Hypertrophic and Dilated Cardiomyopathy-Associated Troponin T Mutations R130C and ΔK210 Oppositely Affect Length-Dependent Calcium Sensitivity of Force Generation

Abstract

Length-dependent activation of calcium-dependent myocardial force generation provides the basis for the Frank-Starling mechanism. To directly compare the effects of mutations associated with hypertrophic cardiomyopathy and dilated cardiomyopathy, the native troponin complex in skinned trabecular fibers of guinea pigs was exchanged with recombinant heterotrimeric, human, cardiac troponin complexes containing different human cardiac troponin T subunits (hcTnT): hypertrophic cardiomyopathy-associated hcTnT^R130C, dilated cardiomyopathy-associated hcTnT^ΔK210 or the wild type hcTnT (hcTnT^WT) serving as control. Force-calcium relations of exchanged fibers were explored at short fiber length defined as 110% of slack length (L ₀) and long fiber length defined as 125% of L ₀ (1.25 L ₀). At short fiber length (1.1 L ₀), calcium sensitivity of force generation expressed by -log [Ca²⁺] required for half-maximum force generation (pCa₅₀) was highest for the hypertrophic cardiomyopathy-associated mutation R130C (5.657 ± 0.019), intermediate for the wild type control (5.580 ± 0.028) and lowest for the dilated cardiomyopathy-associated mutation ΔK210 (5.325 ± 0.038). Lengthening fibers from 1.1 L ₀ to 1.25 L ₀ increased calcium sensitivity in fibers containing hcTnT^R130C (delta-pCa₅₀ = +0.030 ± 0.010), did not alter calcium sensitivity in the wild type control (delta-pCa₅₀ = -0.001 ± 0.010), and decreased calcium sensitivity in fibers containing hcTnT^ΔK210 (delta-pCa₅₀ = -0.034 ± 0.013). Length-dependent activation indicated by the delta-pCa₅₀ was highly significantly (P < 0.001) different between the two mutations. We hypothesize that primary effects of mutations on length-dependent activation contribute to the development of the diverging phenotypes in hypertrophic and dilated cardiomyopathy.

Keywords: calcium sensitivity; cardiomyopathy; contractility; force generation; length dependent activation; sarcomere length; thin filament regulation; troponin.

FIGURE 1

Resting, maximum and calcium-dependent force generation of skinned fibers exchanged for human hcTn containing HCM-associated hcTnT^R130C (n = 18 fibers), hcTnT^WT (n = 19 fibers), or DCM-associated hcTnT^ΔK210 (n = 19 fibers). (A) Resting tension measured in relaxing solution (pCa 7) at short (1.1 L₀) and long (1.25 L₀) fiber length. (B) Maximum tension measured at pCa 4.28. (C) Force-pCa relations at short fiber length (1.1 L₀) and long fiber length (1.25 L₀). Normalized force is scaled as percentage from 0% for resting tension to 100% for maximum tension. ^###Indicates highly significant different to 1.1 L0 in paired Bonferroni post tests.

FIGURE 2

Length-dependent parameters and parameter changes of calcium-dependent force generation of hcTn-exchanged skinned fibers. HCM: fibers containing hcTnT^R130C (black circles and bars), WT: fibers containing hcTnT^WT (white circles and bars), DCM: fibers containing hcTnT^ΔK210 (gray circles and bars). (A) Calcium sensitivity is expressed by the pCa₅₀ required for half-maximum increase of calcium-dependent force generation. (B) Cooperativity of calcium-dependent force generation is expressed by the Hill coefficient n_H of force-pCa relations. (C) Change of pCa₅₀ and Hill coefficient n_H induced by lengthening the fiber from 1.1 L₀ to 1.25 L₀. (D) Change of normalized force at the respective pCa induced by lengthening the fiber from 1.1 L₀ to 1.25 L₀. Change (delta) of parameters in (C,D) are calculated by subtracting for each fiber the parameter value at 1.1 L₀ from the parameter value at 1.25 L₀. Significant change of parameter (delta different from zero) is indicated by ^#P < 0.05, ^##P < 0.01, ^###(P < 0.001). Asterisks indicate significant differences *P < 0.05, **P < 0.01, ***P < 0.001 between the hcTnT-types in Tukey’s multiple comparison post-tests. For sake of clarity, only the P-values for comparisons between mutants and wild type but not the ones comparing the two mutants were plotted in the subfigure (D) (for complete list of P-values see Table 2).