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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

Model of dual gene transfer incorporation of mutant Tm + cTnI and cTnC + cTnI combinations.(A) Native cTnI and αTm is shown in white. This model predicts that mutant Tm (green) has ordered incorporation starting at the pointed end and moving towards the Z-line, while the simultaneous transduction of mutant cTnI (red) will stochastically incorporate along the thin filament. As such double mutant regulatory units containing both mutant Tm and cTnI are located at the pointed end of the thin filament in this dual gene transfer approach. (B) Schematic depicting the predicted population of regulatory units that decorate the thin filament in this model system and in CM patients. Four populations of regulatory units consisting of wild type (WT), mutant cTnI alone, mutant Tm alone, and both mutant cTnI and TM (double mutant) are predicted to constitute the thin filament regulatory system as a whole. The relative proportion of units will be directly related to the percent replacement of native Tm and cTnI that in a patient will reach an even and random distribution overtime as the thin filaments turn-over. In our model system, TmA63V + cTnIR193H myocytes very few double mutant regulatory units are predicted as R193H mutant cTnI will be randomly distributed while only 10% should contain A63V Tm. (C) Native cTnI and cTnC are shown in white. This model predicts that like mutant cTnI (red), mutant cTnC (yellow) will have stochastic incorporation. Regulatory units containing both mutant cTnC and cTnI are randomly decorated along the entire length of the thin filament. The incorporation of mutant cTnC is predicted to follow cTnI. (D) Schematic depicting the predicted population of regulatory units that decorate the thin filament with this dual gene transfer approach. Four populations consisting of wild type (WT), mutant cTnI alone, mutant cTnC alone, and both mutant cTnI and cTnC (double mutant) regulatory units are predicted to constitute the thin filament regulatory system in our model and in CM patients. If mutant cTnC follows cTnI then the population of single mutant regulatory units should approach zero with double mutant and WT regulatory units being more prominent and in a 50∶50 distribution. The relative proportion of double mutant regulatory units is predicted to be directly related to the percent replacement of native cTnC and cTnI.
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pone-0009140-g005: Model of dual gene transfer incorporation of mutant Tm + cTnI and cTnC + cTnI combinations.(A) Native cTnI and αTm is shown in white. This model predicts that mutant Tm (green) has ordered incorporation starting at the pointed end and moving towards the Z-line, while the simultaneous transduction of mutant cTnI (red) will stochastically incorporate along the thin filament. As such double mutant regulatory units containing both mutant Tm and cTnI are located at the pointed end of the thin filament in this dual gene transfer approach. (B) Schematic depicting the predicted population of regulatory units that decorate the thin filament in this model system and in CM patients. Four populations of regulatory units consisting of wild type (WT), mutant cTnI alone, mutant Tm alone, and both mutant cTnI and TM (double mutant) are predicted to constitute the thin filament regulatory system as a whole. The relative proportion of units will be directly related to the percent replacement of native Tm and cTnI that in a patient will reach an even and random distribution overtime as the thin filaments turn-over. In our model system, TmA63V + cTnIR193H myocytes very few double mutant regulatory units are predicted as R193H mutant cTnI will be randomly distributed while only 10% should contain A63V Tm. (C) Native cTnI and cTnC are shown in white. This model predicts that like mutant cTnI (red), mutant cTnC (yellow) will have stochastic incorporation. Regulatory units containing both mutant cTnC and cTnI are randomly decorated along the entire length of the thin filament. The incorporation of mutant cTnC is predicted to follow cTnI. (D) Schematic depicting the predicted population of regulatory units that decorate the thin filament with this dual gene transfer approach. Four populations consisting of wild type (WT), mutant cTnI alone, mutant cTnC alone, and both mutant cTnI and cTnC (double mutant) regulatory units are predicted to constitute the thin filament regulatory system in our model and in CM patients. If mutant cTnC follows cTnI then the population of single mutant regulatory units should approach zero with double mutant and WT regulatory units being more prominent and in a 50∶50 distribution. The relative proportion of double mutant regulatory units is predicted to be directly related to the percent replacement of native cTnC and cTnI.

Mentions: CM sarcomeric proteins can directly cause thin filament deactivation [52]–[55]. It is therefore possible that individuals could inherit one CM mutant myofilament activator allele and one CM mutant deactivator allele. The functional outcomes of such a pairing have not been addressed in any model system to our knowledge. Thus, co-transduction of intact adult cardiac myocytes with CM myofilament deactivator cTnCG159D together with myofilament activator cTnIR193H was examined. Figure 4A is a representative Western blot probed with anti-Tm, anti-cTnI, and anti-cTnC antibodies showing the targeted stoichiometric replacement of native cTnI with R193H cTnI. R193H cTnI replacement reached 58±5% with single gene transfer and was slightly reduced to 50±2% when co-expressed with cTnCG159D (Figure 5A, top panel). The separation between native cTnC and HA-tagged cTnCG159D was resolved on an 18% SDS-page gel as shown in the bottom panel of Figure 5A. G159D cTnC replacement reached 60±4% with single gene transfer and was slightly reduced to 50±2% when co-expressed with cTnIR193H. Representative sarcomere shortening traces (Figure 4B) demonstrate the 2.5 fold difference in myocyte relaxation between cTnCG159D and cTnIR193H mutant myocytes. R193H cTnI significantly slowed relaxation relative to cTnCG159D and myocytes expressing native cTnC/cTnI (Figure 4C and Table 1). Dual gene transfer of cTnCG159D and cTnIR193H resulted in a hybrid phenotype (Figure 4C), in which the 75% relaxation time was 21% faster in G159D+R193H myocytes relative to R193H myocytes (Figure 4C); however, the G159D+R193H myocytes were still slower to relax when compared to cTnCG159D alone and WT myocytes (Figure 4C). The decay time of the Ca2+ transient was also decreased in G159D+R193H myocyte (Figure 4D), but this change was not statistically different from cTnIR193H myocyte Ca2+ decay times.


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)

Model of dual gene transfer incorporation of mutant Tm + cTnI and cTnC + cTnI combinations.(A) Native cTnI and αTm is shown in white. This model predicts that mutant Tm (green) has ordered incorporation starting at the pointed end and moving towards the Z-line, while the simultaneous transduction of mutant cTnI (red) will stochastically incorporate along the thin filament. As such double mutant regulatory units containing both mutant Tm and cTnI are located at the pointed end of the thin filament in this dual gene transfer approach. (B) Schematic depicting the predicted population of regulatory units that decorate the thin filament in this model system and in CM patients. Four populations of regulatory units consisting of wild type (WT), mutant cTnI alone, mutant Tm alone, and both mutant cTnI and TM (double mutant) are predicted to constitute the thin filament regulatory system as a whole. The relative proportion of units will be directly related to the percent replacement of native Tm and cTnI that in a patient will reach an even and random distribution overtime as the thin filaments turn-over. In our model system, TmA63V + cTnIR193H myocytes very few double mutant regulatory units are predicted as R193H mutant cTnI will be randomly distributed while only 10% should contain A63V Tm. (C) Native cTnI and cTnC are shown in white. This model predicts that like mutant cTnI (red), mutant cTnC (yellow) will have stochastic incorporation. Regulatory units containing both mutant cTnC and cTnI are randomly decorated along the entire length of the thin filament. The incorporation of mutant cTnC is predicted to follow cTnI. (D) Schematic depicting the predicted population of regulatory units that decorate the thin filament with this dual gene transfer approach. Four populations consisting of wild type (WT), mutant cTnI alone, mutant cTnC alone, and both mutant cTnI and cTnC (double mutant) regulatory units are predicted to constitute the thin filament regulatory system in our model and in CM patients. If mutant cTnC follows cTnI then the population of single mutant regulatory units should approach zero with double mutant and WT regulatory units being more prominent and in a 50∶50 distribution. The relative proportion of double mutant regulatory units is predicted to be directly related to the percent replacement of native cTnC and cTnI.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0009140-g005: Model of dual gene transfer incorporation of mutant Tm + cTnI and cTnC + cTnI combinations.(A) Native cTnI and αTm is shown in white. This model predicts that mutant Tm (green) has ordered incorporation starting at the pointed end and moving towards the Z-line, while the simultaneous transduction of mutant cTnI (red) will stochastically incorporate along the thin filament. As such double mutant regulatory units containing both mutant Tm and cTnI are located at the pointed end of the thin filament in this dual gene transfer approach. (B) Schematic depicting the predicted population of regulatory units that decorate the thin filament in this model system and in CM patients. Four populations of regulatory units consisting of wild type (WT), mutant cTnI alone, mutant Tm alone, and both mutant cTnI and TM (double mutant) are predicted to constitute the thin filament regulatory system as a whole. The relative proportion of units will be directly related to the percent replacement of native Tm and cTnI that in a patient will reach an even and random distribution overtime as the thin filaments turn-over. In our model system, TmA63V + cTnIR193H myocytes very few double mutant regulatory units are predicted as R193H mutant cTnI will be randomly distributed while only 10% should contain A63V Tm. (C) Native cTnI and cTnC are shown in white. This model predicts that like mutant cTnI (red), mutant cTnC (yellow) will have stochastic incorporation. Regulatory units containing both mutant cTnC and cTnI are randomly decorated along the entire length of the thin filament. The incorporation of mutant cTnC is predicted to follow cTnI. (D) Schematic depicting the predicted population of regulatory units that decorate the thin filament with this dual gene transfer approach. Four populations consisting of wild type (WT), mutant cTnI alone, mutant cTnC alone, and both mutant cTnI and cTnC (double mutant) regulatory units are predicted to constitute the thin filament regulatory system in our model and in CM patients. If mutant cTnC follows cTnI then the population of single mutant regulatory units should approach zero with double mutant and WT regulatory units being more prominent and in a 50∶50 distribution. The relative proportion of double mutant regulatory units is predicted to be directly related to the percent replacement of native cTnC and cTnI.
Mentions: CM sarcomeric proteins can directly cause thin filament deactivation [52]–[55]. It is therefore possible that individuals could inherit one CM mutant myofilament activator allele and one CM mutant deactivator allele. The functional outcomes of such a pairing have not been addressed in any model system to our knowledge. Thus, co-transduction of intact adult cardiac myocytes with CM myofilament deactivator cTnCG159D together with myofilament activator cTnIR193H was examined. Figure 4A is a representative Western blot probed with anti-Tm, anti-cTnI, and anti-cTnC antibodies showing the targeted stoichiometric replacement of native cTnI with R193H cTnI. R193H cTnI replacement reached 58±5% with single gene transfer and was slightly reduced to 50±2% when co-expressed with cTnCG159D (Figure 5A, top panel). The separation between native cTnC and HA-tagged cTnCG159D was resolved on an 18% SDS-page gel as shown in the bottom panel of Figure 5A. G159D cTnC replacement reached 60±4% with single gene transfer and was slightly reduced to 50±2% when co-expressed with cTnIR193H. Representative sarcomere shortening traces (Figure 4B) demonstrate the 2.5 fold difference in myocyte relaxation between cTnCG159D and cTnIR193H mutant myocytes. R193H cTnI significantly slowed relaxation relative to cTnCG159D and myocytes expressing native cTnC/cTnI (Figure 4C and Table 1). Dual gene transfer of cTnCG159D and cTnIR193H resulted in a hybrid phenotype (Figure 4C), in which the 75% relaxation time was 21% faster in G159D+R193H myocytes relative to R193H myocytes (Figure 4C); however, the G159D+R193H myocytes were still slower to relax when compared to cTnCG159D alone and WT myocytes (Figure 4C). The decay time of the Ca2+ transient was also decreased in G159D+R193H myocyte (Figure 4D), but this change was not statistically different from cTnIR193H myocyte Ca2+ decay times.

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