Involvement of sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) in mPRα (PAQR7)-mediated progesterone induction of vascular smooth muscle relaxation.

Progesterone acts directly on vascular smooth muscle cells (VSMCs) through activation of membrane progesterone receptor α (mPRα)-dependent signaling to rapidly decrease cytosolic Ca2+ concentrations and induce muscle relaxation. However, it is not known whether this progesterone action involves uptake of Ca2+ by the sarco/endoplasmic reticulum (SR) and increased sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) activity. The present results show that treatment of cultured human VSMCs with progesterone and the selective mPR agonist Org OD-02-0 (OD 02-0) but not with the nuclear PR agonist R5020 increased SERCA protein expression, which was blocked by knockdown of mPRα with siRNA. Moreover, treatments with progesterone and OD 02-0, but not with R5020, increased phospholamban (PLB) phosphorylation, which would result in disinhibition of SERCA function. Progesterone and OD 02-0 significantly increased Ca2+ levels in the SR and caused VSMC relaxation. These effects were blocked by pretreatment with cyclopiazonic acid (CPA), a SERCA inhibitor, and by knockdown of SERCA2 with siRNA, suggesting that SERCA2 plays a critical role in progesterone induction of VSMC relaxation. Treatment with inhibitors of inhibitory G proteins (Gi, NF023), MAP kinase (AZD 6244), Akt/Pi3k (wortmannin), and a Rho activator (calpeptin) blocked the progesterone- and OD 02-0-induced increase in Ca2+ levels in the SR and SERCA expressions. These results suggest that the rapid effects of progesterone on cytosolic Ca2+ levels and relaxation of VSMCs through mPRα involve regulation of the functions of SERCA2 and PLB through Gi, MAP kinase, and Akt signaling pathways and downregulation of RhoA activity.NEW & NOTEWORTHY The rapid effects of progesterone on cytosolic Ca2+ levels and relaxation of VSMCs through mPRα involve regulation of the functions of SERCA2 and PLB through Gi, MAP kinase, and Akt signaling pathways and downregulation of RhoA activity.

The evolutionarily conserved C-terminal peptide of troponin I is an independently configured regulatory structure to function as a myofilament Ca2+-desensitizer.

The C-terminal end segment of troponin subunit I (TnI) is a structure highly conserved among the three muscle type-specific isoforms and across vertebrate species. Partial deletion or point mutation in this segment impairs cardiac muscle relaxation. In the present study, we characterized the C-terminal 27 amino acid peptide of human cardiac TnI (HcTnI-C27) for its role in modulating muscle contractility. Biologically or chemically synthesized HcTnI-C27 peptide retains an epitope structure in physiological solutions similarly to that in intact TnI as recognized by an anti-TnI C-terminus monoclonal antibody (mAb TnI-1). Protein binding studies found that HcTnI-C27 retains the binding affinity for tropomyosin as previously shown with intact cardiac TnI. A restrictive cardiomyopathy mutation R192H in this segment abolishes the bindings to mAb TnI-1 and tropomyosin, demonstrating a pathogenic loss of function. Contractility studies using skinned muscle preparations demonstrated that addition of HcTnI-C27 peptide reduces the Ca2+-sensitivity of myofibrils without decreasing maximum force production. The results indicate that the C-terminal end segment of TnI is a regulatory element of troponin, which retains the native configuration in the form of free peptide to confer an effect on myofilament Ca2+-desensitization. Without negative inotropic impact, this short peptide may be developed into a novel reagent to selectively facilitate cardiac muscle relaxation at the activated state as a potential treatment for heart failure.

Green tea extract catechin improves internal cardiac muscle relaxation in RCM mice.

BACKGROUND: Diastolic dysfunction refers to an impaired relaxation and an abnormality in a heart's filling during diastole while left ventricular systolic function is preserved. Diastolic dysfunction is commonly observed in patients with primary hypertension, diabetes and cardiomyopathies such as hypertrophic cardiomyopathy or restrictive cardiomyopathy. We have generated a restrictive cardiomyopathy (RCM) mouse model with troponin mutations in the heart to mimic the human RCM patients carrying the same mutations. RESULTS: In the present study, we have investigated the ventricular muscle internal dynamics and pressure developed during systole and diastole by inserting a micro-catheter into the left ventricle of the RCM mice with or without treatment of desensitizer green tea extracts catechins. Our results demonstrate that green tea catechin is able to correct diastolic dysfunction in RCM mainly by improving ventricular compliance and reducing the internal muscle rigidity caused by myofibril hypersensitivity to Ca(2+). CONCLUSION: Green tea extract catechin is effective in correcting diastolic dysfunction and improving ventricular muscle intrinsic compliance in RCM caused by troponin mutations.

Functional role of vasoactive intestinal polypeptide in inhibitory motor innervation in the mouse internal anal sphincter.

There is evidence that vasoactive intestinal polypeptide (VIP) participates in inhibitory neuromuscular transmission (NMT) in the internal anal sphincter (IAS). However, specific details concerning VIP-ergic NMT are limited, largely because of difficulties in selectively blocking other inhibitory neural pathways. The present study used the selective P2Y1 receptor antagonist MRS2500 (1 µm) and the nitric oxide synthase inhibitor N(G)-nitro-l-arginine (l-NNA; 100 µm) to block purinergic and nitrergic NMT to characterize non-purinergic, non-nitrergic (NNNP) inhibitory NMT and the role of VIP in this response. Nerves were stimulated with electrical field stimulation (0.1-20 Hz, 4-60 s) and the associated changes in contractile and electrical activity measured in non-adrenergic, non-cholinergic conditions in the IAS of wild-type and VIP(-/-) mice. Electrical field stimulation gave rise to frequency-dependent relaxation and hyperpolarization that was blocked by tetrodotoxin. Responses during brief trains of stimuli (4 s) were mediated by purinergic and nitrergic NMT. During longer stimulus trains, an NNNP relaxation and hyperpolarization developed slowly and persisted for several minutes beyond the end of the stimulus train. The NNNP NMT was abolished by VIP6-28 (30 µm), absent in the VIP(-/-) mouse and mimicked by exogenous VIP (1-100 nm). Immunoreactivity for VIP was co-localized with neuronal nitric oxide synthase in varicose intramuscular fibres but was not detected in the VIP(-/-) mouse IAS. In conclusion, this study identified an ultraslow component of inhibitory NMT in the IAS mediated by VIP. In vivo, this pathway may be activated with larger rectal distensions, leading to a more prolonged period of anal relaxation.

Ablation of smooth muscle caldesmon affects the relaxation kinetics of arterial muscle.

Smooth muscle caldesmon (h-CaD) is an actin- and myosin-binding protein that reversibly inhibits the actomyosin ATPase activity in vitro. To test the function of h-CaD in vivo, we eliminated its expression in mice. The h-CaD-null animals appeared normal and fertile, although the litter size was smaller. Tissues from the homozygotes lacked h-CaD and exhibited upregulation of the non-muscle isoform, l-CaD, in visceral, but not vascular tonic smooth muscles. While the Ca(2+) sensitivity of force generation of h-CaD-deficient smooth muscle remained largely unchanged, the kinetic behavior during relaxation in arteries was different. Both intact and permeabilized arterial smooth muscle tissues from the knockout animals relaxed more slowly than those of the wild type. Since this difference occurred after myosin dephosphorylation was complete, the kinetic effect most likely resulted from slower detachment of unphosphorylated crossbridges. Detailed analyses revealed that the apparently slower relaxation of h-CaD-null smooth muscle was due to an increase in the amplitude of a slower component of the biphasic tension decay. While the identity of this slower process has not been unequivocally determined, we propose it reflects a thin filament state that elicits fewer re-attached crossbridges. Our finding that h-CaD modulates the rate of smooth muscle relaxation clearly supports a role in the control of vascular tone.

Na⁺/Ca²âº exchanger 1/2 double-heterozygote knockout mice display increased nitric oxide component and altered colonic motility.

The Na⁺/Ca²âº exchanger (NCX) is a plasma membrane transporter involved in regulating intracellular Ca²âº concentrations. NCX is critical for Ca²âº regulation in cardiac muscle, vascular smooth muscle, and nerve fibers. To determine the role of NCX1 and NCX2 in gastrointestinal tissues, we examined electric field stimulation (EFS)-induced responses in the longitudinal smooth muscle of the distal colon in NCX1 and NCX2 double-heterozygote knockoutmice (Double HET). We found that the amplitudes of EFS-induced relaxation that persisted during EFS were greater in Double HET than in wild-type mice (WT). Under the non-adrenergic, non-cholinergic (NANC) condition, EFS-induced relaxation in Double HET was similar in amplitude to that of WT. In the experiments in which l-NNA was added under NANC conditions following the EFS, the magnitudes of EFS-induced relaxation were smaller in Double HET than those in WT. In addition, an NCX inhibitor, SN-6, enhanced EFS-induced relaxation but did not affect EFS-induced relaxation under NANC condition, as in Double HET. Moreover, the magnitudes of relaxation induced by NOR-1, which generates NO, were greater in Double HET compared with WT. Similarly, SN-6 potentiated the magnitudes of NOR-1-induced relaxation. In this study, we demonstrate that NCX regulate colonic motility by altering the sensitivity of the inhibitory component.

Large-conductance Ca2+-activated K+ channel beta1-subunit knockout mice are not hypertensive.

Large-conductance Ca2+-activated K+ (BK) channels are composed of pore-forming α-subunits and accessory ß1-subunits that modulate Ca2+ sensitivity. BK channels regulate arterial myogenic tone and renal Na+ clearance/K+ reabsorption. Previous studies using indirect or short-term blood pressure measurements found that BK channel ß1-subunit knockout (BK ß1-KO) mice were hypertensive. We evaluated 24-h mean arterial pressure (MAP) and heart rate in BK ß1-KO mice using radiotelemetry. BK ß1-KO mice did not have a higher 24-h average MAP when compared with wild-type (WT) mice, although MAP was ∼10 mmHg higher at night. The dose-dependent peak declines in MAP by nifedipine were only slightly larger in BK ß1-KO mice. In BK ß1-KO mice, giving 1% NaCl to mice to drink for 7 days caused a transient (5 days) elevation of MAP (∼5 mmHg); MAP returned to pre-saline levels by day 6. BK ß1-KO mesenteric arteries in vitro demonstrated diminished contractile responses to paxilline, increased reactivity to Bay K 8644 and norepinephrine (NE), and maintained relaxation to isoproterenol. Paxilline and Bay K 8644 did not constrict WT or BK ß1-KO mesenteric veins (MV). BK ß1-subunits are not expressed in MV. The results indicate that BK ß1-KO mice are not hypertensive on normal or high-salt intake. BK channel deficiency increases arterial reactivity to NE and L-type Ca2+ channel function in vitro, but the L-type Ca2+ channel modulation of MAP is not altered in BK ß1-KO mice. BK and L-type Ca(2+) channels do not modulate murine venous tone. It appears that selective loss of BK channel function in arteries only is not sufficient to cause sustained hypertension.

Melatonin improves muscle function of the dystrophic mdx5Cv mouse, a model for Duchenne muscular dystrophy.

Duchenne muscular dystrophy (DMD) is a severe X-linked muscle-wasting disease caused by the absence of the cytoskeletal protein dystrophin. In addition to abnormal calcium handling, numerous studies point to a crucial role of oxidative stress in the pathogenesis of the disease. Considering the impressive results provided by antioxidants on dystrophic muscle structure and function, we investigated whether melatonin can protect the mdx(5Cv) mouse, an animal model for DMD. Male mdx(5Cv) mouse pups were treated with melatonin by daily intraperitoneal (i.p.) injection (30 mg/kg body weight) or by subcutaneous (s.c.) implant(s) (18 or 54 mg melatonin as Melovine® implants) from 17/18 to 28/29 days of age. Isometric force of the triceps surae was recorded at the end of the treatment. The i.p. treatment increased the phasic twitch tension of mdx(5Cv) mice. The maximal tetanic tension was ameliorated by 18 mg s.c. and 30 mg/kg i.p. treatments. Melatonin caused the dystrophic muscle to contract and relax faster. The force-frequency relationship of melatonin-treated dystrophic mice was shifted to the right. In accordance with improved muscle function, melatonin decreased plasma creatine kinase activity, a marker for muscle injury. Melatonin treatment increased total glutathione content and lowered the oxidized/reduced glutathione ratio, indicating a better redox status of the muscle. In light of the present investigation, the therapeutic potential of melatonin should be further considered for patients with DMD.

Crosstalk between Gi and Gq/Gs pathways in airway smooth muscle regulates bronchial contractility and relaxation.

Receptor-mediated airway smooth muscle (ASM) contraction via G(alphaq), and relaxation via G(alphas), underlie the bronchospastic features of asthma and its treatment. Asthma models show increased ASM G(alphai) expression, considered the basis for the proasthmatic phenotypes of enhanced bronchial hyperreactivity to contraction mediated by M(3)-muscarinic receptors and diminished relaxation mediated by beta(2)-adrenergic receptors (beta(2)ARs). A causal effect between G(i) expression and phenotype has not been established, nor have mechanisms whereby G(i) modulates G(q)/G(s) signaling. To delineate isolated effects of altered G(i), transgenic mice were generated overexpressing G(alphai2) or a G(alphai2) peptide inhibitor in ASM. Unexpectedly, G(alphai2) overexpression decreased contractility to methacholine, while G(alphai2) inhibition enhanced contraction. These opposite phenotypes resulted from different crosstalk loci within the G(q) signaling network: decreased phospholipase C and increased PKCalpha, respectively. G(alphai2) overexpression decreased beta(2)AR-mediated airway relaxation, while G(alphai2) inhibition increased this response, consistent with physiologically relevant coupling of this receptor to both G(s) and G(i). IL-13 transgenic mice (a model of asthma), which developed increased ASM G(alphai), displayed marked increases in airway hyperresponsiveness when G(alphai) function was inhibited. Increased G(alphai) in asthma is therefore a double-edged sword: a compensatory event mitigating against bronchial hyperreactivity, but a mechanism that evokes beta-agonist resistance. By selective intervention within these multipronged signaling modules, advantageous G(s)/G(q) activities could provide new asthma therapies.

Ghrelin in the human myometrium.

BACKGROUND: Ghrelin is a 28-amino acid octanolyated peptide, synthesised primarily in the stomach. It stimulates growth hormone release, food intake and exhibits many other diverse effects. Our group have previously determined that ghrelin inhibited human contractility in vitro. The aim of this study therefore, was to investigate the expression of ghrelin, its receptor, the growth hormone secretagogue receptor type 1 (GHS-R1), ghrelin O-acyltransferase (GOAT) which catalyses ghrelin octanoylation, prohormone convertase 1/3 (PC1/3) responsible for pro-ghrelin processing, in human myometrium, during pregnancy prior to labour, during labour and in the non-pregnant state. Modulation of ghrelin and ghrelin receptor expression in cultured myometrial cells was also investigated. METHODS: mRNA and protein were isolated from human myometrium and the myometrial smooth muscle cell line hTERT-HM; and real-time fluorescence RT-PCR, western blotting and fluorescence microscopy performed. The effects of beta-Estradiol and bacterial lipopolysaccharide (LPS) on hTERT-HM gene expression were evaluated by western blotting. RESULTS: We have reported for the first time the expression and processing of ghrelin, GHS-R1, GOAT and PC1/3 expression in human myometrium, and also the down-regulation of ghrelin mRNA and protein expression during labour. Furthermore, GHS-R1 protein expression significantly decreased at labour. Myometrial GOAT expression significantly increased during term non-labouring pregnancy in comparison to both non-pregnant and labouring myometrium. Mature PC1/3 protein expression was significantly decreased at term pregnancy and labour in comparison to non-pregnant myometrium. Ghrelin, GHS-R1, GOAT and PC1/3 mRNA and protein expression was also detected in the hTERT-HM cells. Ghrelin protein expression decreased upon LPS treatment in these cells while beta-Estradiol treatment increased GHS-R1 expression. CONCLUSIONS: Ghrelin processing occurred in the human myometrium at term pregnancy and in the non-pregnant state. GOAT expression which increased during term non-labouring pregnancy demonstrating a similar expression pattern to prepro-ghrelin and GHS-R1, decreased at labour, signifying possible myometrial ghrelin acylation. Moreover, the presence of PC1/3 may contribute to pro-ghrelin processing. These results along with the previous in vitro data suggest that myometrially-produced and processed ghrelin plays a significant autocrine or paracrine role in the maintenance of relaxation in this tissue during pregnancy. Furthermore, the significant uterine modulators LPS and beta-Estradiol are involved in the regulation of ghrelin and ghrelin receptor expression respectively, in the human myometrium.

A2A adenosine receptor deficiency leads to impaired tracheal relaxation via NADPH oxidase pathway in allergic mice.

A(2A) adenosine receptor (A(2A)AR) has been shown to suppress superoxide generation in leukocytes via the cAMP-protein kinase A (PKA) pathway. However, no study has yet explored the role of A(2A)AR in relation to NADPH oxidase in murine tracheas in vitro, which may lead to altered smooth muscle relaxation in asthma. Therefore, the present study evaluated the effects of A(2A)AR deficiency on the NADPH oxidase pathway in tracheas of A(2A) wild-type (WT) and A(2A) knockout (KO) mice. A(2A)WT mice were sensitized with ovalbumin (30 microg i.p.) on days 1 and 6, followed by 5% ovalbumin aerosol challenge on days 11, 12, and 13. A(2A)AR (gene and protein expression), cAMP, and phosphorylated PKA (p-PKA) levels were decreased in A(2A)WT sensitized mice compared with controls. A(2A)KO mice also showed decreased cAMP and p-PKA levels. A(2A)WT sensitized and A(2A)KO control mice had increased gene and protein expression of NADPH oxidase subunits (p47phox and gp91phox) compared with the controls. Tracheal relaxation to specific A(2A)AR agonist, 4-[2-[[6-amino-9-(N-ethyl-beta-d-ribofuranuronamidosyl)-9H-purin-2-yl]amino]ethyl]benzenepropanoic acid hydrochloride (CGS 21680), decreased in A(2A)WT sensitized mice compared with the controls, although it was absent in A(2A)KO mice. Pretreatment with NADPH oxidase inhibitors apocyanin/diphenyliodonium reversed the attenuated relaxation to CGS 21680 in A(2A)WT sensitized tracheas, whereas specific PKA inhibitor (9S,10S,12R)-2,3,9,10,11,12-hexahydro-10-hydroxy-9-methyl-1-oxo-9,12-epoxy-1H-diindolo[1,2,3-fg:3',2',1'-kl]pyrrolo[3,4-i] [1,6]benzodiazocine-10-carboxylic acid hexyl ester (KT 5720) blocked CGS 21680-induced relaxation. Tracheal reactive oxygen species (ROS) generation was also increased in A(2A)WT sensitized and A(2A)KO control mice compared with the controls. In conclusion, this study shows that A(2A)AR deficiency causes increased NADPH oxidase activation leading to decreased tracheal relaxation via altered cAMP-PKA signaling and ROS generation.

The homozygous K280N troponin T mutation alters cross-bridge kinetics and energetics in human HCM.

Hypertrophic cardiomyopathy (HCM) is a genetic form of left ventricular hypertrophy, primarily caused by mutations in sarcomere proteins. The cardiac remodeling that occurs as the disease develops can mask the pathogenic impact of the mutation. Here, to discriminate between mutation-induced and disease-related changes in myofilament function, we investigate the pathogenic mechanisms underlying HCM in a patient carrying a homozygous mutation (K280N) in the cardiac troponin T gene (TNNT2), which results in 100% mutant cardiac troponin T. We examine sarcomere mechanics and energetics in K280N-isolated myofibrils and demembranated muscle strips, before and after replacement of the endogenous troponin. We also compare these data to those of control preparations from donor hearts, aortic stenosis patients (LVHao), and HCM patients negative for sarcomeric protein mutations (HCMsmn). The rate constant of tension generation following maximal Ca2+ activation (k ACT) and the rate constant of isometric relaxation (slow k REL) are markedly faster in K280N myofibrils than in all control groups. Simultaneous measurements of maximal isometric ATPase activity and Ca2+-activated tension in demembranated muscle strips also demonstrate that the energy cost of tension generation is higher in the K280N than in all controls. Replacement of mutant protein by exchange with wild-type troponin in the K280N preparations reduces k ACT, slow k REL, and tension cost close to control values. In donor myofibrils and HCMsmn demembranated strips, replacement of endogenous troponin with troponin containing the K280N mutant increases k ACT, slow k REL, and tension cost. The K280N TNNT2 mutation directly alters the apparent cross-bridge kinetics and impairs sarcomere energetics. This result supports the hypothesis that inefficient ATP utilization by myofilaments plays a central role in the pathogenesis of the disease.

Activation of Nrf2 attenuates delayed gastric emptying in obesity induced diabetic (T2DM) female mice.

Diabetic gastroparesis (GP) is a clinical syndrome characterized by delayed gastric emptying (DGE). Loss of Nrf2 (Nuclear factor (erythroid-derived 2)-like 2) led to reduced nNOSα mediated gastric motility and DGE. The molecular signaling of cinnamaldehyde (CNM) mediated Nrf2 activation and its mechanistic role on DGE were further investigated in obese/T2D female mice. Adult female homozygous Nfe2l2-/- (C57BL/6J) and their wild-type (WT) littermates (Nfe2l2+/+) mice were fed with high fat diet (HFD; Obese/T2D model), or normal diet (ND) with or without CNM (50 mg/kg b.w; i.p). Supplementation of CNM attenuated (p < 0.05) DGE in WT female but not in Nrf2 KO Obese/T2D mice. CNM (1) normalized serum estradiol-17ß levels, (2) induced gastric Nrf2 and phase II antioxidant enzymes through extracellular signal-regulated kinase, (ERK)/c-Jun N-terminal kinase (JNK)/p38 mitogen-activated protein kinase (MAPK), (3) reduced glucose synthase kinase 3 beta (GSK3ß) and aryl hydrocarbon receptor (AhR) and this was associated with (4) increased estrogen receptor expression, BH4 (Cofactor of nNOS) biosynthesis enzyme GCH-1 and nNOSα dimerization in WT Obese/T2 diabetic female mice. In addition, CNM restored impaired nitrergic relaxation in hyperglycemic conditions. These findings emphasize the importance of Nrf2 in maintaining nNOSα mediated GE and may have a translational relevance to treat obese/diabetic gastroparesis in women.

MYBPC3 mutations are associated with a reduced super-relaxed state in patients with hypertrophic cardiomyopathy.

The "super-relaxed state" (SRX) of myosin represents a 'reserve' of motors in the heart. Myosin heads in the SRX are bound to the thick filament and have a very low ATPase rate. Changes in the SRX are likely to modulate cardiac contractility. We previously demonstrated that the SRX is significantly reduced in mouse cardiomyocytes lacking cardiac myosin binding protein-C (cMyBP-C). Here, we report the effect of mutations in the cMyBP-C gene (MYBPC3) using samples from human patients with hypertrophic cardiomyopathy (HCM). Left ventricular (LV) samples from 11 HCM patients were obtained following myectomy surgery to relieve LV outflow tract obstruction. HCM samples were genotyped as either MYBPC3 mutation positive (MYBPC3mut) or negative (HCMsmn) and were compared to eight non-failing donor hearts. Compared to donors, only MYBPC3mut samples display a significantly diminished SRX, characterised by a decrease in both the number of myosin heads in the SRX and the lifetime of ATP turnover. These changes were not observed in HCMsmn samples. There was a positive correlation (p < 0.01) between the expression of cMyBP-C and the proportion of myosin heads in the SRX state, suggesting cMyBP-C modulates and maintains the SRX. Phosphorylation of the myosin regulatory light chain in MYBPC3mut samples was significantly decreased compared to the other groups, suggesting a potential mechanism to compensate for the diminished SRX. We conclude that by altering both contractility and sarcomeric energy requirements, a reduced SRX may be an important disease mechanism in patients with MYBPC3 mutations.

SERCA1 attenuates diaphragm relaxation and uptake rate of SERCA in rats with acute sepsis.

The present study aimed to investigate the effects of acute sepsis on diaphragm contractility and relaxation, via examining the Ca2+­uptake function of sarco/endoplasmic reticulum Ca2+ adenosine triphosphatase (SERCA), and the protein levels of SERCA1, SERCA2 and the ryanodine receptor (RyR) of the sarcoplasmic reticulum (SR). A sepsis rat model was established through cecal ligation and puncture (CLP). A total of 6 and 12 h following CLP, the isometric contractile and relaxation parameters of the diaphragm were measured. In addition, Ca2+ uptake and release from the SR, and the protein expression levels of SERCA1, SERCA2 and RyR in diaphragm muscle tissue were investigated. At 6 and 12 h post­CLP, the diaphragm half­relaxation time was prolonged and the maximum rate of tension decline was decreased and the Ca2+­uptake function of SERCA was markedly reduced. The maximum rate of twitch force development, the maximal twitch and tetanic tension, and the release function of SR were decreased at 12 h post­CLP. A total of 12 h following CLP, the protein expression levels of SERCA1 were significantly downregulated, and its activity was significantly reduced; conversely, the protein levels of SERCA2 remained unaltered. The present findings indicated that at the acute stage of sepsis induced by CLP the contractile and relaxation functions of the diaphragm were significantly compromised. The impairments in relaxation may be a result of the impaired uptake function of the SR and the downregulation in SERCA1 protein expression. Conversely, the compromised contractility may be a result of the impaired release function of the SR and the downregulation in RyR protein levels. This could provide some new insights into the treatment of sepsis. In acute stages of sepsis, the improvement of SERCA function could reduce the disequilibrium of calcium homeostasis to improve the critical illness myopathy and respiratory failure.

Targeting deregulated AMPK/mTORC1 pathways improves muscle function in myotonic dystrophy type I.

Myotonic dystrophy type I (DM1) is a disabling multisystemic disease that predominantly affects skeletal muscle. It is caused by expanded CTG repeats in the 3'-UTR of the dystrophia myotonica protein kinase (DMPK) gene. RNA hairpins formed by elongated DMPK transcripts sequester RNA-binding proteins, leading to mis-splicing of numerous pre-mRNAs. Here, we have investigated whether DM1-associated muscle pathology is related to deregulation of central metabolic pathways, which may identify potential therapeutic targets for the disease. In a well-characterized mouse model for DM1 (HSALR mice), activation of AMPK signaling in muscle was impaired under starved conditions, while mTORC1 signaling remained active. In parallel, autophagic flux was perturbed in HSALR muscle and in cultured human DM1 myotubes. Pharmacological approaches targeting AMPK/mTORC1 signaling greatly ameliorated muscle function in HSALR mice. AICAR, an AMPK activator, led to a strong reduction of myotonia, which was accompanied by partial correction of misregulated alternative splicing. Rapamycin, an mTORC1 inhibitor, improved muscle relaxation and increased muscle force in HSALR mice without affecting splicing. These findings highlight the involvement of AMPK/mTORC1 deregulation in DM1 muscle pathophysiology and may open potential avenues for the treatment of this disease.

Highly sensitive myosin phosphorylation analysis in the renal afferent arteriole.

The regulation of smooth muscle contraction and relaxation involves phosphorylation and dephosphorylation of regulatory proteins, particularly myosin. To elucidate the regulatory mechanisms, analyzing the phosphorylation signal transduction is crucial. Although a pharmacological approach with selective inhibitors is sensitive and a useful technique, it leads to speculation regarding a signaling pathway but does not provide direct evidence of changes at a molecular level. We developed a highly sensitive biochemical technique to analyze phosphorylation by adapting Phos-tag SDS-PAGE. With this technique, we successfully analyzed myosin light chain (LC20) phosphorylation in tiny renal afferent arterioles. In the rat afferent arterioles, endothelin-1 (ET-1) induced diphosphorylation of LC20 at Ser19 and Thr18 as well as monophosphorylation at Ser19 via ET B receptor activation. Considering that LC20 diphosphorylation can decrease the rate of dephosphorylation and thus relaxation, we concluded that LC20 diphosphorylation contributes, at least in part, to the prolonged contraction induced by ET-1 in the renal afferent arteriole.

Divergent abnormal muscle relaxation by hypertrophic cardiomyopathy and nemaline myopathy mutant tropomyosins.

Mutations in tropomyosin (Tm) have been linked to distinct inherited diseases of cardiac and skeletal muscle, hypertrophic cardiomyopathy (HCM), and nemaline myopathy (NM). How HCM and NM mutations in nearly identical Tm proteins produce the vastly divergent clinical phenotypes of heightened, prolonged cardiac muscle contraction in HCM and skeletal muscle weakness in NM is currently unknown. We report here a direct comparison of the effects of HCM (A63V) and NM (M9R) mutant Tm on membrane-intact myocyte contractile function as assessed by adenoviral gene transfer to fully differentiated cardiac muscle cells. Wild-type, and mutant HCM, and mutant NM proteins were expressed at similar levels in myocytes and incorporated into sarcomeres. Interestingly, HCM mutant Tm produced significantly longer contractions by slowing relaxation, whereas NM mutant Tm produced the opposite effect of accelerated muscle relaxation. We propose slowed relaxation caused by HCM mutant Tm can directly contribute to diastolic dysfunction seen in HCM even without secondary cardiac remodeling. Conversely, hastening of relaxation by NM mutant Tm may shift the force-frequency relationship in skeletal muscle and contribute to muscle weakness seen in NM. Together, these results implicate divergent, abnormal "turning off" of muscle contraction as a cellular basis for the differential pathogenesis of mutant Tm-associated HCM and NM.

Impaired relaxation of stomach smooth muscle in mice lacking cyclic GMP-dependent protein kinase I.

1. Guanosine 3', 5'-cyclic monophosphate (cyclic GMP)-dependent kinase I (cGKI) is a major receptor for cyclic GMP in a variety of cells. Mice lacking cGKI exhibit multiple phenotypes, including severe defects in smooth muscle function. We have investigated the NO/cGMP- and vasoactive intestinal polypeptide (VIP)/adenosine 3', 5'-cyclic monophosphate (cyclic AMP)-signalling pathways in the gastric fundus of wild type and cGKI-deficient mice. 2. Using immunohistochemistry, similar staining patterns for NO-synthase, cyclic GMP- and VIP-immunoreactivities were found in wild type and cGKI-deficient mice. 3. In isolated, endothelin-1 (3 nM - 3 microM)-contracted, muscle strips from wild type mice, electrical field stimulation (1 - 16 Hz) caused a biphasic relaxation, one initial rapid, followed by a more slowly developing phase. In preparations from cGKI-deficient mice only the slowly developing relaxation was observed. 4. The responses to the NO donor, SIN-1 (10 nM - 100 microM), and to 8-Br-cyclic GMP (10 nM - 100 microM) were markedly impaired in strips from cGKI-deficient mice, whereas the responses to VIP (0.1 nM - 1 microM) and forskolin (0.1 nM - 1 microM) were similar to those in wild type mice. 5. These results suggest that cGKI plays a central role in the NO/cGMP signalling cascade producing relaxation of mouse gastric fundus smooth muscle. Relaxant agents acting via the cyclic AMP-pathway can exert their effects independently of cGKI.

Regulation of the spontaneous contractile activity of the portal vein by the sarcoplasmic reticulum: evidence from the phospholamban gene-ablated mouse.

The rapid contraction/relaxation cycles of phasic smooth muscles necessitates intracellular calcium cycling at a more rapid rate than that of tonic smooth muscles. Recent studies suggest that sarcoplasmic reticulum calcium handling is an important determinant of portal vein phasic contractions. We evaluated the importance and role of phospholamban, a protein which inhibits the sarcoplasmic reticulum (SR) calcium ATPase (SERCA), in regulating the contractility of the phasic mouse portal vein. PLB gene ablation significantly reduced the basal frequency of spontaneous mechanical activity and increased force development of the portal vein. Cyclopiazonic acid (CPA), an inhibitor of SERCA, did not significantly affect the spontaneous activity of the wild-type (WT) portal vein. CPA (1 microM) eliminated the differences in frequency and force between the PLB-KO and WT, localizing the effects to the SR. The PLB-KO portal vein had a lower resting membrane potential than WT controls. There were no significant differences between WT and KO responses to charybdotoxin (250 nM), indicating that calcium-activated potassium channels do not contribute to altered KO portal vein contractility. While contractile sensitivity to acetylcholine was not different between WT and PLB-KO portal veins, force generated in response to a given concentration of acetylcholine was significantly greater in the PLB-KO portal vein, both in the absence and presence of CPA. Our results confirm that SR activity can play a major role in modulating the frequency of the spontaneous mechanical activity of portal veins and removal of PLB inhibition of the SR calcium ATPase has significant effects on the spontaneous activity of the portal vein.