Length-dependent Activation Research Articles

Sarcomere Length Dependent Effects on Ca2+-Induced Troponin Regulation within Chemically Skinned Cardiac Muscle Fibers

Sarcomere length dependent activation (LDA) of myocardial force development is the cellular basis underlying the Frank-Starling law of the heart, but it is still elusive how sarcomere detects the SL changes and converts it into altered activation of thin filament. Among the myofilament proteins, troponin and myosin are two key components that are likely involved in the LDA. The two components are functionally linked through Ca2+ activation and cross-bridge feedback. Although the active cross-bridge feedback has been strongly implicated in LDA, there was no evidence linking sarcomere length changes and troponin regulation until our recent study showing that Ca2+-cTnC interaction in cardiac muscle can be modulated by sarcomere length through cross-bridge feedback [Biophysical Journal, 107(3), 682-93 (2014)]. In this study, we continue our efforts to understand the role of the C-domain of cTnI in LDA. Specifically, we used in situ time-resolved FRET measurements to determine how the switch region of cTnI is affected by Ca2+, sarcomere length, and cross-bridge in skinned cardiac muscle fibers. To monitor the Ca2+-induced structural transition of the switch region, skinned myocardial fibers were reconstituted with troponin complex containing FRET donor (AEDANS) modified cTnI(167C)AEDANS and acceptor (DDPM) modified cTnC(89C)DDPM. The measured FRET distance changes show that Ca2+, strong cross-bridges and sarcomere length all influence the structural transition of the switching region of cTnI within myocardial fibers. The results provide a mechanism by which sarcomere length can modulate Ca2+-troponin regulation via strong cross-bridge binding and suggest that the sarcomere length dependent cross-bridge effect plays an important role in the Frank-Starling law of the heart.

Crossbridge Recruitment Dynamics are Divergently Affected by α/β-Myosin Heavy Chain Isoforms in Cardiac Muscle Fibers Containing the Hypertrophic Cardiomyopathy Mutation (A30V) and Protein Kinase C Phosphomimetic (T203E) in Mouse Cardiac Troponin T

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.

Cardiac Length-Dependent Activation: Weak Binding Hypothesis Tested by a Computational Sarcomere Model

Experiments suggest that length changes alter activation in cardiac sarcomeres within a few milliseconds (Mateja & deTombe, 2012). Thus, the mechanism producing length-dependent activation must occur early in the contractile cycle, such as during the formation of weakly bound crossbridges. We hypothesized that a decrease in the energy required to form weak crossbridges would lead to greater probability of their formation. Such decrease in energy at longer sarcomere lengths could occur via reduction in both: (1) radial distance a crossbridge stretches to attach to actin, due to lattice spacing; (2) radial elastic crossbridge stiffness, due to influence of sarcomere length on modulators such as titin, myosin light chains, or myosin binding protein-C. We have previously developed a compact kinetic model of cardiac sarcomere dynamics (Hunter, BPS Meeting 2015). Parameters in this model were adjusted to produce the best fit with five experimental measures at constant sarcomere length (from others’ published data): (1) F-pCa relation, (2) alteration in F-pCa with addition of NEM-S1 strong crossbridges, (3) relation between kinetics of tension recovery and calcium concentration [k(TR)-pCa relation], (4) alteration in k(TR)-pCa with addition of NEM-S1, (5) shape of F-Ca loops during twitches. All five of these sets of experimental data were reasonably matched by the model using the best-fit set of parameters. Holding all other best-fit parameters constant, we then modified just the binding constant for weak crossbridges. An increased binding constant (due to lower energy to form a weak crossbridge) reproduced both of the following effects of increased sarcomere length: (1) shifted mid-point of F-pCa relation to lower [Ca++], (2) increased maximum force at saturating [Ca++]. Both of these experimental effects of increased sarcomere length are well known from published data (Dobesh, Konhilas, deTombe, 2002).

Selective phosphorylation of PKA targets after β-adrenergic receptor stimulation impairs myofilament function in Mybpc3-targeted HCM mouse model.

Hypertrophic cardiomyopathy (HCM) has been associated with reduced β-adrenergic receptor (β-AR) signalling, leading downstream to a low protein kinase A (PKA)-mediated phosphorylation. It remained undefined whether all PKA targets will be affected similarly by diminished β-AR signalling in HCM. We aimed to investigate the role of β-AR signalling on regulating myofilament and calcium handling in an HCM mouse model harbouring a gene mutation (G > A transition on the last nucleotide of exon 6) in Mybpc3 encoding cardiac myosin-binding protein C. Cardiomyocyte contractile properties and phosphorylation state were measured in left ventricular permeabilized and intact cardiomyocytes isolated from heterozygous (HET) or homozygous (KI) Mybpc3-targeted knock-in mice. Significantly higher myofilament Ca²⁺sensitivity and passive tension were detected in KI mice, which were normalized after PKA treatment. Loaded intact cardiomyocyte force-sarcomere length relation was impaired in both HET and KI mice, suggesting a reduced length-dependent activation. Unloaded cardiomyocyte function revealed an impaired myofilament contractile response to isoprenaline (ISO) in KI, whereas the calcium-handling response to ISO was maintained. This disparity was explained by an attenuated increase in cardiac troponin I (cTnI) phosphorylation in KI, whereas the increase in phospholamban (PLN) phosphorylation was maintained to wild-type values. These data provide evidence that in the KI HCM mouse model, β-AR stimulation leads to preferential PKA phosphorylation of PLN over cTnI, resulting in an impaired inotropic and lusitropic response.

Effect of exercise training and myocardial infarction on force development and contractile kinetics in isolated canine myocardium.

It is well known that moderate exercise training elicits a small increase in ventricular mass (i.e., a physiological hypertrophy) that has many beneficial effects on overall cardiac health. It is also well known that, when a myocardial infarction damages part of the heart, the remaining myocardium remodels to compensate for the loss of viable functioning myocardium. The effects of exercise training, myocardial infarction (MI), and their interaction on the contractile performance of the myocardium itself remain largely to be determined. The present study investigated the contractile properties and kinetics of right ventricular myocardium isolated from sedentary and exercise trained (10-12 wk progressively increasing treadmill running, begun 4 wk after MI induction) dogs with and without a left ventricular myocardial infarction. Exercise training increased force development, whereas MI decreased force development that was not improved by exercise training. Contractile kinetics were significantly slower in the trained dogs, whereas this impact of training was less or no longer present after MI. Length-dependent activation, both evaluated on contractile force and kinetics, was similar in all four groups. The control exercise-trained group exhibited a more positive force-frequency relationship compared with the sedentary control group while both sedentary and trained post-MI dogs had a more negative relationship. Last, the impact of the β-adrenergic receptor agonist isoproterenol resulted in a similar increase in force and acceleration of contractile kinetics in all groups. Thus, exercise training increased developed force but slowed contractile kinetics in control (noninfarcted animals), actions that were attenuated or completely absent in post-MI dogs.

Instrumentation to study myofibril mechanics from static to artificial simulations of cardiac cycle

Many causes of heart muscle diseases and skeletal muscle diseases are inherited and caused by mutations in genes of sarcomere proteins which play either a structural or contractile role in the muscle cell. Tissue samples from human hearts with mutations can be obtained but often samples are only a few milligrams and it is necessary to freeze them for storage and transportation. Myofibrils are the fundamental contractile components of the muscle cell and retain all structural elements and contractile proteins performing in contractile event; moreover viable myofibrils can be obtained from frozen tissue.•We are describing a versatile technique for measuring the contractility and its Ca2+ regulation in single myofibrils. The control of myofibril length, incubation medium and data acquisition is carried out using a digital acquisition board via computer software. Using computer control it is possible not only to measure contractile and mechanical parameters but also simulate complex protocols such as a cardiac cycle to vary length and medium independently.•This single myofibril force assay is well suited for physiological measurements. The system can be adapted to measure tension amplitude, rates of contraction and relaxation, Ca2+ dependence of these parameters in dose-response measurements, length-dependent activation, stretch response, myofibril elasticity and response to simulated cardiac cycle length changes. Our approach provides an all-round quantitative way to measure myofibrils performance and to observe the effect of mutations or posttranslational modifications. The technique has been demonstrated by the study of contraction in heart with hypertrophic or dilated cardiomyopathy mutations in sarcomere proteins.

The kinetics of cytosolic calcium in the right ventricular myocardium of guinea pigs and rats

The characteristics of tension increase and decline, as well as those of the calcium transients, have been measured in the trabeculae of the right cardiac ventricle of the guinea pig and rat in the isometric contraction mode with different preloads. Measurements were performed at different temperatures of physiological saline and the effects of inhibition of calcium removal from the cytosol mediated by Na+–Ca2+ exchange and the ATP-dependent Ca2+ pump of the sarcoplasmic reticulum (SERCA2a) were analyzed. Emergence of the “bump” phase (a phase of brief deceleration of the decay of the calcium transient) was observed in the guinea pig myocardium as the temperature was increased from 25 to 30°C; earlier observations of this phenomenon were reported only for rats. As the temperature was elevated to 35°C, the “bump” phase in the guinea pig myocardium transformed into a “plateau” phase of the calcium transient. The effect of temperature on the course of the decay of the calcium transient in the rat myocardium was negligible. In contrast, a gradual stretching of the right ventricular trabecula of the rat was accompanied by the emergence of the “bump” phase and a gradual increase of its parameters (amplitude, integral intensity, and duration), whereas preload did not exert a similar effect on the guinea pig myocardium. Selective inhibition of the reverse mode of Na+–Ca2+ exchange did not affect the characteristics of the decay of the calcium transient in guinea pig myocardium. Selective inhibition of SERCA2a in the guinea pig and rat myocardium had a significant modifying effect on the decay phase of the calcium transient and resulted in emergence of the “bump” phase or an increase in the intensity of this phase in the myocardium of these animal species. The characteristics of this phase can be used to quantify the length-dependent activation of myocardial contraction.

Cardiac Myosin-binding Protein C and Troponin-I Phosphorylation Independently Modulate Myofilament Length-dependent Activation

β-Adrenergic stimulation in heart leads to increased contractility and lusitropy via activation of protein kinase A (PKA). In the cardiac sarcomere, both cardiac myosin binding protein C (cMyBP-C) and troponin-I (cTnI) are prominent myofilament targets of PKA. Treatment of permeabilized myocardium with PKA induces enhanced myofilament length-dependent activation (LDA), the cellular basis of the Frank-Starling cardiac regulatory mechanism. It is not known, however, which of these targets mediates the altered LDA and to what extent. Here, we employed two genetic mouse models in which the three PKA sites in cMyBP-C were replaced with either phospho-mimic (DDD) or phospho-null (AAA) residues. AAA- or DDD-permeabilized myocytes (n = 12-17) were exchanged (~93%) for recombinant cTnI in which the two PKA sites were mutated to either phospho-mimic (DD) or phospho-null (AA) residues. Force-[Ca(2+)] relationships were determined at two sarcomere lengths (SL = 1.9 μm and SL = 2.3 μm). Data were fit to a modified Hill equation for each individual cell preparation at each SL. LDA was indexed as ΔEC50, the difference in [Ca(2+)] required to achieve 50% force activation at the two SLs. We found that PKA-mediated phosphorylation of cMyBP-C and cTnI each independently contribute to enhance myofilament length-dependent activation properties of the cardiac sarcomere, with relative contributions of ~67 and ~33% for cMyBP-C for cTnI, respectively. We conclude that β-adrenergic stimulation enhances the Frank-Starling regulatory mechanism predominantly via cMyBP-C PKA-mediated phosphorylation. We speculate that this molecular mechanism enhances cross-bridge formation at long SL while accelerating cross-bridge detachment and relaxation at short SLs.

LKB1/Mo25/STRAD Uniquely Impacts Sarcomeric Contractile Function and Posttranslational Modification

The myocardium undergoes extensive metabolic and energetic remodeling during the progression of cardiac disease. Central to remodeling are changes in the adenine nucleotide pool. Fluctuations in these pools can activate AMP-activated protein kinase (AMPK), the central regulator of cellular energetics. Binding of AMP to AMPK not only allosterically activates AMPK but also promotes phosphorylation of AMPK by an upstream kinase complex, LKB1/Mo25/STRAD (liver kinase B 1, mouse protein 25, STE-related adaptor protein). AMPK phosphorylation by the LKB1 complex results in a substantial increase in AMPK activity. Molecular targeting by the LKB1 complex depends on subcellular localization and transcriptional expression. Yet, little is known about the ability of the LKB1 complex to modulate targeting of AMPK after activation. Accordingly, we hypothesized that differing stoichiometric ratios of LKB1 activator complex to AMPK would uniquely impact myofilament function. Demembranated rat cardiac trabeculae were incubated with varying ratios of the LKB1 complex to AMPK or the LKB1 complex alone. After incubation, we measured the Ca2+ sensitivity of tension, rate constant for tension redevelopment, maximum tension generation, length-dependent activation, cooperativity, and sarcomeric protein phosphorylation status. We found that the Ca2+ sensitivity of tension and cross-bridge dynamics were dependent on the LKB1 complex/AMPK ratio. We also found that the LKB1 complex desensitizes and suppresses myofilament function independently of AMPK. A phospho-proteomic analysis of myofilament proteins revealed site-specific changes in cardiac Troponin I (cTnI) phosphorylation, as well as a unique distribution of cTnI phosphospecies that were dependent on the LKB1 complex/ AMPK ratio. Fibers treated with the LKB1 complex alone did not alter cTnI phosphorylation or phosphospecies distribution. However, LKB1 complex treatment independent of AMPK increased phosphorylation of myosin-binding protein C. Therefore, we conclude that the LKB1/AMPK signaling axis is able to alter muscle function through multiple mechanisms.

Length-Dependent Contractile Dynamics are Blunted Upon Ablation of Cardiac Myosin Binding Protein-C

Enhanced cardiac contractile function due to 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 role of cMyBP-C in modulating cardiac LDA is unclear. To determine the impact of cMyBP-C on LDA, we measured isometric force, myofilament Ca2+-sensitivity (pCa50) and length-dependent crossbridge (XB) contractile dynamics in skinned ventricular muscle fibers 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 fibers. pCa50 was not significantly different between WT and KO fibers 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 fibers at short SL (5.71±0.02 in WT vs. 5.80±0.01 in KO; p<0.05). The rate of force redevelopment (ktr), measured at submaximal Ca2+-activation, was significantly accelerated at short SL in WT fibers (8.74±0.43s-1at 1.9µm vs. 5.71±0.40s-1at 2.1µm, p<0.05). Furthermore, the rates of stretch-induced XB relaxation (krel) and XB recruitment (kdf) were accelerated by 32% and 70%, respectively at short SL in WT fibers. In contrast, ktr was not significantly different between both SL's in KO fibers (8.03±0.54s-1at 1.9µm vs. 8.90±0.37s-1at 2.1µm). Furthermore, KO fibers did not exhibit length-dependent differences in krel and kdf -suggesting that LDA is severely depressed in KO fibers. Collectively, our data indicate that cMyBP-C plays a central role in modulating cardiac LDA.

Exploring cardiac biophysical properties.

The heart is subject to multiple sources of stress. To maintain its normal function, and successfully overcome these stresses, heart muscle is equipped with fine-tuned regulatory mechanisms. Some of these mechanisms are inherent within the myocardium itself and are known as intrinsic mechanisms. Over a century ago, Otto Frank and Ernest Starling described an intrinsic mechanism by which the heart, even ex vivo, regulates its function on a beat-to-beat basis. According to this phenomenon, the higher the ventricular filling is, the bigger the stroke volume. Thus, the Frank-Starling law establishes a direct relationship between the diastolic and systolic function of the heart. To observe this biophysical phenomenon and to investigate it, technologic development has been a pre-requisite to scientific knowledge. It allowed for example to observe, at the cellular level, a Frank-Starling like mechanism and has been termed: Length Dependent Activation (LDA).In this review, we summarize some experimental systems that have been developed and are currently still in use to investigate cardiac biophysical properties from the whole heart down to the single myofibril. As a scientific support, investigation of the Frank-Starling mechanism will be used as a case study.

Direct Evidence for the Effect of Titin Stiffness on Thick-Filament Mechanical Properties in Stretched Muscle Sarcomeres

The titin springs of muscle sarcomeres determine tensile muscle stress. However, they run not strictly in parallel with the sarcomeric axis, being anchored to actin at Z-disks and myosin at A-bands. On tensile sarcomere strain, which stretches the titin springs, a force component arises both longitudinally and laterally. The latter could contribute to increased sarcomeric transversal stiffness, decreased lateral myofilament spacing, and length-dependent activation of stretched muscle. We aimed to quantify by AFM force-mapping the titin contribution to sarcomeric transversal stiffness in comparison to the contribution from electrostatic forces modulated by osmotic compression. Single myofibrils were isolated from skeletal muscles expressing stiff titin-isoform (rabbit psoas) or compliant titin-isoform (rabbit diaphragm). Myofibrils were placed in relaxing buffer under the MFP-3D-BIO-AFM (Asylum Research) and force curves (50x50) were performed over a region-of-interest encompassing a whole sarcomere. Measurements were done in the absence or presence (5%) of dextran, and in the non-stretched (‘slack’) or stretched state (140-150% of slack). Force mapping revealed distinct transversal stiffness patterns along the sarcomere, suggesting relatively higher Z-disk stiffness and somewhat lower A-band stiffness. A-band lateral stiffness did not differ between psoas and diaphragm sarcomeres at slack. With stretch, this stiffness was increased significantly greater in psoas than in diaphragm. Osmotic compression by 5% dextran at slack increased the A-band lateral stiffness to a degree matching (psoas) or exceeding (diaphragm) that seen after stretch in 0% dextran. The stretch-induced and compression-induced contributions to A-band lateral stiffness were additive. Thus, stiff titin contributes more to A-band stiffness than compliant titin, confirming a role for titin in lateral force generation during sarcomere stretching. The electrostatic and the titin-based lateral forces compressing the A-band lattice are of the same order of magnitude in stretched muscle.

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 1.9 μm or 2.1 μm. 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 ± 0.56 s−1 at 1.9 μm vs. 5.71 ± 0.40 s−1 at 2.1 μm, 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 ± 0.54 s−1 at 1.9 μm vs. 8.90 ± 0.37 s−1 at 2.1 μm). Similarly, KO preparations did not exhibit length-dependent changes in krel and kdf. Collectively, our data implicate cMyBP-C as an important regulator of LDA via its impact on dynamic XB behavior due to changes in SL.

Pathomechanisms in heart failure: the contractile connection.

Heart failure is a multi-factorial progressive disease in which eventually the contractile performance of the heart is insufficient to meet the demands of the body, even at rest. A distinction can be made on the basis of the cause of the disease in genetic and acquired heart failure and at the functional level between systolic and diastolic heart failure. Here the basic determinants of contractile function of myocardial cells will be reviewed and an attempt will be made to elucidate their role in the development of heart failure. The following topics are addressed: the tension generating capacity, passive tension, the rate of tension development, the rate of ATP utilisation, calcium sensitivity of tension development, phosphorylation of contractile proteins, length dependent activation and stretch activation. The reduction in contractile performance during systole can be attributed predominantly to a loss of cardiomyocytes (necrosis), myocyte disarray and a decrease in myofibrillar density all resulting in a reduction in the tension generating capacity and likely also to a mismatch between energy supply and demand of the myocardium. This leads to a decline in the ejection fraction of the heart. Diastolic dysfunction can be attributed to fibrosis and an increase in titin stiffness which result in an increase in stiffness of the ventricular wall and hampers the filling of the heart with blood during diastole. A large number of post translation modifications of regulatory sarcomeric proteins influence myocardial function by altering calcium sensitivity of tension development. It is still unclear whether in concert these influences are adaptive or maladaptive during the disease process.

The Dilated Cardiomyopathy-Causing Mutation ACTC E361G in Cardiac Muscle Myofibrils Specifically Abolishes Modulation of Ca2+ Regulation by Phosphorylation of Troponin I

Phosphorylation of troponin I by protein kinase A (PKA) reduces Ca2+ sensitivity and increases the rate of Ca2+ release from troponin C and the rate of relaxation in cardiac muscle. In vitro experiments indicate that mutations that cause dilated cardiomyopathy (DCM) uncouple this modulation, but this has not been demonstrated in an intact contractile system. Using a Ca2+-jump protocol, we measured the effect of the DCM-causing mutation ACTC E361G on the equilibrium and kinetic parameters of Ca2+ regulation of contractility in single transgenic mouse heart myofibrils. We used propranolol treatment of mice to reduce the level of troponin I and myosin binding protein C (MyBP-C) phosphorylation in their hearts before isolating the myofibrils. In nontransgenic mouse myofibrils, the Ca2+ sensitivity of force was increased, the fast relaxation phase rate constant, kREL, was reduced, and the length of the slow linear phase, tLIN, was increased when the troponin I phosphorylation level was reduced from 1.02 to 0.3 molPi/TnI (EC50 P/unP = 1.8 ± 0.2, p < 0.001). Native myofibrils from ACTC E361G transgenic mice had a 2.4-fold higher Ca2+ sensitivity than nontransgenic mouse myofibrils. Strikingly, the Ca2+ sensitivity and relaxation parameters of ACTC E361G myofibrils did not depend on the troponin I phosphorylation level (EC50 P/unP = 0.88 ± 0.17, p = 0.39). Nevertheless, modulation of the Ca2+ sensitivity of ACTC E361G myofibrils by sarcomere length or EMD57033 was indistinguishable from that of nontransgenic myofibrils. Overall, EC50 measured in different conditions varied over a 7-fold range. The time course of relaxation, as defined by tLIN and kREL, was correlated with EC50 but varied by just 2.7- and 3.3-fold, respectively. Our results confirm that troponin I phosphorylation specifically alters the Ca2+ sensitivity of isometric tension and the time course of relaxation in cardiac muscle myofibrils. Moreover, the DCM-causing mutation ACTC E361G blunts this phosphorylation-dependent response without affecting other parameters of contraction, including length-dependent activation and the response to EMD57033.

Tri-modal regulation of cardiac muscle relaxation; intracellular calcium decline, thin filament deactivation, and cross-bridge cycling kinetics.

Cardiac muscle relaxation is an essential step in the cardiac cycle. Even when the contraction of the heart is normal and forceful, a relaxation phase that is too slow will limit proper filling of the ventricles. Relaxation is too often thought of as a mere passive process that follows contraction. However, many decades of advancements in our understanding of cardiac muscle relaxation have shown it is a highly complex and well-regulated process. In this review, we will discuss three distinct events that can limit the rate of cardiac muscle relaxation: the rate of intracellular calcium decline, the rate of thin-filament de-activation, and the rate of cross-bridge cycling. Each of these processes are directly impacted by a plethora of molecular events. In addition, these three processes interact with each other, further complicating our understanding of relaxation. Each of these processes is continuously modulated by the need to couple bodily oxygen demand to cardiac output by the major cardiac physiological regulators. Length-dependent activation, frequency-dependent activation, and beta-adrenergic regulation all directly and indirectly modulate calcium decline, thin-filament deactivation, and cross-bridge kinetics. We hope to convey our conclusion that cardiac muscle relaxation is a process of intricate checks and balances, and should not be thought of as a single rate-limiting step that is regulated at a single protein level. Cardiac muscle relaxation is a system level property that requires fundamental integration of three governing systems: intracellular calcium decline, thin filament deactivation, and cross-bridge cycling kinetics.

Phosphorylation of protein kinase C sites Ser42/44 decreases Ca2+-sensitivity and blunts enhanced length-dependent activation in response to protein kinase A in human cardiomyocytes

Protein kinase C (PKC)-mediated phosphorylation of troponin I (cTnI) at Ser42/44 is increased in heart failure. While studies in rodents demonstrated that PKC-mediated Ser42/44 phosphorylation decreases maximal force and ATPase activity, PKC incubation of human cardiomyocytes did not affect maximal force. We investigated whether Ser42/44 pseudo-phosphorylation affects force development and ATPase activity using troponin exchange in human myocardium. Additionally, we studied if pseudo-phosphorylated Ser42/44 modulates length-dependent activation of force, which is regulated by protein kinase A (PKA)-mediated cTnI-Ser23/24 phosphorylation. Isometric force was measured in membrane-permeabilized cardiomyocytes exchanged with human recombinant wild-type troponin or troponin mutated at Ser42/44 or Ser23/24 into aspartic acid (D) or alanine (A) to mimic phosphorylation and dephosphorylation, respectively. In troponin-exchanged donor cardiomyocytes experiments were repeated after PKA incubation. ATPase activity was measured in troponin-exchanged cardiac muscle strips. Compared to wild-type, 42D/44D decreased Ca2+-sensitivity without affecting maximal force in failing and donor cardiomyocytes. In donor myocardium, 42D/44D did not affect maximal ATPase activity or tension cost. Interestingly, 42D/44D blunted the length-dependent increase in Ca2+-sensitivity induced upon PKA-mediated phosphorylation. Since the drop in Ca2+-sensitivity at physiological Ca2+-concentrations is relatively large phosphorylation of Ser42/44 may result in a decrease of force and associated ATP utilization in the human heart.

Cardiac thin filament regulation and the Frank-Starling mechanism.

The heart has an intrinsic ability to increase systolic force in response to a rise in ventricular filling (the Frank–Starling law of the heart). It is widely accepted that the length dependence of myocardial activation underlies the Frank–Starling law of the heart. Recent advances in muscle physiology have enabled the identification of the factors involved in length-dependent activation, viz., titin (connectin)-based interfilament lattice spacing reduction and thin filament “on–off” regulation, with the former triggering length-dependent activation and the latter determining the number of myosin molecules recruited to thin filaments. Patients with a failing heart have demonstrated reduced exercise tolerance at least in part via depression of the Frank–Starling mechanism. Recent studies revealed that various mutations occur in the thin filament regulatory proteins, such as troponin, in the ventricular muscle of failing hearts, which consequently alter the Frank–Starling mechanism. In this article, we review the molecular mechanisms of length-dependent activation, and the influence of troponin mutations on the phenomenon.

Novel approaches to determine contractile function of the isolated adult zebrafish ventricular cardiac myocyte

The zebrafish (Danio rerio) has been used extensively in cardiovascular biology, but mainly in the study of heart development. The relative ease of its genetic manipulation may indicate the suitability of this species as a cost-effective model system for the study of cardiac contractile biology. However, whether the zebrafish heart is an appropriate model system for investigations pertaining to mammalian cardiac contractile structure-function relationships remains to be resolved. Myocytes were isolated from adult zebrafish hearts by enzymatic digestion, attached to carbon rods, and twitch force and intracellular Ca(2+) were measured. We observed the modulation of twitch force, but not of intracellular Ca(2+), by both extracellular [Ca(2+)] and sarcomere length. In permeabilized cells/myofibrils, we found robust myofilament length-dependent activation. Moreover, modulation of myofilament activation-relaxation and force redevelopment kinetics by varied Ca(2+) activation levels resembled that found previously in mammalian myofilaments. We conclude that the zebrafish is a valid model system for the study of cardiac contractile structure-function relationships.

Length-dependent activation is modulated by cardiac troponin I bisphosphorylation at Ser23 and Ser24 but not by Thr143 phosphorylation

Frank-Starling's law reflects the ability of the heart to adjust the force of its contraction to changes in ventricular filling, a property based on length-dependent myofilament activation (LDA). The threonine at amino acid 143 of cardiac troponin I (cTnI) is prerequisite for the length-dependent increase in Ca(2+) sensitivity. Thr143 is a known target of protein kinase C (PKC) whose activity is increased in cardiac disease. Thr143 phosphorylation may modulate length-dependent myofilament activation in failing hearts. Therefore, we investigated if pseudo-phosphorylation at Thr143 modulates length dependence of force using troponin exchange experiments in human cardiomyocytes. In addition, we studied effects of protein kinase A (PKA)-mediated cTnI phosphorylation at Ser23/24, which has been reported to modulate LDA. Isometric force was measured at various Ca(2+) concentrations in membrane-permeabilized cardiomyocytes exchanged with recombinant wild-type (WT) troponin or troponin mutated at the PKC site Thr143 or Ser23/24 into aspartic acid (D) or alanine (A) to mimic phosphorylation and dephosphorylation, respectively. In troponin-exchanged donor cardiomyocytes experiments were repeated after incubation with exogenous PKA. Pseudo-phosphorylation of Thr143 increased myofilament Ca(2+) sensitivity compared with WT without affecting LDA in failing and donor cardiomyocytes. Subsequent PKA treatment enhanced the length-dependent shift in Ca(2+) sensitivity after WT and 143D exchange. Exchange with Ser23/24 variants demonstrated that pseudo-phosphorylation of both Ser23 and Ser24 is needed to enhance the length-dependent increase in Ca(2+) sensitivity. cTnI pseudo-phosphorylation did not alter length-dependent changes in maximal force. Thus phosphorylation at Thr143 enhances myofilament Ca(2+) sensitivity without affecting LDA, while Ser23/24 bisphosphorylation is needed to enhance the length-dependent increase in myofilament Ca(2+) sensitivity.