A spectral probe mutant (F29W) of chicken skeletal muscle troponin C (TnC) has been prepared in which Phe-29 has been substituted by Trp. Residue 29 is at the COOH-terminal end of the A helix immediately adjacent to the Ca2+ binding loop of site I (residues 30-41) of the regulatory N domain. Since this protein is naturally devoid of Tyr and Trp, spectral features can be assigned unambiguously to the single Trp. The fluorescent quantum yield at 336 nm is increased almost 3-fold in going from the Ca(2+)-free state to the 4Ca2+ state with no change in the wavelength of maximum emission. Comparisons of the Ca2+ titration curves of the change in far-UV CD and fluorescence emission indicated that the latter was associated only with the binding of 2Ca2+ to the regulatory sites I and II. No change in fluorescence was detected by titration with Mg2+. The Ca(2+)-induced transitions of both the N and C domains were highly cooperative. Addition of Ca2+ also produced a red shift in the UV absorbance spectrum and a reduction in positive ellipticity as monitored by near-UV CD measurements. The fluorescent properties of F29W were applied to an investigation of five double mutants: F29W/V45T, F29W/M46Q, F29W/M48A, F29W/L49T, and F29W/M82Q. Ca2+ titration of their fluorescent emissions indicated in each case an increased Ca2+ affinity of their N domains. The magnitude of these changes and the decreased cooperativity observed between Ca2+ binding sites I and II for some of the mutants are discussed in terms of the environment of the mutated residues in the 2Ca2+ and modeled 4Ca2+ states.(ABSTRACT TRUNCATED AT 250 WORDS)

Shifts in myosin heavy chain (MHC) isoforms in cardiac myocytes have been shown to alter cardiac muscle function not only in healthy developing hearts but also in diseased hearts. In guinea pig hearts, there is a large age-dependent shift in MHC isoforms from 80% α-MHC/20% β-MHC at 3 wk to 14% α-MHC/86% β-MHC at 11 wk. Because kinetic differences in α- and β-MHC cross-bridges (XBs) are known to impart different cooperative effects on thin filaments, we hypothesize here that differences in α- and β-MHC expression in guinea pig cardiac muscle impact sarcomere length (SL)–dependent contractile function. We therefore measure steady state and dynamic contractile parameters in detergent-skinned cardiac muscle preparations isolated from the left ventricles of young (3 wk old) or adult (11 wk old) guinea pigs at two different SLs: short (1.9 µm) and long (2.3 µm). Our data show that SL-dependent effects on contractile parameters are augmented in adult guinea pig cardiac muscle preparations. Notably, the SL-mediated increase in myofilament Ca2+ sensitivity (ΔpCa50) is twofold greater in adult guinea pig muscle preparations (ΔpCa50 being 0.11 units in adult preparations but only 0.05 units in young preparations). Furthermore, adult guinea pig cardiac muscle preparations display greater SL-dependent changes than young muscle preparations in (1) the magnitude of length-mediated increase in the recruitment of new force-bearing XBs, (2) XB detachment rate, (3) XB strain-mediated effects on other force-bearing XBs, and (4) the rate constant of force redevelopment. Our findings suggest that increased β-MHC expression enhances length-dependent activation in the adult guinea pig cardiac myocardium.

Alterations in length-dependent activation (LDA) may constitute a mechanism by which cardiomyopathy mutations lead to deleterious phenotypes and compromised heart function, because LDA underlies the molecular basis by which the heart tunes myocardial force production on a beat-to-beat basis (Frank-Starling mechanism). In this study, we investigated the effect of DCM-linked mutation (R173W) in human cardiac troponin T (TnT) on myofilament LDA. R173W mutation is associated with left ventricular dilatation and systolic dysfunction and is found in multiple families. R173W mutation is in the central region (residues 80-180) of TnT, which is known to be important for myofilament cooperativity and cross-bridge (XB) recruitment. Steady-state and dynamic contractile parameters were measured in detergent-skinned guinea pig left ventricular muscle fibers reconstituted with recombinant guinea pig wild-type TnT (TnTWT) or mutant TnT (TnTR174W; guinea pig analog of human R173W mutation) at two different sarcomere lengths (SL): short (1.9 µm) and long (2.3 µm). TnTR174W decreased pCa50 (-log [Ca2+]free required for half-maximal activation) to a greater extent at long than at short SL; for example, pCa50 decreased by 0.12 pCa units at long SL and by 0.06 pCa units at short SL. Differential changes in pCa50 at short and long SL attenuated the SL-dependent increase in myofilament Ca2+ sensitivity (ΔpCa50) in TnTR174W fibers; ΔpCa50 was 0.10 units in TnTWT fibers but only 0.04 units in TnTR174W fibers. Furthermore, TnTR174W blunted the SL-dependent increase in the magnitude of XB recruitment. Our observations suggest that the R173W mutation in human cardiac TnT may impair Frank-Starling mechanism.NEW & NOTEWORTHY This work characterizes the effect of dilated cardiomyopathy mutation in cardiac troponin T (TnTR174W) on myofilament length-dependent activation. TnTR174W attenuates the length-dependent increase in cross-bridge recruitment and myofilament Ca2+ sensitivity.

Recent clinical studies have revealed a new hypertrophic cardiomyopathy-associated mutation (F87L) in the central region of human cardiac troponin T (TnT). However, despite its implication in several incidences of sudden cardiac death in young and old adults, whether F87L is associated with cardiac contractile dysfunction is unknown. Because the central region of TnT is important for modulating the muscle length-mediated recruitment of new force-bearing cross-bridges (XBs), we hypothesize that the F87L mutation causes molecular changes that are linked to the length-dependent activation of cardiac myofilaments. Length-dependent activation is important because it contributes significantly to the Frank-Starling mechanism, which enables the heart to vary stroke volume as a function of changes in venous return. We measured steady-state and dynamic contractile parameters in detergent-skinned guinea pig cardiac muscle fibers reconstituted with recombinant guinea pig wild-type TnT (TnTWT) or the guinea pig analogue (TnTF88L) of the human mutation at two different sarcomere lengths (SLs): short (1.9 µm) and long (2.3 µm). TnTF88L increases pCa50 (-log [Ca2+]free required for half-maximal activation) to a greater extent at short SL than at long SL; for example, pCa50 increases by 0.25 pCa units at short SL and 0.17 pCa units at long SL. The greater increase in pCa50 at short SL leads to the abolishment of the SL-dependent increase in myofilament Ca2+ sensitivity (ΔpCa50) in TnTF88L fibers, ΔpCa50 being 0.10 units in TnTWT fibers but only 0.02 units in TnTF88L fibers. Furthermore, at short SL, TnTF88L attenuates the negative impact of strained XBs on force-bearing XBs and augments the magnitude of muscle length-mediated recruitment of new force-bearing XBs. Our findings suggest that the TnTF88L-mediated effects on cardiac thin filaments may lead to a negative impact on the Frank-Starling mechanism.

The central region of cardiac troponin T (TnT) is important for modulating the dynamics of muscle length-mediated cross-bridge recruitment. Therefore, hypertrophic cardiomyopathy mutations in the central region may affect cross-bridge recruitment dynamics to alter myofilament Ca2+ sensitivity and length-dependent activation of cardiac myofilaments. Given the importance of the central region of TnT for cardiac contractile dynamics, we studied if hypertrophic cardiomyopathy-linked mutation (TnTR94H)-induced effects on contractile function would be differently modulated by sarcomere length (SL). Recombinant wild-type TnT (TnTWT) and the guinea pig analog of the human R94H mutation (TnTR95H) were reconstituted into detergent-skinned cardiac muscle fibers from guinea pigs. Steady-state and dynamic contractile measurements were made at short and long SLs (1.9 and 2.3 µm, respectively). Our results demonstrated that TnTR95H increased pCa50 (-log of free Ca2+ concentration) to a greater extent at short SL; TnTR95H increased pCa50 by 0.11 pCa units at short SL and 0.07 pCa units at long SL. The increase in pCa50 associated with an increase in SL from 1.9 to 2.3 µm (ΔpCa50) was attenuated nearly twofold in TnTR95H fibers; ΔpCa50 was 0.09 pCa units for TnTWT fibers but only 0.05 pCa units for TnTR95H fibers. The SL dependency of rate constants of cross-bridge distortion dynamics and tension redevelopment was also blunted by TnTR95H Collectively, our observations on the SL dependency of pCa50 and rate constants of cross-bridge distortion dynamics and tension redevelopment suggest that mechanisms underlying the length-dependent activation cardiac myofilaments are attenuated by TnTR95HNEW & NOTEWORTHY Mutant cardiac troponin T (TnTR95H) differently affects myofilament Ca2+ sensitivity at short and long sarcomere length, indicating that mechanisms underlying length-dependent activation are altered by TnTR95H TnTR95H enhances myofilament Ca2+ sensitivity to a greater extent at short sarcomere length, thus attenuating the length-dependent increase in myofilament Ca2+ sensitivity.

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.

Omecamtiv mecarbil (OM) is a pharmacological agent that augments cardiac contractile function by enhancing myofilament Ca2+ sensitivity. Given that interventions that increase myofilament Ca2+ sensitivity have the potential to alter length-dependent activation (LDA) of cardiac myofilaments, we tested the influence of OM on this fundamental property of the heart. This is significant not only because LDA is prominent in cardiac muscle but also because it contributes to the Frank-Starling law, a mechanism by which the heart increases stroke volume in response to an increase in venous return. We measured steady-state and dynamic contractile indices in detergent-skinned guinea pig (Cavia porcellus) cardiac muscle fibers in the absence and presence of 0.3 and 3.0 μM OM at two different sarcomere lengths (SLs), short SL (1.9 μm) and long SL (2.3 μm). Myofilament Ca2+ sensitivity, as measured by pCa50 (-log of [Ca2+]free concentration required for half-maximal activation), increased significantly at both short and long SLs in OM-treated fibers when compared to untreated fibers; however, the magnitude of increase in pCa50 was twofold greater at short SL than at long SL. A consequence of this greater increase in pCa50 at short SL was that pCa50 did not increase any further at long SL, suggesting that OM abolished the SL dependency of pCa50. Furthermore, the SL dependency of rate constants of cross-bridge distortion dynamics (c) and force redevelopment (ktr) was abolished in 0.3-μM-OM-treated fibers. The negative impact of OM on the SL dependency of pCa50, c, and ktr was also observed in 3.0-μM-OM-treated fibers, indicating that cooperative mechanisms linked to LDA were altered by the OM-mediated effects on cardiac myofilaments.

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.

Background We hypothesized that the functional effects of R206L—a rat analog of the dilated cardiomyopathy ( DCM ) mutation R205L in human cardiac troponin T (TnT)—were differently modulated by myosin heavy chain ( MHC ) isoforms and T204E, a protein kinase C ( PKC ) phosphomimic of TnT. Our hypothesis was based on two observations: (1) α‐ and β‐ MHC differentially influence the functional effects of TnT; and (2) PKC isoforms capable of phosphorylating TnT are upregulated in failing human hearts. Methods and Results We generated 4 recombinant TnT variants: wild type; R206L; T204E; and R206L+T204E. Functional effects of the TnT variants were tested in cardiac muscle fibers (minimum 14 per group) from normal (α‐ MHC ) and propylthiouracil‐treated rats (β‐ MHC ) using steady‐state and dynamic contractile measurements. Notably, in α‐ MHC fibers, Ca 2+ ‐activated maximal tension was attenuated by R206L (≈32%), T204E (≈63%), and R206L+T204E (≈64%). In β‐ MHC fibers, maximal tension was unaffected by R206L, but was attenuated by T204E (≈33%) and R206L+T204E (≈40%). Thus, β‐ MHC differentially counteracted the attenuating effects of the TnT variants on tension. However, in β‐ MHC fibers, R206L+T204E attenuated tension to a greater extent when compared to T204E alone. In β‐ MHC fibers, R206L+T204E attenuated the magnitude of the length‐mediated recruitment of new cross‐bridges (≈28%), suggesting that the Frank‐Starling mechanism was impaired. Conclusions Our findings are the first (to our knowledge) to demonstrate that the functional effects of a DCM ‐linked TnT mutation are not only modulated by MHC isoforms, but also by the pathology‐associated post‐translational modifications of TnT.

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.

Failing hearts of dilated cardiomyopathy (DCM)-patients reveal systolic dysfunction and upregulation of several Protein Kinase C (PKC) isoforms. Recently, we demonstrated that the functional effects of T204E, a PKC phosphomimic of cardiac troponin T (TnT), were differently modulated by α- and β-myosin heavy chain (MHC) isoforms. Therefore, we hypothesized that the interplay between the effects of T204E and a DCM-linked mutation (K211Δ or R206W) in TnT would modulate contractile parameters linked-to systolic function in an MHC-dependent manner. To test our hypothesis, five TnT variants (wildtype, K211Δ, K211Δ + T204E, R206W, and R206W + T204E) were generated and individually reconstituted into demembranated cardiac muscle fibers from normal (α-MHC) and propylthiouracil-treated (β-MHC) rats. Steady-state and mechano-dynamic measurements were performed on reconstituted fibers. Myofilament Ca2+ sensitivity (pCa50) was decreased by both K211Δ and R206W to a greater extent in α-MHC fibers (~0.15 pCa units) than in β-MHC fibers (~0.06 pCa units). However, T204E exacerbated the attenuating influence of both mutants on pCa50 only in β-MHC fibers. Moreover, the magnitude of muscle length (ML)-mediated crossbridge (XB) recruitment was decreased by K211Δ + T204E (~47 %), R206W (~34 %), and R206W + T204E (~36 %) only in β-MHC fibers. In relevance to human hearts, which predominantly express β-MHC, our data suggest that the interplay between the effects of DCM mutations, PKC phosphomimic in TnT, and β-MHC lead to systolic dysfunction by attenuating pCa50 and the magnitude of ML-mediated XB recruitment.

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.

In mammalian ventricles, two myosin heavy chain (MHC) isoforms have been identified. Small animals express α-MHC, whereas large animals express β-MHC, which contribute to a large difference in the heart rate. Sprague–Dawley rats possessing ~99% α-MHC were treated with propylthiouracil to result in 100% β-MHC. Papillary muscles were skinned, dissected into small fibers, and used for experiments. To understand the functional difference between α-MHC and β-MHC, skinned-fibers were activated under the intracellular ionic conditions: 5 mM MgATP, 1 mM Mg2+, 8 mM Pi, 200 mM ionic strength, pH 7.00 at 25 °C. Small amplitude sinusoidal length oscillations were applied in the frequency range 0.13–100 Hz (corresponding time domain: 1.6–1200 ms), and effects of Ca2+, Pi, and ATP were studied. The results show that Ca2+ sensitivity was slightly less (10–15%) in β-MHC than α-MHC containing fibers. Sinusoidal analysis at pCa 4.66 (full Ca2+ activation) demonstrated that, the apparent rate constants were 2–4× faster in α-MHC containing fibers. The ATP study demonstrated that, in β-MHC containing fibers, K 1 (ATP association constant) was greater (1.7×), k 2 and k −2 (cross-bridge detachment and its reversal rate constants) were smaller (×0.6). The Pi study demonstrated that, in β-MHC containing fibers, k 4 (rate constant of the force-generation step) and k −4 were smaller (0.75× and 0.25×, respectively), resulting in greater K 4 (3×). There were no differences in active tension, rigor stiffness, or K 2 (equilibrium constant of the cross-bridge detachment step). Our study further demonstrated that there were no differences in parameters between fibers obtained from left and right ventricles, but with an exception in K 5 (Pi association constant).

The hypertrophic cardiomyopathy (HCM) mutation A30V is located within the N-terminus of troponin T (TnT), a region known to regulate Ca2+-activated maximal tension and length-dependent activation (LDA) in cardiac muscle. Given how the function of the N-terminus is differently altered by α/β-myosin heavy chain (MHC) isoforms, we believe the mutation-mediated effects are also differently altered by α- and β-MHC isoforms. Likewise, the effects of protein kinase C (PKC)-mediated phosphorylation of TnT (residue T203) are also differently altered by MHC isoforms; phosphorylation of TnT is important to consider because PKC isoforms are upregulated in failing human hearts. We tested the effects of TnTA30V (mouse analog of human A28V), PKC phosphomimetic T203E (TnTT203E), and TnTT203E+A30V in α- and β-MHC backgrounds. In α-MHC fibers, Ca2+-activated maximal tension was attenuated by TnTA30V (∼11%) and TnTT203E+A30V (∼59%). However, in β-MHC fibers, maximal tension was augmented by TnTA30V (∼13%), but was attenuated by TnTT203E+A30V (∼29%) to a lesser extent than in α-MHC fibers. Rate constants of tension redevelopment (ktr) and crossbridge (XB) recruitment dynamics (b) were attenuated by TnTT203E+A30V in α-MHC fibers, but were unaffected in β-MHC fibers. The magnitude of muscle length-mediated recruitment of strong XBs (ER) was attenuated by TnTA30V (∼31%) and TnTT203E+A30V (∼43%) in α-MHC fibers. In contrast, ER was augmented by TnTA30V (∼65%) and TnTT203E+A30V (∼65%) in β-MHC fibers. Inferences drawn from dynamic and steady-state contractile measurements suggest that XB-RU cooperativity - a significant player in LDA - is attenuated by TnTA30V and TnTT203E+A30V in α-MHC, but augmented in β-MHC. These observations demonstrate that α/β-MHC isoforms divergently modify contractile dynamics in TnTA30V and TnTT203E+A30V fibers.

In mammalian heart muscles, two isoforms of MHC were identified. Small animals carry primarily α-MHC (fast), whereas large animals (e.g., bovines and humans) carry mostly β-MHC (slow), resulting in a large difference in the heart rate. Sprague-Dawley rats, which possess 99% α-MHC in their ventricles, were treated with propylthiouracil to result in 100% β-MHC, as demonstrated by SDS-PAGE. Papillary muscles were dissected, skinned, split into small fibers (90-110 µm diameter), and mounted on experimental apparatus. To understand the functional difference between α-MHC and β-MHC, fibers were activated under intracellular ionic conditions of cardiomyocytes: 5mM ATP, 1mM Mg2+, 8mM phosphate (Pi), 200mM ionic strength, and pH 7.0. These measurements were carried out at 25°C. When steady tension developed, small amplitude sinusoidal length oscillations were applied in the frequency range 0.13-100Hz (corresponding time domain: 1.6-1200 ms), and the effects of Ca2+, Pi, and ATP were studied. The results show that Ca2+ sensitivity was slightly less (12%) in β-MHC than α-MHC, while cooperativity was not statistically different. Sinusoidal analysis demonstrated that, in β-MHC containing fibers, K1 (ATP association constant) was greater (1.7x), k2 and k-2 (cross-bridge detachment and its reversal rate constants) were smaller (0.6x); k4 (rate constant of the force-generation step) and k-4 (its reversal step) were smaller (0.75x and 0.25x, respectively), which resulted in greater (3x) K4 (equilibrium constant of the force generation step) than those of α-MHC. There were no differences in K2 (equilibrium constant of the cross-bridge detachment step), K5 (Pi association constant), active tension, or rigor stiffness. Our study also demonstrated that there were no differences in the above mentioned parameters between fibers obtained from left and right ventricles, indicating that there is no difference at the myofilament level in both ventricles.

The primary structure of the H1-helix of troponin T (TnT) varies among different types of striated muscles. Moreover, these muscles also express different myosin heavy chain (MHC) isoforms. Recently, we demonstrated that pseudo-phosphorylation of residue 204 (near the H1-helix) of cardiac TnT affected the functional state of the thin filament differently in fibers that expressed either α- or β-MHC isoforms (Michael et al., Basic Res Cardiol, 109:442, 2014). In this follow-up study, we investigated how the isoform-specific function of the H1-helix of cardiac TnT was influenced by α- and β-MHC isoforms. We generated a mutant rat cardiac TnT (RfsH1) in which the cardiac H1-helix was replaced by the fast skeletal H1-helix. Recombinant RfsH1 was reconstituted into detergent-skinned cardiac muscle fibers from either normal rats (expressing α-MHC) or propylthiouracil treated rats (expressing β-MHC). Steady-state and dynamic measurements were carried out at sarcomere length 2.3 µm. Our results demonstrated that RfsH1 decreased Ca2+-activated maximal ATPase activity differently in α-MHC (∼33%) and β-MHC (∼17%) fibers. Furthermore, RfsH1 decreased tension cost (∼31%) and crossbridge (XB) distortion dynamics (∼25%) in α-MHC but not in β-MHC fibers. Because the above mentioned parameters are indices of the rate of XB detachment, our results suggest that the interplay between the RfsH1- and α-MHC-mediated effects on the thin filament modulates XB detachment kinetics. Our findings suggest that the conformational changes in the H1-helix of TnT are sensitive to MHC isoform-mediated changes in the thin filament.

The N-terminal extension (NTE; residues 42-73) of mouse cardiac troponin T (TnT) desensitizes cardiac myofilaments to Ca2+ by stabilizing thin filaments in the blocked-state. We arrived at this conclusion using detergent-skinned muscle from transgenic (TG) mouse hearts that expressed 54% of chimeric TnT (residues 1-73 of mouse cardiac TnT were replaced by residues 1-41 of mouse fast skeletal TnT). Here, we extended our investigation to include higher dose effects of the modified TnT on cardiac myofilament function/phenotype using detergent-skinned fiber studies and echocardiography measurements in two different TG mouse lines (TG-55 and TG-64 that expressed 55% and 64% of chimeric TnT, respectively). Both TG-55 and TG-64 mice showed a similar increase in myofilament Ca2+ sensitivity at sarcomere lengths (SL) of 1.9 and 2.3 μm. However, Ca2+-activated maximal tension increased significantly only in TG-64 mice at either SL. There was a progressive decrease in the overall heart size and heart-to-body weight ratios in both TG-55 and TG-64 mice. Left ventricular diastolic functional parameters (isovolumic relaxation time and E-wave deceleration time) showed a graded increase in TG-55 and TG-64 mice; however, such effects were only significant in TG-64 mice, suggesting impaired relaxation. Systolic functional parameters (stroke volume, ejection fraction and fractional shortening) were unaffected in TG-55 mice, but significantly decreased in TG-64 mice. Thus, higher levels of chimeric TnT (64%) depressed both diastolic and systolic function significantly in TG-64 mice. We will discuss the link between the effects of the modified N-terminus of TnT on cardiac myofilament function and the resultant pathological remodeling of the heart. Our findings have pathological relevance because a growing number of disease-related mutations are found both in and near the NTE of cardiac TnT.

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.

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 mechanical (static and dynamic) properties of striated muscles (skeletal and cardiac) will be the focus of this article. We will begin with the gross anatomy of striated muscles and then elucidate the structural basis for striated muscle contraction and relaxation. Throughout the article, some important functional differences between skeletal and cardiac muscles will be highlighted.