/cytosol fractions of cardiomyocytes. Discrete expressions of various splice variants in different compartments of cardiomyocytes suggest diverse roles nNOS play in healthy and diseased heart.

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.

A model of damage to fresh concrete in a corrosive sulphate environment was formulated to investigate how and why the strength of corroded concrete changes over time. First, a corroded concrete block was divided into three regions: an expanded and dense region; a crack-development region; and a noncorroded region. Second, based on the thickness of the surface corrosion layer and the rate of loss of compressive strength of the corroding region, a computational model of the concrete blocks’ corrosion-resistance coefficient of compressive strength in a sulphate environment was generated. Third, experimental tests of the corrosion of concrete were conducted by immersing specimens in a corrosive medium for 270 d. A comparison of the experimental results with the computational formulae shows that the calculation results and test results are in good agreement. A parameter analysis reveals that the corrosion reaction plays a major role in the corrosion of fresh concrete containing ordinary Portland cement, but the diffusion of the corrosion medium plays a major role in the corrosion of concrete mixtures containing fly ash and sulphate-resistant cement. Fresh concrete with a high water-to-cement ratio shows high performance during the whole experiment process whereas fresh concrete with a low water-to-cement ratio shows poor performance during the late experiment period.