A8V Mutation Research Articles

Myosin Rod Hypophosphorylation and CB Kinetics in Papillary Muscles from a TnC-A8V KI Mouse Model

The cardiac troponin C (TnC)-A8V mutation is associated with hypertrophic and restrictive cardiomyopathy (HCM and RCM) in human and mice. The residue affected lies in the N-helix, a region known to affect Ca2+-binding affinity to the N-terminal domain. Here we report on the functional effects of this mutation in skinned papillary muscle fibers from homozygous knock-in TnC-A8V mice. Muscle fibers from left ventricle were activated at 25°C under the ionic conditions of working cardiomyocytes. The pCa-tension relationship showed a 3× increase in Ca2+-sensitivity and a decrease (0.8×) in cooperativity (nH) in mutant fibers. The elementary steps of the cross-bridge (CB) cycle were investigated by sinusoidal analysis. The ATP study revealed that there is no significant change in the affinity of ATP (K1) for the myosin head. In TnC-A8V mutant fibers, the CB detachment rate (k2) and its equilibrium constant (K2) increased (1.5×). The phosphate study revealed that rate constant of the force-generation step (k4) decreased (0.5×), reversal step (k−4) increased (2×), and the phosphate-release step (1/K5) increased (2×). Pro-Q Diamond staining of the skinned fibers samples revealed no significant changes in total phosphorylation of multiple sarcomeric proteins. Further investigation using liquid chromatography-tandem mass spectrometry revealed hypophosphorylation of the rod domain of myosin heavy chain in TnC-A8V mutant fibers compared to wild-type. Immunoblotting confirmed the results observed in the mass spectrometry analysis. The results suggest perturbed CB kinetics—possibly caused by changes in the α-myosin heavy chain phosphorylation profile—as a novel mechanism, to our knowledge, by which a mutation in TnC can have rippling effects in the myofilament and contribute to the pathogenesis of HCM/RCM.

A8V Mutation of Cardiac Troponin C Enhances Troponin I Binding

We find in our model that higher affinity for TnI is a potential mechanism by which the A8V mutation renders troponin-C (TnC) capable of causing hypertrophy in hearts carrying TnCA8V. At issue is to explain how muscle fibers reconstituted with TnCA8V are sensitized to Ca2+, i.e. requiring ∼2.3 fold less [Ca2+] to achieve 50% maximum tension compared with fibers reconstituted with wild type TnC (TnCWT). The N-helix of TnC, which is the location of alanine 8, is situated between the EF hand that binds Ca2+ and the hydrophobic patch that binds the switch peptide of troponin-I (TnIsp). Binding measurements rule out a significant change in Ca2+ affinity of TnCA8V, but TnCA8V binds TnIsp ∼1.6 fold more strongly than TnCWT. Hence, we focus on elements of the model in which the TnC-TnIsp interaction competes with the TnI-actin interaction. Tension data are well-fit by specific conditions of the model, namely, with the TnCA8V-TnIsp affinity adjusted ∼1.5 fold higher than TnCWT-TnIsp affinity for all Ca2+ concentrations. Myofibrillar ATPase assays confirm greater activation by TnCA8V at higher Ca2+ concentration, but are inconclusive at low Ca2+ concentration. To test for altered binding in low Ca2+, displacement of the environmentally sensitive fluorescence probe, bis-ANS, from TnI is monitored as a function of added TnC. Whereas Ca2+-bound TnCWT (Ca-TnCWT) displaces significantly more bis-ANS than Ca2+-free TnCWT (Mg-TnCWT) both Ca-TnCA8V and Mg-TnCA8V displace nearly the same amount of probe as Ca-TnCWT. Hence, the mutation predicted to increase hydrophobicity promotes a more intimate association with TnI independent of Ca2+, consistent with the observed increase in activation. Our work suggests that contractility is constantly above normal in hearts made hypertrophic by TnCA8V. Supported by NIH-HL103840 (JRP) and AHA-15GRNT25280004 (JRP).

Structural and Functional Changes in Skeletal Muscles in an A8V-Troponin C Hypertrophic Cardiomyopathy Knock-In Mouse Model

Missense mutations in the TNNC1 gene encoding cardiac and slow skeletal troponin C (c/ssTnC) are associated with phenotypic outcomes of hypertrophic cardiomyopathy (HCM). The impact of HCM c/ssTnC mutations in skeletal muscle structure and function is unknown; therefore, we examined the effect of an HCM-associated c/ssTnC A8V mutation on myosin heavy chain (MHC) isoform expression, contractile properties of different skeletal muscles, and cardiac autonomic function in a homozygous knock-in mouse (KI-TnC-A8V+/+). The distribution of MHC I/II isoforms in various muscles from 3-month old male WT and KI-TnC-A8V+/+ mice were analyzed by glycerol SDS-PAGE and the muscle/body weight ratios (M/BW) were recorded. Mechanics of contraction in isolated intact soleus muscle, running/endurance capacity and heart rate variability (HRV) were also measured. In comparison to the WT mice, MHC isoform switching was observed in diaphragm (increased MHCIIb, decreased MHCI) and soleus (decreased MHCIIa, increased MHCI) muscles of KI-TnC-A8V+/+ mice; while, the M/BW ratio was unchanged for all tested muscles. The force vs frequency relationship at 15Hz and the t1/2 of relaxation increased in the intact soleus muscle of KI-TnC A8V+/+ mice in comparison to the WT; whereas, no changes in maximal force, time-to-twitch peak, velocity and power output were observed. In endurance and HRV tests, KI-TnC-A8V+/+ mice had a lower running distance capacity and their global autonomic, cardiovagal, and baroreflex activities were significantly reduced compared to WT. In spite of the increase in type I fibers in soleus muscle of mice expressing c/ssTnC A8V, they display reduced endurance capacity. These findings suggest that the slow skeletal muscle is trying to compensate for the cardiac dysfunction caused by the c/ssTnC A8V+/+ mutation. Supported by NIH HL103840 (JRP).

DEFINING THE MOLECULAR RELATIONSHIP BETWEEN EX VIVO PROLIFERATED AND IN SITU RESIDENT CARDIAC STEM CELLS

the cardiac troponin (cTn) complex. Understanding these mutations is crucial to the accurate diagnosis and development of novel treatments for FHC. The cTn complex contains three proteins: cTnC, a Ca sensor protein; cTnI, an inhibitory protein; and cTnT, a tether to the thin filament. To date six FHC-linked mutations have been found in cTnC, a highly conserved protein that initiates cardiac contraction in response to the cytosolic level of Ca. Upon Ca binding, a hydrophobic patch on cTnC is exposed, which causes the inhibitory peptide of cTnI to withdraw from the actin filament, initiating muscle contraction. Mutations that alter the ability of cTnC to bind Ca are hypothesized to induce either hypertrophic or dilated cardiomyopathies depending on whether Ca affinity is increased or decreased, respectively. METHODS AND RESULTS: The FHC-associated mutations A8V, L29Q and C84Y in the N-terminal domain were studied. The structural effects of these mutations were modeled through molecular dynamics and their functional impact assessed using free energy calculations of the cTnCCa interaction. In each mutant, the equilibrated structures were very similar to those of wild-type (WT), the site II Ca coordination, size of cTnC-Ca interface, and the stability of the structures were consistent. Changes were noted in the angle between helices A and B, indicative of the exposure of the hydrophobic patch. Additionally, a change in the free energy of Ca binding was calculated between the mutant and WT structures. CONCLUSION: The change in free energy due to the cTnCCa interaction was the highest for the A8V mutation, followed by the WT protein, C84Y and finally, L29Q. Only the A8V mutation caused a free energy change in Ca binding that was greater than WT. This is contrary to the hypothesis that higher affinity for Ca is associated with FHC. Interestingly, the degree to which the hydrophobic patch on cTnC was exposed was greater than WT in each of the mutants. Taken together, these results suggest that while the Ca affinity does not change, the energy barrier to transduction of the Ca-binding signal has been lowered in each of the mutated proteins. CIHR

MOLECULAR DYNAMICS-BASED PREDICTIONS OF THE STRUCTURAL AND FUNCTIONAL EFFECTS OF CARDIAC TROPONIN MUTATIONS ASSOCIATED WITH FAMILIAL HYPERTROPHIC CARDIOMYOPATHY

Familial Hypertrophic cardiomyopathy (FHC) affects one in every 500 people. Many FHC cases occur without overt symptoms and patients may be unaware of their condition prior to sudden cardiac death. FHC is caused by a growing list of largely sarcomeric mutations, including 69 in the cardiac troponin (cTn) complex. Understanding these mutations is crucial to the accurate diagnosis and development of novel treatments for FHC. The cTn complex contains three proteins: cTnC, a Ca2+ sensor protein; cTnI, an inhibitory protein; and cTnT, a tether to the thin filament. To date six FHC-linked mutations have been found in cTnC, a highly conserved protein that initiates cardiac contraction in response to the cytosolic level of Ca2+. Upon Ca2+ binding, a hydrophobic patch on cTnC is exposed, which causes the inhibitory peptide of cTnI to withdraw from the actin filament, initiating muscle contraction. Mutations that alter the ability of cTnC to bind Ca2+ are hypothesized to induce either hypertrophic or dilated cardiomyopathies depending on whether Ca2+ affinity is increased or decreased, respectively. The FHC-associated mutations A8V, L29Q and C84Y in the N-terminal domain were studied. The structural effects of these mutations were modeled through molecular dynamics and their functional impact assessed using free energy calculations of the cTnC-Ca2+ interaction. In each mutant, the equilibrated structures were very similar to those of wild-type (WT), the site II Ca2+ coordination, size of cTnC-Ca2+ interface, and the stability of the structures were consistent. Changes were noted in the angle between helices A and B, indicative of the exposure of the hydrophobic patch. Additionally, a change in the free energy of Ca2+ binding was calculated between the mutant and WT structures. The change in free energy due to the cTnC-Ca2+ interaction was the highest for the A8V mutation, followed by the WT protein, C84Y and finally, L29Q. Only the A8V mutation caused a free energy change in Ca2+ binding that was greater than WT. This is contrary to the hypothesis that higher affinity for Ca2+ is associated with FHC. Interestingly, the degree to which the hydrophobic patch on cTnC was exposed was greater than WT in each of the mutants. Taken together, these results suggest that while the Ca2+ affinity does not change, the energy barrier to transduction of the Ca2+-binding signal has been lowered in each of the mutated proteins.

In Vivo Analysis of Troponin C Knock-In (A8V) Mice: Evidence that TNNC1 is a Hypertrophic Cardiomyopathy Susceptibility Gene

Recently, the TNNC1 gene that encodes cardiac troponin C (cTnC) was found as a target for many hypertrophic cardiomyopathy (HCM) mutations in humans, eliciting alterations in the Ca2+ binding properties of the N-domain of cTnC. We genetically engineered knock-in mice containing the HCM-associated A8V mutation in cTnC (heterozygote = KI-TnC-A8V+/-; homozygote = KI-TnC-A8V+/+) in order to characterize its in vivo, molecular and cellular effects. ECHO revealed that at 3 months old (mo) KI-TnC-A8V+/+ mice display increased IVRT and E/A ∼1 compared to WT, suggesting diastolic dysfunction; whereas KI-TnC-A8V+/- showed signs of cardiac restriction at 14 mo. Histopathology of both genotyped hearts revealed papillary muscle hypertrophy, interstitial fibrosis, and myofibrilar disarray. Real-time PCR analysis at 3 mo demonstrated increases in BNP, α-MHC and β-MHC mRNA levels in the right ventricles of the KI-TnC-A8V mice (only ANP increased in the left ventricle). We identified in intact KI-TnC-A8V+/- and KI-TnC-A8V+/+ cardiomyocytes: a significant decrease in the sarcomere length at several stimulation frequencies; prolonged Ca2+ and contractile transient kinetics at 4Hz; uncoupling between Ca2+ decay (delayed) and contractile (no change) transients at 6Hz; suggesting a mechanical frequency-dependent uncoupling from the Ca2+ transient. Furthermore, a decrease in the baseline Ca2+ fluorescence and in Ca2+ peak percentage was also detected in KI-TnC-A8V+/- and KI-TnC-A8V+/+, indicating increased myofilament Ca2+ buffering. The calcium sensitivity of contraction in skinned fibers increased in a gene dose fashion: KI-TnC-A8V+/+ > KI-TnC-A8V+/- > WT. The rate of relaxation in KI-TnC-A8V+/+ cardiac skinned fibers investigated by flash photolysis was found increased, compared to WT. These results suggest that the A8V mutation in cTnC increases Ca2+ binding affinity to its N-domain eliciting changes in intracellular Ca2+ homeostasis and cellular mechanical function, ultimately leading to diastolic dysfunction.

Myosin Heavy Chain Isoform Switching in Skeletal Muscles in an A8V-Troponin C Hypertrophic Cardiomyopathy Knock-In Mouse Model

Missense mutations in the TNNC1 gene encoding cardiac and slow skeletal troponin C (c/ssTnC) are associated with phenotypic outcomes of hypertrophic cardiomyopathy (HCM). Since the impact of HCM c/ssTnC mutations during skeletal muscle development and function is unknown, we addressed whether changes in the myosin heavy chain (MHC) isoforms can occur outside of the heart. We hypothesized that the ratio of MHC I to MHC II in various skeletal muscles change due to an HCM-associated c/ssTnC A8V mutation in a knock-in homozygous mouse (KI-TnC-A8V+/+). The distribution of MHC I/II isoforms in diaphragm (D), soleus (S), tibialis anterior (TA), extensor digitorum longus (EDL) and quadriceps (Q) isolated from 3- and 9-month (mo) old male WT and KI-TnC-A8V+/+ mice were analyzed by glycerol SDS-PAGE. The muscle/body weight ratio (M/BW) and running capacity were also measured. At 3 mo, MHC isoform switching was observed in D (increased MHCIIb), S (decreased MHCIIa, increased MHCI) and EDL (increased MHCIIa/x, decreased MHCIIb) of KI-TnC-A8V+/+ compared to WT. TA M/BW is increased in 3- and 9-mo KI-TnC-A8V+/+ mice compared to WT at the same age. Pairwise comparison of M/BW from 3- vs 9-mo KI-TnC-A8V+/+ mice showed a significant decrease for TA and Q and an increase for EDL, and no change for any WT muscles. In running tests, KI-TnC-A8V+/+ mice had a lower capacity to run distances compared to WT (281 vs 413 meters). Thus, changes in MHC isoforms in the skeletal muscles of mice expressing c/ssTnC A8V are associated with reduced exercise endurance, suggesting that skeletal muscle remodeling may contribute to the development of HCM phenotype. Future studies will examine MHC isoforms at 9 mo and Ca2+-activated myofibrilar Mg2+-ATPase rates from skeletal muscles in KI-TnC-A8V+/+ mice.

Effects of Calcium Binding and the Hypertrophic Cardiomyopathy A8V Mutation on the Dynamic Equilibrium between Closed and Open Conformations of the Regulatory N-Domain of Isolated Cardiac Troponin C

Troponin C (TnC) is the calcium-binding subunit of the troponin complex responsible for initiating striated muscle contraction in response to calcium influx. In the skeletal TnC isoform, calcium binding induces a structural change in the regulatory N-domain of TnC that involves a transition from a closed to open structural state and accompanying exposure of a large hydrophobic patch for troponin I (TnI) to subsequently bind. However, little is understood about how calcium primes the N-domain of the cardiac isoform (cTnC) for interaction with the TnI subunit as the open conformation of the regulatory domain of cTnC has been observed only in the presence of bound TnI. Here we use paramagnetic relaxation enhancement (PRE) to characterize the closed to open transition of isolated cTnC in solution, a process that cannot be observed by traditional nuclear magnetic resonance methods. Our PRE data from four spin-labeled monocysteine constructs of isolated cTnC reveal that calcium binding triggers movement of the N-domain helices toward an open state. Fitting of the PRE data to a closed to open transition model reveals the presence of a small population of cTnC molecules in the absence of calcium that possess an open conformation, the level of which increases substantially upon Ca(2+) binding. These data support a model in which calcium binding creates a dynamic equilibrium between the closed and open structural states to prime cTnC for interaction with its target peptide. We also used PRE data to assess the structural effects of a familial hypertrophic cardiomyopathy point mutation located within the N-domain of cTnC (A8V). The PRE data show that the Ca(2+) switch mechanism is perturbed by the A8V mutation, resulting in a more open N-domain conformation in both the apo and holo states.

Effect of Hypertrophic Cardiomyopathy-Linked Troponin C Mutations on the Response of Reconstituted Thin Filaments to Calcium upon Troponin I Phosphorylation

The objective of this work was to investigate the effect of hypertrophic cardiomyopathy-linked A8V and E134D mutations in cardiac troponin C (cTnC) on the response of reconstituted thin filaments to calcium upon phosphorylation of cardiac troponin I (cTnI) by protein kinase A. The phosphorylation of cTnI at protein kinase A sites was mimicked by the S22D/S23D double mutation in cTnI. Our results demonstrate that the A8V and E134D mutations had no effect on the extent of calcium desensitization of reconstituted thin filaments induced by cTnI pseudophosphorylation. However, the A8V mutation enhanced the effect of cTnI pseudophosphorylation on the rate of dissociation of calcium from reconstituted thin filaments and on the calcium dependence of actomyosin ATPase. Consequently, while the A8V mutation still led to a slower rate of dissociation of calcium from reconstituted thin filaments upon pseudophosphorylation of cTnI, the ability of the A8V mutation to decrease the rate of calcium dissociation was weakened. In addition, the ability of the A8V mutation to sensitize actomyosin ATPase to calcium was weakened after cTnI was replaced by the phosphorylation mimetic of cTnI. Consistent with the hypothesis that the E134D mutation is benign, it exerted a minor to no effect on the rate of dissociation of calcium from reconstituted thin filaments or on the calcium sensitivity of actomyosin ATPase, regardless of the cTnI phosphorylation status. In conclusion, our study enhances our understanding of how cardiomyopathy-linked cTnC mutations affect the response of reconstituted thin filaments to calcium upon cTnI phosphorylation.

Effect of Hypertrophic Cardiomyopathy Linked Troponin C Mutations on the Calcium Sensitivity of Acto-Myosin ATPase under Different Phosphorylation States of Troponin I

The objective of this work was to investigate the effect of hypertrophic cardiomyopathy linked A8V and E134D cardiac troponin C (cTnC) mutations on the calcium sensitivity of acto-myosin ATPase under different phosphorylation states of cardiac troponin I (cTnI). Consistent with its effect on the calcium sensitivity of cTnC reconstituted into the thin filament, the A8V mutation led to substantially higher calcium sensitivity of acto-myosin ATPase. The effect of the A8V mutation on the calcium sensitivity of acto-myosin ATPase was also evaluated after cTnI was replaced by the PKA phosphorylation mimetic of cTnI (S22D/S23DcTnI). Replacement of cTnI by the PKA phosphorylation mimetic of cTnI led to a larger decrease in the calcium sensitivity of acto-myosin ATPase for the A8VcTnC compared to wild-type cTnC. In fact, the ability of the A8V mutation to sensitize the acto-myosin ATPase to calcium was almost completely eliminated after cTnI was replaced by the PKA phosphorylation mimetic of cTnI. The PKA phosphorylation mimetic of cTnI also had a bigger effect on the rate of calcium dissociation from the A8VcTnC reconstituted into the thin filament than on that from wild-type cTnC. The E134D mutation exerted no effect on the calcium sensitivity of the acto-myosin ATPase regardless of cTnI phosphorylation status. Thus, the ability of hypertrophic cardiomyopathy linked cTnC mutations to affect the calcium sensitivity of acto-myosin ATPase varies depending on phosphorylation status of cTnI.

Effect of Hypertrophic Cardiomyopathy Linked Troponin C Mutations and Troponin I Phosphorylation on the Rate of Calcium Dissociation from the Thin Filament

The objective of this work was to investigate the effect of hypertrophic cardiomyopathy linked A8V, E134D and D145E mutations on the rate of calcium dissociation from cardiac troponin C (cTn)C on the thin filament under different phosphorylation states of cTnI. While the E134D mutation did not affect the rate of calcium dissociation, the A8V and D145E mutations led to significantly slower rates of calcium dissociation from cTnC on the thin filament. The effect of A8V, E134D and D145E mutations on the rate of calcium dissociation from the thin filament was also examined after cTnI was replaced by either PKA (S22D/S23DcTnI) or PKC (S41D/S43DcTnI) phosphorylation mimetic of cTnI. Replacement of cTnI by either the PKA or PKC phosphorylation mimetic of cTnI dramatically accelerated calcium dissociation from wild-type cTnC on the thin filament. The A8V mutation still led to significantly slower rate of calcium dissociation from cTnC on the thin filament after cTnI was replaced by the PKA phosphorylation mimetic of cTnI. However, in the presence of PKC phosphorylation mimetic of cTnI, the ability of A8V mutation to slow the rate of calcium dissociation from the cTnC on thin filament was abolished. The E134D mutation exerted minor to no effect on the rate of calcium dissociation from cTnC on the thin filament regardless of cTnI phosphorylation status. On the other hand, the D145E mutation led to significantly slower rates of calcium dissociation from cTnC on the thin filament after cTnI was replaced by either PKA or PKC phosphorylation mimetic of cTnI. Thus, the ability of hypertrophic cardiomyopathy linked cTnC mutations to affect the rate of calcium dissociation from cTnC on the thin filament varies depending on phosphorylation status of cTnI.

Hypertrophic Cardiomyopathy-Linked Mutation D145E Drastically Alters Calcium Binding by the C-Domain of Cardiac Troponin C

The role of the C-domain sites of cardiac troponin C in the modulation of the calcium signal remains unclear. In this study, we investigated the effects of hypertrophic cardiomyopathy-linked mutations A8V, E134D, and D145E in cardiac troponin C on the properties of the C-domain sites. The A8V mutation had essentially no effect on the calcium or magnesium binding properties of the C-domain sites, while the mutation E134D moderately decreased calcium and magnesium binding affinities. On the other hand, the D145E mutation affected cooperative interactions between sites III and IV, significantly reducing the calcium binding affinity of both sites. Binding of the anchoring region of cardiac troponin I (corresponding to residues 34-71) to cardiac troponin C with the D145E mutation was not able to recover normal calcium binding to the C-domain. Experiments utilizing the fluorescent hydrophobic probe bis-ANS suggest that the D145E mutation dramatically reduced the extent of calcium-induced hydrophobic exposure by the C-domain. At high nonphysiological calcium concentration, A8V, E134D, and D145E mutations minimally affected the affinity of cardiac troponin C for the regulatory region of cardiac troponin I (corresponding to residues 128-180). In contrast, at lower physiological calcium concentration, the D145E mutation led to an approximately 8-fold decrease in the affinity of cardiac troponin C for the regulatory region of cardiac troponin I. Our results suggest that calcium binding properties of the C-domain sites might be important for the proper regulatory function of cardiac troponin C.

N- and C-terminal KCNE1 mutations cause distinct phenotypes of long QT syndrome

Long QT syndromes (LQTS) are inherited diseases involving mutations to genes encoding a number of cardiac ion channels and a membrane adaptor protein. The MinK protein is a cardiac K-channel accessory subunit encoded by the KCNE1 gene, mutations of which are associated with the LQT5 form of LQTS. The purpose of this study was to search for the KCNE1 mutations and clarify the function of those mutations. We conducted a genetic screen of KCNE1 mutations in 151 Japanese LQTS patients using the denaturing high-performance liquid chromatography-WAVE system and direct sequencing. In two LQTS patients, we identified two KCNE1 missense mutations, located in the MinK N- and C-terminal domains. The functional effects of these mutations were examined by heterologous coexpression with KCNQ1 and KCNH2. One mutation, which was identified in a 67-year-old woman, A8V, was novel. Her electrocardiogram (ECG) revealed marked bradycardia and QT interval prolongation. Another mutation, R98W, was identified in a 19-year-old woman. She experienced syncope followed by palpitation in exercise. At rest, her ECG showed bradycardia with mild QT prolongation, which became more prominent during exercise. In electrophysiological analyses, R98W produced reduced I(Ks) currents with a positive shift in the half activation voltages. In addition, when the A8V mutation was coexpressed with KCNH2, this reduced current magnitude, which is suggestive of a modifier effect by the A8V KCNE1 mutation on I(Kr). KCNE1 mutations may be associated with mild LQTS phenotypes, and KCNE1 gene screening is of clinical importance for asymptomatic and mild LQTS patients.