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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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Intact single myocyte shortening from double activating mutations in αTm and cTnI.(A) Representative Western blot showing non-transduced (WT), R146G cTnI, and A63V Tm + R146G cTnI myocytes. Blots were probed with anti-Tm and anti-cTnI antibodies. (B) Sarcomere shortening transients from WT, A63V Tm, R146G cTnI, and A63V+R146G transduced myocytes. Traces were normalized to peak shortening to emphasize the mutant dependent slowing of relaxation. (C) Summary of 75% sarcomere relaxation time. Relaxation time was determined by calculating the difference from the time of peak shortening to 75% relaxation. Values are represented as the mean+SEM and Newman-Keuls post hoc comparisons defined as follows: (*) different from WT, (+) different from R146G cTnI, (#) different from A63V Tm, P<0.05, myocytes were derived from a minimum of 5 different rat heart isolations with n = 45 WT, n = 40 R146G, and n = 40 A63V+R146G myocytes.
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pone-0009140-g002: Intact single myocyte shortening from double activating mutations in αTm and cTnI.(A) Representative Western blot showing non-transduced (WT), R146G cTnI, and A63V Tm + R146G cTnI myocytes. Blots were probed with anti-Tm and anti-cTnI antibodies. (B) Sarcomere shortening transients from WT, A63V Tm, R146G cTnI, and A63V+R146G transduced myocytes. Traces were normalized to peak shortening to emphasize the mutant dependent slowing of relaxation. (C) Summary of 75% sarcomere relaxation time. Relaxation time was determined by calculating the difference from the time of peak shortening to 75% relaxation. Values are represented as the mean+SEM and Newman-Keuls post hoc comparisons defined as follows: (*) different from WT, (+) different from R146G cTnI, (#) different from A63V Tm, P<0.05, myocytes were derived from a minimum of 5 different rat heart isolations with n = 45 WT, n = 40 R146G, and n = 40 A63V+R146G myocytes.

Mentions: We tested three different CM mutant alleles shown previously and confirmed here to cause weak, moderate, or strong myofilament activation when tested individually (Table 1). The primary functional effects of these CM mutants, when expressed together at the working myocyte level, are presently unknown. We first tested the functional effects of co-expressing a moderate myofilament activator, αTmA63V, with a weak activator, cTnIR146G. Unloaded sarcomere shortening in intact adult rat cardiac myocytes at physiologic temperatures (37°C) was examined four days after dual adenoviral gene transfer. A representative Western blot in Figure 2A demonstrates the targeted stoichiometric replacement of αTm and cTnI achieved with dual gene transfer of TmA63V and cTnI R146G. In this pairing, A63V has 15±7% replacement of native αTm, and cTnIR146G achieves approximately 20±5% replacement of native cTnI. This level of replacement is less than previous reports of single gene transfer with these particular mutants [31], [32], [45]. Representative raw sarcomere shortening traces normalized to peak shortening amplitude illustrate the pronounced slowing of relaxation characteristic of single TmA63V and cTnIR146G mutant myocytes (Figure 2B). Dual gene transfer of these mutants (TmA63V + cTnIR146G) had an additive effect when compared to either mutant alone in which 75% relaxation time was significantly slowed by 30% (Figure 2C).


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)

Intact single myocyte shortening from double activating mutations in αTm and cTnI.(A) Representative Western blot showing non-transduced (WT), R146G cTnI, and A63V Tm + R146G cTnI myocytes. Blots were probed with anti-Tm and anti-cTnI antibodies. (B) Sarcomere shortening transients from WT, A63V Tm, R146G cTnI, and A63V+R146G transduced myocytes. Traces were normalized to peak shortening to emphasize the mutant dependent slowing of relaxation. (C) Summary of 75% sarcomere relaxation time. Relaxation time was determined by calculating the difference from the time of peak shortening to 75% relaxation. Values are represented as the mean+SEM and Newman-Keuls post hoc comparisons defined as follows: (*) different from WT, (+) different from R146G cTnI, (#) different from A63V Tm, P<0.05, myocytes were derived from a minimum of 5 different rat heart isolations with n = 45 WT, n = 40 R146G, and n = 40 A63V+R146G myocytes.
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Related In: Results  -  Collection

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pone-0009140-g002: Intact single myocyte shortening from double activating mutations in αTm and cTnI.(A) Representative Western blot showing non-transduced (WT), R146G cTnI, and A63V Tm + R146G cTnI myocytes. Blots were probed with anti-Tm and anti-cTnI antibodies. (B) Sarcomere shortening transients from WT, A63V Tm, R146G cTnI, and A63V+R146G transduced myocytes. Traces were normalized to peak shortening to emphasize the mutant dependent slowing of relaxation. (C) Summary of 75% sarcomere relaxation time. Relaxation time was determined by calculating the difference from the time of peak shortening to 75% relaxation. Values are represented as the mean+SEM and Newman-Keuls post hoc comparisons defined as follows: (*) different from WT, (+) different from R146G cTnI, (#) different from A63V Tm, P<0.05, myocytes were derived from a minimum of 5 different rat heart isolations with n = 45 WT, n = 40 R146G, and n = 40 A63V+R146G myocytes.
Mentions: We tested three different CM mutant alleles shown previously and confirmed here to cause weak, moderate, or strong myofilament activation when tested individually (Table 1). The primary functional effects of these CM mutants, when expressed together at the working myocyte level, are presently unknown. We first tested the functional effects of co-expressing a moderate myofilament activator, αTmA63V, with a weak activator, cTnIR146G. Unloaded sarcomere shortening in intact adult rat cardiac myocytes at physiologic temperatures (37°C) was examined four days after dual adenoviral gene transfer. A representative Western blot in Figure 2A demonstrates the targeted stoichiometric replacement of αTm and cTnI achieved with dual gene transfer of TmA63V and cTnI R146G. In this pairing, A63V has 15±7% replacement of native αTm, and cTnIR146G achieves approximately 20±5% replacement of native cTnI. This level of replacement is less than previous reports of single gene transfer with these particular mutants [31], [32], [45]. Representative raw sarcomere shortening traces normalized to peak shortening amplitude illustrate the pronounced slowing of relaxation characteristic of single TmA63V and cTnIR146G mutant myocytes (Figure 2B). Dual gene transfer of these mutants (TmA63V + cTnIR146G) had an additive effect when compared to either mutant alone in which 75% relaxation time was significantly slowed by 30% (Figure 2C).

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