Length-dependent changes in contractile dynamics are blunted due to cardiac myosin binding protein-C ablation

Enhanced cardiac contractile function with increased sarcomere length (SL) is, in part, mediated by a decrease in the radial distance between myosin heads and actin. The radial disposition of myosin heads relative to actin is modulated by cardiac myosin binding protein-C (cMyBP-C), suggesting that cMyBP-C contributes to the length-dependent activation (LDA) in the myocardium. However, the precise roles of cMyBP-C in modulating cardiac LDA are unclear. To determine the impact of cMyBP-C on LDA, we measured isometric force, myofilament Ca2+-sensitivity (pCa50) and length-dependent changes in kinetic parameters of cross-bridge (XB) relaxation (krel), and recruitment (kdf) due to rapid stretch, as well as the rate of force redevelopment (ktr) in response to a large slack-restretch maneuver in skinned ventricular multicellular preparations isolated from the hearts of wild-type (WT) and cMyBP-C knockout (KO) mice, at SL’s 1.9µm or 2.1µm. Our results show that maximal force was not significantly different between KO and WT preparations but length-dependent increase in pCa50 was attenuated in the KO preparations. pCa50 was not significantly different between WT and KO preparations at long SL (5.82±0.02 in WT vs. 5.87±0.02 in KO), whereas pCa50 was significantly different between WT and KO preparations at short SL (5.71±0.02 in WT vs. 5.80±0.01 in KO; p<0.05). The ktr, measured at half-maximal Ca2+-activation, was significantly accelerated at short SL in WT preparations (8.74±0.56s-1at 1.9µm vs. 5.71±0.40s-1at 2.1µm, p<0.05). Furthermore, krel and kdf were accelerated by 32% and 50%, respectively at short SL in WT preparations. In contrast, ktr was not altered by changes in SL in KO preparations (8.03±0.54s-1at 1.9µm vs. 8.90±0.37s-1at 2.1µm). Similarly, KO preparations did not exhibit length-dependent changes in krel and kdf. Collectively, our data implicate cMyBP-C is an important regulator of LDA via its impact on dynamic XB behavior due to changes in SL

Role of the N-terminal region of troponin T in cardiac muscle contractile activation

Cardiac muscle contraction is the result of coordinated interactions between the contractile regulatory proteins of the thick and thin filaments, and a detailed understanding of how they interact is central to advancing treatments for human heart diseases. Coordinated interactions between the regulatory proteins mediate Ca2+-, crossbridge (XB)-, and sarcomere length (SL)-dependent cardiac contractile activation. The thick filaments contain myosin, while the thin filament proteins include actin, tropomyosin (Tm), cardiac troponin C, cardiac troponin I, and cardiac troponin T (cTnT). cTnT is a key player in cardiac contraction since it interacts with all other proteins on the thin filament. In particular, cTnT has an N-terminal end that extends into the neighboring structural unit (SU; 7actin:1Tm:1Tn), indicating that this region has a cardiac-specific functional role. However, there is a lack of knowledge regarding the role of the N-terminal end in cardiac contraction. Thus, the overall objective of this dissertation is to determine the role of specific regions in the N-terminal end of cTnT in regulating Ca2+-, XB-, and SL-mediated cardiac contraction. The central hypothesis is that the functional role of specific regions in the N-terminal end of cTnT is modulated by shifts in the isoforms of Tm and myosin heavy chain (MHC). We tested our hypothesis by deleting specific regions in the N-terminal end of cTnT and measuring the contractile function in fibers reconstituted with the cTnT variants. Specific Aim 1 was designed to determine the cardiac-specific role of the N-terminal end of cTnT. Specific Aim 2 was designed to determine how changes in Tm affect the role of the N-terminal end of cTnT. Specific Aim 3 was designed to determine how shifts in MHC isoforms affect the role of the N-terminal end of cTnT. Our findings reveal that specific regions of in the N-terminal end of cTnT have distinct functional impact, and that changes in Tm and MHC modify the impact of the N-terminal end of cTnT in cardiac contraction. New insights from our study will have a positive impact because they further our understanding regarding the role of the N-terminal end of cTnT under physiological and diseased states

Interplay between the overlapping ends of tropomyosin and the N terminus of cardiac troponin T affects tropomyosin states on actin

The functional significance of the molecular swivel at the head-to-tail overlapping ends of contiguous tropomyosin (Tm) dimers in striated muscle is unknown. Contractile measurements were made in muscle fibers from transgenic (TG) mouse hearts that expressed a mutant α-Tm (Tm H276N ). We also reconstituted mouse cardiac troponin T (McTnT) N-terminal deletion mutants, McTnT 1–44 Δ and McTnT 45–74 Δ , into muscle fibers from Tm H276N . For controls, we used the wild-type (WT) McTnT because altered effects could be correlated with the mutant forms of McTnT. Tm H276N slowed crossbridge (XB) detachment rate ( g ) by 19%. McTnT 1–44 Δ attenuated Ca 2+ -activated maximal tension against Tm WT (36%) and Tm H276N (38%), but sped g only against Tm H276N by 35%. The rate of tension redevelopment decreased (17%) only in McTnT 1–44 Δ + Tm H276N fibers. McTnT 45–74 Δ attenuated tension (19%) and myofilament Ca 2+ sensitivity (pCa 50 =5.93 vs. 6.00 in the control fibers) against Tm H276N , but not against Tm WT background. Thus, altered XB cycling kinetics decreased the fraction of strongly bound XBs in McTnT 1–44 Δ + Tm H276N fibers, whereas diminished thin-filament cooperativity attenuated tension in McTnT 45–74 Δ + Tm H276N fibers. In summary, our study is the first to show that the interplay between the N terminus of cTnT and the overlapping ends of contiguous Tm effectuates different states of Tm on the actin filament. —Mamidi, R., Michael, J. J., Muthuchamy, M., Chandra, M. Interplay between the overlapping ends of tropomyosin and the N terminus of cardiac troponin T affects tropomyosin states on actin

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

Structural and Kinetic Effects of PAK3 Phosphorylation Mimic of cTnI(S151E) on the cTnC-cTnI Interaction in the Cardiac Thin Filament

Residue Ser151 of cardiac troponin I (cTnI) is known to be phosphorylated by p21-activated kinase 3 (PAK3). It has been found that PAK3-mediated phosphorylation of cTnI induces an increase in the sensitivity of myofilament to Ca 2+, but the detailed mechanism is unknown. We investigated how the structural and kinetic effects mediated by pseudo-phosphorylation of cTnI (S151E) modulates Ca 2+-induced activation of cardiac thin filaments. Using steady-state, time-resolved Förster resonance energy transfer (FRET) and stopped-flow kinetic measurements, we monitored Ca 2+-induced changes in cTnI–cTnC interactions. Measurements were done using reconstituted thin filaments, which contained the pseudo-phosphorylated cTnI(S151E). We hypothesized that the thin filament regulation is modulated by altered cTnC–cTnI interactions due to charge modification caused by the phosphorylation of Ser151 in cTnI. Our results showed that the pseudo-phosphorylation of cTnI (S151E) sensitizes structural changes to Ca 2+ by shortening the intersite distances between cTnC and cTnI. Furthermore, kinetic rates of Ca 2+ dissociation-induced structural change in the regulatory region of cTnI were reduced significantly by cTnI (S151E). The aforementioned effects of pseudo-phosphorylation of cTnI were similar to those of strong crossbridges on structural changes in cTnI. Our results provide novel information on how cardiac thin filament regulation is modulated by PAK3 phosphorylation of cTnI