Limits...
Micromechanical thermal assays of Ca2+-regulated thin-filament function and modulation by hypertrophic cardiomyopathy mutants of human cardiac troponin.

Brunet NM, Mihajlović G, Aledealat K, Wang F, Xiong P, von Molnár S, Chase PB - J. Biomed. Biotechnol. (2012)

Bottom Line: Microfabricated thermoelectric controllers can be employed to investigate mechanisms underlying myosin-driven sliding of Ca(2+)-regulated actin and disease-associated mutations in myofilament proteins.Tn-Tm enhanced sliding speed at pCa 5 and increased a transition temperature (T(t)) between a high activation energy (E(a)) but low temperature regime and a low E(a) but high temperature regime.This was modulated by factors that alter cross-bridge number and kinetics.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Science, The Florida State University, Tallahassee, FL 32306, USA.

ABSTRACT
Microfabricated thermoelectric controllers can be employed to investigate mechanisms underlying myosin-driven sliding of Ca(2+)-regulated actin and disease-associated mutations in myofilament proteins. Specifically, we examined actin filament sliding-with or without human cardiac troponin (Tn) and α-tropomyosin (Tm)-propelled by rabbit skeletal heavy meromyosin, when temperature was varied continuously over a wide range (~20-63°C). At the upper end of this temperature range, reversible dysregulation of thin filaments occurred at pCa 9 and 5; actomyosin function was unaffected. Tn-Tm enhanced sliding speed at pCa 5 and increased a transition temperature (T(t)) between a high activation energy (E(a)) but low temperature regime and a low E(a) but high temperature regime. This was modulated by factors that alter cross-bridge number and kinetics. Three familial hypertrophic cardiomyopathy (FHC) mutations, cTnI R145G, cTnI K206Q, and cTnT R278C, cause dysregulation at temperatures ~5-8°C lower; the latter two increased speed at pCa 5 at all temperatures.

Show MeSH

Related in: MedlinePlus

Correction for temperature-dependent effects of solvent viscosity (solid, cyan lines) on filament sliding speed of regulated thin filaments (solid symbols) and unregulated F-actin (open symbols). Data from Figure 1(c) were replotted in Arrhenius form (symbols as in Figure 1), with data omitted for regulated thin filaments above ∼54°C and filament sliding speeds expressed in μm s−1. Dashed lines (black) are linear least squares regressions on the data before correction for temperature-dependent changes in solvent viscosity. Note the change in slope at ∼38°C (Tt) for regulated thin filaments. Effects of temperature-dependent changes in solvent viscosity (normalized to that at body temperature, 37°C) were removed by assuming that speed varies inversely with viscosity, as demonstrated experimentally [43]; the net result of this correction in all instances is a decrease in apparent Ea.
© Copyright Policy - open-access
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3303698&req=5

fig3: Correction for temperature-dependent effects of solvent viscosity (solid, cyan lines) on filament sliding speed of regulated thin filaments (solid symbols) and unregulated F-actin (open symbols). Data from Figure 1(c) were replotted in Arrhenius form (symbols as in Figure 1), with data omitted for regulated thin filaments above ∼54°C and filament sliding speeds expressed in μm s−1. Dashed lines (black) are linear least squares regressions on the data before correction for temperature-dependent changes in solvent viscosity. Note the change in slope at ∼38°C (Tt) for regulated thin filaments. Effects of temperature-dependent changes in solvent viscosity (normalized to that at body temperature, 37°C) were removed by assuming that speed varies inversely with viscosity, as demonstrated experimentally [43]; the net result of this correction in all instances is a decrease in apparent Ea.

Mentions: Arrhenius analysis of the data in Figure 1(c) reveals that the activation energy (Ea) for regulated thin-filament speed at pCa 5 exhibited two distinct values, with a change in the slope at Tt ~ 38°C (Figure 3, solid symbols and black solid lines). For regulated thin filaments (Figure 3, solid symbols and black solid lines), Ea at temperatures below physiological (100.4 kJ/mol) was >2-fold greater than Ea for the higher-temperature regime (41.8 kJ/mol), while the latter value was closer to that of unregulated F-actin (61.9 kJ/mol) (Figure 3, open symbols and black dashed line). Temperature has a dramatic, nonlinear effect on the viscosity of water [55], and the speed of filament sliding varies inversely with solvent viscosity [43, 56]; we therefore asked whether temperature-dependent changes in solvent viscosity could explain the nonlinear Arrhenius relation (Figure 3). The data in Figure 3 were processed to remove the temperature dependence of solvent viscosity by first normalizing viscosity with respect to that of water at 37°C (i.e., body temperature), and second assuming that speed varies inversely with solvent viscosity [43, 56]. After removing the effects of viscosity, slopes of the Arrhenius plots were reduced (Figure 3, blue lines). Ea for unregulated F-actin decreased from 61.9 kJ mol−1 to 47.0 kJ mol−1 (Figure 3, blue dashed line). For regulated thin filaments, Ea decreased from 100.4 to 83.9 kJ mol−1 for T < 38°C, and from 41.8 to 26.6 kJ mol−1 for T > 38°C (Figure 3, black versus blue solid lines). The latter value of Ea is suggestive of a diffusion-limited process, in accord with the previously observed inverse relationship between speed and viscosity [43, 56]. Despite the reduction of slopes for regulated thin-filaments, the ratio of Ea's between the two temperature regimes increased from 2.4 to 3.1. Below 38°C, the ratio of Ea's for regulated thin-filament sliding speed with respect to that of unregulated F-actin increased from 1.6 to 1.8. It is clear from this analysis that it is important to consider temperature-dependent changes in solvent viscosity when evaluating actin filament sliding speed. Nonlinearities in the Arrhenius plot (Figure 3), however, cannot be explained by temperature-dependent changes in solvent viscosity.


Micromechanical thermal assays of Ca2+-regulated thin-filament function and modulation by hypertrophic cardiomyopathy mutants of human cardiac troponin.

Brunet NM, Mihajlović G, Aledealat K, Wang F, Xiong P, von Molnár S, Chase PB - J. Biomed. Biotechnol. (2012)

Correction for temperature-dependent effects of solvent viscosity (solid, cyan lines) on filament sliding speed of regulated thin filaments (solid symbols) and unregulated F-actin (open symbols). Data from Figure 1(c) were replotted in Arrhenius form (symbols as in Figure 1), with data omitted for regulated thin filaments above ∼54°C and filament sliding speeds expressed in μm s−1. Dashed lines (black) are linear least squares regressions on the data before correction for temperature-dependent changes in solvent viscosity. Note the change in slope at ∼38°C (Tt) for regulated thin filaments. Effects of temperature-dependent changes in solvent viscosity (normalized to that at body temperature, 37°C) were removed by assuming that speed varies inversely with viscosity, as demonstrated experimentally [43]; the net result of this correction in all instances is a decrease in apparent Ea.
© Copyright Policy - open-access
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3303698&req=5

fig3: Correction for temperature-dependent effects of solvent viscosity (solid, cyan lines) on filament sliding speed of regulated thin filaments (solid symbols) and unregulated F-actin (open symbols). Data from Figure 1(c) were replotted in Arrhenius form (symbols as in Figure 1), with data omitted for regulated thin filaments above ∼54°C and filament sliding speeds expressed in μm s−1. Dashed lines (black) are linear least squares regressions on the data before correction for temperature-dependent changes in solvent viscosity. Note the change in slope at ∼38°C (Tt) for regulated thin filaments. Effects of temperature-dependent changes in solvent viscosity (normalized to that at body temperature, 37°C) were removed by assuming that speed varies inversely with viscosity, as demonstrated experimentally [43]; the net result of this correction in all instances is a decrease in apparent Ea.
Mentions: Arrhenius analysis of the data in Figure 1(c) reveals that the activation energy (Ea) for regulated thin-filament speed at pCa 5 exhibited two distinct values, with a change in the slope at Tt ~ 38°C (Figure 3, solid symbols and black solid lines). For regulated thin filaments (Figure 3, solid symbols and black solid lines), Ea at temperatures below physiological (100.4 kJ/mol) was >2-fold greater than Ea for the higher-temperature regime (41.8 kJ/mol), while the latter value was closer to that of unregulated F-actin (61.9 kJ/mol) (Figure 3, open symbols and black dashed line). Temperature has a dramatic, nonlinear effect on the viscosity of water [55], and the speed of filament sliding varies inversely with solvent viscosity [43, 56]; we therefore asked whether temperature-dependent changes in solvent viscosity could explain the nonlinear Arrhenius relation (Figure 3). The data in Figure 3 were processed to remove the temperature dependence of solvent viscosity by first normalizing viscosity with respect to that of water at 37°C (i.e., body temperature), and second assuming that speed varies inversely with solvent viscosity [43, 56]. After removing the effects of viscosity, slopes of the Arrhenius plots were reduced (Figure 3, blue lines). Ea for unregulated F-actin decreased from 61.9 kJ mol−1 to 47.0 kJ mol−1 (Figure 3, blue dashed line). For regulated thin filaments, Ea decreased from 100.4 to 83.9 kJ mol−1 for T < 38°C, and from 41.8 to 26.6 kJ mol−1 for T > 38°C (Figure 3, black versus blue solid lines). The latter value of Ea is suggestive of a diffusion-limited process, in accord with the previously observed inverse relationship between speed and viscosity [43, 56]. Despite the reduction of slopes for regulated thin-filaments, the ratio of Ea's between the two temperature regimes increased from 2.4 to 3.1. Below 38°C, the ratio of Ea's for regulated thin-filament sliding speed with respect to that of unregulated F-actin increased from 1.6 to 1.8. It is clear from this analysis that it is important to consider temperature-dependent changes in solvent viscosity when evaluating actin filament sliding speed. Nonlinearities in the Arrhenius plot (Figure 3), however, cannot be explained by temperature-dependent changes in solvent viscosity.

Bottom Line: Microfabricated thermoelectric controllers can be employed to investigate mechanisms underlying myosin-driven sliding of Ca(2+)-regulated actin and disease-associated mutations in myofilament proteins.Tn-Tm enhanced sliding speed at pCa 5 and increased a transition temperature (T(t)) between a high activation energy (E(a)) but low temperature regime and a low E(a) but high temperature regime.This was modulated by factors that alter cross-bridge number and kinetics.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Science, The Florida State University, Tallahassee, FL 32306, USA.

ABSTRACT
Microfabricated thermoelectric controllers can be employed to investigate mechanisms underlying myosin-driven sliding of Ca(2+)-regulated actin and disease-associated mutations in myofilament proteins. Specifically, we examined actin filament sliding-with or without human cardiac troponin (Tn) and α-tropomyosin (Tm)-propelled by rabbit skeletal heavy meromyosin, when temperature was varied continuously over a wide range (~20-63°C). At the upper end of this temperature range, reversible dysregulation of thin filaments occurred at pCa 9 and 5; actomyosin function was unaffected. Tn-Tm enhanced sliding speed at pCa 5 and increased a transition temperature (T(t)) between a high activation energy (E(a)) but low temperature regime and a low E(a) but high temperature regime. This was modulated by factors that alter cross-bridge number and kinetics. Three familial hypertrophic cardiomyopathy (FHC) mutations, cTnI R145G, cTnI K206Q, and cTnT R278C, cause dysregulation at temperatures ~5-8°C lower; the latter two increased speed at pCa 5 at all temperatures.

Show MeSH
Related in: MedlinePlus