Ameliorative effect of flavocoxid on cyclophosphamide-induced cardio and neurotoxicity via targeting the GM-CSF/NF-κB signaling pathway.

Cyclophosphamide (Cyclo) is a chemotherapeutic agent used as an immunosuppressant and as a treatment for many cancerous diseases. Many previous pieces of literature proved the marked cardio and neurotoxicity of the drug. Thus, this research provides evidence on the alleviative effect of flavocoxid on the cardiac and brain toxicity of cyclophosphamide in mice and determines its underlying mechanisms. Flavocoxid (Flavo) is a potent antioxidant and anti-inflammatory agent that inhibits the peroxidase activity of cyclooxygenase (COX-1 and COX-2) enzymes and 5-lipooxygenase (5-LOX). Flavo was administered orally (20 mg/kg) for 2 weeks, followed by Cyclo (100 mg/kg, i.p.) on day 14. Higher heart and brain weight indices, serum lactate dehydrogenase (LDH), creatine kinase (CK-MB), and nitric oxide (NO) were mitigated following Flavo administration. Flavo modulated oxidative stress biomarkers (malonaldehyde (MDA), glutathione (GSH), and superoxide dismutase (SOD)), tumor necrosis factor-α (TNF-α), and interleukin (IL)-1ß. Additionally, cardiac troponin I (cTn-I), nuclear factor kappa B (NF-κB), brain amyloid precursor protein (APP), and granulocyte macrophage colony-stimulating factor (GM-CSF) were decreased by Flavo administration. Moreover, Flavo ameliorated heart and brain histopathological changes and caspase-3 levels. Collectively, Flavo (20 mg/kg) for 14 days showed significant cardio and neuroprotective effects due to its antioxidant, anti-inflammatory, and antiapoptotic activities via modulation of oxidative stress, inflammation, and the GM-CSF/NF-κB signaling pathway.

Effects of Sarcomere Activators and Inhibitors Targeting Myosin Cross-Bridges on Ca2+-Activation of Mature and Immature Mouse Cardiac Myofilaments.

We tested the hypothesis that isoform shifts in sarcomeres of the immature heart modify the effect of cardiac myosin-directed sarcomere inhibitors and activators. Omecamtiv mecarbil (OM) activates tension and is in clinical trials for the treatment of adult acute and chronic heart failure. Mavacamten (Mava) inhibits tension and is in clinical trials to relieve hypercontractility and outflow obstruction in advanced genetic hypertrophic cardiomyopathy (HCM), which is often linked to mutations in sarcomeric proteins. To address the effect of these agents in developing sarcomeres, we isolated heart fiber bundles, extracted membranes with Triton X-100, and measured tension developed over a range of Ca2+ concentrations with and without OM or Mava treatment. We made measurements in fiber bundles from hearts of adult nontransgenic (NTG) controls expressing cardiac troponin I (cTnI), and from hearts of transgenic (TG-ssTnI) mice expressing the fetal/neonatal form, slow skeletal troponin I (ssTnI). We also compared fibers from 7- and 14-day-old NTG mice expressing ssTnI and cTnI. These studies were repeated with 7- and 14-day-old transgenic mice (TG-cTnT-R92Q) expressing a mutant form of cardiac troponin T (cTnT) linked to HCM. OM increased Ca2+-sensitivity and decreased cooperative activation in both ssTnI- and cTnI-regulated myofilaments with a similar effect: reducing submaximal tension in immature and mature myofilaments. Although Mava decreased tension similarly in cTnI- and ssTnI-regulated myofilaments controlled either by cTnT or cTnT-R92Q, its effect involved a depressed Ca2+-sensitivity in the mature cTnT-R92 myofilaments. Our data demonstrate an influence of myosin and thin-filament associated proteins on the actions of myosin-directed agents such as OM and Mava. SIGNIFICANCE STATEMENT: The effects of myosin-targeted activators and inhibitors on Ca2+-activated tension in developing cardiac sarcomeres presented here provide novel, ex vivo evidence as to their actions in early-stage cardiac disorders. These studies advance understanding of the molecular mechanisms of these agents, which are important in preclinical studies employing sarcomere Ca2+-response as a screening approach. The data also inform the use of commonly immature cardiac myocytes generated from human-inducible pluripotent stem cells in screening for sarcomere activators and inhibitors.

Interleukin-37 and Dendritic Cells Treated With Interleukin-37 Plus Troponin I Ameliorate Cardiac Remodeling After Myocardial Infarction.

BACKGROUND: Excessive immune-mediated inflammatory reactions play a deleterious role in postinfarction ventricular remodeling. Interleukin-37 (IL-37) emerges as an inhibitor of both innate and adaptive immunity. However, the exact role of IL-37 and IL-37 plus troponin I (TnI)-treated dendritic cells (DCs) in ventricular remodeling after myocardial infarction (MI) remains elusive. METHODS AND RESULTS: MI was induced by permanent ligation of the left anterior descending artery. Our results showed that treatment with recombinant human IL-37 significantly ameliorated ventricular remodeling after MI, as demonstrated by decreased infarct size, better cardiac function, lower mortality, restricted inflammatory responses, decreased myocardial fibrosis, and inhibited cardiomyocyte apoptosis. In vitro, we examined the phenotype of IL-37 plus TnI-conditioned DCs of male C57BL/6 mice and their capacity to influence the number of regulatory T cells. Our results revealed that IL-37 plus TnI-conditioned DCs obtained the characteristics of tolerogenic DCs (tDCs) and expanded the number of regulatory T cells when co-cultured with splenic CD4+ T cells. Interestingly, we also found that adoptive transfer of these antigen-loaded tDCs markedly increased the number of regulatory T cells in the spleen, attenuated the infiltration of inflammatory cells in the infarct hearts, decreased myocardial fibrosis, and improved cardiac function. CONCLUSIONS: Our results reveal a beneficial role of IL-37 or tDCs treated with IL-37 plus TnI in post-MI remodeling that is possibly mediated by reestablishing a tolerogenic immune response, indicating that IL-37 or adoptive transfer of IL-37 plus TnI-treated tDCs may be a novel therapeutic strategy for ventricular remodeling after MI.

Sitagliptin attenuates cardiomyopathy by modulating the JAK/STAT signaling pathway in experimental diabetic rats.

Sitagliptin, a dipeptidyl peptidase-4 inhibitor, has been reported to promote cardioprotection in diabetic hearts by limiting hyperglycemia and hyperlipidemia. However, little is known about the involvement of the Janus kinase/signal transducer and activator of transcription (JAK/STAT) pathway modulation in the cardioprotective effects of sitagliptin. The current study aimed to investigate the protective effects of sitagliptin against diabetic cardiomyopathy (DCM), focusing on the modulation of the JAK/STAT pathway. Diabetes was induced by streptozotocin injection, and rats received sitagliptin orally and daily for 90 days. Diabetic rats exhibited hyperglycemia, hyperlipidemia, and a significant increase in heart-to-body weight (HW/BW) ratio. Serum troponin I and creatine kinase MB, cardiac interleukin-6 (IL-6), lipid peroxidation, and nitric oxide levels showed significant increase in diabetic rats. In contrast, both enzymatic and nonenzymatic antioxidant defenses were significantly declined in the heart of diabetic rats. Histopathological study revealed degenerations, increased collagen deposition in the heart of diabetic rats. Sitagliptin alleviated hyperglycemia, hyperlipidemia, HW/BW ratio, histological architecture, oxidative stress, and inflammation, and rejuvenated the antioxidant defenses. In addition, cardiac levels of pJAK2 and pSTAT3 were increased in diabetic rats, an effect which was remarkably decreased after sitagliptin treatment. In conclusion, these results confer an evidence that sitagliptin has great therapeutic potential on DCM through down-regulation of the JAK/STAT signaling pathway.

The troponin I: inhibitory peptide uncouples force generation and the cooperativity of contractile activation in mammalian skeletal muscle.

Hodges and his colleagues identified a 12 amino acid fragment of troponin I (TnI-ip) that inhibits Ca(2+)-activated force and reduces the effectiveness Ca(2+) as an activator. To understand the role of troponin C (TnC) in the extended cooperative interactions of thin filament activation, we compared the effect of TnI-ip with that of partial troponin TnC extraction. Both methods reduce maximal Ca(2+)-activated force and increase [Ca(2+)] required for activation. In contrast to TnC extraction, TnI-ip does not reduce the extended cooperative interactions between adjacent thin filament regulatory units as assessed by the slope of the pCa/force relationship. Additional evidence that TnI-ip does not interfere with extended cooperativity comes from studies that activate muscle by rigor crossbridges (RXBs). TnI-ip increases both the cooperativity of activation and the concentration of RXBs needed for maximal force. This shows that TnI-ip binding to TnC increases the stability of the relaxed state of the thin filament. TnI-ip, therefore, uncouples force generation from extended cooperativity in both Ca(2+) and RXB activated muscle contraction. Because maximum force can be reduced with no change-or even an increase-in cooperativity, force-generating crossbridges do not appear to be the primary activators of cooperativity between thin filament regulatory units of skeletal muscle.

Cellular proliferative response to cardiac troponin-I in patients with idiopathic dilated cardiomyopathy.

BACKGROUND: Approximately 20% of patients with idiopathic dilated cardiomyopathy (iDCM) have autoantibodies (AAbs) specific to cardiac troponin-I (cTnI). However, there has been no work evaluating active cellular autoimmunity. We aimed to identify a cTnI-stimulated cellular autoimmune response and to correlate our findings with cTnI AAb production. METHODS: Samples were obtained from stable ambulatory iDCM patients and healthy controls. Peripheral blood monocytes were incubated with cTnI, and cellular proliferation was measured using flow cytometry. AAbs against cTnI were detected by enzyme-linked immunosorbent assay. RESULTS: A positive cellular proliferative response to cTnI was identified in 20.5% (9/44) patients with iDCM and 5.7% (2/35) of healthy controls (p < 0.05). Positive cTnI AAbs were identified in 20% (7/35) of healthy controls and 13.6% (6/44) of patients with iDCM (p = NS). The presence of cTnI AAbs did not correlate with a positive cellular proliferative response. However, patients with iDCM who had an AAb response to cTnI were less likely to be taking a statin (p < 0.05). CONCLUSIONS: A cellular autoimmune response to cTnI is demonstrated in a subset of patients with iDCM. However, the presence of a cellular response did not correlate with the presence of AAbs to the same antigen.

Antiinflammatory autoimmune cellular responses to cardiac troponin I in idiopathic dilated cardiomyopathy.

BACKGROUND: Autoimmune mechanisms, particularly through generation of autoantibodies, may contribute to the pathophysiology of idiopathic dilated cardiomyopathy (iDCM). The precise role of cellular autoimmune responses to cardiac-specific antigens has not been well described in humans. The purpose of this study was to characterize the cellular autoimmune response to cardiac troponin I (cTnI), specifically, the release of cytokines by peripheral blood mononuclear cells (PBMCs), in subjects with iDCM and healthy control subjects. METHODS AND RESULTS: We performed enzyme-linked immunospot assays on PBMCs isolated from subjects with iDCM and healthy control subjects to examine the ex vivo interferon-gamma (IFN-γ) and interleukin-10 (IL-10) production in response to cTnI exposure. Thirty-five consecutive subjects with iDCM (mean age 53 ± 11 years, 60% male, left ventricular ejection fraction 23 ± 7%) and 26 control subjects (mean age 46 ± 13 years, 46% male) were prospectively enrolled. IFN-γ production in response to cTnI did not differ between the groups (number of secreting cells 26 ± 49 vs 38 ± 53, respectively; P = .1). In contrast, subjects with iDCM showed significantly higher IL-10 responses to cTnI compared with control subjects (number of secreting cells 386 ± 428 vs 152 ± 162, respectively; P < .05). Among iDCM subjects, heightened IL-10 response to cTnI was associated with reduced systemic inflammation and lower prevalence of advanced diastolic dysfunction compared with those with normal IL-10 response to cTnI. CONCLUSIONS: Our preliminary findings suggest that a heightened cellular autoimmune IL-10 response to cTnI is detectable in a subset of patients with iDCM, which may be associated with reduced systemic levels of high-sensitivity C-reactive protein and lower prevalence of advanced diastolic dysfunction.

Increased cross-bridge cycling kinetics after exchange of C-terminal truncated troponin I in skinned rat cardiac muscle.

The precise mechanism of cardiac troponin I (cTnI) proteolysis in myocardial stunning is not fully understood. Accordingly, we determined the effect of cTnI C terminus truncation on chemo-mechanical transduction in isolated skinned rat trabeculae. Recombinant troponin complex (cTn), containing either mouse cTnI-(1-193) or human cTnI-(1-192) was exchanged into skinned cardiac trabeculae; Western blot analysis confirmed that 60-70% of the endogenous cTn was replaced by recombinant Tn. Incorporation of truncated cTnI induced significant reductions ( approximately 50%) in maximum force and cooperative activation as well as increases ( approximately 50%) in myofilament Ca(2+) sensitivity and tension cost. Similar results were obtained with either mouse or human truncated cTn. Presence of truncated cTnI increased maximum actin-activated S1 ATPase activity as well as its Ca(2+) sensitivity in vitro. Partial exchange (50%) for truncated cTnI resulted in similar reductions in maximum force and cooperativity; tension cost was increased in proportion to truncated cTnI content. In vitro, to determine the molecular mechanism responsible for the enhanced myofilament Ca(2+) sensitivity, we measured Ca(2+) binding to cTn as reported using a fluorescent probe. Incorporation of truncated cTnI did not affect Ca(2+) binding affinity to cTn alone. However, when cTn was incorporated into thin filaments, cTnI truncation induced a significant increase in Ca(2+) binding affinity to cTn. We conclude that cTnI truncation induces depressed myofilament function. Decreased cardiac function after ischemia/reperfusion injury may directly result, in part, from proteolytic degradation of cTnI, resulting in alterations in cross-bridge cycling kinetics.

Tick troponin I-like molecule is a potent inhibitor for angiogenesis.

This is the first report identifying the anti-angiogenesis saliva molecule of the ixodid tick. In our previous study, we identified a troponin I-like molecule (HLTnI) of the ixodid hard tick Haemaphysalis longicornis, a vector for various pathogens. To investigate its potential inhibitory effects for angiogenesis, we expressed and purified recombinant HLTnI in Escherichia coli. In a vascular endothelial growth factor (VEGF) competitive angiogenesis assay, the recombinant HLTnI significantly inhibited the capillary formation of human vascular endothelial cells (HUVEC) in vitro. The inhibition was dose-dependent with an IC(50) of 18.95 nM. These results indicated that HLTnI is a potent angiogenesis inhibitor.

Troponina como indicador de gravidade angiográfica em pacientes com síndrome coronariana aguda sem supradesnível de segmento ST / Use of troponin level as an indicator of angiographic gravity in patients with acute coronary syndrome without ST - segment elevation

Objetivos:Avaliar se os níveis aumentados de tropina I nos pacientes com sídrome coronariana aguda sem supradesnível de segmento ST são indicadores de gravidade coronariográfica, comparar os achados coronariográficos dos pacientes com e sem elevação da troponina e correlacionar os níveis de troponina I com os seguintes achados angiográficos: número de vasos com doença coronariana obstrutiva (maior ou igual 70 por cento), fluxo coronariano, aspecto da lesão e a presença de trombo na lesão culpada.Métodos:Foram incluídos 90 pacientes admitidos com diagnóstico de spindrome coronariana aguda sem supradesnível de segmento ST, submetidos a coronariografia nas primeiras 48 horas com dosagens séricas de troponina I nas primeiras 12 horas

Synthetic peptides of actin-tropomyosin binding region of troponin I and heat shock protein 20 modulate the relaxation process of skinned preparations of taenia caeci from guinea pig.

To explore the possible role of the thin filament-linked regulation of cross-bridge cycling in living smooth muscle contraction, we studied the effects of TnIp and HSP20p, a synthetic peptide originating from an actin tropomyosin binding region of rabbit cardiac troponin I (residues 136-147; GKFKRPTLRRVR), and that of human heat shock protein 20 (residues 110-121; GFVAREFHRRYR) on the relaxation of skinned (cell membrane ilized) preparations from guinea pig taenia caeci. An active stress of the skinned preparations, resulting from actin-myosin interaction, rapidly decayed following Ca(2+) removal (relaxation). TnIp accelerated the initial rapid phase and slowed the following slow phase of the relaxation. On the other hand, HSP20p only slowed the whole process of the relaxation. The relaxation time courses were well fitted in a double exponential manner, and the double exponential decay of the stress could be explained as a portion of fast-detaching cross bridges not to dissociate rapidly by Ca(2+) removal, but to transfer to latch bridges dissociating very slowly. Our present results suggested that (i) TnIp and HSP20p accelerated transferring from fast-detaching cross bridges to slow-detaching (latch) bridges, and (ii) TnIp accelerated dissociation of the fast-detaching cross bridges and the latch bridges, while HSP20p slowed dissociation the fast-detaching cross bridges. Since TnIp and HSP20p are thought to bind to actin and tropomyosin, but not to myosin, we concluded that through thin-filament-dependent mechanisms these peptides regulated the formation and/or deformation of latch bridges in smooth muscle. The thin-filament-dependent regulation might physiologically control the stress maintenance and relaxation in smooth muscle cells.

Augmented systolic response to the calcium sensitizer EMD-57033 in a transgenic model with troponin I truncation.

Myocardial stunning is a form of acute reversible cardiac dysfunction that occurs after brief periods of ischemia and reperfusion. In several animal models, stunning is associated with proteolytic truncation of troponin I (TnI). Mice expressing the same proteolytic TnI fragment [TnI-(1-193)] demonstrate cardiac depression with a decreased maximal calcium-activated tension. We therefore hypothesized preferential improvement in mice expressing TnI-(1-193) treated with the calcium-sensitizing drug EMD-57033. TnI-(1-193) and nontransgenic myofibrils exhibited significant sensitization to calcium in Mg-ATPase assays after EMD-57033 exposure. However, only transgenic myofibrils exhibited an increase in maximal activity (P = 0.023). EMD-57033 also increased maximal calcium-activated force in TnI-(1-193) muscle, such that it was comparable to nontransgenic cardiac muscle. EMD-57033 enhanced in vivo systolic function modestly in controls but had a marked effect in transgenic mice, with an almost threefold greater leftward shift of the end-systolic pressure-volume relation (P = 0.0005). These data indicate a targeted efficacy of EMD-57033 in offsetting the contractile defect in TnI-(1-193) mice, and this may have therapeutic implications in models displaying this myofilament defect.

Inherited cardiomyopathies as a troponin disease.

Troponin, one of the sarcomeric proteins, plays a central role in the Ca(2+) regulation of contraction in vertebrate skeletal and cardiac muscles. It consists of three subunits with distinct structure and function, troponin T, troponin I, and troponin C, and their accurate and complex intermolecular interaction in response to the rapid rise and fall of Ca(2+) in cardiomyocytes plays a key role in maintaining the normal cardiac pump function. More than 200 mutations in the cardiac sarcomeric proteins, including myosin heavy and light chains, actin, troponin, tropomyosin, myosin-binding protein-C, and titin/connectin, have been found to cause various types of cardiomyopathy in human since 1990, and more than 60 mutations in human cardiac troponin subunits have been identified in dilated, hypertrophic, and restrictive forms of cardiomyopathy. In this review, we have focused on the mutations in the genes for human cardiac troponin subunits and discussed their functional consequences that might be involved in the primary mechanisms for the pathogenesis of these different types of cardiomyopathy.

Troponin I peptide (Glu94-Leu123), a cartilage-derived angiogenesis inhibitor: in vitro and in vivo effects on human endothelial cells and on pancreatic cancer.

Several inhibitors of angiogenesis have been identified in bovine and shark cartilage. One of them is troponin I, which is the molecule responsible for the inhibition of the actomyosin ATPase during muscle contraction. In this study we sought to investigate if the active site of troponin I (peptide Glu94-Leu123; pTnI) is also the one responsible for the antiangiogenic properties of this protein. The effects of pTnI on endothelial cell tube formation and endothelial cell division were investigated using human umbilical vein endothelial cells, Matrigel, light microscopy, carboxyfluorescein diacetate, succinimidyl esterlabeling, and flow cytometry. Its effects on induction of ICAM-1 and production of vascular endothelial growth factor by pancreatic cancer cells (CAPAN-1) were also investigated, as was its efficacy in a mouse model of pancreatic cancer metastases. Our results show that concentrations as low as 1 pg/ml of pTnI significantly inhibit endothelial cell tube formation, and that endothelial cell division was inhibited at 96 hours by 3 microg/ml pTnI (P=0.0001). No effects were seen using troponin peptide 124-181 as a control. pTnI-treated supernatant from the pancreatic cancer cell line CAPAN-1 downregulated ICAM-1 expression on human umbilical vein endothelial cells up to 10 ng/ml pTnI, and a significant reduction in vascular endothelial growth factor production was seen by treating CAPAN-1 cells with up to 1 microg/ml pTnI. After intrasplenic injection of CAPAN-1 cells, mice treated with pTnI had fewer liver metastases compared to control mice (liver/body weight 5.5 vs. 11.1; P=0.03). The active region of troponin I is the one responsible for its antiangiogenic effect. The mechanism of action of this peptide is probably multifactorial.

Calpain-1-dependent degradation of troponin I mutants found in familial hypertrophic cardiomyopathy.

The mechanism by which mutations of the cardiac troponin I (cTnI) gene evoke familial hypertrophic cardiomyopathy (fHCM) is unknown. In this investigation the potential effects of three fHCM-related cTnI mutations on Calpain-1-induced cTnI degradation were tested, and a study was made of whether additional conformational changes due to troponin complex formation and protein kinase A-induced phosphorylation affect the intensity of cTnI proteolysis. Purified recombinant wild-type cTnI and three of its fHCM-related missense mutants (R145G, G203S and K206Q), alone or in the troponin complex (i.e. together with troponin C and troponin T), in the non-phosphorylated or protein kinase A-bisphosphorylated forms were proteolyzed in vitro in the presence of Calpain-1 (0.05-2.5 U) at 30 degrees C. Following incubation with Calpain-1 for 0.5, 30, 60 or 120 min, the extent of protein degradation was evaluated through the use of Western immunoblotting and densitometry. The results indicated that both the wild-type and the mutant cTnI molecules were susceptible to Calpain-1. However, the degradation of the cTnI molecules in the troponin complex was less intense than that of the non-complexed forms. Moreover, phosphorylation by protein kinase A conferred effective protection against cTnI proteolysis. The data suggested that mutations in the central inhibitory domain (R145G) and in the C-terminal region (G203S and K206Q) of cTnI do not affect its Calpain-1-mediated degradation, or the phosphorylation-induced protection against proteolysis.

Troponin I binds polycystin-L and inhibits its calcium-induced channel activation.

Polycystin-L (PCL) is an isoform of polycystin-2, the product of the second gene associated with autosomal dominant polycystic kidney disease, and functions as a Ca(2+)-regulated nonselective cation channel. We recently demonstrated that polycystin-2 interacts with troponin I, an important regulatory component of the actin microfilament complex in striated muscle cells and an angiogenesis inhibitor. In this study, using the two-microelectrode voltage-clamp technique and Xenopus oocyte expression system, we showed that the calcium-induced PCL channel activation is substantially inhibited by the skeletal and cardiac troponin I (60% and 31% reduction, respectively). Reciprocal co-immunoprecipitation experiments demonstrated that PCL physically associates with the skeletal and cardiac troponin I isoforms in overexpressed Xenopus oocytes and mouse fibroblast NIH 3T3 cells. Furthermore, both native PCL and cardiac troponin I were present in human heart tissues where they indeed associate with each other. GST pull-down and microtiter binding assays showed that the C-terminus of PCL interacts with the troponin I proteins. The yeast two-hybrid assay further verified this interaction and defined the corresponding interacting domains of the PCL C-terminus and troponin I. Taken together, this study suggests that troponin I acts as a regulatory subunit of the PCL channel complex and provides the first direct evidence that PCL is associated with the actin cytoskeleton through troponin I.

Cooperative inhibition of actin filaments in the absence of tropomyosin.

We measured the inhibition of actin activated myosin subfragment-1 MgATPase activity in a solution containing no added KCl (5 mM PIPES.K2 (pH 7.1), 2.5 mM MgCl2, 1 mM DTT, 1 mM NaN3, 5 mM MgATP). Maximal inhibition was observed with substoichiometric concentrations of caldesmon, caldesmon domain 4, troponin and troponin I. In six experiments using different preparations of actin, S-1 and caldesmon 50% inhibition required 0.09 +/- 0.01 (sem) caldesmon added per actin. This compares with 0.66 +/- 0.32 (sem, n = 5) caldesmon per actin for 50% inhibition in the presence of 60 mM KCl. With caldesmon domain 4, 50% inhibition was achieved with 0.17 +/- 0.08 (n = 11) domain 4 added per actin. We measured the amount of caldesmon bound at the same time as inhibition. Complete inhibition of actin activated ATPase needed only one caldesmon bound per 5.0 +/- 0.5 (sem, n = 5) actin monomers or one caldesmon domain 4 bound per 3.9 +/- 0.6 (sem, n = 3) actin monomers at zero KCl. We conclude that under these conditions inhibition of actin is cooperative despite the absence of tropomyosin. We measured the effect of caldesmon inhibition upon S-1 binding to actin. S-1.ADP.Pi (weak binding) was not affected by caldesmon concentrations giving 80% inhibition, however S-1.ADP (strong binding) was highly cooperative, being very weak at <0.3 microM but indistinguishable from uninhibited actin at >2 microM S-1.ADP. We conclude that actin can exist in two activity states corresponding to the 'on' and 'off' states of actin-tropomyosin and inhibitory proteins function as allosteric-cooperative inhibitors of actin. The implications of these findings for the role of tropomyosin in thin filament regulation are discussed.

Effects of T142 phosphorylation and mutation R145G on the interaction of the inhibitory region of human cardiac troponin I with the C-domain of human cardiac troponin C.

Cardiac troponin I (cTnI) is the inhibitory component of the troponin complex, and its interaction with cardiac troponin C (cTnC) plays a critical role in transmitting the Ca(2+) signal to the other myofilament proteins in heart muscle contraction. The switch between contraction and relaxation involves a movement of the inhibitory region of cTnI (cIp) from cTnC to actin-tropomyosin. This region of cTnI is prone to missense mutations in heart disease, and a specific mutation, R145G, has been associated with familial hypertrophic cardiomyopathy. It also contains the unique cardiac PKC phosphorylation site at residue T142. To determine the structural consequences of the mutation R145G and the T142 phosphorylation on the interaction of cIp with cTnC, we have utilized 2D [(1)H, (15)N]-HSQC NMR spectroscopy to monitor the binding of native cIp, cIp-R (R145G), and cIp-P (phosphorylated T142), respectively, to the Ca(2+)-saturated C-domain of cTnC (cCTnC.2Ca(2+)). We also report a strategy for cloning, expression, and purification of cTnI peptide, and both synthetic and recombinant peptides are used in this study. NMR chemical shift mapping indicates that the binding epitope of cIp on cCTnC.2Ca(2+) is not greatly affected, but the affinity is reduced by approximately 14-fold by the T142 phosphorylation and approximately 4-fold by the mutation R145G, respectively. This suggests that these modifications of cIp have an adverse effect on the binding of cIp to cCTnC.2Ca(2+). These perturbations may correlate with the impairment or loss of cTnI function in heart muscle contraction.

Troponin I converts the skeletal muscle ryanodine receptor into a rectifying calcium release channel.

The goal of our present studies has been to find novel ryanodine receptor (RyR1) interacting polypeptides that modulate the channel activity from the luminal side of RyR1. Using K(+) as charge carrier for recording of single channel events here we demonstrate a very unexpected observation that troponin I substantially alters RyR's gating behavior, and that RyR1 in association with troponin I becomes a rectifying Ca(2+) release channel. Troponin I rapidly locks the RyR1 in a non-conducting state only at a negative holding potential, and only when applied to the luminal side; switching to a positive holding potential results in the channel returning to its original activity, immediately. A hypothesis is proposed to account for how an intraluminally located, positively charged molecule might function as a RyR1 regulator under physiological conditions.