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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

Intact single myocyte shortening and Ca2+ transients from a deactivating cTnC and activating cTnI mutant combination.(A) Representative Western blot from myocytes transduced with R193H cTnI, G159D cTnC, or both R193H cTnI and G159D cTnC. Non-transduced myocytes served as controls. Blots were probed with anti-cTnI (top panel) and anti-cTnC (bottom panel) antibodies and anti-Tm was used as a loading control. (B) Representative sarcomere shortening transients from G159D cTnC, R193H cTnI, and G159D cTnC + R193H cTnI transduced myocytes. Traces were normalized to peak shortening to emphasize the mutant dependent slowing of relaxation. (C) Summary of 75% sarcomere relaxation time and (D) Ca2+ transient decay time. Relaxation and decay times were determined by calculating the difference from the time of peak shortening/fluorescence to 75% relaxation/decay. (E) Summary of baseline sarcomere lengths and (F) resting Ca2+ for all of the experimental groups. Values are represented as the mean + SEM, and Newman-Keuls post hoc comparsions defined as follows: (*) different from WT, (+) different from R193H cTnI, (#) different from G159D cTnC, P<0.05, myocytes were derived from a minimum of 5 different rat heart isolations with n = 63/43 WT, n = 64/44 R193H, n = 36/19 G159D,and n = 36/26 G159D+R193H myocytes used for shortening/Ca2+ cycling experiments.
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pone-0009140-g004: Intact single myocyte shortening and Ca2+ transients from a deactivating cTnC and activating cTnI mutant combination.(A) Representative Western blot from myocytes transduced with R193H cTnI, G159D cTnC, or both R193H cTnI and G159D cTnC. Non-transduced myocytes served as controls. Blots were probed with anti-cTnI (top panel) and anti-cTnC (bottom panel) antibodies and anti-Tm was used as a loading control. (B) Representative sarcomere shortening transients from G159D cTnC, R193H cTnI, and G159D cTnC + R193H cTnI transduced myocytes. Traces were normalized to peak shortening to emphasize the mutant dependent slowing of relaxation. (C) Summary of 75% sarcomere relaxation time and (D) Ca2+ transient decay time. Relaxation and decay times were determined by calculating the difference from the time of peak shortening/fluorescence to 75% relaxation/decay. (E) Summary of baseline sarcomere lengths and (F) resting Ca2+ for all of the experimental groups. Values are represented as the mean + SEM, and Newman-Keuls post hoc comparsions defined as follows: (*) different from WT, (+) different from R193H cTnI, (#) different from G159D cTnC, P<0.05, myocytes were derived from a minimum of 5 different rat heart isolations with n = 63/43 WT, n = 64/44 R193H, n = 36/19 G159D,and n = 36/26 G159D+R193H myocytes used for shortening/Ca2+ cycling experiments.

Mentions: The following set of experiments tested the combination of a strong myofilament activator, cTnIR193H, with a moderate activator, TmA63V, on myocyte function. Figure 3A is a representative Western blot probed with anti-Tm and anti-cTnI antibodies showing the targeted stoichiometric replacement of cTnIR193H, TmA63V, and A63V+R193H (dual gene transfer group) relative to WT myocytes (Figure 3A) three days after gene transfer. With single gene transfer, TmA63V achieved 18±5% replacement and cTnIR193H achieved 58±3%, and with dual gene transfer the replacement of both mutant proteins was reduced by 6–8% (Figure 3A). In unloaded functional assays representative sarcomere shortening transients illustrate the effects of αTmA63V, cTnIR193H, and A63V+R193H on cardiac myocyte relaxation (Figure 3B). In this assay direct comparisons of myocyte shortening demonstrated that R193H myocytes are slower to relax than A63V mutants at 50, 75, and 90% relaxation times (Table 1). Dual gene transfer of TmA63V and cTnIR193H produced an additive slowing of relaxation time that was 40% greater than that of R193H alone (Figure 3C). Ca2+ transients were simultaneously measured yielding results similar to mechanical relaxation (Table 1, Figure 3D). Single gene transfer of A63V and R193H slowed Ca2+ transient decay time to similar extents, relative to WT myocytes (Figure 4C, Table 1); whereas, co-expression of TmA63V and cTnIR193H slowed Ca2+ transient decay by 39% relative to cTnIR193H alone (Figure 3C, Table 1). These data show that within the context of the physiologically intact unloaded myocyte, the dual incorporation of two myofilament activators from different sarcomeric loci directly and additively increase the magnitude of cellular diastolic dysfunction beyond the phenotype of the strongest activating allele within the pairing. A comparison of results obtained from the moderate and weak activator pairing (A63V+R146G) versus the moderate and strong activator pairing (A63V+R193H) also underscores the relationship between the nature of the activators (weak, moderate, or strong) and the resulting degree of diastolic dysfunction, which worsens in combinations with stronger activating alleles like the A63V+R193H.


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 and Ca2+ transients from a deactivating cTnC and activating cTnI mutant combination.(A) Representative Western blot from myocytes transduced with R193H cTnI, G159D cTnC, or both R193H cTnI and G159D cTnC. Non-transduced myocytes served as controls. Blots were probed with anti-cTnI (top panel) and anti-cTnC (bottom panel) antibodies and anti-Tm was used as a loading control. (B) Representative sarcomere shortening transients from G159D cTnC, R193H cTnI, and G159D cTnC + R193H cTnI transduced myocytes. Traces were normalized to peak shortening to emphasize the mutant dependent slowing of relaxation. (C) Summary of 75% sarcomere relaxation time and (D) Ca2+ transient decay time. Relaxation and decay times were determined by calculating the difference from the time of peak shortening/fluorescence to 75% relaxation/decay. (E) Summary of baseline sarcomere lengths and (F) resting Ca2+ for all of the experimental groups. Values are represented as the mean + SEM, and Newman-Keuls post hoc comparsions defined as follows: (*) different from WT, (+) different from R193H cTnI, (#) different from G159D cTnC, P<0.05, myocytes were derived from a minimum of 5 different rat heart isolations with n = 63/43 WT, n = 64/44 R193H, n = 36/19 G159D,and n = 36/26 G159D+R193H myocytes used for shortening/Ca2+ cycling experiments.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2818843&req=5

pone-0009140-g004: Intact single myocyte shortening and Ca2+ transients from a deactivating cTnC and activating cTnI mutant combination.(A) Representative Western blot from myocytes transduced with R193H cTnI, G159D cTnC, or both R193H cTnI and G159D cTnC. Non-transduced myocytes served as controls. Blots were probed with anti-cTnI (top panel) and anti-cTnC (bottom panel) antibodies and anti-Tm was used as a loading control. (B) Representative sarcomere shortening transients from G159D cTnC, R193H cTnI, and G159D cTnC + R193H cTnI transduced myocytes. Traces were normalized to peak shortening to emphasize the mutant dependent slowing of relaxation. (C) Summary of 75% sarcomere relaxation time and (D) Ca2+ transient decay time. Relaxation and decay times were determined by calculating the difference from the time of peak shortening/fluorescence to 75% relaxation/decay. (E) Summary of baseline sarcomere lengths and (F) resting Ca2+ for all of the experimental groups. Values are represented as the mean + SEM, and Newman-Keuls post hoc comparsions defined as follows: (*) different from WT, (+) different from R193H cTnI, (#) different from G159D cTnC, P<0.05, myocytes were derived from a minimum of 5 different rat heart isolations with n = 63/43 WT, n = 64/44 R193H, n = 36/19 G159D,and n = 36/26 G159D+R193H myocytes used for shortening/Ca2+ cycling experiments.
Mentions: The following set of experiments tested the combination of a strong myofilament activator, cTnIR193H, with a moderate activator, TmA63V, on myocyte function. Figure 3A is a representative Western blot probed with anti-Tm and anti-cTnI antibodies showing the targeted stoichiometric replacement of cTnIR193H, TmA63V, and A63V+R193H (dual gene transfer group) relative to WT myocytes (Figure 3A) three days after gene transfer. With single gene transfer, TmA63V achieved 18±5% replacement and cTnIR193H achieved 58±3%, and with dual gene transfer the replacement of both mutant proteins was reduced by 6–8% (Figure 3A). In unloaded functional assays representative sarcomere shortening transients illustrate the effects of αTmA63V, cTnIR193H, and A63V+R193H on cardiac myocyte relaxation (Figure 3B). In this assay direct comparisons of myocyte shortening demonstrated that R193H myocytes are slower to relax than A63V mutants at 50, 75, and 90% relaxation times (Table 1). Dual gene transfer of TmA63V and cTnIR193H produced an additive slowing of relaxation time that was 40% greater than that of R193H alone (Figure 3C). Ca2+ transients were simultaneously measured yielding results similar to mechanical relaxation (Table 1, Figure 3D). Single gene transfer of A63V and R193H slowed Ca2+ transient decay time to similar extents, relative to WT myocytes (Figure 4C, Table 1); whereas, co-expression of TmA63V and cTnIR193H slowed Ca2+ transient decay by 39% relative to cTnIR193H alone (Figure 3C, Table 1). These data show that within the context of the physiologically intact unloaded myocyte, the dual incorporation of two myofilament activators from different sarcomeric loci directly and additively increase the magnitude of cellular diastolic dysfunction beyond the phenotype of the strongest activating allele within the pairing. A comparison of results obtained from the moderate and weak activator pairing (A63V+R146G) versus the moderate and strong activator pairing (A63V+R193H) also underscores the relationship between the nature of the activators (weak, moderate, or strong) and the resulting degree of diastolic dysfunction, which worsens in combinations with stronger activating alleles like the A63V+R193H.

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