Sampath K. Gollapudi

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 Ca(2+) 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.

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.

The functional effect of dilated cardiomyopathy mutation (R144W) in mouse cardiac troponin T is differently affected by α- and β-myosin heavy chain isoforms

Given the differential impact of α- and β-myosin heavy chain (MHC) isoforms on how troponin T (TnT) modulates contractile dynamics, we hypothesized that the effects of dilated cardiomyopathy (DCM) mutations in TnT would be altered differently by α- and β-MHC. We characterized dynamic contractile features of normal (α-MHC) and transgenic (β-MHC) mouse cardiac muscle fibers reconstituted with a mouse TnT analog (TnTR144W) of the human DCM R141W mutation. TnTR144W did not alter maximal tension but attenuated myofilament Ca(2+) sensitivity (pCa50) to a similar extent in α- and β-MHC fibers. TnTR144W attenuated the speed of cross-bridge (XB) distortion dynamics (c) by 24% and the speed of XB recruitment dynamics (b) by 17% in α-MHC fibers; however, both b and c remained unaltered in β-MHC fibers. Likewise, TnTR144W attenuated the rates of XB detachment (g) and tension redevelopment (ktr) only in α-MHC fibers. TnTR144W also decreased the impact of strained XBs on the recruitment of new XBs (γ) by 30% only in α-MHC fibers. Because c, b, g, ktr, and γ are strongly influenced by thin filament-based cooperative mechanisms, we conclude that the TnTR144W- and β-MHC-mediated changes in the thin filament interact to produce a less severe functional phenotype, compared with that brought about by TnTR144W and α-MHC. These observations provide a basis for lower mortality rates of humans (β-MHC) harboring the TnTR141W mutant compared with transgenic mouse studies. Our findings strongly suggest that some caution is necessary when extrapolating data from transgenic mouse studies to human hearts.

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 both Ca2+ 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, and cTnCG159D mutants. cTnCL29Q fibers demonstrated a significant decrease in Ca2+ sensitivity, but cTnCG159D fibers did not. Both mutants had no effect on Ca2+-activated maximal tension. The rate of XB recruitment dynamics increased in cTnCL29Q (26%) and cTnCG159D (25%) fibers. The rate of XB distortion dynamics increased in cTnCG159D fibers (15%). Thus, the cTnCL29Q mutant modulates the equilibrium between the non-cycling and cycling pool of XB by affecting the on/off kinetics of the regulatory units (Tropomyosin-Troponin); whereas, the cTnCG159D mutant increases XB cycling rate. Different effects on contractile dynamics may offer clue regarding how cTnCL29Q and cTnCG159D cause divergent effects on cardiac phenotypes.

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

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 Ca(2+)-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.

Rat cardiac troponin T mutation (F72L)-mediated impact on thin filament cooperativity is divergently modulated by α- and β-myosin heavy chain isoforms

The primary causal link between disparate effects of human hypertrophic cardiomyopathy (HCM)-related mutations in troponin T (TnT) and α- and β-myosin heavy chain (MHC) isoforms on cardiac contractile phenotype remains poorly understood. Given the divergent impact of α- and β-MHC on the NH2-terminal extension (44-73 residues) of TnT, we tested if the effects of the HCM-linked mutation (TnTF70L) were differentially altered by α- and β-MHC. We hypothesized that the emergence of divergent thin filament cooperativity would lead to contrasting effects of TnTF70L on contractile function in the presence of α- and β-MHC. The rat TnT analog of the human F70L mutation (TnTF72L) or the wild-type rat TnT (TnTWT) was reconstituted into demembranated muscle fibers from normal (α-MHC) and propylthiouracil-treated (β-MHC) rat hearts to measure steady-state and dynamic contractile function. TnTF72L-mediated effects on tension, myofilament Ca(2+) sensitivity, myofilament cooperativity, rate constants of cross-bridge (XB) recruitment dynamics, and force redevelopment were divergently modulated by α- and β-MHC. TnTF72L increased the rate of XB distortion dynamics by 49% in α-MHC fibers but had no effect in β-MHC fibers; these observations suggest that TnTF72L augmented XB detachment kinetics in α-MHC, but not β-MHC, fibers. TnTF72L increased the negative impact of strained XBs on the force-bearing XBs by 39% in α-MHC fibers but had no effect in β-MHC fibers. Therefore, TnTF72L leads to contractile changes that are linked to dilated cardiomyopathy in the presence of α-MHC. On the other hand, TnTF72L leads to contractile changes that are linked to HCM in the presence of β-MHC.

The effect of cardiomyopathy mutation (R97L) in mouse cardiac troponin T on the muscle length-mediated recruitment of crossbridges is modified divergently by α- and β-myosin heavy chain.

Hypertrophic cardiomyopathy mutations in cardiac troponin T (TnT) lead to sudden cardiac death. Augmented myofilament Ca(2+) sensitivity is a common feature in TnT mutants, but such observations fail to provide a rational explanation for severe cardiac phenotypes. To better understand the mutation-induced effect on the cardiac phenotype, it is imperative to determine the effects on dynamic contractile features such as the muscle length (ML)-mediated activation against α- and β-myosin heavy chain (MHC) isoforms. α- and β-MHC are not only differentially expressed in rodent and human hearts, but they also modify ML-mediated activation differently. Mouse analog of human TnTR94L (TnTR97L) or wild-type TnT was reconstituted into de-membranated muscle fibers from normal (α-MHC) and transgenic (β-MHC) mouse hearts. TnTR97L augmented myofilament Ca(2+) sensitivity by a similar amount in α- and β-MHC fibers. However, TnTR97L augmented the negative impact of strained crossbridges on other crossbridges (γ) by 22% in α-MHC fibers, but attenuated γ by 21% in β-MHC fibers. TnTR97L decreased the magnitude of ML-mediated recruitment of crossbridges (ER) by 37% in α-MHC fibers, but increased ER by 35% in β-MHC fibers. We provide a mechanistic basis for the TnTR97L-induced effects in α- and β-MHC fibers and discuss the relevance to human hearts.

Cardiomyopathy-related mutation (A30V) in mouse cardiac troponin T divergently alters the magnitude of stretch activation in α- and β-myosin heavy chain fibers

The present study investigated the functional consequences of the human hypertrophic cardiomyopathy (HCM) mutation A28V in cardiac troponin T (TnT). The A28V mutation is located within the NH2 terminus of TnT, a region known to be important for full activation of cardiac thin filaments. The functional consequences of the A28V mutation in TnT remain unknown. Given how α- and β-myosin heavy chain (MHC) isoforms differently alter the functional effect of the NH2 terminus of TnT, we hypothesized that the A28V-induced effects would be differently modulated by α- and β-MHC isoforms. Recombinant wild-type mouse TnT (TnTWT) and the mouse equivalent of the human A28V mutation (TnTA30V) were reconstituted into detergent-skinned cardiac muscle fibers extracted from normal (α-MHC) and transgenic (β-MHC) mice. Dynamic and steady-state contractile parameters were measured in reconstituted muscle fibers. Step-like length perturbation experiments demonstrated that TnTA30V decreased the magnitude of the muscle length-mediated recruitment of new force-bearing cross bridges (ER) by 30% in α-MHC fibers. In sharp contrast, TnTA30V increased ER by 55% in β-MHC fibers. Inferences drawn from other dynamic contractile parameters suggest that directional changes in ER in TnTA30V + α-MHC and TnTA30V + β-MHC fibers result from a divergent impact on cross bridge-regulatory unit (troponin-tropomyosin complex) cooperativity. TnTA30V-mediated effects on Ca2+-activated maximal tension and instantaneous muscle fiber stiffness (ED) were also divergently affected by α- and β-MHC. Our study demonstrates that TnTA30V + α-MHC and TnTA30V + β-MHC fibers show contrasting contractile phenotypes; however, only the observations from β-MHC fibers are consistent with the clinical data for A28V in humans. NEW & NOTEWORTHY The differential impact of α- and β-myosin heavy chain (MHC) on contractile dynamics causes a mutant cardiac troponin T (TnTA30V) to differently modulate cardiac contractile function. TnTA30V attenuated Ca2+-activated maximal tension and length-mediated cross-bridge recruitment against α-MHC but augmented these parameters against β-MHC, suggesting divergent contractile phenotypes.

L71F mutation in rat cardiac troponin T augments crossbridge recruitment and detachment dynamics against α-myosin heavy chain, but not against β-myosin heavy chain

The N-terminal extension of human cardiac troponin T (TnT), which modulates myofilament Ca2+ sensitivity, contains several hypertrophic cardiomyopathy (HCM)-causing mutations including S69F. However, the functional consequence of S69F mutation is unknown. The human analog of S69F in rat TnT is L71F (TnTL71F). Because the functional consequences due to structural changes in the N-terminal extension are influenced by the type of myosin heavy chain (MHC) isoform, we hypothesized that the TnTL71F-mediated effect would be differently modulated by α- and β-MHC isoforms. TnTL71F and wild-type rat TnT were reconstituted into de-membranated muscle fibers from normal (α-MHC) and propylthiouracil-treated rat hearts (β-MHC) to measure steady-state and dynamic contractile parameters. The magnitude of the TnTL71F-mediated attenuation of Ca2+-activated maximal tension was greater in α- than in β-MHC fibers. For example, TnTL71F attenuated maximal tension by 31% in α-MHC fibers but only by 10% in β-MHC fibers. Furthermore, TnTL71F reduced myofilament Ca2+ sensitivity by 0.11 pCa units in α-MHC fibers but only by 0.05 pCa units in β-MHC fibers. TnTL71F augmented rate constants of crossbridge recruitment and crossbridge detachment dynamics in α-MHC fibers but not in β-MHC fibers. Collectively, our data demonstrate that TnTL71F induces greater contractile deficits against α-MHC than against β-MHC background.

Dilated Cardiomyopathy Mutation (R134W) in Mouse Cardiac Troponin T Induces Greater Contractile Deficits against α-Myosin Heavy Chain than against β-Myosin Heavy Chain

Many studies have demonstrated that depressed myofilament Ca2+ sensitivity is common to dilated cardiomyopathy (DCM) in humans. However, it remains unclear whether a single determinant—such as myofilament Ca2+ sensitivity—is sufficient to characterize all cases of DCM because the severity of disease varies widely with a given mutation. Because dynamic features dominate in the heart muscle, alterations in dynamic contractile parameters may offer better insight on the molecular mechanisms that underlie disparate effects of DCM mutations on cardiac phenotypes. Dynamic features are dominated by myofilament cooperativity that stem from different sources. One such source is the strong tropomyosin binding region in troponin T (TnT), which is known to modulate crossbridge (XB) recruitment dynamics in a myosin heavy chain (MHC)-dependent manner. Therefore, we hypothesized that the effects of DCM-linked mutations in TnT on contractile dynamics would be differently modulated by α- and β-MHC. After reconstitution with the mouse TnT equivalent (TnTR134W) of the human DCM mutation (R131W), we measured dynamic contractile parameters in detergent-skinned cardiac muscle fiber bundles from normal (α-MHC) and transgenic mice (β-MHC). TnTR134W significantly attenuated the rate constants of tension redevelopment, XB recruitment dynamics, XB distortion dynamics, and the magnitude of length-mediated XB recruitment only in α-MHC fiber bundles. TnTR134W decreased myofilament Ca2+ sensitivity to a greater extent in α-MHC (0.14 pCa units) than in β-MHC fiber bundles (0.08 pCa units). Thus, our data demonstrate that TnTR134W induces a more severe DCM-like contractile phenotype against α-MHC than against β-MHC background.