Intermolecular And Dynamic Investigation of The Mechanism of Action of Reldesemtiv on Fast Skeletal Muscle Troponin Complex Toward the Treatment of Impaired Muscle Function.

Muscle weakness as a secondary feature of attenuated neuronal input often leads to disability and sometimes death in patients with neurogenic neuromuscular diseases. These impaired muscle function has been observed in several diseases including amyotrophic lateral sclerosis, Charcot-Marie-Tooth, spinal muscular atrophy and Myasthenia gravis. This has spurred the search for small molecules which could activate fast skeletal muscle troponin complex as a means to increase muscle strength. Discovered small molecules have however been punctuated by off-target and side effects leading to the development of the second-generation small molecule, Reldesemtiv. In this study, we investigated the impact of Reldesemtiv binding to the fast skeletal troponin complex and the molecular determinants that condition the therapeutic prowess of Redesemtiv through computational techniques. It was revealed that Reldesemtiv binding possibly potentiates troponin C compacting characterized by reduced exposure to solvent molecules which could favor the slow release of calcium ions and the resultant sensitization of the subunit to calcium. These conformational changes were underscored by conventional and carbon hydrogen bonds, pi-alkyl, pi-sulfur and halogen interactions between Reldesemtiv the binding site residues. Arg113 (-3.96 kcal/mol), Met116 (-2.23 kcal/mol), Val114 (-1.28 kcal/mol) and Met121 (-0.63 kcal/mol) of the switch region of the inhibitory subunit were among the residues that contributed the most to the total free binding energy of Reldesemtiv highlighting their importance. These findings present useful insights which could lay the foundation for the development of fast skeletal muscle small molecule activators with high specificity and potency.

HSF-1-mediated cytoskeletal integrity determines thermotolerance and life span.

The conserved heat shock transcription factor-1 (HSF-1) is essential to cellular stress resistance and life-span determination. The canonical function of HSF-1 is to regulate a network of genes encoding molecular chaperones that protect proteins from damage caused by extrinsic environmental stress or intrinsic age-related deterioration. In Caenorhabditis elegans, we engineered a modified HSF-1 strain that increased stress resistance and longevity without enhanced chaperone induction. This health assurance acted through the regulation of the calcium-binding protein PAT-10. Loss of pat-10 caused a collapse of the actin cytoskeleton, stress resistance, and life span. Furthermore, overexpression of pat-10 increased actin filament stability, thermotolerance, and longevity, indicating that in addition to chaperone regulation, HSF-1 has a prominent role in cytoskeletal integrity, ensuring cellular function during stress and aging.

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.

Delivery and visualization of proteins conjugated to quantum dots in cardiac myocytes.

The design of a novel transduction complex has permitted the introduction of protein-quantum dot conjugates into the cytoplasm of living cells. Appropriate subcellular localization of quantum dot-conjugated cardiac troponin C to the myofibrils and a nuclear peptide to the nucleus was attained in living cardiac myocytes using this approach. This new methodology opens the possibility for live tracking of exogenous proteins and study of protein dynamics.

Functional effects of the DCM mutant Gly159Asp troponin C in skinned muscle fibres.

We recently reported a dilated cardiomyopathy (DCM) causing mutation in a novel disease gene, TNNC1, which encodes cardiac troponin C (TnC). We have determined how this mutation, Gly159Asp, affects contractile regulation when incorporated into muscle fibres. Endogenous troponin in rabbit skinned psoas fibres was partially replaced by recombinant human cardiac troponin containing either wild-type or Gly159Asp TnC. We measured both the force-pCa relationship of these fibres and the activation rate using the caged-Ca(2+) compound nitrophenyl-EGTA. Gly159Asp TnC had no significant effect on either the Ca(2+) sensitivity or cooperativity of force generation when compared to wild type. However, the mutation caused a highly significant (ca. 50%) decrease in the rate of activation. This study shows that whilst not affecting the force-pCa relationship, the mutation Gly159Asp causes a significant decrease in the rate of force production and a change in the relationship between the rate of force production and generated force. In vivo, this mutation may cause both a slowing of force generation and reduction in total systolic force. This represents a novel mechanism by which a cardiomyopathy-causing mutation can affect contractility.

Left ventricular myofilament dysfunction in rat experimental hypertrophy and congestive heart failure.

It is currently unclear whether left ventricular (LV) myofilament function is depressed in experimental LV hypertrophy (LVH) or congestive heart failure (CHF). To address this issue, we studied pressure overload-induced LV hypertrophy (POLVH) and myocardial infarction-elicited congestive heart failure (MICHF) in rats. LV myocytes were isolated from control, POLVH, and MICHF hearts by mechanical homogenization, skinned with Triton, and attached to micropipettes that projected from a sensitive force transducer and high-speed motor. A subset of cells was treated with either unphosphorylated, recombinant cardiac troponin (cTn) or cTn purified from either control or failing ventricles. LV myofilament function was characterized by the force-[Ca(2+)] relation yielding Ca(2+)-saturated maximal force (F(max)), myofilament Ca(2+) sensitivity (EC(50)), and cooperativity (Hill coefficient, n(H)) parameters. POLVH was associated with a 35% reduction in F(max) and 36% increase in EC(50). Similarly, MICHF resulted in a 42% reduction in F(max) and a 30% increase in EC(50). Incorporation of recombinant cTn or purified control cTn into failing cells restored myofilament Ca(2+) sensitivity toward levels observed in control cells. In contrast, integration of cTn purified from failing ventricles into control myocytes increased EC(50) to levels observed in failing myocytes. The F(max) parameter was not markedly affected by troponin exchange. cTnI phosphorylation was increased in both POLVH and MICHF left ventricles. We conclude that depressed myofilament Ca(2+) sensitivity in experimental LVH and CHF is due, in part, to a decreased functional role of cTn that likely involves augmented phosphorylation of cTnI.

Differential activation of nitric-oxide synthase isozymes by calmodulin-troponin C chimeras.

The interactions of neuronal nitric-oxide synthase (nNOS) with calmodulin (CaM) and mutant forms of CaM, including CaM-troponin C chimeras, have been previously reported, but there has been no comparable investigation of CaM interactions with the other constitutively expressed NOS (cNOS), endothelial NOS (eNOS), or the inducible isoform (iNOS). The present study was designed to evaluate the role of the four CaM EF hands in the activation of eNOS and iNOS. To assess the role of CaM regions on aspects of enzymatic function, three distinct activities associated with NOS were measured: NADPH oxidation, cytochrome c reduction, and nitric oxide (*NO) generation as assessed by the oxyhemoglobin capture assay. CaM activates the cNOS enzymes by a mechanism other than stimulating electron transfer into the oxygenase domain. Interactions with the reductase moiety are dominant in cNOS activation, and EF hand 1 is critical for activation of both nNOS and eNOS. Although the activation patterns for nNOS and eNOS are clearly related, effects of the chimeras on all the reactions are not equivalent. We propose that cytochrome c reduction is a measure of the release of the FMN domain from the reductase complex. In contrast, cytochrome c reduction by iNOS is readily activated by each of the chimeras examined here and may be constitutive. Each of the chimeras were co-expressed with the human iNOS enzyme in Escherichia coli and subsequently purified. Domains 2 and 3 of CaM contain important elements required for the Ca2+/CaM independence of *NO production by the iNOS enzyme. The disparity between cytochrome c reduction and *NO production at low calcium can be attributed to poor association of heme and FMN domains when the bound CaM constructs are depleted of Ca2+. In general cNOSs are much more difficult to activate than iNOS, which can be attributed to their extra sequence elements, which are adjacent to the CaM-binding site and associated with CaM control.

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.

Thin filament activation and unloaded shortening velocity of rabbit skinned muscle fibres.

The unloaded shortening velocity of skinned rabbit psoas muscle fibres is sensitive to [Ca2+]. To determine whether Ca2+ affects the unloaded shortening velocity via regulation of crossbridge kinetics or crossbridge number, the shortening velocity was measured following changes in either [Ca2+] or the number of active thin filament regulatory units. The native troponin C (TnC) was extracted and replaced with either cardiac TnC (cTnC) or a mixture of cTnC and an inactive mutant cardiac TnC (CBMII TnC). The unloaded shortening velocity of the cTnC-replaced fibres was determined at various values of [Ca2+] and compared with different cTnC:CBMII TnC ratios at a saturating [Ca2+]. If Ca2+ regulates the unloaded shortening velocity via kinetic modulation, differences in the velocity-tension relationship between the cTnC fibres and the cTnC:CBMII TnC fibres would be apparent. Alternatively, Ca2+ control of the number of active crossbridges would yield similar velocity-tension relationships when comparing the cTnC and cTnC:CBMII TnC fibres. The results show a decline in the unloaded shortening velocity that is determined by the relative tension, defined as the level of thin filament activation, rather than the [Ca2+]. Furthermore, at lower levels of relative tension, the reduction in unloaded shortening is not the result of changes in any cooperative effects of myosin on Ca2+ binding to the thin filament. Rather, it may be related to a decrease in crossbridge-induced activation of the thin filament at the level of the individual regulatory unit. In summary, the results suggest that Ca2+ regulates the unloaded shortening velocity in skinned fibres by reducing the number of crossbridges able to productively bind to the thin filament without affecting any inherent property of the myosin.

Myosin light chain 2 modulates MgADP-induced contraction in rabbit skeletal and bovine cardiac skinned muscle.

Skinned skeletal and cardiac muscle fibres can be activated by MgADP in the presence of MgATP without Ca2+; the isometric tension is developed in a sigmoidal manner with the addition of MgADP under relaxing conditions. The critical concentrations of MgADP for this MgADP-induced contraction are about 7.5 and 2.6 mM for skeletal and cardiac muscle fibres, respectively. To investigate whether muscle regulatory proteins, myosin light chain 2 (LC2) and troponin C (TnC), play a part in the MgADP-induced contraction, these proteins were partly extracted by treatment with trans-1,2-cyclohexanediamine-N,N,N',N'-tetraacetic acid (CDTA), a chelater of divalent cations, and the MgADP-tension relationship was examined in rabbit psoas and bovine cardiac skinned fibres. We found that the sigmoidal MgADP-tension relationship became hyperbolic after a partial extraction of LC2 (about 30 %) and TnC (about 70 %). Reconstitution with LC2 restored the sigmoidal MgADP-tension relationship of control fibres almost fully in both skeletal and cardiac fibres, whereas reconstitution with TnC alone had no effect. Furthermore, cardiac fibres reconstituted with skeletal LC2 exhibited an MgADP-tension relationship intermediate between skeletal and cardiac fibres. The partial extraction of LC2 and TnC resulted in a reduction of the inhibitory effect of inorganic phosphate (P(i)) on the MgADP-activated tension. Reconstitution with LC2 restored the original P(i)-tension relationship, whereas reconstitution with TnC had no effect. In other words, extraction of LC2 apparently increased the affinity of myosin for MgADP but decreased the affinity for P(i). These results demonstrate that LC2 modulates MgADP-induced activation of actomyosin interaction.

Protein kinase A increases the rate of relaxation but not the rate of tension development in skinned rat cardiac muscle.

To clarify the contribution of cross-bridge kinetics to the contraction profile of cardiac twitch during beta-adrenergic stimulation, we studied the rate of tension development and relaxation following laser flash photolysis of caged compounds in rat-skinned ventricular trabeculae before and after treatment with the catalytic subunit of protein kinase A (PKA, 0.5 U/microl, 40 min). Tension development following nitrophenyl (NP)-EGTA photolysis was fitted with a single exponential function. The rate constant increased with an increase in postphotolysis steady tension, and the relation between the rate constant and the tension was not influenced by PKA. The rate of relaxation following diazo-2 photolysis was fitted with a double exponential function. The rate of both initial rapid and subsequent slow relaxation was independent of the extent of relaxation. PKA increased the rate of initial rapid relaxation by about twofold, but showed no significant effect on the rate of subsequent slow relaxation. These results suggest that in beta-receptor stimulated rat cardiac muscle, the increased rate of tension development and the facilitated relaxation rate during twitch can be partly explained as being due to the combined effects of decreased Ca(2+) affinity of troponin C and increased cycling rate of cross-bridges (subtractive combination for tension development and additive combination for tension relaxation).

Effects of sarcomere length and Ca(2+) binding on h reactivity of myofilament bound troponin C in porcine skinned cardiac muscle fibers.

Length dependence of cardiac Ca(2+) activation is an essential component of the Frank-Starling relation. The aim of this study is to examine the length effects on the Ca(2+)-induced conformational changes of filament-bound cTnC in skinned cardiac muscle fibers. The two cysteine residues (Cys-35 and Cys-84) in the regulatory domain of cTnC allow for the attachment of conformational probes to this region. Their incorporation with the fluorescent probe, 7-diethylamino-3-[4'-maleimidylphenyl]-4-methylcoumarin (CPM), was used to determine the varying cTnC conformations in cardiac fibers. The data obtained show that the length-dependent Ca(2+)-mediated conformational changes require strong-binding cross-bridges for cardiac activation.

Functional consequences of the deletion mutation deltaGlu160 in human cardiac troponin T.

To explore the functional consequences of a deletion mutation of troponin T (DeltaGlu160) found in familial hypertrophic cardiomyopathy, the mutant human cardiac troponin T, and wild-type troponins T, I, and C were expressed in Escherichia coli and directly incorporated into isolated porcine cardiac myofibrils using our previously reported troponin exchange technique. The mutant troponin T showed a slightly reduced potency in replacing the endogenous troponin complex in myofibrils and did not affect the inhibitory action of troponin I but potentiated the neutralizing action of troponin C, suggesting that the deletion of a single amino acid, Glu-160, in the strong tropomyosin-binding region affects the tropomyosin binding affinity of the entire troponin T molecule and alters the interaction between troponin I and troponin C within ternary troponin complex in the thin filament. This mutation also increased the Ca(2+) sensitivity of the myofibrillar ATPase activity, as in the case of other mutations in troponin T with clinical phenotypes of poor prognosis similar to that of Glu160. These results provide strong evidence that the increased Ca(2+) sensitivity of cardiac myofilament is a typical functional consequence of the troponin T mutation associated with a malignant form of hypertrophic cardiomyopathy.

A novel role for calmodulin: Ca2+-independent inhibition of type-1 inositol trisphosphate receptors.

Calmodulin inhibits both inositol 1,4,5-trisphosphate (IP3) binding to, and IP3-evoked Ca2+ release by, cerebellar IP3 receptors [Patel, Morris, Adkins, O'Beirne and Taylor (1997) Proc. Natl. Acad. Sci. U. S.A. 94, 11627-11632]. In the present study, full-length rat type-1 and -3 IP3 receptors were expressed at high levels in insect Spodoptera frugiperda 9 cells and the effects of calmodulin were examined. In the absence of Ca2+, calmodulin caused a concentration-dependent and reversible inhibition of [3H]IP3 binding to type-1 IP3 receptors by decreasing their apparent affinity for IP3. The effect was not reproduced by high concentrations of troponin C, parvalbumin or S-100. Increasing the medium free [Ca2+] ([Ca2+]m) inhibited [3H]IP3 binding to type-1 receptors, but the further inhibition caused by a submaximal concentration of calmodulin was similar at each [Ca2+]m. In the absence of Ca2+, 125I-calmodulin bound to a single site on each type-1 receptor subunit and to an additional site in the presence of Ca2+. There was no detectable binding of 125I-calmodulin to type-3 receptors and binding of [3H]IP3 was insensitive to calmodulin at all [Ca2+]m. Both peptide and conventional Ca2+-calmodulin antagonists affected neither [3H]IP3 binding directly nor the inhibitory effect of calmodulin in the absence of Ca2+, but each caused a [Ca2+]m-dependent reversal of the inhibition of [3H]IP3 binding caused by calmodulin. Camstatin, a peptide that binds to calmodulin equally well in the presence or absence of Ca2+, reversed the inhibitory effects of calmodulin on [3H]IP3 binding at all [Ca2+]m. We conclude that calmodulin specifically inhibits [3H]IP3 binding to type-1 IP3 receptors: the first example of a protein regulated by calmodulin in an entirely Ca2+-independent manner. Inhibition of type-1 IP3 receptors by calmodulin may dynamically regulate their sensitivity to IP3 in response to the changes in cytosolic free calmodulin concentration thought to accompany stimulation of neurones.

Neuronal nitric-oxide synthase interaction with calmodulin-troponin C chimeras.

Calmodulin (CaM) binding activates neuronal nitric-oxide synthase (nNOS) catalytic functions and also up-regulates electron transfer into its flavin and heme centers. Here, we utilized seven tight binding CaM-troponin C chimeras, which variably activate nNOS NO synthesis to examine the relationship between CaM domain structure, activation of catalytic functions, and control of internal electron transfer at two points within nNOS. Chimeras that were singly substituted with troponin C domains 4, 3, 2, or 1 were increasingly unable to activate NO synthesis, but all caused some activation of cytochrome c reduction compared with CaM-free nNOS. The magnitude by which each chimera activated NO synthesis was approximately proportional to the rate of heme iron reduction supported by each chimera, which varied from 0% to approximately 80% compared with native CaM and remained coupled to NO synthesis in all cases. In contrast, chimera activation of cytochrome c reduction was not always associated with accelerated reduction of nNOS flavins, and certain chimeras activated cytochrome c reduction without triggering heme iron reduction. We conclude: 1) CaM effects on electron transfer at two points within nNOS can be functionally separated. 2) CaM controls NO synthesis by governing heme iron reduction, but enhances reductase activity by two mechanisms, only one of which is associated with an increased rate of flavin reduction.

Utilization of troponin C as a model calcium-binding protein for mapping of the calmodulin-binding sites of caldesmon.

Troponin C, a structural analogue of calmodulin, was used for mapping the calmodulin-binding sites of caldesmon. The apparent Kd values for the formation of the caldesmon-calcium-binding-protein complex as determined by native gel electrophoresis were 0.5, 1.2 and 3.9 microM for calmodulin, rabbit skeletal muscle troponin C and bovine cardiac troponin C respectively. Troponin C induced a 4-6 nm blue shift of the Trp fluorescence of caldesmon without affecting the amplitude of fluorescence. In the presence of Ca2+, troponin C induced partial displacement of caldesmon from actin tropomyosin complexes. Addition of 5,5'-dithiobis(nitrobenzoic) acid to an equimolar complex of caldesmon and troponin C induced disulphide cross-linking between Cys-98 of rabbit skeletal muscle troponin C and the single Cys residue of duck gizzard caldesmon, located in a position analogous to Cys-580 of the chicken gizzard protein. The cross-linked caldesmon-troponin C complex was ineffective in inhibiting actomyosin ATPase activity. It is concluded that Cys-580 of caldesmon can be located close to both the central helix of calcium-binding proteins and the C-terminal domain of actin. This may be important for the regulation of actomyosin ATPase activity by caldesmon.

Effects of troponin C isoforms on pH sensitivity of contraction in mammalian fast and slow skeletal muscle fibres.

1. The effects of troponin C (TnC) isoforms on the acidic pH-induced rightward shift in the tension-pCa (-log[Ca2+]) relationship were examined in slow soleus and fast psoas skeletal muscle fibers. Endogenous TnC was partially extracted from skinned single fibres and the extracted fibres were subsequently reconstituted with purified TnC. The pCa producing one-half maximal tension (pCa50) was determined at pH 7.00 and 6.20 in each fibre and then the pH-induced shift in pCa50 (delta pCa50) was calculated. 2. In control fast fibres which express fast skeletal TnC (sTnC), the delta pCa50 was 0.64 +/- 0.02 pCa units (n = 10), and this increased significantly to 0.78 +/- 0.04 pCa units (n = 8) following extraction and reconstitution with cardiac TnC (cTnC). In each fibre, the reconstituted delta pCa50 was subtracted from the control delta pCa50 which yielded a significant shift of -0.13 +/- 0.05 pCa units (n = 8; P < 0.05). Thus, the pH sensitivity of contraction was increased in the cTnC-reconstituted psoas fibres. 3. In extracted psoas fibres that were reconstituted with fast sTnC the pH sensitivity of contraction was unchanged, indicating that the above effects were related to the TnC isoform and not a non-specific effect of the extraction procedure. 4. In a second series of experiments cTnC was specifically extracted from slow soleus fibres which were subsequently reconstituted with purified fast sTnC. Skeletal TnC reconstituted soleus fibres demonstrated a significant decrease in pH sensitivity. In each fibre, the reconstituted delta pCa50 (mean, 0.58 +/- 0.02 pCa units) was subtracted from the control delta pCa50 (mean, 0.63 +/- 0.02 pCa units) which yielded a significant shift of 0.05 +/- 0.01 pCa units (n = 4; P < 0.05). The pH sensitivity was not altered in cTnC-reconstituted soleus fibres (-0.01 +/- 0.01 pCa units, n = 4). 5. These findings indicate that TnC isoforms alter the pH sensitivities of contraction in slow and fast skeletal muscle fibres. However, the magnitude of the change in pH sensitivity is muscle lineage dependent, indicating that differential expression of other myofilament protein isoforms, together with TnC, is necessary to confer full pH sensitivity of contraction in striated muscles.