Sensitivity Of Force Development Research Articles

Характеристика основних біомаркерів ішемічного ураження м’язової тканини

Ішемія становить основу патогенетичних механізмів гострих і хронічних захворювань серця й судин. Найвищу смертність спричинено саме ішемічною хворобою серця. Важливу роль у відповіді на ішемічне пошкодження відіграє активація низки кіназ, які встановлюють складні зв’язки між клітинами для досягнення біологічних ефектів. Через каскад реакцій, індукований кіназами, синтезуються та виділяються в кров’яне русло різні фактори білкової природи. Найбільш вивченими є натрійуретичні пептиди, кардіальні тропоніни, кінази, різні фактори росту. Вони використовуються як діагностичні маркери ушкодження міокарда. Найбільш точними маркерами вважаються натрійуретичні пептиди та кардіальні тропоніни. Проте на сьогодні важливе використання комплексу маркерів для більш точної діагностики ішемічного ураження. Концентрація цих білкових факторів зростає вже в перші години пошкодження й залежить від його ступеня. Деякі серед наведених білкових факторів не є специфічними до серцевої тканини та впливають також на скелетну мускулатуру. Систематизація літературних даних дає змогу зрозуміти основні ланки в механізмі дії цих факторів і їх впливу на ішемізовану тканину. Виявлені особливості є актуальними для проведення подальших досліджень щодо впливу білкових факторів на параметри скорочення м’язової тканини.

3-Chlorodiphenylamine activates cardiac troponin by a mechanism distinct from bepridil or TFP.

Despite extensive efforts spanning multiple decades, the development of highly effective Ca2+ sensitizers for the heart remains an elusive goal. Existing Ca2+ sensitizers have other targets in addition to cardiac troponin (cTn), which can lead to adverse side effects, such as hypotension or arrhythmias. Thus, there is a need to design Ca2+-sensitizing drugs with higher affinity and selectivity for cTn. Previously, we determined that many compounds based on diphenylamine (DPA) were able to bind to a cTnC-cTnI chimera with moderate affinity (Kd ∼10-120 µM). Of these compounds, 3-chlorodiphenylamine (3-Cl-DPA) bound most tightly (Kd of 10 µM). Here, we investigate 3-Cl-DPA further and find that it increases the Ca2+ sensitivity of force development in skinned cardiac muscle. Using NMR, we show that, like the known Ca2+ sensitizers, trifluoperazine (TFP) and bepridil, 3-Cl-DPA is able to bind to the isolated N-terminal domain (N-domain) of cTnC (Kd of 6 µM). However, while the bulky molecules of TFP and bepridil stabilize the open state of the N-domain of cTnC, the small and flexible 3-Cl-DPA molecule is able to bind without stabilizing this open state. Thus, unlike TFP, which drastically slows the rate of Ca2+ dissociation from the N-domain of isolated cTnC in a dose-dependent manner, 3-Cl-DPA has no effect on the rate of Ca2+ dissociation. On the other hand, the affinity of 3-Cl-DPA for a cTnC-TnI chimera is at least an order of magnitude higher than that of TFP or bepridil, likely because 3-Cl-DPA is less disruptive of cTnI binding to cTnC. Therefore, 3-Cl-DPA has a bigger effect on the rate of Ca2+ dissociation from the entire cTn complex than TFP and bepridil. Our data suggest that 3-Cl-DPA activates the cTn complex via a unique mechanism and could be a suitable scaffold for the development of novel treatments for systolic heart failure.

Desensitizing mouse cardiac troponin C to calcium converts slow muscle towards a fast muscle phenotype.

The Ca2+ -desensitizing D73N mutation in slow skeletal/cardiac troponin C caused dilatated cardiomyopathy in mice, but the consequences of this mutation in skeletal muscle were not known. The D73N mutation led to a rightward shift in the force versus pCa (-log [Ca]) relationship in slow-twitch mouse fibres. The D73N mutation led to a rightward shift in the force-stimulation frequency relationship and reduced fatigue resistance of mouse soleus muscle. The D73N mutation led to reduced cross-sectional area of slow-twitch fibres in mouse soleus muscle without affecting fibre type composition of the muscle. The D73N mutation resulted in significantly shorter times to peak force and to relaxation during isometric twitches and tetani in mouse soleus muscle. The D73N mutation led to major changes in physiological properties of mouse soleus muscle, converting slow muscle toward a fast muscle phenotype. The missense mutation, D73N, in mouse cardiac troponin C has a profound impact on cardiac function, mediated by a decreased myofilament Ca2+ sensitivity. Mammalian cardiac muscle and slow skeletal muscle normally share expression of the same troponin C isoform. Therefore, the objective of this study was to determine the consequences of the D73N mutation in skeletal muscle, as a potential mechanism that contributes to the morbidity associated with heart failure or other conditions in which Ca2+ sensitivity might be altered. Effects of the D73N mutation on physiological properties of mouse soleus muscle, in which slow-twitch fibres are prevalent, were examined. The mutation resulted in a rightward shift of the force-stimulation frequency relationship, and significantly faster kinetics of isometric twitches and tetani in isolated soleus muscle. Furthermore, soleus muscles from D73N mice underwent a significantly greater reduction in force during a fatigue test. The mutation significantly reduced slow fibre mean cross-sectional area without affecting soleus fibre type composition. The effects of the mutation on Ca2+ sensitivity of force development in soleus skinned slow and fast fibres were also examined. As expected, the D73N mutation did not affect the Ca2+ sensitivity of force development in fast fibres but resulted in substantially decreased Ca2+ sensitivity in slow fibres. The results demonstrate that a point mutation in a single constituent of myofilaments (slow/cardiac troponin C) led to major changes in physiological properties of skeletal muscle and converted slow muscle toward a fast muscle phenotype with reduced fatigue resistance and Ca2+ sensitivity of force generation.

Hypertrophic Cardiomyopathy Cardiac Troponin C Mutations Differentially Affect Slow Skeletal and Cardiac Muscle Regulation.

Mutations in TNNC1—the gene encoding cardiac troponin C (cTnC)—that have been associated with hypertrophic cardiomyopathy (HCM) and cardiac dysfunction may also affect Ca2+-regulation and function of slow skeletal muscle since the same gene is expressed in both cardiac and slow skeletal muscle. Therefore, we reconstituted rabbit soleus fibers and bovine masseter myofibrils with mutant cTnCs (A8V, C84Y, E134D, and D145E) associated with HCM to investigate their effects on contractile force and ATPase rates, respectively. Previously, we showed that these HCM cTnC mutants, except for E134D, increased the Ca2+ sensitivity of force development in cardiac preparations. In the current study, an increase in Ca2+ sensitivity of isometric force was only observed for the C84Y mutant when reconstituted in soleus fibers. Incorporation of cTnC C84Y in bovine masseter myofibrils reduced the ATPase activity at saturating [Ca2+], whereas, incorporation of cTnC D145E increased the ATPase activity at inhibiting and saturating [Ca2+]. We also tested whether reconstitution of cardiac fibers with troponin complexes containing the cTnC mutants and slow skeletal troponin I (ssTnI) could emulate the slow skeletal functional phenotype. Reconstitution of cardiac fibers with troponin complexes containing ssTnI attenuated the Ca2+ sensitization of isometric force when cTnC A8V and D145E were present; however, it was enhanced for C84Y. In summary, although the A8V and D145E mutants are present in both muscle types, their functional phenotype is more prominent in cardiac muscle than in slow skeletal muscle, which has implications for the protein-protein interactions within the troponin complex. The C84Y mutant warrants further investigation since it drastically alters the properties of both muscle types and may account for the earlier clinical onset in the proband.

Amino Acid Changes at Arginine 204 of Troponin I Result in Increased Calcium Sensitivity of Force Development.

Mutations in human cardiac troponin I (cTnI) have been associated with restrictive, dilated, and hypertrophic cardiomyopathies. The most commonly occurring residue on cTnI associated with familial hypertrophic cardiomyopathy (FHC) is arginine (R), which is also the most common residue at which multiple mutations occur. Two FHC mutations are known to occur at cTnI arginine 204, R204C and R204H, and both are associated with poor clinical prognosis. The R204H mutation has also been associated with restrictive cardiomyopathy (RCM). To characterize the effects of different mutations at the same residue (R204) on the physiological function of cTnI, six mutations at R204 (C, G, H, P, Q, W) were investigated in skinned fiber studies. Skinned fiber studies showed that all tested mutations at R204 caused significant increases in Ca2+ sensitivity of force development (ΔpCa50 = 0.22–0.35) when compared to wild-type (WT) cTnI. Investigation of the interactions between the cTnI mutants and WT cardiac troponin C (cTnC) or WT cardiac troponin T (cTnT) showed that all the mutations investigated, except R204G, affected either or both cTnI:cTnT and cTnI:cTnC interactions. The R204H mutation affected both cTnI:cTnT and cTnI:cTnC interactions while the R204C mutation affected only the cTnI:cTnC interaction. These results suggest that different mutations at the same site on cTnI could have varying effects on thin filament interactions. A mutation in fast skeletal TnI (R174Q, homologous to cTnI R204Q) also significantly increased Ca2+ sensitivity of force development (ΔpCa50 = 0.16). Our studies indicate that known cTnI mutations associated with poor prognosis (R204C and R204H) exhibit large increases in Ca2+ sensitivity of force development. Therefore, other R204 mutations that cause similar increases in Ca2+ sensitivity are also likely to have poor prognoses.

Abstract 257: Determining the Potential Role of cTnT Isoform Switching in the Development of Early Childhood Tropomyosin-linked DCM

An oft noted component of sarcomeric DCM is the observation that patients within families carrying the same primary mutation exhibit significant phenotypic variability. This lack of a distinct link between genotype and phenotype has complicated clinical management. Recently two unrelated multigenerational families were identified with the tropomyosin (Tm) mutation Asp230Asn (D230N), exhibiting a striking “bimodal” distribution of severity. In these families, many children (<1 year) with D230N Tm presented with a severe form of DCM that led to sudden, often fatal CHF, while adults developed a mild to moderate DCM in mid-life. Of note, children who survived the initial presentation often recovered significant systolic function into young adulthood. To explain this improvement, despite the persistence of D230N Tm, we hypothesized that the phenotype is modified by other thin filament (TF) isoforms. Thus we propose the age-dependent remodeling seen in children with D230N Tm is a result of temporal isoform switches involving a closely linked Tm binding partner cardiac Troponin T (cTnT). We have shown that D230N Tm leads to a more stable (rigid) filament primarily at the C-terminus of Tm near the Tm overlap, a crucial region for TF regulatory function that cTnT modulates. Myofilaments from D230N Tm mice exhibited a small decrease in calcium sensitivity of force development that was significantly reduced in the presence of cTnT1, supportive of a modulatory role. We assessed cardiac performance in our novel D230N Tm x cTnT1 double transgenic (DTg) mice, % FS was similarly reduced for D230N Tm and DTg mice at 2 months likely due to persistent endogenous expression of cTnT1. Divergent cardiac remodeling occurred at 4 months at which point DTg mice had significantly reduced % FS compared to D230N, indicating that additive exposure to cTnT1 is detrimental to the function of D230N hearts. Additionally, In vitro studies on non-failing and failing human heart tissue found that RNA levels of cTnT1 are significantly higher in failing hearts. Thus modulation by cTnT1 could be a more general mechanism for the progressive remodeling seen in heart failure.

Knock-in mice harboring a Ca(2+) desensitizing mutation in cardiac troponin C develop early onset dilated cardiomyopathy.

The physiological consequences of aberrant Ca2+ binding and exchange with cardiac myofilaments are not clearly understood. In order to examine the effect of decreasing Ca2+ sensitivity of cTnC on cardiac function, we generated knock-in mice carrying a D73N mutation (not known to be associated with heart disease in human patients) in cTnC. The D73N mutation was engineered into the regulatory N-domain of cTnC in order to reduce Ca2+ sensitivity of reconstituted thin filaments by increasing the rate of Ca2+ dissociation. In addition, the D73N mutation drastically blunted the extent of Ca2+ desensitization of reconstituted thin filaments induced by cTnI pseudo-phosphorylation. Compared to wild-type mice, heterozygous knock-in mice carrying the D73N mutation exhibited a substantially decreased Ca2+ sensitivity of force development in skinned ventricular trabeculae. Kaplan-Meier survival analysis revealed that median survival time for knock-in mice was 12 weeks. Echocardiographic analysis revealed that knock-in mice exhibited increased left ventricular dimensions with thinner walls. Echocardiographic analysis also revealed that measures of systolic function, such as ejection fraction (EF) and fractional shortening (FS), were dramatically reduced in knock-in mice. In addition, knock-in mice displayed electrophysiological abnormalities, namely prolonged QRS and QT intervals. Furthermore, ventricular myocytes isolated from knock-in mice did not respond to β-adrenergic stimulation. Thus, knock-in mice developed pathological features similar to those observed in human patients with dilated cardiomyopathy (DCM). In conclusion, our results suggest that decreasing Ca2+ sensitivity of the regulatory N-domain of cTnC is sufficient to trigger the development of DCM.

Biochemical characterisation of Troponin C mutations causing hypertrophic and dilated cardiomyopathies.

Cardiac muscle contraction occurs through an interaction of the myosin head with the actin filaments, a process which is regulated by the troponin complex together with tropomyosin and is Ca(2+) dependent. Mutations in genes encoding sarcomeric proteins are a common cause of familial hypertrophic and dilated cardiomyopathies. The scope of this review is to gather information from studies regarding the in vitro characterisation of six HCM and six DCM mutations on the cardiac TnC gene and to suggest, if possible, how they may lead to dysfunction. Since TnC is the subunit responsible for Ca(2+) binding, mutations in the TnC could possibly have a strong impact on Ca(2+) binding affinities. Furthermore, the interactions of mutant TnCs with their binding partners could be altered. From the characterisation studies available to date, we can conclude that the HCM mutations on TnC increase significantly the Ca(2+) sensitivity of force development or of ATPase activity, producing large pCa shifts in comparison to WT TnC. In contrast, the DCM mutations on TnC have a tendency to decrease the Ca(2+) sensitivity of force development or of ATPase activity in comparison to WT TnC. Furthermore, the DCM mutants of TnC are not responsive to the TnI phosphorylation signal resulting in filaments that preserve their Ca(2+) sensitivity in contrast to WT filaments that experience a decrease in Ca(2+) sensitivity upon TnI phosphorylation.

Dcm-Causing Mutation E361G in Actin Uncouples Myofibril Ca2+ Sensitivity from Protein Phosphorylation

Experiments using the in vitro motility assay have shown that the myofilament Ca2+-sensitivity is modulated by phosphorylation of troponin I and that the DCM-causing mutation ACTC E361G uncouples this relationship. Here, we determine whether this also happens in heart muscle myofibrils producing isometric force. We know that TnI and MyBP-C phosphorylation levels are high in normal mouse heart (1.1 mol Pi/mol TnI). To reduce the phosphorylation level of TnI and MyBP-C, mice were injected with a high dose of propranolol which reduced of both TnI bisphosphorylation and MyBP-C phosphorylation by at least 95%. Then we measured contraction in single myofibrils with a Ca2+-jump protocol using a range of Ca2+concentrations. The Ca2+-sensitivity of isometric force and the kinetics of tension development and relaxation for the ACTC E361G TG mouse myofibrils were compared with WT. Modulation of the Ca2+-sensitivity by changes in sarcomere length and by the Ca2+-sensitizer EMD 57033 was further investigated.The maximum isometric force and the tension development rate was the same for phosphorylated and dephosphorylated WT and ACTC E361G mouse myofibrils. The ACTC E361G myofibrils had higher Ca2+-sensitivity of force development then WT. Despite that, the magnitude of length dependent activation was well preserved in ACTC E361G myofibrils. Essentially, while the Ca2+-sensitivity of isometric force was increased 1.4-fold when WT myofibrils were dephosphorylated, the ACTC E361G Ca2+-sensitivity was not altered by dephosphorylation. Additionally, comparing dephosphorylated WT vs WT, the relaxation rate was 1.3-fold slower but dephosphorylation did not change the kinetics of E361G myofibril. Changes in Ca2+-sensitivity were correlated with changes in relaxation rate. Thus the ACTC E361G mutation uncoupled Ca2+-sensitivity and lusitropy from the level of TnI and MyBP-C phosphorylation in intact myofibrils.

Altered Contractile Phenotypes of Intestinal Smooth Muscle in Mice Deficient in Myosin Phosphatase Target Subunit 1

The regulatory subunit of myosin light chain phosphatase, MYPT1, has been proposed to control smooth muscle contractility by regulating phosphorylation of the Ca(2+)-dependent myosin regulatory light chain. We generated mice with a smooth muscle-specific deletion of MYPT1 to investigate its physiologic role in intestinal smooth muscle contraction. We used the Cre-loxP system to establish Mypt1-floxed mice, with the promoter region and exon 1 of Mypt1 flanked by 2 loxP sites. These mice were crossed with SMA-Cre transgenic mice to generate mice with smooth muscle-specific deletion of MYPT1 (Mypt1(SMKO) mice). The phenotype was assessed by histologic, biochemical, molecular, and physiologic analyses. Young adult Mypt1(SMKO) mice had normal intestinal motility in vivo, with no histologic abnormalities. On stimulation with KCl or acetylcholine, intestinal smooth muscles isolated from Mypt1(SMKO) mice produced robust and increased sustained force due to increased phosphorylation of the myosin regulatory light chain compared with muscle from control mice. Additional analyses of contractile properties showed reduced rates of force development and relaxation, and decreased shortening velocity, compared with muscle from control mice. Permeable smooth muscle fibers from Mypt1(SMKO) mice had increased sensitivity and contraction in response to Ca(2+). MYPT1 is not essential for smooth muscle function in mice but regulates the Ca(2+) sensitivity of force development and contributes to intestinal phasic contractile phenotype. Altered contractile responses in isolated tissues could be compensated by adaptive physiologic responses in vivo, where gut motility is affected by lower intensities of smooth muscle stimulation for myosin phosphorylation and force development.

The functional properties of human slow skeletal troponin T isoforms in cardiac muscle regulation.

Human slow skeletal troponin T (HSSTnT) shares a high degree of homology with cardiac TnT (CTnT). Although the presence of HSSTnT has not been confirmed in the heart at the protein level, detectable levels of HSSTnT mRNA have been found. Whether HSSTnT isoforms are expressed transiently remains unknown. Because transient re-expression of HSSTnT may be a potential mechanism of regulating function, we explored the effect of HSSTnT on the regulation of cardiac muscle. At least three HSSTnT isoforms have been found to exist in slow skeletal muscle: HSSTnT1 (+exons 5 and 12), HSSTnT2 (+exon 5, -exon 12), and HSSTnT3 (-exons 5 and 12). Another isoform, HSSTnT hypothetical (Hyp) (-exon 5, +exon 12), has only been found at the mRNA level. Compared with HCTnT3 (adult isoform), Tn complexes containing HSSTnT1, -2, and -3 did not alter the actomyosin ATPase activation and inhibition in the presence and absence of Ca(2+), respectively. HSSTnTHyp was not evaluated as it did not form a Tn complex under a variety of conditions. Porcine papillary skinned fibers displaced with HSSTnT1, -2, or -3 and reconstituted with human cardiac troponin I and troponin C (HCTnI·TnC) complex showed a decrease in the Ca(2+) sensitivity of force development and an increase in maximal recovered force (HSSTnT1 and -3) compared with HCTnT3. In contrast, HSSTnTHyp showed an increase in the Ca(2+) sensitivity of force development. This suggests that re- or overexpression of specific SSTnT isoforms might have therapeutic potential in the failing heart because they increase the maximal force of contraction. In addition, circular dichroism and proteolytic digestion experiments revealed structural differences between HSSTnT isoforms and HCTnT3 and that HSSTnT1 is more susceptible to calpain and trypsin proteolysis than the other HSSTnTs. Overall, HSSTnT isoforms despite being homologues of CTnT may display distinct functional properties in muscle regulation.

Pseudo-Phosphorylation of Cardiac Troponin I Containing the RCM Troponin T Deletion Glutamic Acid 96 is not an Accurate Indicator of the Phosphorylation Effects by PKA in Skinned Fibers

The cardiac TnT deletion (HcTnT-ΔE96) linked to Restrictive Cardiomyopathy (RCM) dramatically increases the Ca2+ sensitivity of force development in skinned fibers (J. Biol. Chem. 283(4):2156–66). This study explores whether HcTnT-ΔE96 interferes with convergent regulation by Protein Kinase A (PKA) which decreases myofilament Ca2+ sensitivity. Native cTnT of porcine cardiac skinned fibers was displaced by HcTnT-WT or HcTnT-ΔE96 and reconstituted with binary complex containing pseudo non-phosphorylatable cTnI (cTnI-SS/AA.cTnC) or pseudo-phosphorylated (cTnI-SS/DD.cTnC). For comparison, skinned fibers were also reconstituted with binary cTnI.cTnC phosphorylated by PKA in vitro (cTnI-2P.cTnC) to assess effects of HcTnT-ΔE96 on Ca2+ sensitivity of force development. As expected, fibers displaced with HcTnT-WT reconstituted with cTnI-SS/DD.cTnC decreased Ca2+ sensitivity of force compared to control reconstituted with cTnI-SS/AA.cTnC. In contrast, fibers displaced with HCTnT-ΔE96 and reconstituted with cTnI-SS/DD.cTnC or cTnI-SS/AA.cTnC had similar Ca2+ sensitivities. When fibers were displaced with HcTnT3-WT or HcTnT3-ΔE96 and reconstituted with cTnI-2P.cTnC, the expected decrease in Ca2+ sensitivity was observed for both. These results suggest that phosphorylation mimetic cTnI-SS/DD may not always mimic phosphorylation by PKA. Furthermore, the mutant HcTnT-ΔE96 protein revealed reduced α-helical content by CD spectroscopy when compared to WT. Thermo-denaturation studies indicated that the mutant protein unfolded earlier than WT and was partially unfolded at physiological temperatures. Fluorescence studies containing CTnC-IAANS demonstrated that a high order of complexity (thin filament + myosin S1) is needed to better approach changes in Ca2+ affinity produced by the RCM cTnT mutation in skinned fibers. The effects of the HcTnT mutation on Ca2+ sensitivity properties were likely due to altered interactions of TnT with other thin filament proteins. Supported AHA 09POST2300030 (MSP), NIH R01-HL42325 (JDP) and J&E King 1KN13-34001 (JRP).

Functional Characterization of TNNC1 Rare Variants Identified in Dilated Cardiomyopathy

TNNC1, which encodes cardiac troponin C (cTnC), remains elusive as a dilated cardiomyopathy (DCM) gene. Here, we report the clinical, genetic, and functional characterization of four TNNC1 rare variants (Y5H, M103I, D145E, and I148V), all previously reported by us in association with DCM (Hershberger, R. E., Norton, N., Morales, A., Li, D., Siegfried, J. D., and Gonzalez-Quintana, J. (2010) Circ. Cardiovasc. Genet. 3, 155-161); in the previous study, two variants (Y5H and D145E) were identified in subjects who also carried MYH7 and MYBPC3 rare variants, respectively. Functional studies using the recombinant human mutant cTnC proteins reconstituted into porcine papillary skinned fibers showed decreased Ca(2+) sensitivity of force development (Y5H and M103I). Furthermore, the cTnC mutants diminished (Y5H and I148V) or abolished (M103I) the effects of PKA phosphorylation on Ca(2+) sensitivity. Only M103I decreased the troponin activation properties of the actomyosin ATPase when Ca(2+) was present. CD spectroscopic studies of apo (absence of divalent cations)-, Mg(2+)-, and Ca(2+)/Mg(2+)-bound states indicated that all of the cTnC mutants (except I148V in the Ca(2+)/Mg(2+) condition) decreased the α-helical content. These results suggest that each mutation alters the function/ability of the myofilament to bind Ca(2+) as a result of modifications in cTnC structure. One variant (D145E) that was previously reported in association with hypertrophic cardiomyopathy and that produced results in vivo in this study consistent with prior hypertrophic cardiomyopathy functional studies was found associated with the MYBPC3 P910T rare variant, likely contributing to the observed DCM phenotype. We conclude that these rare variants alter the regulation of contraction in some way, and the combined clinical, molecular, genetic, and functional data reinforce the importance of TNNC1 rare variants in the pathogenesis of DCM.

Reducing Thin Filament Ca 2+ Affinity with a cTnC Variant (L57Q) Reduces Force but Enhances Cross-Bridge Dependence of Cooperative Activation in Demembranated Rat Trabeculae

Activation of cardiac contraction is initiated by binding of Ca2+ to troponin C (cTnC) and regulated by cooperative strong cross-bridge binding. We previously showed that passive exchange with cTn containing a cTnC variant, L48Q, increased Ca2+ sensitivity of force development and eliminated sarcomere length (SL) dependence of Ca2+ sensitivity of force in rat cardiac trabeculae. This was shown to be due to decreased reliance on strong cross-bridge binding for full thin-filament activation, possibly due to stronger cTnC-cTnI interaction. We also showed that PKA phosphorylation decreased Ca2+ sensitivity of force and eliminated SL dependence of force-pCa relations, by unknown specific mechanisms. Here we test the hypothesis that incorporation of a cTnC variant with decreased Ca2+ binding affinity, L57Q cTnC, will result in increased reliance on cross-bridge binding for full activation, thus increasing SL-dependence of Ca2+ sensitivity. As expected, results indicate trabeculae passively exchanged with L57Q cTnC-cTn displayed decreased Ca2+ sensitivity and rate of force production compared to WT cTnC at a given [Ca2+]. Interestingly, preliminary results indicate L57Q cTnC-cTn mildly increased the SL-dependence of Ca2+ sensitivity of force and also significantly decreased maximal force. Both of these effects were not observed in PKA-treated trabeculae, which had a comparable reduction in Ca2+ sensitivity of force. These findings suggest that reducing cTnC Ca2+ affinity per se can reduce Ca2+ sensitivity of contractile activation to the point of limited overall force production, which may enhance cross-bridge dependence of cooperative thin filament activation. Current experiments aim to increase force in L57Q cTnC (to near WT cTnC) by increasing cross-bridge attachment using 2’-deoxy-ATP, which has previously been shown to increase force and Ca2+ sensitivity while maintaining SL dependence. NIH-HL65497(MR), AHA-2310117(FSK).

The HCM-Linked Ala13thr Mutation in the Cardiac Myosin Regulatory Light Chain Increases Isometric Force Production

The myosin regulatory light chain (RLC) is attached to the α-helical neck region of the myosin head, the so called lever arm, which connects the catalytic and actin binding domains with the thick filament backbone thus participating in the transmission of external forces to the myosin active site. It is understandable that mutations in the RLC associated with hypertrophic cardiomyopathy (HCM) may lead to alterations in force generation affecting cardiac muscle performance. Here, we studied the physiological consequences of an Alanine to Threonine (A13T) mutation in the N-domain of myosin RLC, found in population studies to cause HCM with a specific disease phenotype characterized by mid-ventricular obstruction. We observed an A13T-induced 30-50% increase in maximal force measured in skinned cardiac muscle fibers from transgenic Tg-A13T mice compared to control, Tg-WT and non-Tg littermates. Furthermore, a mutation-mediated 1.3-fold decrease in Vmax and a 1.5-fold increase in Km were observed in the actin-activated myosin ATPase activity compared with myosin from the healthy controls. The binding of Tg-myosin to pyrene-actin was similar for all groups of mice. No changes in the maximal myofibrillar ATPase or in the Ca2+-sensitivity were noted. The same was true for the force-pCa relationship and the mutation did not introduce any alterations in the Ca2+-sensitivity of force development. Gross morphological evaluation revealed enlarged inter-ventricular septa and left ventricles in the hearts from Tg-A13T mice, a phenotype observed in patients harboring the A13T mutation. Our results indicate that the A13T mutation may result in a hypertrophic response through abnormally increased force that may exceed the tolerance of a healthy myocardium. A decreased rate of cross-bridge turnover further demonstrates inadequate energy generation in Tg-A13T mice adding to impaired sarcomeric function. Supported by NIH-HL071778 (DSC).

Novel Role for p90 Ribosomal S6 Kinase in the Regulation of Cardiac Myofilament Phosphorylation

In myocardium, the 90-kDa ribosomal S6 kinase (RSK) is activated by diverse stimuli and regulates the sarcolemmal Na(+)/H(+) exchanger through direct phosphorylation. Only limited information is available on other cardiac RSK substrates and functions. We evaluated cardiac myosin-binding protein C (cMyBP-C), a sarcomeric regulatory phosphoprotein, as a potential RSK substrate. In rat ventricular myocytes, RSK activation by endothelin 1 (ET1) increased cMyBP-C phosphorylation at Ser(282), which was inhibited by the selective RSK inhibitor D1870. Neither ET1 nor D1870 affected the phosphorylation status of Ser(273) or Ser(302), cMyBP-C residues additionally targeted by cAMP-dependent protein kinase (PKA). Complementary genetic gain- and loss-of-function experiments, through the adenoviral expression of wild-type or kinase-inactive RSK isoforms, confirmed RSK-mediated phosphorylation of cMyBP-C at Ser(282). Kinase assays utilizing as substrate wild-type or mutated (S273A, S282A, S302A) recombinant cMyBP-C fragments revealed direct and selective Ser(282) phosphorylation by RSK. Immunolabeling with a Ser(P)(282) antibody and confocal fluorescence microscopy showed RSK-mediated phosphorylation of cMyBP-C across the C-zones of sarcomeric A-bands. In chemically permeabilized mouse ventricular muscles, active RSK again induced selective Ser(282) phosphorylation in cMyBP-C, accompanied by significant reduction in Ca(2+) sensitivity of force development and significant acceleration of cross-bridge cycle kinetics, independently of troponin I phosphorylation at Ser(22)/Ser(23). The magnitudes of these RSK-induced changes were comparable with those induced by PKA, which phosphorylated cMyBP-C additionally at Ser(273) and Ser(302). We conclude that Ser(282) in cMyBP-C is a novel cardiac RSK substrate and its selective phosphorylation appears to regulate cardiac myofilament function.

Effect of troponin I Ser23/24 phosphorylation on Ca2+-sensitivity in human myocardium depends on the phosphorylation background

Protein kinase A (PKA)-mediated phosphorylation of Ser23/24 of cardiac troponin I (cTnI) causes a reduction in Ca2+-sensitivity of force development. This study aimed to determine whether the PKA-induced modulation of the Ca2+-sensitivity is solely due to cTnI phosphorylation or depends on the phosphorylation status of other sarcomeric proteins. Endogenous troponin (cTn) complex in donor cardiomyocytes was partially exchanged (up to 66±1%) with recombinant unphosphorylated human cTn and in failing cells similar exchange was achieved using PKA-(bis)phosphorylated cTn complex. Cardiomyocytes immersed in exchange solution without complex added served as controls. Partial exchange of unphosphorylated cTn complex in donor tissue significantly increased Ca2+-sensitivity (pCa50) to 5.50±0.02 relative to the donor control value (pCa50=5.43±0.04). Exchange in failing tissue with PKA-phosphorylated cTn complex did not change Ca2+-sensitivity relative to the failing control (pCa50=5.60±0.02). Subsequent treatment of the cardiomyocytes with the catalytic subunit of PKA significantly decreased Ca2+-sensitivity in donor and failing tissue. Analysis of phosphorylated cTnI species revealed the same distribution of un-, mono- and bis-phosphorylated cTnI in donor control and in failing tissue exchanged with PKA-phosphorylated cTn complex. Phosphorylation of myosin-binding protein-C in failing tissue was significantly lower compared to donor tissue. These differences in Ca2+-sensitivity in donor and failing cells, despite similar distribution of cTnI species, could be abolished by subsequent PKA-treatment and indicate that other targets of PKA are involved the reduction of Ca2+-sensitivity. Our findings suggest that the sarcomeric phosphorylation background, which is altered in cardiac disease, influences the impact of cTnI Ser23/24 phosphorylation by PKA on Ca2+-sensitivity.

Does the DCM Functional Phenotype Predominate over that of HCM and RCM?

In vitro investigations into Hypertrophic Cardiomyopathy (HCM) and Restrictive Cardiomyopathy (RCM) show that mutations in cardiac Troponin T (cTnT) produce a pathogenic state via increase in myofilament Ca2+ sensitivity, whereas mutations in cTnT that cause Dilated Cardiomyopathy (DCM) decrease Ca2+ sensitivity and maximal force. Our aim was to determine whether combinatory mutations of an HCM, RCM and a DCM in cTnT yield an intermediate or dominant functional phenotype that could be correlated with the clinical condition seen in patients. Standard laboratory methods were used for cloning, expression and purification of the WT and mutants: ΔK210 (DCM), I79N (HCM), ΔE96 (RCM), ΔK210/I79N, ΔK210/ΔE96 and ΔE96/I79N. Porcine papillary skinned fibers were displaced with cTnT WT or mutant and reconstituted with HCTnI-TnC. The Ca2+ sensitivity of force development, maximal force and basal force were evaluated. The extent of TnT displacement was analyzed by measuring the unregulated tension at pCa 8.0 after cTnT treatment and none of the mutants showed an inability to displace the native cTn complex. Both double mutants (ΔK210/I79N and ΔK210/ΔE96) containing the DCM mutant showed a rightward shift in the Ca2+ sensitivity with a decrease in maximal force. In addition, the ΔK210 mutation rescued the impaired relaxation produced by the RCM mutation (ΔE96). From the skinned fiber data, ΔK210 has a dominant effect over I79N and DE96 mutations in cTnT. Circular dichroism measurements demonstrated that all three double mutants had lower alpha helical content than WT. In contrast, single mutants I79N (significantly) and DK210 (showed a tendency) to increase alpha helical content. These results suggest that cTnT can exist in multiple conformations that may be responsible for these distinct functional phenotypes. NIH HL-67415 and HL-42325 (JDP), AHA 0825368E (JRP).

Comparison of the Binding of the Switch Regions of Cardiac Troponin-I and Skeletal Troponin-I to the Functional N-Domain of Human Cardiac Troponin-C

In the heart, there are two isoforms of troponin-I (TnI) that are developmentally regulated. The slow skeletal TnI (ssTnI) is the sole isoform expressed in the embryonic or neonatal heart, while cardiac TnI (cTnI) is expressed exclusively in the adult heart. One important distinction between ssTnI and cTnI relates to differences in myofilament Ca2+-sensitivity of force development under acidic pH conditions. Hearts expressing ssTnI show heightened Ca2+-sensitivity compared with hearts expressing cTnI under basal conditions. This isoform-specific difference is greatly accentuated during acidosis, which is often associated with ischemia of myocardial infarction. In vitro and in vivo functional studies have shown that replacement of cTnI with ssTnI results in a marked enhancement of myofilament Ca2+-sensitivity at acidic pH conditions. Recent reports have indicated that this effect can be ascribed to amino acid sequence differences in ssTnI and cTnI and in particular to a critical A162H substitution in the switch region. In this study, we have used NMR spectroscopy to examine the binding of the switch regions of ssTnI (sTnI115-131) and cTnI (cTnI147-163) to the N-domain of cardiac troponin-C (cNTnC) at physiological and acidic pH conditions. The results show that the affinity of sTnI115-131 for cNTnC•Ca2+ (KD ∼50uM) is ∼3-fold stronger than that of cTnI147-163 (KD ∼150uM), but neither are affected by a pH change from 7 to 6. The pKa of H130 in sTnI115-131 is 6.2 when free and 6.7 when bound to cNTnC•Ca2+. We have also used {1H, 15N}-HSQC NMR spectroscopy to monitor the pKa changes of cNTnC•Ca2+ from peptide free to peptide bound states. The implications of these results will be discussed in the context of structure and function of myofilament protein interactions.

A Functional and Structural Study of Troponin C Mutations Related to Hypertrophic Cardiomyopathy

Recently four new hypertrophic cardiomyopathy mutations in cardiac troponin C (cTnC) (A8V, C84Y, E134D, and D145E) were reported, and their effects on the Ca(2+) sensitivity of force development were evaluated (Landstrom, A. P., Parvatiyar, M. S., Pinto, J. R., Marquardt, M. L., Bos, J. M., Tester, D. J., Ommen, S. R., Potter, J. D., and Ackerman, M. J. (2008) J. Mol. Cell. Cardiol. 45, 281-288). We performed actomyosin ATPase and spectroscopic solution studies to investigate the molecular properties of these mutations. Actomyosin ATPase activity was measured as a function of [Ca(2+)] utilizing reconstituted thin filaments (TFs) with 50% mutant and 50% wild type (WT) and 100% mutant cardiac troponin (cTn) complexes: A8V, C84Y, and D145E increased the Ca(2+) sensitivity with only A8V demonstrating lowered Ca(2+) sensitization at the 50% ratio when compared with 100%; E134D was the same as WT at both ratios. Of these four mutants, only D145E showed increased ATPase activation in the presence of Ca(2+). None of the mutants affected ATPase inhibition or the binding of cTn to the TF measured by co-sedimentation. Only D145E increased the Ca(2+) affinity of site II measured by 2-(4'-(2''-iodoacetamido)phenyl)aminonaphthalene-6-sulfonic acid fluorescence in isolated cTnC or the cTn complex. In the presence of the TF, only A8V was further sensitized to Ca(2+). Circular dichroism measurements in different metal-bound states of the isolated cTnCs showed changes in the secondary structure of A8V, C84Y, and D145E, whereas E134D was the same as WT. PyMol modeling of each cTnC mutant within the cTn complex revealed potential for local changes in the tertiary structure of A8V, C84Y, and D145E. Our results indicate that 1) three of the hypertrophic cardiomyopathy cTnC mutants increased the Ca(2+) sensitivity of the myofilament; 2) the effects of the mutations on the Ca(2+) affinity of isolated cTnC, cTn, and TF are not sufficient to explain the large Ca(2+) sensitivity changes seen in reconstituted and fiber assays; and 3) changes in the secondary structure of the cTnC mutants may contribute to modified protein-protein interactions along the sarcomere lattice disrupting the coupling between the cross-bridge and Ca(2+) binding to cTnC.