RESUMO
Mavacamten is a FDA-approved small-molecule therapeutic designed to regulate cardiac function at the sarcomere level by selectively but reversibly inhibiting the enzymatic activity of myosin. It shifts myosin toward ordered off states close to the thick filament backbone. It remains elusive whether these myosin heads in the off state(s) can be recruited in response to physiological stimuli when required to boost cardiac output. We show that cardiac myosins stabilized in these off state(s) by mavacamten are recruitable by 1) Ca2+, 2) increased chronotropy [heart rate (HR)], 3) stretch, and 4) ß-adrenergic (ß-AR) stimulation, all known physiological inotropic interventions. At the molecular level, we show that Ca2+ increases myosin ATPase activity by shifting mavacamten-stabilized myosin heads from the inactive super-relaxed state to the active disordered relaxed state. At the myofilament level, both Ca2+ and passive lengthening can shift mavacamten-ordered off myosin heads from positions close to the thick filament backbone to disordered on states closer to the thin filaments. In isolated rat cardiomyocytes, increased stimulation rates enhanced shortening fraction in mavacamten-treated cells. This observation was confirmed in vivo in telemetered rats, where left-ventricular dP/dtmax, an index of inotropy, increased with HR in mavacamten-treated animals. Finally, we show that ß-AR stimulation in vivo increases left-ventricular function and stroke volume in the setting of mavacamten. Our data demonstrate that the mavacamten-promoted off states of myosin in the thick filament are at least partially activable, thus preserving cardiac reserve mechanisms.
Assuntos
Miócitos Cardíacos , Miosinas , Uracila/análogos & derivados , Animais , Ratos , Benzilaminas/farmacologia , Contração MuscularRESUMO
A hallmark feature of myosin-II is that it can spontaneously self-assemble into bipolar synthetic thick filaments (STFs) in low-ionic-strength buffers, thereby serving as a reconstituted in vitro model for muscle thick filaments. Although these STFs have been extensively used for structural characterization, their functional evaluation has been limited. In this report, we show that myosins in STFs mirror the more electrostatic and cooperative interactions that underlie the energy-sparing super-relaxed (SRX) state, which are not seen using shorter myosin subfragments, heavy meromyosin (HMM) and myosin subfragment 1 (S1). Using these STFs, we show several pathophysiological insults in hypertrophic cardiomyopathy, including the R403Q myosin mutation, phosphorylation of myosin light chains, and an increased ADP:ATP ratio, destabilize the SRX population. Furthermore, WT myosin containing STFs, but not S1, HMM, or STFs-containing R403Q myosin, recapitulated the ADP-induced destabilization of the SRX state. Studies involving a clinical-stage small-molecule inhibitor, mavacamten, showed that it is more effective in not only increasing myosin SRX population in STFs than in S1 or HMM but also in increasing myosin SRX population equally well in STFs made of healthy and disease-causing R403Q myosin. Importantly, we also found that pathophysiological perturbations such as elevated ADP concentration weakens mavacamten's ability to increase the myosin SRX population, suggesting that mavacamten-bound myosin heads are not permanently protected in the SRX state but can be recruited into action. These findings collectively emphasize that STFs serve as a valuable tool to provide novel insights into the myosin SRX state in healthy, diseased, and therapeutic conditions.
Assuntos
Benzilaminas/química , Benzilaminas/metabolismo , Miosinas/metabolismo , Uracila/análogos & derivados , Trifosfato de Adenosina/metabolismo , Animais , Humanos , Músculo Esquelético/metabolismo , Contração Miocárdica/fisiologia , Cadeias Leves de Miosina/química , Cadeias Leves de Miosina/metabolismo , Subfragmentos de Miosina/química , Subfragmentos de Miosina/metabolismo , Miosinas/química , Fosforilação/fisiologia , Uracila/química , Uracila/metabolismoRESUMO
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.
Assuntos
Cálcio/metabolismo , Sarcômeros/efeitos dos fármacos , Sarcômeros/metabolismo , Ureia/análogos & derivados , Animais , Fenômenos Biomecânicos/efeitos dos fármacos , Relação Dose-Resposta a Droga , Cobaias , Contração Miocárdica/efeitos dos fármacos , Fosforilação/efeitos dos fármacos , Sarcômeros/fisiologia , Ureia/farmacologiaRESUMO
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 ß-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 ß-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 (ß-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 ß-MHC fibers. Inferences drawn from other dynamic contractile parameters suggest that directional changes in ER in TnTA30V + α-MHC and TnTA30V + ß-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 ß-MHC. Our study demonstrates that TnTA30V + α-MHC and TnTA30V + ß-MHC fibers show contrasting contractile phenotypes; however, only the observations from ß-MHC fibers are consistent with the clinical data for A28V in humans. NEW & NOTEWORTHY: The differential impact of α- and ß-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 ß-MHC, suggesting divergent contractile phenotypes.
Assuntos
Cálcio/metabolismo , Cardiomiopatia Hipertrófica/genética , Contração Miocárdica/genética , Miócitos Cardíacos/metabolismo , Cadeias Pesadas de Miosina/metabolismo , Tropomiosina/metabolismo , Troponina T/genética , Troponina/metabolismo , Animais , Western Blotting , Masculino , Camundongos , Camundongos Transgênicos , Mutação , Contração Miocárdica/fisiologia , Miócitos Cardíacos/fisiologia , Cadeias Pesadas de Miosina/genética , Isoformas de ProteínasRESUMO
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 ß-myosin heavy chain (MHC) isoforms. α- and ß-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 (ß-MHC) mouse hearts. TnTR97L augmented myofilament Ca(2+) sensitivity by a similar amount in α- and ß-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 ß-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 ß-MHC fibers. We provide a mechanistic basis for the TnTR97L-induced effects in α- and ß-MHC fibers and discuss the relevance to human hearts.
Assuntos
Cardiomiopatia Hipertrófica/genética , Mutação , Cadeias Pesadas de Miosina/metabolismo , Troponina T/genética , Adenosina Trifosfatases/metabolismo , Animais , Cardiomiopatia Hipertrófica/metabolismo , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Contração Miocárdica , Miofibrilas/metabolismo , Proteínas Recombinantes/metabolismo , Estresse Mecânico , Miosinas Ventriculares/metabolismoRESUMO
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 ß-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 (ß-MHC) rats. Steady-state and mechano-dynamic measurements were performed on reconstituted fibers. Myofilament Ca(2+) sensitivity (pCa50) was decreased by both K211Δ and R206W to a greater extent in α-MHC fibers (~0.15 pCa units) than in ß-MHC fibers (~0.06 pCa units). However, T204E exacerbated the attenuating influence of both mutants on pCa50 only in ß-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 ß-MHC fibers. In relevance to human hearts, which predominantly express ß-MHC, our data suggest that the interplay between the effects of DCM mutations, PKC phosphomimic in TnT, and ß-MHC lead to systolic dysfunction by attenuating pCa50 and the magnitude of ML-mediated XB recruitment.
Assuntos
Cardiomiopatia Dilatada/metabolismo , Miócitos Cardíacos/metabolismo , Cadeias Pesadas de Miosina/metabolismo , Proteína Quinase C/metabolismo , Troponina T/metabolismo , Animais , Humanos , Mutação , RatosRESUMO
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 ß-MHC isoforms. TnTL71F and wild-type rat TnT were reconstituted into de-membranated muscle fibers from normal (α-MHC) and propylthiouracil-treated rat hearts (ß-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 ß-MHC fibers. For example, TnTL71F attenuated maximal tension by 31% in α-MHC fibers but only by 10% in ß-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 ß-MHC fibers. TnTL71F augmented rate constants of crossbridge recruitment and crossbridge detachment dynamics in α-MHC fibers but not in ß-MHC fibers. Collectively, our data demonstrate that TnTL71F induces greater contractile deficits against α-MHC than against ß-MHC background.
Assuntos
Mutação/genética , Cadeias Pesadas de Miosina/genética , Troponina T/genética , Miosinas Ventriculares/genética , Animais , Cálcio/metabolismo , Masculino , Contração Miocárdica/genética , Miofibrilas/genética , Isoformas de Proteínas/genética , Ratos , Ratos Sprague-DawleyRESUMO
The primary causal link between disparate effects of human hypertrophic cardiomyopathy (HCM)-related mutations in troponin T (TnT) and α- and ß-myosin heavy chain (MHC) isoforms on cardiac contractile phenotype remains poorly understood. Given the divergent impact of α- and ß-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 ß-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 ß-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 (ß-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 ß-MHC. TnTF72L increased the rate of XB distortion dynamics by 49% in α-MHC fibers but had no effect in ß-MHC fibers; these observations suggest that TnTF72L augmented XB detachment kinetics in α-MHC, but not ß-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 ß-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 ß-MHC.
Assuntos
Mutação , Contração Miocárdica , Miofibrilas/metabolismo , Cadeias Pesadas de Miosina/metabolismo , Músculos Papilares/metabolismo , Troponina T/genética , Animais , Sinalização do Cálcio , Cardiomiopatia Dilatada/genética , Cardiomiopatia Dilatada/metabolismo , Cardiomiopatia Dilatada/fisiopatologia , Cardiomiopatia Hipertrófica/genética , Cardiomiopatia Hipertrófica/metabolismo , Cardiomiopatia Hipertrófica/fisiopatologia , Predisposição Genética para Doença , Cinética , Masculino , Força Muscular , Contração Miocárdica/efeitos dos fármacos , Miofibrilas/efeitos dos fármacos , Músculos Papilares/efeitos dos fármacos , Músculos Papilares/fisiopatologia , Fenótipo , Fosforilação , Propiltiouracila/farmacologia , Ligação Proteica , Ratos Sprague-Dawley , Troponina T/metabolismo , Função Ventricular EsquerdaRESUMO
Given the differential impact of α- and ß-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 ß-MHC. We characterized dynamic contractile features of normal (α-MHC) and transgenic (ß-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 ß-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 ß-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 ß-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 (ß-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.
Assuntos
Miosinas Cardíacas/metabolismo , Cardiomiopatia Dilatada/genética , Mutação de Sentido Incorreto , Miócitos Cardíacos/metabolismo , Cadeias Pesadas de Miosina/metabolismo , Troponina T/metabolismo , Animais , Cardiomiopatia Dilatada/metabolismo , Células Cultivadas , Camundongos , Camundongos Endogâmicos C57BL , Contração Miocárdica , Miócitos Cardíacos/fisiologia , Isoformas de Proteínas/metabolismo , Troponina T/genéticaRESUMO
Interplay between the protein kinase C (PKC)-mediated phosphorylation of troponin T (TnT)- and myosin heavy chain (MHC)-mediated effects on thin filaments takes on a new significance because: (1) there is significant interaction between the TnT- and MHC-mediated effects on cardiac thin filaments; (2) although the phosphorylation of TnT by PKC isoforms is common to both human and rodent hearts, human hearts predominantly express ß-MHC while rodent hearts predominantly express α-MHC. Therefore, we tested how α- and ß-MHC isoforms differently affected the functional effects of phosphorylated TnT. Contractile measurements were made on cardiac muscle fibers from normal rats (α-MHC) and propylthiouracil-treated rats (ß-MHC), reconstituted with the recombinant phosphomimetic-TnT (T204E; threonine 204 replaced by glutamate). Ca2+ -activated maximal tension decreased differently in α-MHC + T204E (~68%) and ß-MHC + T204E (~35%). However, myofilament Ca2+ sensitivity decreased similarly in α-MHC + T204E and ß-MHC + T204E, demonstrating that a decrease in Ca2+ sensitivity alone cannot explain the greater attenuation of tension in α-MHC + T204E. Interestingly, dynamic contractile parameters (rates of tension redevelopment, crossbridge (XB) recruitment dynamics, XB distortion dynamics, and XB detachment kinetics) decreased only in α-MHC + T204E. Thus, the transition of thin filaments from the blocked- to closed-state was attenuated in α-MHC + T204E and ß-MHC + T204E, but the closed- to open-state transition was attenuated only in α-MHC + T204E. Our study demonstrates that the effects of phosphorylated TnT and MHC isoforms interact to bring about different functional states of cardiac thin filaments.
Assuntos
Miocárdio/metabolismo , Cadeias Pesadas de Miosina/fisiologia , Troponina T/metabolismo , Animais , Masculino , Fosforilação , Isoformas de Proteínas , Ratos , Ratos Sprague-DawleyRESUMO
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.
Assuntos
Cálcio/metabolismo , Acoplamento Excitação-Contração , Deleção de Genes , Contração Miocárdica , Miócitos Cardíacos/metabolismo , Miofibrilas/metabolismo , Cadeias Leves de Miosina/metabolismo , Sequência de Aminoácidos , Animais , Fenômenos Biomecânicos , Feminino , Humanos , Cinética , Camundongos , Camundongos Transgênicos , Força Muscular , Cadeias Leves de Miosina/química , Cadeias Leves de Miosina/genética , Sarcômeros/metabolismoRESUMO
Hypertrophic cardiomyopathy (HCM) is a primary myocardial disorder characterized by left ventricular hypertrophy, hyperdynamic contraction, and impaired relaxation of the heart. These functional derangements arise directly from altered sarcomeric function due to either mutations in genes encoding sarcomere proteins, or other defects such as abnormal energetics. Current treatment options do not directly address this causal biology but focus on surgical and extra-sarcomeric (sarcolemmal) pharmacological symptomatic relief. Mavacamten (formerly known as MYK-461), is a small molecule designed to regulate cardiac function at the sarcomere level by selectively but reversibly inhibiting the enzymatic activity of myosin, the fundamental motor of the sarcomere. This review summarizes the mechanism and translational progress of mavacamten from proteins to patients, describing how the mechanism of action and pharmacological characteristics, involving both systolic and diastolic effects, can directly target pathophysiological derangements within the cardiac sarcomere to improve cardiac structure and function in HCM. Mavacamten was approved by the Food and Drug Administration in April 2022 for the treatment of obstructive HCM and now goes by the commercial name of Camzyos. Full information about the risks, limitations, and side effects can be found at www.accessdata.fda.gov/drugsatfda_docs/label/2022/214998s000lbl.pdf.
Assuntos
Cardiomiopatia Hipertrófica , Medicina de Precisão , Estados Unidos , Humanos , Cardiomiopatia Hipertrófica/tratamento farmacológico , Cardiomiopatia Hipertrófica/genética , Benzilaminas/efeitos adversos , Benzilaminas/química , MiosinasRESUMO
Mavacamten is a novel, FDA-approved, small molecule therapeutic designed to regulate cardiac function by selectively but reversibly inhibiting the enzymatic activity of myosin. It shifts myosin towards ordered off states close to the thick filament backbone. It remains unresolved whether mavacamten permanently sequesters these myosin heads in the off state(s) or whether these heads can be recruited in response to physiological stimuli when required to boost cardiac output. We show that cardiac myosins stabilized in these off state(s) by mavacamten are recruitable by Ca2+, increased heart rate, stretch, and ß-adrenergic (ß-AR) stimulation, all known physiological inotropic effectors. At the molecular level, we show that, in presence of mavacamten, Ca2+ increases myosin ATPase activity by shifting myosin heads from the reserve super-relaxed (SRX) state to the active disordered relaxed (DRX) state. At the myofilament level, both Ca2+ and passive lengthening can shift ordered off myosin heads from positions close to the thick filament backbone to disordered on states closer to the thin filaments in the presence of mavacamten. In isolated rat cardiomyocytes, increased stimulation rates enhanced shortening fraction in mavacamten-treated cells. This observation was confirmed in vivo in telemetered rats, where left-ventricular dP/dtmax, an index of inotropy, increased with heart rate in mavacamten treated animals. Finally, we show that ß-AR stimulation in vivo increases left-ventricular function and stroke volume in the setting of mavacamten. Our data demonstrate that the mavacamten-promoted off states of myosin in the thick filament are activable, at least partially, thus leading to preservation of cardiac reserve mechanisms.
RESUMO
Cardiac troponin T (cTnT) is a key component of contractile regulatory proteins. cTnT is characterized by a â¼32 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 µm. 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.
Assuntos
Miocárdio/citologia , Miocárdio/metabolismo , Miofibrilas/metabolismo , Troponina T/química , Troponina T/metabolismo , Adenosina Trifosfatases/metabolismo , Sequência de Aminoácidos , Substituição de Aminoácidos , Animais , Fenômenos Biomecânicos , Cálcio/metabolismo , Feminino , Regulação da Expressão Gênica , Cinética , Camundongos , Camundongos Transgênicos , Dados de Sequência Molecular , Mutagênese , Fosforilação , Conformação Proteica , Troponina T/genéticaRESUMO
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.
Assuntos
Alanina/metabolismo , Substituição de Aminoácidos , Ácido Aspártico/metabolismo , Fibras Musculares Esqueléticas/metabolismo , Serina/genética , Troponina I/química , Troponina I/metabolismo , Adenosina Trifosfatases/metabolismo , Alanina/genética , Animais , Ácido Aspártico/genética , Cálcio/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Cinética , Masculino , Contração Muscular , Fibras Musculares Esqueléticas/citologia , Fibras Musculares Esqueléticas/fisiologia , Miocárdio/citologia , Miocárdio/metabolismo , Miofibrilas/metabolismo , Fosforilação , Ratos , Troponina I/genéticaRESUMO
In addition to a conventional relaxed state, a fraction of myosins in the cardiac muscle exists in a low-energy consuming super-relaxed (SRX) state, which is kept as a reserve pool that may be engaged under sustained increased cardiac demand. The conventional relaxed and the super-relaxed states are widely assumed to correspond to a structure where myosin heads are in an open configuration, free to interact with actin, and a closed configuration, inhibiting binding to actin, respectively. Disruption of the myosin SRX population is an emerging model in different heart diseases, such as hypertrophic cardiomyopathy, which results in excessive muscle contraction, and stabilizing them using myosin inhibitors is budding as an attractive therapeutic strategy. Here we examined the structure-function relationships of two myosin ATPase inhibitors, mavacamten and para-nitroblebbistatin, and found that binding of mavacamten at a site different than para-nitroblebbistatin populates myosin into the SRX state. Para-nitroblebbistatin, binding to a distal pocket to the myosin lever arm near the nucleotide-binding site, does not affect the usual myosin SRX state but instead appears to render myosin into a new, perhaps much more inhibited, 'ultra-relaxed' state. X-ray scattering-based rigid body modeling shows that both mavacamten and para-nitroblebbistatin induce novel conformations in human ß-cardiac heavy meromyosin that diverge significantly from the hypothetical open and closed states, and furthermore, mavacamten treatment causes greater compaction than para-nitroblebbistatin. Taken together, we conclude that mavacamten and para-nitroblebbistatin stabilize myosin in different structural states, and such states may give rise to different functional energy-sparing states.
Assuntos
Benzilaminas/química , Modelos Moleculares , Conformação Proteica , Uracila/análogos & derivados , Miosinas Ventriculares/química , Benzilaminas/farmacologia , Miosinas/antagonistas & inibidores , Miosinas/química , Domínios e Motivos de Interação entre Proteínas , Estabilidade Proteica , Análise Espectral , Relação Estrutura-Atividade , Uracila/química , Uracila/farmacologia , Miosinas Ventriculares/antagonistas & inibidoresRESUMO
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% ß-MHC at 3 wk to 14% α-MHC/86% ß-MHC at 11 wk. Because kinetic differences in α- and ß-MHC cross-bridges (XBs) are known to impart different cooperative effects on thin filaments, we hypothesize here that differences in α- and ß-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 ß-MHC expression enhances length-dependent activation in the adult guinea pig cardiac myocardium.
Assuntos
Miocárdio/metabolismo , Cadeias Pesadas de Miosina/metabolismo , Sarcômeros/metabolismo , Citoesqueleto de Actina/metabolismo , Animais , Cálcio/metabolismo , Feminino , Cobaias , Ventrículos do Coração/metabolismo , Cinética , Contração Miocárdica/fisiologia , Miócitos Cardíacos/metabolismo , Fosforilação/fisiologia , Isoformas de Proteínas/metabolismo , Suínos , Troponina T/metabolismoRESUMO
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 ß-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 (ß-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 ß-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 ß-MHC background.
RESUMO
Direct measurement of the in vivo contractile properties of an individual muscle cannot be made in humans. The objective of this study was to predict the force-velocity (F-V) properties of slow human skeletal muscle for the in vivo temperature of 37 °C from F-V measurements in type I myofibers. Specifically, to quantitatively link myofiber measurements, which must be conducted at relatively low temperatures, to in vivo properties, the temperature-dependence of contractile properties must be modeled. We estimated the kinetic parameters of a crossbridge model within 15-30 °C from F-V measurements recorded in the myofibers of one subject, extrapolated their values at 37 °C, and then predicted the in vivo shortening and lengthening F-V curves. The prediction for maximal shortening velocity was 2.2 ± 0.2 fiber lengths per second and that for saturation force during lengthening was 2.3 ± 0.2 times isometric force. These estimates agree with previously reported in vivo measurements but are substantially different than those used in muscle models for many musculoskeletal simulations. The results from this study indicate that during low levels of muscle activation when slow motor units are primarily recruited, musculoskeletal models should consider having F-V properties that reflect the contractile properties of type I myofibers.
Assuntos
Modelos Biológicos , Contração Muscular/fisiologia , Fibras Musculares de Contração Lenta/fisiologia , Miofibrilas/fisiologia , Células Cultivadas , Simulação por Computador , Humanos , Técnicas In Vitro , Estresse MecânicoRESUMO
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.