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Combinatorial effects of double cardiomyopathy mutant alleles in rodent myocytes: a predictive cellular model of myofilament dysregulation in disease.

Davis J, Metzger JM - PLoS ONE (2010)

Bottom Line: These results were qualitatively similar to a combination of moderate and strong activating CM mutant alleles alphaTmA63V and cTnI R193H, which approached a functional threshold.This is evidence of neutralizing effects of activating/deactivating mutant alleles in combination.Taken together, this combinatorial mutant allele functional analysis lends molecular insight into disease severity and forms the foundation for a predictive model to deconstruct the myriad of possible CM double mutations in presenting patients.

View Article: PubMed Central - PubMed

Affiliation: Department of Integrative Biology and Physiology, University of Minnesota, Minneapolis, Minnesota, United States of America.

ABSTRACT
Inherited cardiomyopathy (CM) represents a diverse group of cardiac muscle diseases that present with a broad spectrum of symptoms ranging from benign to highly malignant. Contributing to this genetic complexity and clinical heterogeneity is the emergence of a cohort of patients that are double or compound heterozygotes who have inherited two different CM mutant alleles in the same or different sarcomeric gene. These patients typically have early disease onset with worse clinical outcomes. Little experimental attention has been directed towards elucidating the physiologic basis of double CM mutations at the cellular-molecular level. Here, dual gene transfer to isolated adult rat cardiac myocytes was used to determine the primary effects of co-expressing two different CM-linked mutant proteins on intact cardiac myocyte contractile physiology. Dual expression of two CM mutants, that alone moderately increase myofilament activation, tropomyosin mutant A63V and cardiac troponin mutant R146G, were shown to additively slow myocyte relaxation beyond either mutant studied in isolation. These results were qualitatively similar to a combination of moderate and strong activating CM mutant alleles alphaTmA63V and cTnI R193H, which approached a functional threshold. Interestingly, a combination of a CM myofilament deactivating mutant, troponin C G159D, together with an activating mutant, cTnIR193H, produced a hybrid phenotype that blunted the strong activating phenotype of cTnIR193H alone. This is evidence of neutralizing effects of activating/deactivating mutant alleles in combination. Taken together, this combinatorial mutant allele functional analysis lends molecular insight into disease severity and forms the foundation for a predictive model to deconstruct the myriad of possible CM double mutations in presenting patients.

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Related in: MedlinePlus

Working model of additive and neutralizing outcomes for double heterozygous genotypes based on the combine effects of allele-specific thin filament activation.In the models depicted in all panels the arrows represent the individual allele as a linear vector. The length of the arrow represent the magnitude of activation/deactivation of the thin filament caused by the mutant allele alone, which is classified as weak, moderate, or strong. The arrow's direction represents the allele's alteration of thin filament activation in which a rightward pointing arrow (positive direction) represents activation and a leftward pointing (negative direction) arrow represents deactivation. (A) Double Activating Allele Model: the positive sloping gradient represents the additive slowing of relaxation and heightened severity of diastolic dysfunction that occurs with the combination of two activating mutant alleles from different sarcomeric loci. The combination of two activating alleles is represented by adding two activating allele vectors together, which yields a higher degree of activation and thus more severe diastolic dysfunction. Some double activating allele combinations may cause the thin filament to become saturated thereby approaching a threshold of activation in which the combination of two vectors is not additive on a one to one basis. (B) Double Deactivating Allele Model: the negative sloping gradient represents the predicted additive consequence to accelerate relaxation and reduce contractility that occurs with the combination of two deactivating mutant alleles from different sarcomeric loci. The combination of two deactivating alleles is represented by adding two activating allele vectors together but in the negative direction, which yields a higher degree of deactivation and thus altered contractility with enhanced relaxation. Some double deactivating allele combinations may also result in saturation thereby approaching a threshold of deactivation. (C) Mixed Model of Activating + Deactivating Alleles: the combination of two alleles with different activation properties is depicted by the addition of a positive gradient and vector (activating allele) and a negative gradient and vector (deactivating allele). This combination results in mitigating effects, in which the primary phenotypes of each individual mutant are neutralized (represented by the trapezoid) when the opposing alleles are co-expressed.
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pone-0009140-g006: Working model of additive and neutralizing outcomes for double heterozygous genotypes based on the combine effects of allele-specific thin filament activation.In the models depicted in all panels the arrows represent the individual allele as a linear vector. The length of the arrow represent the magnitude of activation/deactivation of the thin filament caused by the mutant allele alone, which is classified as weak, moderate, or strong. The arrow's direction represents the allele's alteration of thin filament activation in which a rightward pointing arrow (positive direction) represents activation and a leftward pointing (negative direction) arrow represents deactivation. (A) Double Activating Allele Model: the positive sloping gradient represents the additive slowing of relaxation and heightened severity of diastolic dysfunction that occurs with the combination of two activating mutant alleles from different sarcomeric loci. The combination of two activating alleles is represented by adding two activating allele vectors together, which yields a higher degree of activation and thus more severe diastolic dysfunction. Some double activating allele combinations may cause the thin filament to become saturated thereby approaching a threshold of activation in which the combination of two vectors is not additive on a one to one basis. (B) Double Deactivating Allele Model: the negative sloping gradient represents the predicted additive consequence to accelerate relaxation and reduce contractility that occurs with the combination of two deactivating mutant alleles from different sarcomeric loci. The combination of two deactivating alleles is represented by adding two activating allele vectors together but in the negative direction, which yields a higher degree of deactivation and thus altered contractility with enhanced relaxation. Some double deactivating allele combinations may also result in saturation thereby approaching a threshold of deactivation. (C) Mixed Model of Activating + Deactivating Alleles: the combination of two alleles with different activation properties is depicted by the addition of a positive gradient and vector (activating allele) and a negative gradient and vector (deactivating allele). This combination results in mitigating effects, in which the primary phenotypes of each individual mutant are neutralized (represented by the trapezoid) when the opposing alleles are co-expressed.

Mentions: Using experimental data from activating and deactivating sarcomeric CM mutants obtained here and elsewhere as foundations, a working model was developed to promote predictive insights into myocyte function of the theoretically possible tens of thousands of compound CM combinations. The model was constructed by categorizing single mutant CM alleles by their functional properties as myofilament activators or deactivators, and by the strength with which these properties have been altered. The activation properties of numerous single sarcomeric mutants in both the thick and thin filament have already been characterized [22], [29], [30], [36], [37]. We propose all CM sarcomeric can be incorporated into the model as they all fundamentally effect myofilament regulation [8], [29], [36], [37], [70]–[73]. The model employs vector addition to illustrate the merging of the primary functional effects of each distinct mutant allele in a given double heterozygous combination. Here a linear vector represents a single mutant allele (Figure 6). The length of the vector reflects the individual strength of activation/deactivation (weak, moderate, or strong; Figure 6), and the direction of the vector represents the activation properties of a mutant (right arrow  =  activator, left arrow  =  deactivation; Figure 6A and B). The single mutant vectors of a double heterozygous combination are then added head to tail. Two vectors (alleles) in the same direction are summated to give the net outcome. If the net direction points towards activation results in more severe diastolic dysfunction as represented by the blue positive sloping gradient (Figure 6A). By contrast, summated myofilament deactivators are predicted to hasten relaxation and alter contractility [52], [55], [74] as represented by the red negative sloping gradient (Figure 6A). Support for this model comes from the data presented here in which a weak/strong and a moderate/strong activator combination both additively worsened diastolic function. This model is further supported by the heightened pathology and increased mortality reported in a CM double heterozygous mouse model that expresses both strong (R403Q Myosin) and moderate activator (G203S cTnI) alleles [28]. The model incorporates functional ceiling effects obtained by combining two strong activator alleles (or two strong deactivating alleles) where the myofilament activation (or deactivaiton) reaches a functional threshold and is no longer additive on a one to one basis.


Combinatorial effects of double cardiomyopathy mutant alleles in rodent myocytes: a predictive cellular model of myofilament dysregulation in disease.

Davis J, Metzger JM - PLoS ONE (2010)

Working model of additive and neutralizing outcomes for double heterozygous genotypes based on the combine effects of allele-specific thin filament activation.In the models depicted in all panels the arrows represent the individual allele as a linear vector. The length of the arrow represent the magnitude of activation/deactivation of the thin filament caused by the mutant allele alone, which is classified as weak, moderate, or strong. The arrow's direction represents the allele's alteration of thin filament activation in which a rightward pointing arrow (positive direction) represents activation and a leftward pointing (negative direction) arrow represents deactivation. (A) Double Activating Allele Model: the positive sloping gradient represents the additive slowing of relaxation and heightened severity of diastolic dysfunction that occurs with the combination of two activating mutant alleles from different sarcomeric loci. The combination of two activating alleles is represented by adding two activating allele vectors together, which yields a higher degree of activation and thus more severe diastolic dysfunction. Some double activating allele combinations may cause the thin filament to become saturated thereby approaching a threshold of activation in which the combination of two vectors is not additive on a one to one basis. (B) Double Deactivating Allele Model: the negative sloping gradient represents the predicted additive consequence to accelerate relaxation and reduce contractility that occurs with the combination of two deactivating mutant alleles from different sarcomeric loci. The combination of two deactivating alleles is represented by adding two activating allele vectors together but in the negative direction, which yields a higher degree of deactivation and thus altered contractility with enhanced relaxation. Some double deactivating allele combinations may also result in saturation thereby approaching a threshold of deactivation. (C) Mixed Model of Activating + Deactivating Alleles: the combination of two alleles with different activation properties is depicted by the addition of a positive gradient and vector (activating allele) and a negative gradient and vector (deactivating allele). This combination results in mitigating effects, in which the primary phenotypes of each individual mutant are neutralized (represented by the trapezoid) when the opposing alleles are co-expressed.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2818843&req=5

pone-0009140-g006: Working model of additive and neutralizing outcomes for double heterozygous genotypes based on the combine effects of allele-specific thin filament activation.In the models depicted in all panels the arrows represent the individual allele as a linear vector. The length of the arrow represent the magnitude of activation/deactivation of the thin filament caused by the mutant allele alone, which is classified as weak, moderate, or strong. The arrow's direction represents the allele's alteration of thin filament activation in which a rightward pointing arrow (positive direction) represents activation and a leftward pointing (negative direction) arrow represents deactivation. (A) Double Activating Allele Model: the positive sloping gradient represents the additive slowing of relaxation and heightened severity of diastolic dysfunction that occurs with the combination of two activating mutant alleles from different sarcomeric loci. The combination of two activating alleles is represented by adding two activating allele vectors together, which yields a higher degree of activation and thus more severe diastolic dysfunction. Some double activating allele combinations may cause the thin filament to become saturated thereby approaching a threshold of activation in which the combination of two vectors is not additive on a one to one basis. (B) Double Deactivating Allele Model: the negative sloping gradient represents the predicted additive consequence to accelerate relaxation and reduce contractility that occurs with the combination of two deactivating mutant alleles from different sarcomeric loci. The combination of two deactivating alleles is represented by adding two activating allele vectors together but in the negative direction, which yields a higher degree of deactivation and thus altered contractility with enhanced relaxation. Some double deactivating allele combinations may also result in saturation thereby approaching a threshold of deactivation. (C) Mixed Model of Activating + Deactivating Alleles: the combination of two alleles with different activation properties is depicted by the addition of a positive gradient and vector (activating allele) and a negative gradient and vector (deactivating allele). This combination results in mitigating effects, in which the primary phenotypes of each individual mutant are neutralized (represented by the trapezoid) when the opposing alleles are co-expressed.
Mentions: Using experimental data from activating and deactivating sarcomeric CM mutants obtained here and elsewhere as foundations, a working model was developed to promote predictive insights into myocyte function of the theoretically possible tens of thousands of compound CM combinations. The model was constructed by categorizing single mutant CM alleles by their functional properties as myofilament activators or deactivators, and by the strength with which these properties have been altered. The activation properties of numerous single sarcomeric mutants in both the thick and thin filament have already been characterized [22], [29], [30], [36], [37]. We propose all CM sarcomeric can be incorporated into the model as they all fundamentally effect myofilament regulation [8], [29], [36], [37], [70]–[73]. The model employs vector addition to illustrate the merging of the primary functional effects of each distinct mutant allele in a given double heterozygous combination. Here a linear vector represents a single mutant allele (Figure 6). The length of the vector reflects the individual strength of activation/deactivation (weak, moderate, or strong; Figure 6), and the direction of the vector represents the activation properties of a mutant (right arrow  =  activator, left arrow  =  deactivation; Figure 6A and B). The single mutant vectors of a double heterozygous combination are then added head to tail. Two vectors (alleles) in the same direction are summated to give the net outcome. If the net direction points towards activation results in more severe diastolic dysfunction as represented by the blue positive sloping gradient (Figure 6A). By contrast, summated myofilament deactivators are predicted to hasten relaxation and alter contractility [52], [55], [74] as represented by the red negative sloping gradient (Figure 6A). Support for this model comes from the data presented here in which a weak/strong and a moderate/strong activator combination both additively worsened diastolic function. This model is further supported by the heightened pathology and increased mortality reported in a CM double heterozygous mouse model that expresses both strong (R403Q Myosin) and moderate activator (G203S cTnI) alleles [28]. The model incorporates functional ceiling effects obtained by combining two strong activator alleles (or two strong deactivating alleles) where the myofilament activation (or deactivaiton) reaches a functional threshold and is no longer additive on a one to one basis.

Bottom Line: These results were qualitatively similar to a combination of moderate and strong activating CM mutant alleles alphaTmA63V and cTnI R193H, which approached a functional threshold.This is evidence of neutralizing effects of activating/deactivating mutant alleles in combination.Taken together, this combinatorial mutant allele functional analysis lends molecular insight into disease severity and forms the foundation for a predictive model to deconstruct the myriad of possible CM double mutations in presenting patients.

View Article: PubMed Central - PubMed

Affiliation: Department of Integrative Biology and Physiology, University of Minnesota, Minneapolis, Minnesota, United States of America.

ABSTRACT
Inherited cardiomyopathy (CM) represents a diverse group of cardiac muscle diseases that present with a broad spectrum of symptoms ranging from benign to highly malignant. Contributing to this genetic complexity and clinical heterogeneity is the emergence of a cohort of patients that are double or compound heterozygotes who have inherited two different CM mutant alleles in the same or different sarcomeric gene. These patients typically have early disease onset with worse clinical outcomes. Little experimental attention has been directed towards elucidating the physiologic basis of double CM mutations at the cellular-molecular level. Here, dual gene transfer to isolated adult rat cardiac myocytes was used to determine the primary effects of co-expressing two different CM-linked mutant proteins on intact cardiac myocyte contractile physiology. Dual expression of two CM mutants, that alone moderately increase myofilament activation, tropomyosin mutant A63V and cardiac troponin mutant R146G, were shown to additively slow myocyte relaxation beyond either mutant studied in isolation. These results were qualitatively similar to a combination of moderate and strong activating CM mutant alleles alphaTmA63V and cTnI R193H, which approached a functional threshold. Interestingly, a combination of a CM myofilament deactivating mutant, troponin C G159D, together with an activating mutant, cTnIR193H, produced a hybrid phenotype that blunted the strong activating phenotype of cTnIR193H alone. This is evidence of neutralizing effects of activating/deactivating mutant alleles in combination. Taken together, this combinatorial mutant allele functional analysis lends molecular insight into disease severity and forms the foundation for a predictive model to deconstruct the myriad of possible CM double mutations in presenting patients.

Show MeSH
Related in: MedlinePlus