Cardiomyopathy-Related Mutations in Cardiac Troponin C, L29Q and G159D, Have Divergent Effects on Rat Cardiac Myofiber Contractile Dynamics

Previous studies of cardiomyopathy-related mutations in cardiac troponin C (cTnC)—L29Q and G159D—have shown diverse findings. The link between such mutant effects and their divergent impact on cardiac phenotypes has remained elusive due to lack of studies on contractile dynamics. We hypothesized that a cTnC mutant-induced change in the thin filament will affect global myofilament mechanodynamics because of the interactions of thin filament kinetics with bothCa2+binding and crossbridge (XB) cycling kinetics. We measured pCa-tension relationship and contractile dynamics in detergent-skinned rat cardiac papillary muscle fibers reconstituted with the recombinant wild-type rat cTnC (cTnCWT),cTnCL29Q, andcTnCG159Dmutants.cTnCL29Qfibers demonstrated a significant decrease inCa2+sensitivity, butcTnCG159Dfibers did not. Both mutants had no effect onCa2+-activated maximal tension. The rate of XB recruitment dynamics increased incTnCL29Q(26%) andcTnCG159D(25%) fibers. The rate of XB distortion dynamics increased incTnCG159Dfibers (15%). Thus, thecTnCL29Qmutant modulates the equilibrium between the non-cycling and cycling pool of XB by affecting theon/offkinetics of the regulatory units (Tropomyosin-Troponin); whereas, thecTnCG159Dmutant increases XB cycling rate. Different effects on contractile dynamics may offer clue regarding howcTnCL29QandcTnCG159Dcause divergent effects on cardiac phenotypes

The Tropomyosin Binding Region of Cardiac Troponin T Modulates Crossbridge Recruitment Dynamics in Rat Cardiac Muscle Fibers

The cardiac muscle comprises dynamically interacting components that use allosteric/cooperative mechanisms to yield unique heart-specific properties. An essential protein in this allosteric/cooperative mechanism is cardiac muscle troponin T (cTnT), the central region (CR) and the T2 region of which differ significantly from those of fast skeletal muscle troponin T (fsTnT). To understand the biological significance of such sequence heterogeneity, we replaced the T1 or T2 domain of rat cTnT (RcT1 or RcT2) with its counterpart from rat fsTnT (RfsT1or RfsT2) to generate RfsT1-RcT2 and RcT1-RfsT2 recombinant proteins. In addition to contractile function measurements, dynamic features of RfsT1-RcT2- and RcT1-RfsT2-reconstituted rat cardiac muscle fibers were captured by fitting the recruitment–distortion model to the force response of small-amplitude (0.5%) muscle length changes. RfsT1-RcT2 fibers showed a 40% decrease in tension and a 44% decrease in ATPase activity, but RcT1-RfsT2 fibers were unaffected. The magnitude of length-mediated increase in crossbridge (XB) recruitment (E0) decreased by ~33% and the speed of XB recruitment (b) increased by ~100% in RfsT1-RcT2 fibers. Our data suggest the following: (1) the CR of cTnT modulates XB recruitment dynamics; (2) the N-terminal end region of cTnT has a synergistic effect on the ability of the CR to modulate XB recruitment dynamics; (3) the T2 region is important for tuning the Ca2+ regulation of cardiac thin filaments. The combined effects of CR–tropomyosin interactions and the modulating effect of the N-terminal end of cTnT on CR–tropomyosin interactions may lead to the emergence of a unique property that tunes contractile dynamics to heart rates. [Display omitted] ► The biological significance of sequence heterogeneity in cTnT is unknown. ► Myofilament cooperativity is modulated by the CR of cTnT. ► The CR of cTnT modulates the speed of XB recruitment dynamics. ► The N-terminus of cTnT affects the ability of the CR to interact with tropomyosin. ► A unique heart-specific feature emerges from the synergistic effect of N-terminus on CR

Alanine or aspartic acid substitutions at serine23/24 of cardiac troponin I decrease thin filament activation, with no effect on crossbridge detachment kinetics

► Homologous proteins were used for reconstitution in our functional assay. ► Ala/Asp substitutions were introduced at Ser23/24 of cardiac troponin I (cTnI). ► Protein kinase A phosphorylation was mimicked using Asp substitutions in cTnI. ► Ala/Asp substitutions at Ser23/24 of cTnI decreased the thin filament activation. ► Ca2+ sensitivity decreased with Asp substitutions, but not with Ala substitutions. Ala/Asp substitutions at Ser23/24 have been employed to investigate the functional impact of cardiac troponin I (cTnI) phosphorylation by protein kinase A (PKA). Some limitations of previous studies include the use of heterologous proteins and confounding effects arising from phosphorylation of cardiac myosin binding protein-C. Our goal was to probe the effects of cTnI phosphorylation using a homologous assay, so that altered function could be solely attributed to changes in cTnI. We reconstituted detergent-skinned rat cardiac papillary fibers with homologous rat cardiac troponin subunits to study the impact of Ala and Asp substitutions at Ser23/24 of rat cTnI (RcTnI S23A/24A and RcTnI S23D/24D). Both RcTnI S23A/24A and RcTnI S23D/24D showed a ∼36% decrease in Ca2+-activated maximal tension. Both RcTnI S23A/24A and RcTnI S23D/24D showed a ∼18% decrease in ATPase activity. Muscle fiber stiffness measurements suggested that the decrease in thin filament activation observed in RcTnI S23A/24A and RcTnI S23D/24D was due to a decrease in the number of strongly-bound crossbridges. Another major finding was that Ala and Asp substitutions in cTnI did not affect crossbridge detachment kinetics

The N-Terminal Extension of Cardiac Troponin T Stabilizes the Blocked State of Cardiac Thin Filament

Cardiac troponin T (cTnT) is a key component of contractile regulatory proteins. cTnT is characterized by a ∼32 amino acid N-terminal extension (NTE), the function of which remains unknown. To understand its function, we generated a transgenic (TG) mouse line that expressed a recombinant chimeric cTnT in which the NTE of mouse cTnT was removed by replacing its 1–73 residues with the corresponding 1–41 residues of mouse fast skeletal TnT. Detergent-skinned papillary muscle fibers from non-TG (NTG) and TG mouse hearts were used to measure tension, ATPase activity, Ca 2+ sensitivity (pCa 50 ) of tension, rate of tension redevelopment, dynamic muscle fiber stiffness, and maximal fiber shortening velocity at sarcomere lengths (SLs) of 1.9 and 2.3 μ m. Ca 2+ sensitivity increased significantly in TG fibers at both short SL (pCa 50 of 5.96 vs. 5.62 in NTG fibers) and long SL (pCa 50 of 6.10 vs. 5.76 in NTG fibers). Maximal cross-bridge turnover and detachment kinetics were unaltered in TG fibers. Our data suggest that the NTE constrains cardiac thin filament activation such that the transition of the thin filament from the blocked to the closed state becomes less responsive to Ca 2+ . Our finding has implications regarding the effect of tissue- and disease-related changes in cTnT isoforms on cardiac muscle function

Deletion of 1–43 amino acids in cardiac myosin essential light chain blunts length dependency of Ca2+ sensitivity and cross-bridge detachment kinetics

The role of cardiac myosin essential light chain (ELC) in the sarcomere length (SL) dependency of myofilament contractility is unknown. Therefore, mechanical and dynamic contractile properties were measured at SL 1.9 and 2.2 μm in cardiac muscle fibers from two groups of transgenic (Tg) mice: 1 ) Tg-wild-type (WT) mice that expressed WT human ventricular ELC and 2 ) Tg-Δ43 mice that expressed a mutant ELC lacking 1–43 amino acids. In agreement with previous studies, Ca 2+ -activated maximal tension decreased significantly in Tg-Δ43 fibers. pCa 50 (−log 10 [Ca 2+ ] free required for half maximal activation) values at SL of 1.9 μm were 5.64 ± 0.02 and 5.70 ± 0.02 in Tg-WT and Tg-Δ43 fibers, respectively. pCa 50 values at SL of 2.2 μm were 5.70 ± 0.01 and 5.71 ± 0.01 in Tg-WT and Tg-Δ43 fibers, respectively. The SL-mediated increase in the pCa 50 value was statistically significant only in Tg-WT fibers ( P < 0.01), indicating that the SL dependency of myofilament Ca 2+ sensitivity was blunted in Tg-Δ43 fibers. The SL dependency of cross-bridge (XB) detachment kinetics was also blunted in Tg-Δ43 fibers because the decrease in XB detachment kinetics was significant ( P < 0.001) only at SL 1.9 μm. Thus the increased XB dwell time at the short SL augments Ca 2+ sensitivity at short SL and thus blunts SL-mediated increase in myofilament Ca 2+ sensitivity. Our data suggest that the NH 2 -terminal extension of cardiac ELC not only augments the amplitude of force generation, but it also may play a role in mediating the SL dependency of XB detachment kinetics and myofilament Ca 2+ sensitivity

Deletion of 1–43 amino acids in cardiac myosin essential light chain blunts length dependency of Ca 2+

The role of cardiac myosin essential light chain (ELC) in the sarcomere length (SL) dependency of myofilament contractility is unknown. Therefore, mechanical and dynamic contractile properties were measured at SL 1.9 and 2.2 μm in cardiac muscle fibers from two groups of transgenic (Tg) mice: 1) Tg-wild-type (WT) mice that expressed WT human ventricular ELC and 2) Tg-Δ43 mice that expressed a mutant ELC lacking 1–43 amino acids. In agreement with previous studies, Ca(2+)-activated maximal tension decreased significantly in Tg-Δ43 fibers. pCa(50) (−log(10) [Ca(2+)](free) required for half maximal activation) values at SL of 1.9 μm were 5.64 ± 0.02 and 5.70 ± 0.02 in Tg-WT and Tg-Δ43 fibers, respectively. pCa(50) values at SL of 2.2 μm were 5.70 ± 0.01 and 5.71 ± 0.01 in Tg-WT and Tg-Δ43 fibers, respectively. The SL-mediated increase in the pCa(50) value was statistically significant only in Tg-WT fibers (P < 0.01), indicating that the SL dependency of myofilament Ca(2+) sensitivity was blunted in Tg-Δ43 fibers. The SL dependency of cross-bridge (XB) detachment kinetics was also blunted in Tg-Δ43 fibers because the decrease in XB detachment kinetics was significant (P < 0.001) only at SL 1.9 μm. Thus the increased XB dwell time at the short SL augments Ca(2+) sensitivity at short SL and thus blunts SL-mediated increase in myofilament Ca(2+) sensitivity. Our data suggest that the NH(2)-terminal extension of cardiac ELC not only augments the amplitude of force generation, but it also may play a role in mediating the SL dependency of XB detachment kinetics and myofilament Ca(2+) sensitivity