Effect of Calcium-Sensitizing Mutations on Calcium Binding and Exchange with Troponin C in Increasingly Complex Biochemical Systems

Tikunova, Svetlana B.; Liu, Bin; Swindle, Nicholas; Little, Sean C.; Gomes, Aldrin V.; Swartz, Darl R.; Davis, Jonathan P.

doi:10.1021/bi901867s

Cited by 43 publications

(110 citation statements)

References 39 publications

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“…2ϩ Binding Studies-Our laboratory has developed a fluorescent troponin C, TnC IAANS T53C , which minimally affects cTnC function and reports the structural changes that occur in the regulatory domain of cTnC upon Ca 2ϩ binding and dissociation on the thin filament (17,32). This fluorescent TnC enabled the Ca 2ϩ binding studies reported here.…”

Section: Thin Filament Camentioning

confidence: 99%

“…A Ca 2ϩ -sensitizing TnC will be required to correct the DCM thin filament behavior. By mutating the hydrophobic pocket of the regulatory domain of TnC, Ca 2ϩ -sensitizing TnC constructs can be engineered (32,33). For instance, the TnC M45Q mutation increased thin filament Ca 2ϩ sensitivity ϳ8-fold when compared with the control Tn IAANS T53C ( Fig.…”

Section: Thin Filament Camentioning

confidence: 99%

“…Unfortunately, there are no pharmacological compounds that just target TnC (31). However, by directly engineering TnC, our laboratory has developed several TnC constructs, which behave as Ca 2ϩ sensitizers or desensitizers in biochemical model systems and in muscle (16,32,33). By utilizing different design principles (32,33), the intrinsic Ca 2ϩ binding properties of TnC can be finely or grossly tuned.…”

mentioning

confidence: 99%

“…However, by directly engineering TnC, our laboratory has developed several TnC constructs, which behave as Ca 2ϩ sensitizers or desensitizers in biochemical model systems and in muscle (16,32,33). By utilizing different design principles (32,33), the intrinsic Ca 2ϩ binding properties of TnC can be finely or grossly tuned. We have rationally engineered TnC to test whether the Ca 2ϩ dependence of biochemical and physiological systems harboring disease-associated protein modifications could be reset.…”

mentioning

confidence: 99%

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Engineered Troponin C Constructs Correct Disease-related Cardiac Myofilament Calcium Sensitivity

Liu

Lee

Biesiadecki

et al. 2012

Journal of Biological Chemistry

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Background: Improved myofilament Ca 2ϩ sensitivity alleviates defects in thin filament bearing disease-causing mutations. Results: By engineering the cardiac muscle Ca 2ϩ sensor troponin C, aberrant myofilament Ca 2ϩ sensitivity can be corrected in vitro. Conclusion: Engineered TnC provides a novel and versatile avenue to reset disease-related myofilament Ca 2ϩ sensitivity. Significance: Engineered TnC could be a new therapeutic strategy for cardiac muscle diseases.

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Section: Thin Filament Camentioning

confidence: 99%

Section: Thin Filament Camentioning

confidence: 99%

mentioning

confidence: 99%

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confidence: 99%

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Engineered Troponin C Constructs Correct Disease-related Cardiac Myofilament Calcium Sensitivity

Liu

Lee

Biesiadecki

et al. 2012

Journal of Biological Chemistry

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“…To slow CaM's N‐terminal rate of Ca dissociation, we stabilized its Ca binding induced open conformation by mutating a key hydrophobic residue, Met 37, into a polar residue Gln. Based on our previous studies on CaM's evolutionary relative troponin C, a similar mutation in troponin C facilitates the Ca‐induced structural transition of the protein, thus stabilizing Ca binding 42, 43. As shown in Figure 5A, mutating Met 37 to Gln (CaM M37Q) drastically slowed the rate of N‐terminal Ca dissociation from CaM.…”

Section: Resultsmentioning

confidence: 90%

Gene Transfer of Engineered Calmodulin Alleviates Ventricular Arrhythmias in a Calsequestrin‐Associated Mouse Model of Catecholaminergic Polymorphic Ventricular Tachycardia

Liu

Walton

et al. 2018

JAHA

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BackgroundCatecholaminergic polymorphic ventricular tachycardia (CPVT) is a familial arrhythmogenic syndrome characterized by sudden death. There are several genetic forms of CPVT associated with mutations in genes encoding the cardiac ryanodine receptor (RyR2) and its auxiliary proteins including calsequestrin (CASQ2) and calmodulin (CaM). It has been suggested that impairment of the ability of RyR2 to stay closed (ie, refractory) during diastole may be a common mechanism for these diseases. Here, we explore the possibility of engineering CaM variants that normalize abbreviated RyR2 refractoriness for subsequent viral‐mediated delivery to alleviate arrhythmias in non–CaM‐related CPVT.Methods and ResultsTo that end, we have designed a CaM protein (GSH‐M37Q; dubbed as therapeutic CaM or T‐CaM) that exhibited a slowed N‐terminal Ca dissociation rate and prolonged RyR2 refractoriness in permeabilized myocytes derived from CPVT mice carrying the CASQ2 mutation R33Q. This T‐CaM was introduced to the heart of R33Q mice through recombinant adeno‐associated viral vector serotype 9. Eight weeks postinfection, we performed confocal microscopy to assess Ca handling and recorded surface ECGs to assess susceptibility to arrhythmias in vivo. During catecholamine stimulation with isoproterenol, T‐CaM reduced isoproterenol‐promoted diastolic Ca waves in isolated CPVT cardiomyocytes. Importantly, T‐CaM exposure abolished ventricular tachycardia in CPVT mice challenged with catecholamines.ConclusionsOur results suggest that gene transfer of T‐CaM by adeno‐associated viral vector serotype 9 improves myocyte Ca handling and alleviates arrhythmias in a calsequestrin‐associated CPVT model, thus supporting the potential of a CaM‐based antiarrhythmic approach as a therapeutic avenue for genetically distinct forms of CPVT.

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Structural and functional consequences of cardiac troponin C L57Q and I61Q Ca2+-desensitizing variants

Wang

McCully

Luo

et al. 2013

Archives of Biochemistry and Biophysics

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Two cTnC variants, L57Q and I61Q, both of which are located on helix C within the N domain of cTnC, were originally reported in the skeletal muscle system [Tikunova and Davis (2004) J Biol Chem 279, 35341-35352], as the analogous L58Q and I62Q sTnC, and demonstrated a decreased Ca2+ binding affinity. Here, we provide detailed characterization of structure-function relationships for these two cTnC variants, to determine if they behave differently in the cardiac system and as a framework for determining similarities and differences with other cTnC mutations that have been associated with DCM. We have used an integrative approach to study the structure and function of these cTnC variants both in solution and in silico, to understand how the L57Q and I61Q mutations influence Ca2+ binding at site II, the subsequent effects on the interaction with cTnI, and the structural changes which are associated with these changes. Steady-state and stopped flow fluorescence spectroscopy confirmed that a decrease in Ca2+ affinity for recombinant cTnC and cTn complexes containing the L57Q or I61Q variants. The L57Q variant was intermediate between WT and I61Q cTnC and also did not significantly alter cTnC-cTnI interaction in the absence of Ca2+, but did decrease the interaction in the presence of Ca2+. In contrast, I61Q decreased the cTnC-cTnI interaction in both the absence and presence of Ca2+. This difference in the absence of Ca2+ suggests a greater structural change in cNTnC may occur with the I61Q mutation than the L57Q mutation. MD simulations revealed that the decreased Ca2+ binding induced by I61Q may result from destabilization of the Ca2+ binding site through interruption of intra-molecular interactions when residue 61 forms new hydrogen bonds with G70 on the Ca2+ binding loop. The experimentally observed interruption of the cTnC-cTnI interaction caused by L57Q or I61Q is due to the disruption of key hydrophobic interactions between helices B and C in cNTnC. This study provides a molecular basis of how single mutations in the C helix of cTnC can reduce Ca2+ binding affinity and cTnC-cTnI interaction, which may provide useful insights for a better understanding of cardiomyopathies and future gene-based therapies.

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Effect of Calcium-Sensitizing Mutations on Calcium Binding and Exchange with Troponin C in Increasingly Complex Biochemical Systems

Cited by 43 publications

References 39 publications

Engineered Troponin C Constructs Correct Disease-related Cardiac Myofilament Calcium Sensitivity

Engineered Troponin C Constructs Correct Disease-related Cardiac Myofilament Calcium Sensitivity

Gene Transfer of Engineered Calmodulin Alleviates Ventricular Arrhythmias in a Calsequestrin‐Associated Mouse Model of Catecholaminergic Polymorphic Ventricular Tachycardia

Structural and functional consequences of cardiac troponin C L57Q and I61Q Ca2+-desensitizing variants

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